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The Use of Stem Cells in Burn Wound Healing: A Review – Hindawi

By daniellenierenberg

Burn wound healing involves a series of complex processes which are subject to intensive investigations to improve the outcomes, in particular, the healing time and the quality of the scar. Burn injuries, especially severe ones, are proving to have devastating effects on the affected patients. Stem cells have been recently applied in the field to promote superior healing of the wounds. Not only have stem cells been shown to promote better and faster healing of the burn wounds, but also they have decreased the inflammation levels with less scar progression and fibrosis. This review aims to highlight the beneficial therapeutic effect of stem cells in burn wound healing and to discuss the involved pathways and signaling molecules. The review covers various types of burn wound healing like skin and corneal burns, along with the alternative recent therapies being studied in the field of burn wound healing. The current reflection of the attitudes of people regarding the use of stem cells in burn wound healing is also stated.

The use of stem cell therapy is the yet to be discovered gold mine of science. A myriad of studies using stem cells are being done with promising results in various fields ranging from oncologic and hematologic diseases to organ transplants and wound healing. In the field of wound healing, the use of different types of stem cells has been reported for different types of wounds [13]. Burn wounds were of special interest due to the large number of cases of burns encountered nowadays, especially in the Middle Eastern Region and specifically in those areas with armed conflicts. Burn wounds have proven to be capable of having a devastating effect both functionally and cosmetically, necessitating the search for a better and more efficient cure. Being a very hot topic in the present field of research with constant studies and updates necessitated an updated review that encompasses the recent advances in stem cell therapy for burn wound healing in addition to relevant experimental studies. The literature was searched using the key words burn, stem cells, and wound healing. CINAHL, PubMed, EMBASE, and Medline were used as search engines to broaden the resources. The studies reported were not limited neither to humans nor by language and were mostly on animals unless otherwise specified. They are mostly reported in a chronological order of their publication dates, except when found relevant to group and mentioning some related studies consecutively.

Stem cells are undifferentiated pluripotential cells that are capable of producing other types of cells, including new stem cells identical to mother cells [4]. Stem cells can be of embryonal origin or adult origin, depending on the type of tissue they are derived from [4]. Embryonal stem cells are derived from either embryonal tissue or from germ cells in adults [4]. On the other hand, adult stem cells are derived from adult tissues of different organs, especially those with a high turnover rate such as intestines and bone marrow [4].

Stem cells have been implicated in the healing of wounds in general. However, the methods of application of the stem cells in burn wound healing are diverse, including topical application, local injection, intravenous or systemic injection, and dermal or carrier application. Several studies have shown the efficacy of stem cells in promoting faster and superior wound healing. Alexaki et al. [5] successfully used adipose derived mesenchymal stem cells in wound healing in mice and compared their effect with dermal fibroblasts. The application of stem cells in wounds promoted more efficient reepithelialization by their proliferative effect on keratinocytes [5]. Moreover, this effect of stem cells was found to be mediated by keratinocyte growth factor-1 (KGF-1) and platelet derived growth factor-BB (PDGF-BB) [5]. Amniotic fluid derived stem cells have also been used in wound healing. Skardal et al. [6] tested the effect of amniotic fluid derived stem cells in wound healing in a mouse model. Wound closure, reepithelialization, and angiogenesis were more rapid in mice treated with the stem cells in comparison to those treated with fibrin collagen gel only [6]. Additionally, stem cells did not integrate permanently in the tissue, thus, suggesting that their effect is due to released factors and not by direct interaction [6]. Additionally, bone marrow derived mesenchymal stem cells have also been used in wound healing. Leonardi et al. [7] utilized bone marrow derived stem cells in artificial dermal substitutes to promote wound healing. These stem cells were shown to increase vascular density in the wounds along with the rate of reepithelialization [7]. A study by Zhang et al. [8] examined the effect of activin signaling on the homing of stem cells to wound sites. It was also found that JNK and ERK signaling pathways were involved in activin signaling and eventually the homing of stem cells [8].

Concerning the physiology by which stem cells enhance the process of burn wound healing, several studies have been reported. Mansilla et al. [9] found evidence of cells in the bloodstream with identical phenotypes to mesenchymal bone marrow stem cells after acute large skin burns. Hence, it was concluded that these stem cells may have a role in promoting wound healing in burns. In a similar study, Fox et al. [10] reported increased levels of bone marrow derived endothelial progenitor cells in burn patients. These levels were proportional to the extent of the burn. The study also showed increased levels of angiogenic cytokines which may be involved in the signaling pathway for promoting the release of bone marrow derived stem cells. Focusing on the role of cytokines in burn wound healing, Payne et al. [11] studied the role of amnion derived cellular cytokine solution. In the study, Payne et al. used amnion derived multipotent progenitor cells to harvest cytokines and apply them in burn wound healing. Amnion derived cellular cytokine solution showed statistically significant improvement in the epithelialization of the burn wounds and the appearance of hair growth compared to controls [11]. In addition, the results demonstrated a faster epithelialization in burn wounds with increased frequency of application of the cytokines, further strengthening the role of stem cell derived cytokines in burn wound healing [11]. Furthermore, Foresta et al. [12] reported a positive linear correlation between endothelial progenitor cell blood levels and the total body surface area burnt. There was an increased level of endothelial progenitor cells in the bloodstream after escharectomy, posing a possible role of escharectomy in burn wound healing [12]. Additionally, stem cells could work by the release of bioactive peptides as proposed by Cabrera et al. [13] in their study where they showed that stem cells have an active role in burn wound healing by producing bioactive peptides, such as thymosin 4 and others.

More recent studies have also highlighted the role of stem cells in the process of wound healing in general and burn wound healing in specific. Koenen et al. [14] isolated acute wound fluids and chronic wound fluids and compared their effects on adipose tissue derived stem cell function in wounds. They came to the conclusion that acute wound fluids had a positive effect on the proliferation of adipose derived stem cells in wounds [14] while chronic wound fluids had a negative effect; the mentioned findings might explain the insufficient and slow healing process in chronic wounds due to a stem cell deficiency [14]. Furthermore, stem cells have been shown to decrease dermal fibrosis development in burn wound healing in mice [15]. Wu et al. performed a series of experiments which showed that bone marrow derived mesenchymal stem cells stimulate the formation of a basket weave organization of collagen in bleomycin treated skin, similar to normal skin [15]. Additionally, stem cell treatment of the skin decreased markers of myofibroblasts and downregulated type I collagen, leading to a decrease in the fibrosis that could have occurred to the skin [15]. Consequently, the role of stem cells in decreasing bleomycin induced fibrosis may be extrapolated to decrease fibrosis in burn wounds and improve their healing with less scar formation. Moreover, Lough et al. [16] performed a study in mice which showed a role for intestine derived human alpha defensin 5 in enhancing wound healing and decreasing its bacterial load. It induces leucine-rich repeat-containing G-protein-coupled receptors which are markers of adult epithelial stem cells both in skin and intestine [16]. Also implicated in the role of stem cells in burn wound healing is the role of SDF-1/CXCR4 signaling; Ding et al. [17] used interferon a2b in patients with burn wounds to suppress SDF-1/CXCR4 signaling. They found out that the decreased levels of signaling lead to better remodeling of hypertrophic scarring in the wounds [17]. Additional studies on the CXCR4 signaling pathway were done by Yang et al. [18] on irradiated mice. The mice having an overexpression of CXCR4, a receptor involved in the homing and migration of several stem cell types, showed an accelerated wound healing time [18]. Furthermore, Hu et al. [19] injected bone marrow derived mesenchymal stem cells into mice and studied the effect of blocking CXCR4 receptors. They found out that blocking the CXCL12/CXCR4 pathway, leading to activation of CXCR4, caused delayed wound closure in inflicted burn wounds. Moreover, CXCL12 levels were elevated in the burn wound one week after injury [19]. Hence, stem cells seem to be attracted to and attach to the burnt injury site by the CXCL12/CXCR4 pathway involving the CXCR4 receptors [18, 19]. The role of the ligand for the CXCR4 receptors, stromal cell derived factor-1 alpha (SDF-1a), has also been studied. L et al. [20] performed a study on the role of SDF-1a and its relation to the expression of miR-27b. It was found that SDF-1a expression was suppressed by direct binding of the miRNA to its 39UTR site [20]. As expected, miRNA expression was suppressed in wounds hence allowing better SDF-1a signaling and more homing of stem cells to the burn wounds [20]. In particular, miR-27b was found to be involved in the burn margins of wounds and in the mobilization of stem cells to the epidermis [20]. Chen et al. [21] performed experiments using porcine acellular dermal matrix on rats with inflicted 2nd degree burns. It stimulated collagen synthesis and stem cell proliferation and differentiation; porcine acellular matrix treated rats had a better and faster healing of the wounds.

Thus, in brief, the process of burn wound healing involves different types of growth factors, receptors, and cytokines. These factors are related to stem cell homing, differentiation, and proliferation. Additionally, when applied to burn wounds, they led to a better and faster healing process.

The use of stem cells for burn wound healing, as reported in the literature, dates back to 2003 with Shumakov et al. [22]. Shumakov et al. were the first to use mesenchymal bone marrow derived stem cells (BMSC) in burn wound healing and compared them to embryonic fibroblasts [22]. The experiments were done on rats where mesenchymal bone marrow derived stem cells were applied to wounds showing decreased cell infiltration of the wound and an accelerated formation of new vessels and granulation tissue in comparison with embryonic fibroblasts and controls (burn wounds with no transplanted cells) [22]. Hence, this study marked a new era in the research of burn wound healing by being the first to test the use of stem cells in this complex process. Following this, a study by Chunmeng et al. [23] found that systemic transplantation of dermis derived multipotent cells promoted the healing of wounds in irradiated rats compared to controls with no transplantation, noting that topical transplantation of the cells had no superior effect. In 2004, Rasulov et al. [24] were the first to report using bone marrow mesenchymal stem cells in humans; a female patient with extensive skin burns (IIIB 30% of body surface area) had the stem cells applied onto the burn surface. The application of stem cells caused faster wound healing and active neoangiogenesis [24]. Another study done by Rasulov et al. on rats also showed the superiority of stem cells in burn wound healing [25]. In the rat study, the application of mesenchymal stem cells on burns reduced cell infiltration, improved neoangeogenesis, and reduced the formation of granulation tissue [25]. The aforementioned conditions created a better medium for wound healing in burns. In a similar effort, Liu et al. [26] performed experiments on pigs where they applied collagen scaffolds with seeded mesenchymal stem cells onto the surface of inflicted burns; the latter were found to induce better burn wound healing with less contraction and better vascularization and keratinization. Moreover, in human cutaneous radiation wounds, Lataillade et al. [27, 28] reported two cases where stem cells where used to aid in burn wound healing. Mesenchymal stem cells were applied, in addition to surgical excision, flaps, and grafts, to burn wounds of cutaneous radiation patients. In these patients, the application of the mesenchymal stem cells decreased the levels of inflammation and promoted a better healing [27, 28]. Further on the role of stem cells in irradiated skin were the studies conducted by Dong et al. [29, 30], where they additionally inserted a vector of human beta defensin 2 into the stem cells. The mentioned studies showed a positive role for stem cells transfected with beta defensin 2 in burn wound healing by exhibiting antibacterial properties in infected burn wounds [29, 30]. In a similar experiment, Ha et al. [31] transfected mesenchymal stem cells with vectors of hepatocyte growth factor. The experiment, done on rats, compared the wound healing of a partial thickness burn treated with stem cells alone or stem cells transfected with hepatocyte growth factor [31]. The group treated with the transfected stem cells showed a significantly larger range of reepidermalization starting the first week, along with a thicker epidermis and lower content of collagen I at 3 weeks after burn [31]. In the same year (2010), Agay et al. [32] performed experimental studies by inflicting pigs with cutaneous radiation and studying the role of stem cells in the healing of the wounds. Intradermal mesenchymal stem cell injections were given locally in the affected area. They led to the accumulation of lymphocytes in the wound with better vascularization compared to controls (pigs with no injections of mesenchymal stem cells) [32]. Later on, Riccobono et al. [33] studied, in another experiment, the role of adipose tissue derived stem cells in the treatment of cutaneous radiation. Autologous, allogeneic, and acellular (empty, control) vehicles of adipose derived stem cells were grafted onto the burn wound areas [33]. Autologous but not allogeneic adipose derived stem cells were found to promote superior burn wound healing with no necrosis and decreased pain [33].

Aside to direct stem cell application to burn wounds, Kinoshita et al. [34] inflicted cutaneous radiation wounds to pigs and used expanders with and without basic fibroblast growth factor to determine their effect on burn wound healing. The group with basic fibroblast growth factor and expander showed greater proliferation of the dermis and epidermis along with increased neoangiogenesis [34]. Thus, basic fibroblast growth factor, which is known to promote the proliferation of mesenchymal stem cells, improved burn wound healing [34, 35].

In 2010, Yan et al. [36] studied the efficacy of porcine bone marrow derived mesenchymal stem cells combined with skin derived keratinocytes, both infected with recombinant retrovirus expressing human (h) platelet derived growth factor-A, in the healing of irradiated skin. The cells were loaded onto a cultured cutaneous substitute and compared their effect on healing with a cell-free cultured cutaneous substitute [36]. The substitute with cells stimulated faster healing, epithelialization, angiogenesis, and better granulation of the burn wound [36]. In another experiment, Collawn et al. [37] inflicted laser burn wounds to organotypic raft cultures. The burn wounds were treated with dermal grafts with and without adipose derived stromal cells [37]. The adipose derived stromal cell-containing grafts showed complete healing of the epidermis after two days, whereas the cell-free grafts still had areas of injury; hence, those stem cells had a role in promoting faster healing of the burnt areas [37]. More on cutaneous radiation treatment came from Xia et al. [38] who transfected human vascular endothelial growth factor 165 and human beta defensin 3 into bone marrow derived mesenchymal stem cells and used the cells to treat irradiated skin. The stem cell treated area, in comparison with cell-free controls, showed shorter healing times with better granulation and collagen deposition [38]. Additionally, Xue et al. [39] examined the effect of human mesenchymal stem cells in mouse models. Mice with inflicted burn wounds were injected locally, in the burn area, with the stem cells (controls injected cell-free injections) [39]. Wound healing was significantly faster when stem cells were included in the injection with an increased and denser neoangiogenesis [39]. Stem cell injections also had a role in resuming activity and regaining body weight more rapidly [39]. Similarly, Mansilla et al. [40] used mesenchymal stem cells in burn wound healing in pigs through an acellular dermal matrix embedded with anti-CD44 antibodies to promote homing and attachment of the stem cells [40]. This study concluded that the use of these dermal matrices with stem cells not only promoted better healing of the burn wound, but also stimulated the formation of hair follicles and regeneration of muscles and ribs [40].

Concerning stem cells from human umbilical cords, Liu et al. [41] studied the effect of human umbilical cord derived mesenchymal stem cells in the healing of severe burns inflicted in rats. The stem cells were intravenously injected into the affected rats [41]. Liu et al. found that the injection of the stem cells into the rats accelerated the wound healing compared to controls, decreased the count of inflammatory cells, downregulated interleukins 1 and 6, and increased the levels of interleukin 10 and TSG-6 [41]. Moreover, stem cell injected rats had increased neovascularization and VEGF levels [41]. Not only do stem cells promote faster wound healing in burns, but also they prevent the progression of burn injuries as showed by Singer et al. [42]. The latter performed an experiment while inflicting thermal burns to rats, with several rectangular burns on each rat separated by unburned interspaces [42]. Some of the rats received tail vein injections of mesenchymal stem cells, while others received saline injections [42]. After 7 days, all of the unburned spaces in the controls were necrotic [42]. However, 20% of the unburned spaces in rats with stem cells injections did not necrose [42]. Consequently, stem cells were also shown to play a possible role in the prevention of progression of burn injuries. Furthermore, in a study by Xu et al. [43], applying autologous bone marrow derived mesenchymal stem cells to grafted burn wounds, they demonstrated decreased contraction of the grafts.

In 2014, Yang et al. [44] attempted to integrate mesenchymal stem cells with fibrin glue into the dressing of burn wounds. They inflicted scald wounds on the back of rats and applied dressing with fibrin glue and stem cells in one group, fibrin glue only in the second, and no intervention in the third [44]. One month later, the treatment group with fibrin glue and stem cells showed significantly faster healing than the other two; moreover, this group had more proliferation of sebaceous glands and the appearance of hair follicle-like structures which were not present in the other groups [44]. In another experiment, Lough et al. [45] isolated leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6+) epithelial stem cells from the adnexal compartment of the skin of mice. They injected the harvested stem cells locally into inflicted burn wounds [45]. The wounds injected with stem cells showed a better healing along with increased vascular endothelial growth factor, platelet derived growth factor, and epidermal growth factor levels [45]. Stem cell injection also promoted the formation of nascent hair follicles and better neoangiogenesis in the wounds of the affected mice [45].

On the other hand, it is very pertinent to report another study in 2014 by Loder et al. [46] where they also tested the effect of adipose derived stem cells in the treatment of burns. The mice with inflicted burns that received stem cells injections showed no significant difference in comparison to controls (received saline injections) with respect to proliferation and vascularization [46]. Nevertheless, the role of stem cells in burn wound healing is a dynamic field and still under extensive research.

Another area of particular interest in the field of burn wound healing is the chemical burns of the cornea. In the year 2000, Dua and Azuara-Blanco [47] used autologous limbal stem cells for ocular surface reconstruction of the contralateral eye. It resulted in the formation of a better corneal surface with significant improvement in the vision and symptoms of the patients [47]. Several other experiments and trials using limbal stem cells showed similar results in inducing improvement of corneal healing and decreased neovascularization in both human (adults and children) and animal subjects [4853]. In 2007, Oh et al. [54] studied the therapeutic effects of mesenchymal stem cells on corneas with chemical burns. They reported that mesenchymal stem cell media and mesenchymal stem cell culture media (without the stem cells) reduced the inflammation and promoted neovascularization of the corneas [54]. They were also found to reduce the infiltration of CD4 cells, as well as IL-6, IL-10, and TGF-B1 levels. It is to be noted that the direct application of the stem cells provided superior results in the healing process in comparison with the stem cell culture media [54]. Another study by Ye et al. [55] utilized cyclophosphamide to suppress inflammatory reactions and the release of bone marrow stem cells into circulation. In this study, rabbits were inflicted with corneal alkali injuries. It was found that rabbits with an unsuppressed bone marrow had significantly greater reepithelialization of the corneas with clearer surfaces [55]. Thus, this study revealed the role of bone marrow cells in enhancing the healing of corneal chemical wounds. Furthermore, Sel et al. [56] inflicted alkali wounds on the corneal surfaces of mice and treated the corneas with bone marrow derived stem cells, CD117+ cells, or medium only as control. Reepithelialization of the wounds in the treatment groups was significantly faster than the control, with no difference in corneal transparency. Stem cells and CD117+ cells were absent from corneas after healing, thus suggesting that soluble factors may be responsible for the effect of the applied cells [56]. In a different study by Rama et al. [57], limbal stem cells were cultured on fibrin and used in corneal burns; not only did stem cells promote a better healing but also they had maintained a superior healing at a follow-up of 10 years later. Several other studies showed comparable results where mesenchymal derived or adipose derived stem cells promoted faster recovery of the corneal epithelium and decreased neovascularization, inflammation, and oxidative injury; moreover, stem cells stimulated the formation of clearer cornea media in some experiments [5861]. Additionally, Basu et al. [6264], in 2011 and 2012, reported a series of studies concerning the use of limbal stem cells in corneal burn wound healing. In the first study, Basu et al. [62] used limbal stem cells in corneal burn wound healing and followed them by penetrating keratoplasty procedures. Good results were observed but they were not compared to controls. However, in the second study, Basu et al. [63] observed that 66% of patients who failed primary procedures of corneal repair and who were subjected to a secondary limbal stem cell transplant on the affected cornea had successful improvement of the corneal surface with no neovascularization at a follow-up of two years. Later on, Sangwan et al. [64] used limbal biopsies from unaffected eyes and cultured them on amniotic membranes as substrates. Similar results to previous experiments were obtained with avascular epithelialization of the new corneal surfaces [64]. Furthermore and as demonstrated by Huang et al. [65], the use of allograft transplants of limbal stem cells in corneal burn wound healing also resulted in improved avascular corneal healing without the need for systemic immunosuppression. Pellegrini et al. [66] studied the biological factors that affected the stem cells role in corneal burn wound healing; the accurate number of stem cells used expressing high levels of the p63 transcription factor was shown to have important influence.

Stem cells do seem to have a very promising role in the treatment of burn wounds; however, other therapies are being developed to improve the treatment. For example, Klinger et al. [67] used fat injections in severe burn wounds as a trial to improve burn wound healing in humans. They did get results showing scar improvement and enhancement of tissue regeneration, but their study was limited to a small population [67]. In other studies, Auxenfans et al. [68] investigated the role of keratinocytes in improving wound healing in burns. They reported that keratinocytes induced a more rapid burn wound healing [68]. On the other hand, stromal vascular fraction has been also shown to play a possible role in enhancing burn wound healing [69]. Atalay et al. used isolated stromal vascular fraction in burn wound healing. It stimulated an increase in vascular endothelial growth factor and reduced the inflammation with an improved fibroblastic activity [69]. Additionally, Hussein et al. [70] studied the effect of Botox injections on burn wounds healing and found that Botox increased fibroblasts, TGF-B, and TNF-alpha levels and decreased inflammation, thus improving burn wound healing. Another recent study by Zhang et al. [71] showed a beneficial effect of heat shock protein 90 alpha on burn wound healing. It promoted faster healing and less inflammation. In addition, several other studies have examined the effects of different factors and substances such as curcumin, mast cell chymase, and phenytoin with hypericin on burn wound healing with promising results and better wound healing [7274].

Stem cells are commonly derived either from bone marrow, umbilical cord, adipose tissue, or skin. Natesan et al. [75] have even used debrided skin from severe burns as a source of stem cells for wound healing and regeneration. Hence, the adipose tissue that is discarded from burn wound debridement may now be of use for better wound healing. In addition, Natesan et al. [76], in another study, used isolated stem cells from debrided skin with fibrin and collagen based scaffolds. The dermal equivalents, created in the study, decreased wound contraction leading to a better matrix deposition and epithelialization [76]. Along the same line, van der Veen et al. [77] isolated mesenchymal stem cells from excised burn wound eschar. These stem cells showed similar abilities to adipose derived stem cells in differentiating into osteocytes, chondroblasts, and adipocytes [77].

A relatively recent approach by Li et al. [78] studied the role of electric fields in the migration of stem cells. They proved that epithelial stem cells migrate to the cathode in an induced electric field, knowing that endogenous electric fields exist naturally in wounds [78]. The migration of the stem cells was found to be proportional to the strength of the electric field and its duration, with the involvement of epidermal growth factor receptor and mitogen activated protein kinase-PI3K [78]. Hence, in addition to the use of stem cells in burn wounds, electric fields can be applied to the wounds to better direct their migration [78].

The role of stem cells in wound healing has been shown to be performed through several pathways, such as JNK and ERK59, and with the involvement of different factors and mediators, such as KGF-1 and PDGF-BB [5, 8]. Additionally, this role could also be carried out by the released factors and not only by direct integration of the stem cells into the wound scaffold or matrix [6].

Stem cells in burn wound healing have been found to follow the same mechanisms. The increased levels of stem cells in burn wounds suggested a possible enhancing role in aiding in the healing process [9, 10, 12, 13]. However, a lack of consistency of the outcome was documented. Different experiments may have used different amounts of purified stem cells, or stem cells at different stages of replication or differentiation in vitro, leading to what may seem different results. In brief, this review depicted the improved healing with stem cells qualitatively rather than quantitatively. To really demonstrate the value of different stem cells in the process of burn wound healing, more studies need to be done under optimal and well controlled conditions, aiming to measure a quantifiable improvement. Additionally, the excess use of stem cells may lead to unwanted results, such as increased fibrosis and thicker healed epithelium. Whether the effect is observed as a result of direct stem cell proliferation, or other induced substances and cells, needs to be studied in the future. Moreover, the role of cytokines released by stem cells along with bioactive peptides such as thymosin 4 has been documented to mediate the beneficial effect of the stem cell application in burn wound healing. Further data refer the superior healing probably not to the direct integration of the stem cells into the wound [11, 13]. Acute wound fluids were also shown to have a role in promoting faster healing of burn wounds, similarly reinforcing the role of mediators released by stem cells. Additionally, human alpha defensin 5 and the CXCL12/CXCR4 pathway with its signal SDF-1a were found to be inducers of stem cells in burn wounds [16, 18, 20].

In addition, stem cells have been shown to decrease cell infiltration, wound contraction, fibrosis, scar progression, and inflammation of burn wounds. Moreover, they have been found to promote faster burn wound healing and angiogenesis along with better granulation and the formation of hair follicles and sebaceous glands [15, 2245]. The studies reviewed showed positive results in both animal experiments and human trials, both in partial and full thickness injury burns. In addition, different ways of stem cell application have been used ranging from using stem cell scaffolds to systemic and intradermal injection [23, 25, 32]. Furthermore, the sources of stem cells used are multiple. They are derived from bone marrow, dermis, adipose tissue, and umbilical cords, among others [22, 23, 33, 41]. Stem cells have proved to be efficient not only in skin burns but also in corneal chemical burns, thus increasing the multiplicity of their use [5461].

Patients with burn wounds, especially those severely injured, tend to have lower quality of life [79]. The injury they suffer is not only physical but also psychological, affecting their jobs and relations with other people, especially their families [80, 81]. With the advance of burn wound treatment with time, patients self-esteem and quality of life have been improving [82, 83]. The hope is that the use of stem cells will open up a new arena of possibilities to improve the wound healing in burn patients, allowing patients to have faster healing, better scars, and a higher quality of life.

Stem cells have attracted many controversial public opinions over time. Many people argue that embryonic stem cell harvesting would be done by killing embryos which would be unethical [84]. Others would argue that even if embryos are used for stem cell research, it is not wrong. However, the path that this may lead to would be wrong such as embryo production for research purposes [84]. The public view towards the therapeutic use of stem cells has become more tolerant over time [85]. The role of educating people about the colossal potential for the use of stem cell has thus proven beneficial. People are now more educated about the different sources of stem cells and have become supportive of their use [85]. Regarding the acceptance of stem cells as an efficient therapy for burn wound healing in specific, a study done by Clover et al. [86] showed a very positive opinion. The biggest majority of people were willing to accept autologous stem cells, though a big percentage was also welcoming the idea of using allogeneic stem cells. These percentages did not differ between the use of stem cells for burn wounds or for the treatment of other diseases such as diabetes or Parkinsons [86].

In brief, the use of stem cells in burn wound healing appears to be very promising. While most studies were performed on animals, the application to humans is yet at its start. Hence, what is needed is more studies. Additionally, the signaling pathways followed by stem cells involved in the burn wound healing along with their factors and signals constitute a very dynamic and promising research field.

The authors declare that there is no conflict of interests regarding the publication of this paper.

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Kaytlyn Gerbin is blazing trails in cell science and as an ultrarunner who has conquered Mount Rainier – GeekWire

By daniellenierenberg

Kaytlyn Gerbin, left, runs the Wonderland Trail around Mount Rainier. She completed the 93-mile loop in just under 19 hours. Her friend Tara Fraga helped with pacing between miles 30-55. (Ryan Thrower Photo)

When Kaytlyn Gerbin moved to Seattle 10 years ago to attend graduate school at the University of Washington, a friend took her to Kerry Park in the Queen Anne neighborhood on her first visit. The celebrated viewpoint offered Gerbin a glimpse of Mount Rainier that ignited an ongoing passion.

At the time, I had absolutely no idea there was a trail all the way around it, and didnt know the first thing that went into climbing to the summit or running even a few miles on the trails, Gerbin said. Since then, Ive climbed Rainier 10 times, and spent countless hours on the mountain and trails in that park.

Along with her drive to get to know Washington states most famous landmark more intimately, Gerbin achieved her PhD in bioengineering at UW, where her research was focused on the therapeutic and regenerative potential of cardiac cells. For the past four years shes been a scientist at Allen Institute for Cell Science, where she studies stem cells and cardiomyocytes, or cardiac muscle cells.

Our latest Geek of the Week, Gerbin is an accomplished ultrarunner, and she now knows a lot more about that trail that encircles Mount Rainier.

With COVID-19 lockdowns impacting her international race season last summer, Gerbin, a sponsored athlete for The North Face, went after the fastest known time, or FKT, for a run around the Wonderland Trail. Together with teammate Dylan Bowman of Portland and a small crew of local filmmakers, they made Summer of Wonder, a short film about the experience, which you can watch in full here:

The average thru-hiker takes 10-14 days to complete the 93-mile Wonderland Trail, with its 24,000 feet of elevation gain. Gerbin did it in 18 hours, 41 minutes, 53 seconds, and the film is a breathtaking look at her endurance feat.

Gerbins passion for running started with 3-mile commutes back and forth between her apartment, her research lab, and campus during grad school. Eventually she started trail running,essentially as a life hack to see if she could squeeze a five-day backpacking route into a weekend between experiments.

It turned out I was actually pretty good at that, and that opened up opportunities to start racing at some of the most competitive trail races in the U.S. and Europe, Gerbin said.

Shes since raced with Team USA at the Trail World Championships, reached the podium at the iconic Western States 100, and won races such as the Canary Islands Transgrancanaria and Cascade Crest 100 in Washington. She also still holds the womens self-supported FKT for the Rainier Infinity Loop (set in 2019), which combines the Wonderland Trail with two summits and descents of Mount Rainier.

Her preferred racing distance is anything between 50-100 miles long, the more elevation gain and technical the trail, the better. During peak training, Gerbin is usually hitting between 70-90 miles with over 20,000 feet of elevation gain each week. She calls the Pacific Northwest the best outdoor playground there is.

Although I love running fast, Im also really excited about pushing myself on more challenging terrain. So many of my other FKT goals and route ideas are along these lines, with more technical traveling than actual running, she said.

COVID permitting, her highest race priority this year is Ultra Trail du Mont Blanc, which is the most competitive world-stage for ultrarunning, at the end of August. The race circumnavigates Mont Blanc, passing through France, Italy, and Switzerland and covering around 105 miles and 33,000 feet of elevation gain.

While Gerbins experience as a scientist does inform her appreciation for what shes putting her body through during ultrarunning, shes equally passionate in the lab. At the Allen Institute shes seeking answers to broad questions about how cells work, including how single cells and all of their components are integrated into a functional system, while using imaging to build predictive models of cell behavior.

I get the opportunity to work with a multidisciplinary team of badass scientists, biologists, and engineers on really cool problems in cell biology, she said.

Learn more about our latest Geek of the Week, Kaytlyn Gerbin:

What do you do, and why do you do it? Science and ultrarunning for me have always come down to problem solving.

As a scientist, problem solving is inherent to experimental design, data analysis, and interpreting results. By asking hard questions, Im interested in pushing the field of cell biology forward, and challenging the current way of thinking.

As an ultrarunner, its a different kind of problem solving, but I lean on the same mindset to figure out how to push my athletic limits further and faster.

One thing that always amazes me is how adaptable the human body is. My training in cell science gives me context for how all of these stressors and inputs were putting on our bodies are fundamentally happening at the single cell level, and it keeps me thinking about the cells response to external cues in my research.

Whats the single most important thing people should know about your field? Yes, I do think about science and when Im running, and no, I do not geek out on heart rate monitors and training zones and all those numbers when Im running.

Where do you find your inspiration? Im inspired by brilliant women that are pushing whats possible in both science and in sports. I think we often set boundaries for ourselves about what we think is possible, without ever letting ourselves really hit that limit. Im inspired by women who set bold goals and bring others up and along for the ride, redefining whats possible.

Whats the one piece of technology you couldnt live without, and why? My Garmin 935. I use this watch daily to track miles run, elevation gain, etc. The battery life has lasted me for 100 miles of running and ~24 hrs, but its small enough to wear every day.

Whats your workspace like, and why does it work for you? Prior to 2020, I was splitting my time between the tissue culture hood (passaging cells, differentiating cardiomyocytes, setting up experiments), conference rooms (team science and collaboration means a lot of group discussions!), and my computer for writing and analysis. Since then, Ive shifted my work to be more remote while I work on a few different manuscripts. I have an office set up at home with a window, some good tunes, plenty of coffee, and a chair for my dog to wait impatiently on.

Your best tip or trick for managing everyday work and life. (Help us out, we need it.) I have always been a to-do list person. Most mornings start with me listing out tasks (and breaking those down into many sub-tasks). I feel productive as I cross things off, and it also helps me prioritize and plan ahead to make sure I can also fit my training runs in.

Mac, Windows or Linux? Mac as a personal preference, Windows for my work computer (I do work at the Paul Allen Institute 🙂

Transporter, Time Machine or Cloak of Invisibility? Transporter. I just promise not to use it in races.

Greatest game in history: Lode Runner. I havent played it since I was a kid, but the memories of yelling at the computer with my sister frantically hitting up-down-up-down arrows make me feel like it was just yesterday.

Best gadget ever: Garmin inReach mini satellite messaging and SOS call, all in a device small enough to throw in the bottom of a pack (or shorts pocket) and forget its there. I bring this with me anytime Im headed out into the wilderness/mountains, but I hope I never need to use it.

First computer: iMac G3.

Current phone: iPhone 11.

Favorite app: I have a love/hate relationship with Strava. Ive also been using DuoLingo during the pandemic and have a strong daily streak going!

Most important technology of 2021: COVID vaccines!!

Most important technology of 2023: Advancements in remote/low-resource medical care.

Final words of advice for your fellow geeks: Most problems can be solved with more snacks and some time (works for science and running).

Twitter: @kaytlyn_gerbin

LinkedIn: Kaytlyn Gerbin

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Kaytlyn Gerbin is blazing trails in cell science and as an ultrarunner who has conquered Mount Rainier - GeekWire

categoriaCardiac Stem Cells commentoComments Off on Kaytlyn Gerbin is blazing trails in cell science and as an ultrarunner who has conquered Mount Rainier – GeekWire dataApril 19th, 2021
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[Full text] Successful Use of Nivolumab in a Patient with Head and Neck Cancer Aft | OTT – Dove Medical Press

By daniellenierenberg

Introduction

Head and neck squamous cell carcinoma (HNSCC) is one of the major causes of cancer-associated illness and death, with more than 600,000 newly diagnosed cases worldwide each year1 and a continuously increasing incidence rate.2 HNSCC includes cancers of the oral cavity, pharynx, and larynx. The anatomical structures of the head and neck can be damaged by the tumor itself or treatments such as surgical resection and chemoradiotherapy, which sometimes cause speech, swallowing, and breathing impairments.3,4 Patients with HNSCC have been shown to bear greater psychological distress than those with other types of cancer.5

Despite the currently available therapies, patients with advanced HNSCC still experience poor outcomes.68 For example >50% of patients with locoregionally advanced HNSCC experience recurrence or metastases development within 3 years of treatment.911 Treatment options for patients with the recurrent and metastatic disease following progression after a platinum-based regimen are limited, and the median overall survival of such patients is less than 7 months.1215

The recurrence and metastasis of HNSCC are facilitated by immune evasion;16 therefore, as one of the methods to inhibit immune evasion, the use of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway inhibitors is considered effective in the treatment of recurrent HNSCC.1719 Nivolumab, a fully human IgG4 antiPD-1 monoclonal antibody, has shown remarkable antitumor efficacy and safety when administered to patients with recurrent HNSCC whose disease had progressed within 6 months of platinum-based chemotherapy;19 Furthermore, nivolumab treatment has been shown to improve the quality of life of these patients.20 However, PD-1 inhibitors can upregulate T cells in vivo, which may lead to the development of graft-versus-host disease (GVHD) in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).2123 To the best of the authors knowledge, no studies have investigated the safety and efficacy of nivolumab in patients with HNSCC after allo-HSCT. Here, we report the case of a patient who experienced excellent control of left buccal squamous cell carcinoma with nivolumab after the failure of platinum-based chemotherapy despite receiving allogeneic bone marrow transplantation.

Without any family history of tumor, a 33-year-old man was diagnosed with Philadelphia chromosome-positive T cell acute lymphoblastic leukemia on March 19, 2014. He received one course of vincristine and prednisone therapy and four courses of vincristine, daunorubicin, cyclophosphamide, and prednisone therapy. He was in complete remission at the end of therapy. Subsequently, allogeneic bone marrow transplantation was performed; the donor was his human leukocyte antigen (HLA)-haploidentical sibling (sister). He experienced chronic GVHD (c GVHD) of the oral cavity and skin 3 months after transplantation, for which he was treated with steroid hormone- and cyclosporine-based therapies. Skin rejection lasted for more than 3 years. Imatinib mesylate was administered for 2 years after transplantation, and his leukemia was well controlled.

In August 2018, the patient developed an ulcer of approximately 0.5 0.5 cm size in the left buccal mucosa; the ulcer was slightly painful and covered with white moss. In September 2018, the patient was admitted to Peking University Stomatological Hospital, where a biopsy of the buccal mucosa was performed. The pathology results showed the presence of squamous cell carcinoma in the left cheek. Unfortunately, this patient was not a right candidate for HNSCC in terms of exposure to risk factors, such as long terms of smoking and drinking. On October 10, 2018, 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (CT) showed that the mass in the left cheek was metabolically active, which is consistent with the activity of a malignant tumor. One course of an adjuvant therapy regimen (nimotuzumab [200 mg d0] + docetaxel [60 mg d1, 8]+ nedaplatin [60 mg d2, 3]) was administered on October 26, 2018. Following this, the patient developed degree II thrombocytopenia and redness, swelling, and ulceration of the cheek, which had discharge with a peculiar smell. On November 29, 2018, a head and neck CT scan showed a left buccal malignant tumor with the destruction of the neighboring mandibular bone and lymph node enlargement in the left submaxillary region and right carotid sheath. The CT examination revealed disease progression. Following a multidisciplinary consultation in our hospital, surgery was not recommended; instead, a chemotherapy-based comprehensive treatment was recommended as a better option for the patient. The patient received chemotherapy with albumin paclitaxel (200 mg d1, 8)+ bleomycin (15,000 units d2, 9) from November 30, 2018 to January 9, 2019. On another CT scan, the curative effect was evaluated as partial remission (showed in Video 1, Figure 1A); subsequently, two courses of a chemotherapy regimen comprising nivolumab (140 mg d1) + albumin paclitaxel (200 mg d1, d8) were administered. A CT examination showed stable disease (SD) on March 12, 2019, following which the patient was administered 120 mg of nivolumab once every 2 weeks from March 15 to May 23, 2019. Another CT examination was performed on May 28, 2019 (showed in Video 2, Figure 1B). During the therapy course, the related tumor markers showed an overall downward trend, the new metastases did not appear, the patients status became better than before. Subsequently, another CT examination performed in August 02, 2019 showed the extent of the tumor was obvious reduction than before (showed in video 3, Figure 1C). And the corresponding CT report in August 02, 2019 was described as follows Compared with the CT on 28 May, 2019, the extent of the tumor in the left cheek became obviously smaller, the tubercle in the left submandibular and the lymph nodes in the left neck also became smaller. There were no other significant changes in this image. Most importantly, the patient did not develop any form of GVHD following nivolumab administration.

Figure 1 Head and neck CT images showing tumor before (A) and after treatment with nivolumab (B, C, respectively).

Abbreviation: CT, computed tomography.

Note: The arrows indicate the maximum length diameter of tumor or tumor site.

Reliable data on the clinical safety and efficacy of nivolumab in the treatment of recurrent or metastatic HNSCC have been obtained in a Phase III randomized clinical trial (CheckMate 141).19 In this trial, 361 patients with recurrent HNSCC for whom disease had progressed within 6 months after platinum-based chemotherapy were enrolled between May 29, 2014, and July 31, 2015. The median follow-up duration for overall survival (OS) was 5.1 months (range, 016.8 months). OS was significantly greater in patients randomized to receive nivolumab than in those who received standard second-line, single-agent systemic therapy with either methotrexate, docetaxel, or cetuximab (hazard ratio, 0.70; 97.73% confidence interval (CI), 0.510.96; P = 0.01). The median OS was 7.5 months (95% CI, 5.59.1) in the nivolumab group versus 5.1 months (95% CI, 4.06.0) in the standard therapy group. The one-year survival was also greater in patients who received nivolumab than in those who received standard therapy (36.0%vs. 16.6%). Furthermore, the response rate was higher in those who received nivolumab than in those who received standard therapy (13.3% vs 5.8%); however, the median progression-free survival was not significantly different between the groups (2.0 vs 2.3 months; P=0.32). In this study, patients who were treated with nivolumab had a longer OS than those treated with standard therapy, regardless of tumor PD-L1 expression or p16 status. Grade 3 or 4 treatment-related adverse events occurred in 13.1% of patients who received nivolumab and 35.1% of those who received standard therapy. Physical function, role functioning, and social functioning were stable in the nivolumab group, whereas they were substantially worse in the standard therapy group.20 Moreover, among Asian patients, the survival benefits were consistent with the global group.24

It was unclear whether nivolumab could be used in patients with recurrent HNSCC after allo-HSCT, though Khaddour et al proved the efficacy and safety of Pembrolizumab in patients who underwent allo-HSCT after relapsed and refractory Szary Syndrome and cutaneous squamous cell carcinoma.25 However, some case reports (Table 1) and clinical trials (Table 2) have reported the efficacy and safety of nivolumab when administrated to patients with recurrent hematological malignancies (mostly Hodgkins lymphoma) after allo-HSCT.

Table 1 Case Reports of Nivolumab Use After Allo-HSCT

Table 2 Studies on Nivolumab Use After Allo-HSCT

In Herbaux et al, nivolumab (3 mg/kg, once every 2 weeks) was administered to 20 patients with Hodgkins lymphoma who experienced relapse after allo-HSCT. The overall response rate was 95%, the 1-year progression-free survival rate was 58.2%, and the 1-year OS rate was 78.8%.26 Compared with other treatment options, nivolumab was more effective in these patients.2730 Haverkos et al reported results after a median follow-up duration was 428 days (range, 133833 days). After treatment with PD-1 inhibitors [nivolumab 3 mg/kg, once every 2 weeks (n = 28) and pembrolizumab (n =3)], the overall response rate of 31 patients with relapsed lymphoma after allo-HSCT was 77%, the median progression-free survival was 591 days (range,400644 days), and 68% of the patients survived to the end of the study.23 These two studies showed that nivolumab is effective when administered to patients with recurrent blood cancers after allo-HSCT, which is consistent with the results of several other case reports3134 and case series.35,36 The PD-1/PD-L1 pathway plays a key role in the regulation of the balance among T cell activation, T-cell tolerance, and immune-mediated tissue damage. This pathway protects healthy cells from excessive inflammatory or autoimmune responses.37,38 Some studies have shown that the activation of the PD-1/PD-L1 pathway can reduce acute and chronic GVHD, whereas its blockade can accelerate the graft-versus-host response and increase the associated mortality.21,22,39 It is unclear whether the PD-1 inhibitor nivolumab increases the risk of GVHD and the associated mortality in patients after allo-HSCT.23,26 Some clinical studies and case reports have shown that nivolumab treatment-related GVHD and consequent death in patients after allo-HSCT might be affected by the following factors. First, GVHD after antiPD-1 treatment has been observed most frequently in matched sibling donor transplants; for which Haverkos et al reported an incidence of 75%.23 In a Phase I pilot study, without GVHD or G3/G4 immune toxicity after receiving multiple doses of nivolumab was only among one patient whose donor source was Haploidentical+cord blood Fludarabine.40 Second, a history of GVHD, especially for the acute GVHD, may lead to an increased risk of nivolumab treatment-related GVHD after allo-HSCT. In a French cohort, all patients who presented with acute GVHD after nivolumab treatment had a prior history of acute GVHD, among which three patients presented with steroid-refractory nivolumab-induced GVHD, and GVHD was not observed among patients without a history of GVHD.26 This phenomenon was also observed in Steinerovs medical report.41 In the study by Haverkos et al, 63% of patients with a history of GVHD prior to antiPD-1 treatment developed treatment-emergent GVHD after receiving antiPD-1.23 Third, the shorter the interval between transplantation and nivolumab use, the greater the risk of GVHD. In the study by Herbaux et al, the median intervals between transplantation and nivolumab use in cases with the presence and absence of GVHD were 8.5 months and 28.5 months, respectively.26 In another study by Wang et al, the reported four patients all experienced immune-related adverse events following nivolumab treatment and the median time from transplantation to nivolumab use was 7.8 months.40 Fourth, dose is a risk factor for nivolumab treatment-related GVHD. In a case report, chronic skin GVHD was observed when the dose of nivolumab was adjusted from 0.5 mg/kg to 2 mg/kg.33 Other factors, such as immunosuppressive therapy at the time of nivolumab administration, may also influence nivolumab treatment-related GVHD. Recently, a comprehensive literature review was launched by Awais et al to assess the safety and efficacy of the use of checkpoint inhibitors (ipilimumab, nivolumab and pembrolizumab) in blood cancers before and after allo-HSCT. Collective data showed that checkpoint inhibitors use after allo-HSCT for post-transplant relapse had higher efficacy but the risk of GVHD was significant. Moreover, the investigation indicated that higher drug doses, shorter intervals between checkpoint inhibitors exposure and allo-HSCT and prior history of GVHD had a positive correlation with the risk of GVHD.42

In the present case, HNSCC was effectively controlled without any nivolumab treatment-related acute or chronic GVHD after nivolumab administration, while the weight loss being the only adverse event. After comprehensive analysis, we found that many factors may impede the development of nivolumab treatment-related GVHD in our patient. On one hand, the appropriate donor, no use of checkpoint inhibitors prior to allo-HSCT, the long interval between nivolumab administration and allo-HSCT (36 months) and the standard dose use of nivolumab were the negative factors for GVHD development. On the other hand, the chronic GVHD of the oral cavity and skin before nivolumab use might lead to the development of GVHD. However, it remained unknown what role the immunosuppressant therapy played in the occurrence of GVHD, though we definitely known that immunosuppressant was administered more than 2 years after allo-HSCT and discontinued for 2 years before treatment with nivolumab in our patient. Finally, whether the two primary cancers in our case affected the efficacy and safety of nivolumab by some unknown pathways were unclear, which needed further exploration.

Nivolumab has been shown to be effective in patients with HNSCC for whom platinum-based therapy has failed. However, little is known about the efficacy and safety of nivolumab in patients with HNSCC who have undergone allo-HSCT. Our case report shows that nivolumab could be used effectively and safely in such patients, however, more clinical trials are required to confirm these results.

This study was approved by the Medical Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. The authors state that they have obtained verbal and written informed consent from the patient for the inclusion of their medical and treatment history within this case report.

This work was supported by the Tianjin Science and Technology Commission (18ZXXYSY00070), Key Task Project of Tianjin Health and Family Planning Commission (16KG128), Anticancer Key Technologies R&D Program of Tianjin (12ZCDZSY16200), and Natural Science Foundation of Tianjin (18JCYBJC91600).

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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25. Khaddour K, Musiek A, Cornelius LA, et al. Rapid and sustained response to immune checkpoint inhibition in cutaneous squamous cell carcinoma after allogenic hematopoietic cell transplant for szary syndrome. J Immunol Cancer. 2019;7:338. doi:10.1186/s40425-019-0801-z

26. Herbaux C, Gauthier J, Brice P, et al. Efficacy and tolerability of nivolumab after allogeneic transplantation for relapsed hodgkin lymphoma. Blood. 2017;129:24712478. doi:10.1182/blood-2016-11-749556

27. Peggs KS, Kayani I, Edwards N, et al. Donor lymphocyte infusions modulate relapse risk in mixed chimeras and induce durable salvage in relapsed patients after T-cell-depleted allogeneic transplantation for hodgkins lymphoma. J Clin Oncol. 2011;29:971978. doi:10.1200/JCO.2010.32.1711

28. Anastasia A, Carlo-Stella C, Corradini P, et al. Bendamustine for Hodgkin lymphoma patients failing autologous or autologous and allogeneic stem cell transplantation: a retrospective study of the fondazione Italiana linfomi. Br J Haematol. 2014;166:140142. doi:10.1111/bjh.12821

29. Carlo-Stella C, Ricci F, Dalto S, et al. Brentuximab vedotin in patients with hodgkin lymphoma and a failed allogeneic stem cell transplantation: results from a named patient program at four Italian centers. Oncologist. 2015;20:323328. doi:10.1634/theoncologist.2014-0420

30. Tsirigotis P, Danylesko I, Gkirkas K, et al. Brentuximab vedotin in combination with or without donor lymphocyte infusion for patients with hodgkin lymphoma after allogeneic stem cell transplantation. Bone Marrow Transplant. 2016;51:13131317. doi:10.1038/bmt.2016.129

31. Angenendt L, Schliemann C, Lutz M, et al. Nivolumab in a patient with refractory Hodgkins lymphoma after allogeneic stem cell transplantation. Bone Marrow Transplant. 2016;51:443445. doi:10.1038/bmt.2015.266

32. Yared JA, Hardy N, Singh Z, et al. Major clinical response to nivolumab in relapsed/refractory hodgkin lymphoma after allogeneic stem cell transplantation. Bone Marrow Transplant. 2016;51:850852. doi:10.1038/bmt.2015.346

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[Full text] Successful Use of Nivolumab in a Patient with Head and Neck Cancer Aft | OTT - Dove Medical Press

categoriaSkin Stem Cells commentoComments Off on [Full text] Successful Use of Nivolumab in a Patient with Head and Neck Cancer Aft | OTT – Dove Medical Press dataFebruary 10th, 2021
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The Anti-aging Benefits of Using Growth Factors Plus Retinol – Coveteur

By daniellenierenberg

Sciences most powerful ingredients are better together.

Every skin-care enthusiast has heard of retinol, the dermatological gold standard in anti-aging and vitamin A derivative that lessens the appearance of fine lines and wrinkles, promotes collagen production, minimizes breakouts, and resurfaces your skin. But the famed ingredient is also known for some of its less desirable side effectsnamely, irritation, redness, flaking, and photosensitivity, which can make it intolerable for sensitive skin types and unsafe for use in pregnant and nursing women.

Even as most dermatologists laud retinol for its extensive benefits, holistic aestheticians remain wary of the actives corrosive effects on the skin barrier. Because the skin barrier is key for retaining moisture and protecting the dermis from external irritants, aggressors, pollutants, and more, compromising it can sensitize skin, making it more susceptible to inflammation and irritation.

Growth factors have piqued our interest for being a sensitive-skin-friendly alternative to retinol, as these pro-healing proteins are safe for more continued use and are non-corrosive to the stratum corneumthe outermost layer of the epidermisand are gentle enough for sensitive skin types. But if youre not quite ready to let go of your retinol, it turns out that they can be used together to enhance the potency of your anti-aging regimen, even buffering the skin from some of retinols less desirable side effects.

Keep reading to get schooled on the use of growth factors in skin care according to dermatologists, and some guidance on whether the controversial ingredient could be right for you to try next.

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As we grow older, our skins natural production of collagen decreases (starting around age 25), the elastin fibers begin to stiffen, and our skin produces lower quantities of growth factors, resulting in various signs of aging, like fine lines, crepiness, thinness, and sagging. This is where adding a topical growth factor into your routine can help. Says Lily Talakoub, MD, dermatologist and owner of dermtodoor.com, A growth factor is a large protein derived from human cells that targets the crucial turnover of cells, [promoting the increase] of [structural proteins] such as collagen and elastin.

Although they were first discovered in the 1950sand the scientists who did it were awarded the Nobel Prize for their research in 1986there was still some confusion as to how growth factors worked and their efficacy. But in the last few years, there has been a flurry of new anti-aging products that use growth factors like EGF (epidermal growth factor).

As Dr. Talakoub explains, growth factors stimulate the regeneration of cells that fight various signs of aging by prompting the cells to produce the structural components of the skin that are responsible for elasticity, firmness, and bounce. Specifically, ultra-healing EGF binds to the EGF receptor in the cell, aiding with cell proliferation, survival, and promoting DNA synthesis. By fortifying the presence of the structural tissues in the skin matrix, research shows that we can expect fewer fine lines and wrinkles over time. Think of growth factors as the key that unlocks and activates the cell to produce collagen and elastinboth of which give us that youthful lookand also help promote cell turnover and fight against inflammation.

As board-certified dermatologist Dr. Hadley King points out, growth factors like EGF can also be used to increase skin thickness and to improve tone and texture, particularly for sensitive skin types. If your skin is too sensitive to tolerate retinoids, then growth factors can be a substitute to stimulate collagen, even tone, and decrease roughness with much less irritation.

Dr. King explains that these proteins are almost universally tolerable, likely due to their natural presence in the body, and through inducing the bodys own natural mechanisms for repair, which in turn promotes skin healing. When they bind to receptors on cell surfaces, King continues, they send commands to repair, rejuvenate, and replicate. This makes them an excellent option for skin concerns ranging from inflammation and acne to fading scarring and post-inflammatory hyperpigmentation.

Thanks to the Kardashians, youve undoubtedly heard of the trendy vampire facial, which uses your bodys own PRPplatelet-rich plasma that is derived from your own bloodto treat acne scars and smooth skin texture. This makes them a fantastic addition to your post-laser or post-microneedling treatment at the dermatologist. Adding a growth factor serum like PRP can also enhance the efficacy of the treatment, as well as reduce healing time.

While a PRP serum created in a centrifuge from your own blood is the ultimate way to go for an in-office encounter with growth factors, you can also follow up at home with a growth-factor-based serum to maximize your treatments effects. Just be sure to perform a patch test before applying it to the area that was treated, as that skin might still be extra sensitive and prone to inflammation.

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One of the most important things to consider when selecting a growth factor comes down to the source. When we think of epidermal growth factor (EGF), there are two common methods of sourcing: bio (i.e., human- or animal-derived) and phyto (i.e., plant-derived). The general consensus within the scientific community seems to be that human-derived growth factors, especially PRP, are the most effective. Says Dr. Talakoub, Plant-based growth factors do not stimulate the growth of in vivo cells [in] the same [way] as ones derived from human cells.

However, not everyone is comfortable using a biological source for their skin-care products, even if they might deliver superior results. In products like the wildly popular growth-factor-based serums from SkinMedica and NeoCutis, the initial cell samples were obtained from neonatal fetal foreskin and fetal fibroblast cells, respectively, before being grown in the lab for use in their products. Depending on your ethical and political views, this might be an issue to consider before you try using these types of formulas.

And while these holy grail serums do use human stem cells, keep in mind that there are no body partswe repeat, absolutely no penises!in these serums, despite what Cate Blanchett says.

If youd rather bypass the ethical controversy of a human- or animal-derived growth factor altogether, rest assured that there are plant-based options. There are some engineered options that have been well tested and shown to be effective, says Dr. King, noting that the brands BIOEFFECT and DNA Renewal have both exhibited promising results related to skin repair, rejuvenation, and enhancement via a humanlike epidermal growth factor made in bio-engineered barley seeds.

Dr. Bjrn rvar, co-founder and chief scientific officer of BIOEFFECT, the makers of the worlds first bio-engineered, plant-based EGF, strongly believes that phyto-EGF is the new frontier in terms of tolerability, efficacy, and even safety. EGF has previously been grown in bacteria, which poses a risk of endotoxins, or extracted from human or animal cells, which presents ethical, moral, and legal issues as well as safety issues.

There are also upsides to selecting a phyto-sourced, synthetic EGF that go beyond ethics and speak to precision and measurable outcomes. The advantage to plant-derived growth factors is that there is a more defined concentration of known synthetic growth factors, and so it is theoretically easier to measure and predict outcomes, says dermatologist Dr. Mamina Turegano. And now with studies pointing to phyto sources of growth factor improving epidermal thicknesswhich thins as we agethese phyto-sourced options could be the new frontier in terms of bypassing the controversy altogether.

Because these two anti-aging actives work differently, with retinol promoting the turnover of keratinocytes (the primary type of cell in your epidermis) and growth factors targeting the formation of cells that produce collagen and elastin and strengthening your skin barrier, EGF and retinol can be used together to maximize your anti-aging skin-care routine.

So what happens when you use a growth factor along with a retinol product? Most growth factors are extremely large proteins [which] have a very difficult time penetrating the outer lipid bilayer of the skin, says Talakoub. Using a retinoid in combination with a growth factor allows [it] to penetrate the outer layers of the skin. She suggests using a vitamin C product during the daytime, and then layering your EGF with your retinol at night to see the best results.

If you do choose to use an EGF serum in conjunction with your favorite retinol, rvar recommends a particular process. We recommend always applying the EGF serum first, on clean skin, and allowing 510 minutes before applying anything else on top [to] give it time to activate the skin cells and do its magic, he notes. As he explains, EGF will help to boost hydration and counter the thinning of the skins outer layer that can occur with retinol. Their synergistic effects are the perfect complement to each other and will help make your anti-aging skin-care routine even more effective.

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The Anti-aging Benefits of Using Growth Factors Plus Retinol - Coveteur

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The Strangest Beauty Treatments Celebrities Have Tried – Marie Claire

By daniellenierenberg

Jodie Turner-Smith: Breast Milk Skincare

"Ever since I had my baby, my current beauty secret is that I put breast milk in all of my face serums," Turner-Smith shared with British Vogue. "My skin is very sensitive, so I use a light cleanser, and then I put on a serum with aloe and breast milk that I literally squeeze right into my hands from my boob. I think it's the lactic acid. I've just found that the milk has been revolutionary."

Blanchett refers to this, er, unique treatment as the penis facial (which she says Sandra Bullock introduced her to) but its more officially known as the Hollywood EGF Facial. "The foreskin is collected during circumcision and the stem cells are then harvested and extracted through a centrifuge," Georgia Louise, the facialist behind the treatment toldThe Hollywood Reporter. "I am always very mindful to explain radical serums and potions that I carry in my back bar, so I always explain that EGF is derived from newborn baby foreskin, but cells were taken and from that, new cells are cloned from a laboratory."

Kardashian Wests infamous blood-covered selfie caused quite the stir back in 2013, but it seems she didnt deem the vampire facial (a treatment where your own blood is spun in a centrifuge to extract the plasma within and then re-applied to your face) worth the pain, admitting that the treatment wasnt for her understandable considering her pregnancy meant painkillers were out of the question.

Unique beauty endeavours arent anything new in Paltrows case but this one has to be the most extreme yet. "I've been stung by bees. It's a thousands of years old treatment called apitherapy. People use it to get rid of inflammation and scarring. It's actually pretty incredible if you research it," she told The New York Times.

"I'm using this amazing snail [essence], yes, snail..." Ratajkowski recently revealed via social media. But while it may sound a little freaky, it certainly isnt fruitless snail mucin is a natural source of hyaluronic acid and has the ability to soothe, repair and diminish a variety of skin issues from dryness and breakouts to pigmentation.

The face and the rear end are two areas people generally like to keep pretty separate, but Bullock says the key to fighting crows feet lies in hemorrhoid care. "My favourite beauty pageant secret: I didn't realise that putting hemorrhoid ointment on your face is acceptable in the beauty business," Bullockshared back in her Miss Congeniality days. "But apparently butt cream does help lines around the eyes."

Lopez always looks a million bucks, which is fitting considering how much she invests in her complexion. In fact, its alleged that she spends approximately $1,200 per week on a texture-refining facial treatment that involves the skin-smoothing abilities of human placenta.

It might sound a little more glamorous than Lopezs placenta-centric option but the price tag is also a lot higher; yes, indeed, Kunis shelled out $7,000 for a sole precious stone treatment that uses diamonds and rubies to enhance radiance. We hope her skin was shining just as brightly as the stones after that cash splash.

Moore relies on medical leeches to keep her complexion looking tight, firm and youthful. "I feel like I've always been someone looking for the cutting edge of things that optimise your health and healing," she toldDavid Letterman in 2008. "I was in Austria doing a cleanse and part of the treatment was leech therapy. These aren't just swamp leeches thoughwe are talking about highly trained medical leeches. These are not some low level scavengerswe're talking high level blood suckers."

Beckhams topical treatment of choice? One composed of nightingale droppings. But while experts do claim that the brightening and exfoliating abilities are impressive, were simply not sure we can stomach this one. However, Beckhams perennial glow may just be enough to get us on board...

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The Strangest Beauty Treatments Celebrities Have Tried - Marie Claire

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3D bioprinting dual-factor releasing and gradient-structured constructs ready to implant for anisotropic cartilage regeneration – Science Advances

By daniellenierenberg

INTRODUCTION

Articular cartilage is an elastic connective tissue in the joint (1). Cartilage injury is extremely common, yet cartilage has limited self-healing capacity because of its low cellularity and avascular nature. Because damage to cartilage leads to knee joint dysfunction, resulting in substantial pain and disability in the arthritic joint, cartilage or joint reconstruction remains a considerable challenge.(1). Arthritic joints in clinical practice are replaced by total joint arthroplasty using metallic and synthetic prosthesis (2, 3). Existing joint prostheses do not remodel with host joint tissue and can lead to long-term failure by aseptic loosening or infection (4), which could only be addressed by biological regeneration of the joint. Recently, using mesenchymal stem cell (MSC) transplants and then stimulating the directional differentiation into chondrocytes is becoming the method of choice for cartilage repair (5, 6). Clinical studies have shown that joint cartilage damage always extends deeply into the subchondral bone and, thus, causes osteochondral defects in the knee joint, which can alter the joints biomechanical properties and influence the long-term performance of the cartilage tissue (7), indicating the significance of simultaneous repair of whole-layer anisotropic articular cartilage in successful knee repair. As articular cartilage transitions from the superficial zone to the deep zone, the extracellular matrix (ECM) of the cartilage is characterized by increased oxygen tension and nutrient availability, lower amounts of ECM constituents such as glycosaminoglycans (GAG), and increased presence of a different phenotype of chondrocyte population with hypertrophic and ossification markers such as RUNX2 (Runt-related transcription factor 2) and type X collagen (8, 9). The gradient and anisotropic structure in ECM deposition and cell type provides excellent permeability in deep zone (vessel ingrowth) as well as desired mechanical support (10). However, developing biomimetic constructs mimicking the gradient anisotropic structure and the signaling approaches in different layers to induce zonal-dependent chondrogenic differentiation and ECM deposition is very challenging in cartilage repair. Previous studies showed that scaffolds with small pore size (100 to 200 m) could better promote chondrogenesis in osteochondral regeneration (11). However, osteogenesis and angiogenesis were inhibited in these scaffolds with small pore sizes, showing less nutrient diffusion and worse tissue integration by decreased microvessel ingrowth in these scaffolds (12). Hydrogel has been reported for cartilage regeneration in many studies (13, 14), yet it is still difficult to construct large-scale tissue structures with hydrogel owing to inadequate structural integrity, mechanical stability, and printability (12). Here, we report developing three-dimensional (3D) bioprinted dual-factor releasing and gradient-structured MSC-laden constructs ready to implant for whole-layer cartilage regeneration.

Different joint tissue constructs for joint reconstruction were fabricated using 3D bioprinting as previously reported with organ printing united system (OPUS; Novaprint) (15). To better mimic the native cartilage, we incorporated biochemical stimulus (BCS) with different growth factor releasing, and biomechanical stimulus (BMS) with small pore sizes to induce better chondrogenesis to create the dual-factor releasing and gradient-structured cartilage construct in the double stimulus (DS) group. We chose to test the combination of bone morphogenetic protein 4 (BMP4) and transforming growth factor3 (TGF3) in the cartilage construct in an established knee cartilage defect model given its potential generalizability in the regeneration of complex, inhomogeneous joint tissues. Poly(lactic-co-glycolic acid) (PLGA) (50:50 PLA/PGA) microspheres (S) were used to deliver TGF3 and BMP4 in hydrogel (Fig. 1, A and B). Briefly, poly(-caprolactone) (PCL) was molten to fabricate the physically gradient supporting structure for the scaffold, while MSC-laden hydrogel encapsulating PLGA microparticles carrying TGF3 or BMP4 in different layers was bioprinted into the microchannels between PCL fibers from different syringes (fig. S1). During plotting, the needle diameter, layer thickness, and speed for PCL printing were kept constant at 200 m, 200 m, and 180 mm/min, respectively. The fiber spacing was kept constant at 150 m (BMS group) or 750 m (BCS group) for nongradient (NG) scaffolds and varied gradually from 150 to 750 m throughout the gradient scaffolds (DS group) (fig. S1). The gradient microchannels between PCL range gradually from 150 m wide from the superficial zone of the cartilage, providing enough mechanical properties and smaller compartments favoring articular chondrocyte differentiation (11, 16), to 750 m wide in the deepest zone of the cartilage construct, maximizing diffusion of nutrients with better microvessel ingrowth and offering higher oxygen stress in the deep zone (Fig. 1B) (12). The fiber spacing was changed by 200 m every millimeter. The scaffolds were plotted in blocks of 4 4 4 mm for rabbit cartilage construct and 14 14 14 mm for human cartilage construct (Fig. 2A and movie S1).

(A) Schematic Illustration of the study design with 3D bioprinted dual-factor releasing and gradient-structured MSC-laden constructs for articular cartilage regeneration in rabbits. Schematic diagram of construction of the anisotropic cartilage scaffold and study design. (B) A computer-aided design (CAD) model was used to design the four-layer gradient PCL scaffolding structure to offer BMS for anisotropic chondrogenic differentiation and nutrient supply in deep layers (left). Gradient anisotropic cartilage scaffold was constructed by one-step 3D bioprinting gradient polymeric scaffolding structure and dual protein-releasing composite hydrogels with bioinks encapsulating BMSCs with BMP4 or TGF3 S as BCS for chondrogenesis (middle). The anisotropic cartilage construct provides structural support and sustained release of BMSCs and differentiative proteins for biomimetic regeneration of the anisotropic articular cartilage when transplanted in the animal model (right). Different components in the diagram are depicted at the bottom. HA, hyaluronic acid.

(A) Gross appearance of (a) human-scale and (b and c) rabbit-scale cartilage scaffold (b, NG with 150-m spacing; c, NG with 750-m spacing). Top view of the rabbit cartilage scaffold is also shown (d, NG with 150-m spacing; e, NG with 750-m spacing; f, gradient scaffold with 150- to 750-m spacing) atop of the SEM images (g, horizontal section; h, vertical section) taken for the 150-m NG scaffold to demonstrate the precise alignment of the PCL fibers in the printed scaffold. (B) Deconstruction of the gradient scaffold. The structure of the gradient scaffold was deconstructed into four layers. Microscopic appearance of the hydrogel-PCL composite structure in each layer demonstrated good interconnectivity and delicate, orderly aligned structure for each layer. (C and D) Good cell viability is shown respectively for superficial and deep layers after printing with live/dead assay (green, live cells; red, dead cells) (C) under a microscope and (D) under a confocal microscope. DAPI, 4,6-diamidino-2-phenylindole. (E) Cell spreading in superficial and deep layers with cytoskeleton staining. (F) Immunostaining for cartilage markers in superficial and deep layers. Expression of COL2A1 and PRG4, the lubrication markers, was significantly higher in the superficial layers with small pore size (a and b), while the chondrogenic cells in the deep layers (c and d) mostly presented with hypertrophic phenotype (COL10A1 expression). Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University.

Recombinant human TGF3 (rhTGF3) and rhBMP4 were microencapsulated in PLGA S (fig. S1) (17). TGF3 and BMP4 S were mixed in the cell-laden hydrogel (table S1), respectively, and printed into the microchannels between PCL fibers with different syringes (Fig. 2B and fig. S1). To chemically simulate the hypertrophic layer in native cartilage, we used PLGABMP4-encapsulated MSC-laden hydrogel in the deepest layer with a 750-m PCL fiber spacing, while PLGATGF3 was used for the other three layers of the cartilage construct. Scanning electron microscopy (SEM) images of PLGA S were taken, showing a less than 2-m diameter for most of the PLGA S. The PLGA-encapsulated MSC-laden hydrogel also showed nice printability as demonstrated (Fig. 2B and fig. S1A).

The final product of the human and rabbit cartilage construct demonstrated good interconnectivity and delicate, orderly aligned structure under the microscope, SEM, and in gross appearance for both PCL fibers and the printed hydrogel in between (Fig. 2, B to D). To validate S distribution in MSC-laden hydrogel, fluorophore-conjugated rhodamine was encapsulated into PLGA S and delivered to the hydrogel. At day 7, PLGArhodamine S showed well-proportioned distribution and minimal cell toxicity in the hydrogel printed between the PCL fibers under a confocal microscope (Fig. 2, C and D, and fig. S1B). Immunostaining for cartilage markers in the gradient scaffold was performed (Fig. 2, E and F). Resembling the native cartilage, the expression of COL2A1 (Collagen Type II Alpha 1 Chain) and PRG4 (Proteoglycan 4), the lubrication marker, was significantly higher in the superficial layers with small pore size, while the chondrogenic cells in the deep layers mostly presented with hypertrophic phenotype (COL10A1 expression) (Fig. 2F and fig. S2). Moreover, the compressive Youngs modulus of the NG-150 scaffold and the gradient scaffold were similar to that of the native cartilage and significantly higher than that of the NG-750 scaffold (fig. S3), demonstrating that smaller PCL fiber spacing plays an important role in enhancing the mechanical properties of the PCL-hydrogel composite scaffolds. In biomimetic regeneration of native articular cartilage, the gradient scaffold could provide anisotropic chondrogenesis in different layers and structural support for the newly formed cartilage tissue in compression, and allow nutrient supply and vessel ingrowth in the deep layers.

To examine the effects of BMP4, TGF3, and their S on bone marrow stromal cell (BMSC) viability and proliferation, we cultured BMSCs in the composite hydrogel for 7 days (fig. S4). Spheres showed controlled release of TGF3 first, followed by BMP4. Relatively rapid TGF3 release in the three layers with smaller PCL fiber spacing and slower release of BMP4 in the deepest layer were sustained over 60 days in vitro (fig. S5). Similar viability and proliferation rate of BMSCs were demonstrated for BMP4 and TGF3 compared with control through 7 days in the hydrogel (fig. S4, A, C, and D). Compared with empty S, S encapsulating BMP4 and TGF3 also showed minimal toxicity to BMSC viability and proliferation in the hydrogel (fig. S4, B, E, and F). Cell viability and proliferation were further examined in the printed scaffolds (Fig. 3, A to E). Scaffold fabrication with gradient structure (Fig. 3A, left) and delicate alignment of hydrogel printing (Fig. 3A, right) were separately conducted. Printed cell-laden hydrogel causes cell alignment in a longitudinal direction of the printed paths, forming a reticular network with cell interaction (Fig. 3B). The PCL pillar structure in the final construct further stabilized the 3D printed BMSC organization, inducing a compaction phenomenon of the patterns of cell alignment in the cell-laden hydrogel (Fig. 3C). Survival of BMSCs throughout the final cartilage construct with gradient structure was examined at 60 min (day 0), 1 day, 7 days, and 21 days after printing (Fig. 3, I to K). Live/dead cell assays showed 95% cell viability on day 0, which was maintained over 75% through days 3 to 21 (Fig. 3D). Cell proliferation, assessed using the alamarBlue assay system, increased over a 21-day period, similar to the proliferation of control cells encapsulated in a fibrin construct (Fig. 3E). Immunostaining of cytoskeleton showed cell spreading, both in the hydrogel and the PCL fibers throughout the four layers of the construct (Fig. 3C). At day 21, good 3D anchoring to the PCL fiber cylinder was observed for the BMSCs released from the hydrogel (Fig. 3F). These data indicate that the one-step 3D bioprinted dual-factor releasing and gradient-structurally optimized cartilage scaffold preserved cell viability during the printing process and provided a favorable microenvironment for BMSC proliferation, spreading, and condensation for differentiation into chondrocytes in vitro.

(A) Schematic of anisotropic cartilage scaffold construction with fabrication of gradient scaffolding structure (left) and large-scale printing of aligned protein-releasing BMSC-laden hydrogel (right). Scale bar, 1 mm. (B) Gross appearance of PLGA Sencapsulated BMSC-laden hydrogel under a microscope (top). Printed cell-laden hydrogel causes cell alignment in a longitudinal direction of the printed paths, forming a reticular network with cell interaction (bottom). (C) Live/dead cell assays showed 95% cell viability maintained through day 1 to 21 for all four layers with gradient spacing (4th row, 150-m spacing; 3rd row, 350-m spacing; 2nd row, 550-m spacing; 1st row, 750-m spacing). Immunostaining of cytoskeleton (rightmost column) showed cell spreading both in the hydrogel and on the PCL fibers throughout the four layers of the construct. Scale bar, 500 m. (D and E) Quantified cell viability and proliferation in the printed scaffolds. (F) Cell anchoring in the scaffolds. (a to c) At day 21, good 3D anchoring to the PCL fiber cylinder was observed for the MSC cells released from the hydrogel. (d to f) Similar cell anchoring was observed for PCL fibers in adjacent layers. (b), (c), (e), and (f) are 3D demonstration of cell anchoring in (a) and (d), respectively. Scale bars, 100 m. Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University.

Before in vivo application of the scaffold, we ascertained whether spatiotemporal delivery of rhTGF3 and rhBMP4 induced layer-specific BMSC differentiation into chondrocytes that present with hyaline articular and hypertrophic phenotype. Articular chondrocytes with hyaline and hypertrophic phenotype were first derived from rabbit BMSCs in vitro. Hyaline chondrocytes concurrently produced both aggrecan and type II collagens, while hypertrophic chondrocytes produced type I collagen and type X collagen. Sequential application of rhTGF3 for 2 weeks in culture, followed by rhTGF3 for another 4 weeks (TGF3 group), induced differentiation of BMSCs into chondrocytes that synthesized aggrecan and type II collagens, suggesting hyaline articular chondrocyte-like cells. BMSCs sequentially treated with rhTGF3 and rhBMP4 demonstrated significantly higher type I collagen, type X collagen, and aggrecan protein expressions than the control (Fig. 4A and fig. S6). Moreover, cells in the TGF3-induced tissue were fibroblastic, whereas those induced with BMP4 were larger and arranged in a cobblestone pattern (Fig. 4A), similar to hypertrophic chondrocytes previously generated in culture (5). Condensation of BMSCs that indicated differentiation was observed at 4 weeks (fig. S6B). Both treatments induced BMSC differentiation and yielded a cartilaginous matrix that stained positively for toluidine blue and alcian blue in condensed BMSCs, indicative of a proteoglycan-rich, cartilage-like ECM.

(A) Chondrogenic differentiation of condensed rMSCs with toluidine blue (TB) and alcian blue (AB) staining. (B) Scaffolds were transplanted subcutaneously for 12 weeks. (C) To validate the cartilage-generating capability, scaffolds were incubated and observed for 12 weeks in vitro, indicating better cartilage-generating potential for the physically gradient protein-releasing scaffold (movie S2). (D) Youngs modulus of the scaffolds compared with native cartilage after 12 weeks. Data are presented as averages SD (n = 6). *P < 0.05 between the NG-750 group and other groups; #P < 0.05 between the native cartilage group and other groups. (E) In the generated cartilage tissues, spatiotemporally released dual-factors induced zone-specific expression of PRG4, aggrecan, and collagens II and X and showed resemblance with native joint cartilage. (F) (a to c) Toluidine blue staining of the 3D printed cartilage constructs (a, top view; b, side view; c, bottom view) after culture in chondrogenic medium for 6 weeks in vitro. (d to g) Toluidine blue and (h to k) alcian blue staining was applied for each layer of the gradient scaffold. (l to p) Safranin O (SO) and (q to t) toluidine blue staining of cartilage tissue between PCL fibers (green curved line) in different layers of the 3D printed cartilage constructs after subcutaneous implantation. Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University.

Cartilage scaffolds incorporating rhTGF3 and rhBMP4 for spatiotemporally controlled release were also examined in different groups of scaffolds transplanted in vivo subcutaneously for 12 weeks (Fig. 4, B to F). To validate the cartilage-generating capability of the composite scaffold, the protein-carrying scaffolds were incubated and observed for 12 weeks in vitro (Fig. 4C). All scaffolds, physically gradient or NG, showed cartilage-like tissue development surrounding the scaffolds, whereas the BCS and BMS scaffolds developed 1/4 to 1/3 thickness cartilage tissue, while the DS scaffold showed almost full-thickness coverage of cartilage-like tissue around the construct (movie S2), indicating a significantly better cartilage-generating potential in vitro and a better prospect of its cartilage matrix integration in vivo for the physically gradient protein-releasing scaffold (Fig. 4C). The compressive Youngs modulus of the BMS scaffold and the DS scaffold were similar to that of the native cartilage and significantly higher than that of the BCS scaffold with large pore sizes (Fig. 4D), demonstrating that smaller PCL fiber spacing plays an important role in enhancing the mechanical properties of the PCL-hydrogel composite scaffolds. The enhanced mechanical properties are promising for biomimetic regeneration of native articular cartilage and provide structural support for the newly formed cartilage tissue.

After 12 weeks in vivo, spatiotemporally released rhTGF3 and rhBMP4 in the DS scaffold induced zone-specific expression of PRG4, aggrecan, and collagen II and X assayed with immunofluorescence, showing resemblance with native joint cartilage (Fig. 4E). Superficial zone marker PRG4, with a gradient manner throughout the four layers, was presented mainly in the superficial layer with the smallest PCL compartments (Fig. 4E, first column, 150 m 150 m). Abundant cartilaginous matrix with collagen type II and aggrecan was present in a gradient manner primarily in the superficial layers with TGF3 delivery, whereas hypertrophic marker collagen type X was primarily expressed in the deepest zone (Fig. 4E, second to fourth columns). Cartilaginous matrix was demonstrated and stained positive for toluidine blue for the scaffold (Fig. 4F, a to c). To determine the production of GAG in each layer of the gradient scaffold, we applied toluidine blue staining (Fig. 4F, d to g) and alcian blue staining (Fig. 4F, h to k). The whole gradient scaffold body stained positive (Fig. 4F, a to c), with a gradient staining intensity from the superficial layer to the deepest layer (Fig. 4F, d to k), indicating a gradient cartilaginous matrix formation resembling the native cartilage matrix. Safranin O staining and toluidine blue staining of the generated cartilage tissue sections showed the production of a cartilaginous matrix between PCL fibers in different layers of the 3D printed cartilage constructs after subcutaneous implantation in vivo (Fig. 4F, j to s). The chondrocytes in the newly formed tissue demonstrated similar morphological characteristics to those in native cartilage. A large fraction of generated chondrocytes in the TGF3-induced tissue were fibroblastic, whereas those induced with BMP4 in the deepest layers were larger and arranged in a cobblestone pattern, similar to hypertrophic chondrocytes generated in the culture plate (Fig. 4F, l to t). All cells located within typical chondrocyte lacunae, surrounded by cartilaginous matrix.

Rabbits were used as animal models to evaluate the knee repair capacity of the cartilage scaffolds. Cartilage scaffolds were constructed by one-step 3D bioprinting gradient polymeric supporting structure and different protein-releasing composite hydrogels with bioinks encapsulating BMSCs with BMP4 or TGF3, providing structural support and sustained release of BMSCs and differentiative proteins for biomimetic regeneration of the native articular cartilage (Fig. 5). As shown in Fig. 5A (first row), a full-thickness cartilage defect was created in the knee joint. The scaffold was implanted into the defect to test for cartilage tissue regeneration. Cartilage repair with the DS scaffold showed much better gross appearance at 8, 12, and 24 weeks compared with the BCS and BMS scaffolds (Fig. 5A, second to fourth rows). During the 24-week posttransplantation period, magnetic resonance imaging (MRI) was made for the operated knee joint, demonstrating significantly better resolution of subchondral edema and healing of the articular surface after 24 weeks for the DS group (Fig. 5A, fifth row). In addition, the chondroprotective effects of the scaffolds were compared (18). The gradient scaffold group showed better chondroprotective effects with a significantly higher histological grading compared with the NG groups over the 24 weeks in vivo (Fig. 5, B to E). Better repairing effects were demonstrated with gradient scaffolds compared with NG groups over 24 weeks (Fig. 5, B to E). Compared with the control group, the gradient group also showed better cartilage regeneration capabilities (fig. S7) and chondroprotection with significantly minor damage to the femoral condyle and tibial plateau (Fig. 5, D and E). Examination of intra-articular inflammatory response showed no significant difference in interleukin-1 and tumor necrosis factor level among different groups, maintaining at a relatively low level during the 24-week cartilage healing (fig. S8, A and B, and table S2). After the 24-week healing, histomorphological analysis was conducted for the generated cartilage. As shown in Fig. 5B, the DS scaffold regenerated fully hyaline-like cartilage in the defect site as evidenced by intense staining for GAGs and better cell filling in hematoxylin and eosin (H&E) staining (Fig. 5B). Type 1 and III collagens were also demonstrated in the regenerated cartilage with picrosirius red staining and compared with the native cartilage (Fig. 5B). Immunohistochemical staining of markers (PRG4 and type II and X collagens) for chondrocyte phenotype was conducted in the generated cartilage tissue sections in different groups compared with the native cartilage (fig. S8C). In the superficial zone, only the DS scaffolds showed PRG4 staining in the superficial chondrocytes in the generated cartilage tissue. Meanwhile, gradient expression of type II and X collagens, resembling the native cartilage, was also demonstrated from the superficial zone to the deep zone of the newly formed cartilage in the DS group, indicating successful construction of the anisotropic layered cartilage with different chondrocyte phenotypes and gradient ECM deposition by the 3D bioprinted dual-factor releasing and gradient-structured MSC-laden scaffold. Furthermore, neocartilage in the DS group showed more similar appearance to normal cartilage than other groups (Fig. 5B and fig. S8C). The above results indicated that the DS anisotropic scaffold had a better cartilage-repairing effect than the BCS or BMS groups and maintained better joint function after transplantation.

(A) Scaffold implantation process and gross appearance of the repair cartilage at 8, 12, and 24 weeks. MRI was made for the operated knee joint (fifth row), demonstrating significant better resolution of subchondral edema and healing of the articular surface (white arrowheads) for joint transplanted with DS scaffolds. (B to F) Chondroprotective effects of the scaffolds were compared by (B) histological scoring evaluation of the repaired cartilage tissue during in vivo implantation. (C) Mankin score and (D) ICRS (International Cartilage Repair Society) histological score of articular cartilage in the femoral condyle (FC) and tibial plateau (TP) in both groups with scaffold implantation. *P < 0.05 between the native group and other groups. #P < 0.05 between the BCS group and the DS group. Data are presented as averages SD (N = 6). (A) Histomorphological analysis of the neocartilage tissue at 24 weeks. PR, picrosirius red. The left bottom panels are higher-resolution pictures of the formed neocartilage outline in the colored square boxes. (a to e) Sections were stained with (a) H&E, (b) Safranin O, (c) TB, and (d) AB staining to indicate the presence of proteoglycans in different groups compared with native cartilage. (e) Picrosirius red was used to stain collagens I and III. The brown irregular area at the interface under the formed neocartilage was undegraded PCL material as supporting structure for the scaffolds. Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University.

As native articular cartilage transitions from the superficial zone to the deep zone, different phenotypes of chondrocyte population were presented with higher lubrication and GAGs (PRG4, ACAN expression) in the superficial layers and ossification (RUNX2, COL10A1 expression) in the deep layers. In the present study, we further tested the anisotropic properties of the generated cartilage and compared it with the native cartilage. In the superficial layer, immunostaining demonstrated greater PRG4 and ACAN expression in the DS group and the native cartilage compared with other two groups (Fig. 6, A to C). Meanwhile, higher expression of ossification markers (RUNX2 and COL10A1) were also observed for the group with implanted dual-factor releasing and gradient-structured scaffold (Fig. 6, D to F). These results indicate that the dual-factor releasing and gradient-structured scaffold could better restore the anisotropic properties of the native cartilage with different chondrogenic and ossification markers in specific layers. Moreover, resembling the ingrown microvessels in the deep layers of the native cartilage, the DS scaffold could better promote microvessel ingrowth compared with the group with small pore sizes, indicating better nutrient supply and tissue integration with large pore sizes in the deep zone (Fig. 6, G and H).

(A to C) In the superficial layer, immunostaining demonstrated greater PRG4 and ACAN expression in the DS group and the native cartilage compared with other two groups. (D to F) Meanwhile, higher expression of ossification markers (RUNX2 and COL10A1) were also observed for the group with implanted dual-factor releasing and gradient-structured scaffold in deep layers. (G and H) Moreover, the DS scaffold could better promote microvessel ingrowth compared with the group with small pore sizes, indicating better nutrient supply and tissue integration with large pore sizes in the deep zone. *P < 0.05 between the native group and other groups. #P < 0.05 between the DS group and other groups. BC, biochemical stimulus; BS, biomechanical stimulus. **P < 0.01; ##P < 0.01.

In conclusion, we have generated 3D bioprinted anisotropic constructs with structural integrity for joint reconstruction and articular cartilage regeneration and further tested the functional knee articular cartilage construct in a rabbit cartilage defect model with 6-month follow-up. Human-scale cartilage constructs with the structural integrity needed and that are ready for surgical implantation were created by sequentially printing protein-releasing and MSC-laden hydrogels with synthetic PCL polymer with gradient structures, a technique that could also be applied to the regeneration of the whole joint. In previous studies, relative nonuniformity was possible when hydrogel was printed alone without PCL as scaffolding support. Although hydrogel could serve as a carrier of cells and growth factors, it alone was quite not suitable for construction of complex biomimetic tissues with required mechanical properties. The combined printing with PCL scaffolding offered the uniformity for the hydrogel and the mechanical properties needed for in vivo study. In the present study, the cell-laden hydrogel allows well-proportioned distribution of MSCs and the protein-encapsulated S and thus protects cell viability and promotes its differentiation and expansion in the scaffold (17). Meanwhile, the adjacent PCL scaffolding provides adequate mechanical support and architectural integrity, offering a stable microenvironment for the 3D anchored MSC cells within the hydrogel to differentiate and form the tissue with their secreted cartilage matrix that replaces the hydrogel as it slowly degrades (15).

However, the release of the growth factors from the embedded S was not tracked in vivo after the scaffold transplantation. The intra-articular environment in vivo would definitely lead to faster disintegration of the S in the hydrogel. In this case, the PCL scaffolding would offer a much more stable microenvironment for cell and growth factor release than hydrogel alone. Lineage tracing studies have provided compelling evidence that articular chondrocytes derive from interzone cells in regions of condensing chondrogenic mesenchyme (19), similar to our observations that the MSCs, in the presence of TGF3 and BMP4, condense in the small compartments with surrounding PCL fibers as supporting structure and develop into articular chondrocytes that express genes expressed in cartilage layers. The MSC-derived articular chondrocytes were able to generate and maintain stable cartilage phenotype in vivo when transplanted into the knee defect site. The ECM composition of TGF3- or BMP4-induced cartilage tissues in the bioprinted scaffold shared many characteristics of native articular cartilage, including the gradient expression of type II collagen, superficial localization of PRG4, and abundant presence of type X collagen in the deep zone, indicative of regenerated superficial zone articular cartilage and deep zone hypertrophic cartilage in the constructs. In summary, we have generated 3D bioprinted constructs with structural integrity for joint reconstruction and articular cartilage regeneration and further tested the functional knee articular cartilage construct in a rabbit cartilage defect model with 6-month follow-up. Generating 3D bioprinted functional constructs as prosthesis for joint replacement or cartilage repair provides an opportunity to integrate the feasibility of MSC- and 3D bioprintingbased therapy for injured or degenerative joints. Evaluation will be needed to assess the function of the joint constructs in animal experiments and whether the functional cartilage phenotypes could be sustained in daily function. For translation, we envision the surgeons could incorporate surgery and 3D bioprinting by performing a mini-invasive arthroscopy procedure to replace the damaged or degenerated articular cartilage with 3D bioprinted cartilage scaffold or by performing joint replacement surgery using 3D bioprinted joint scaffolds.

BMSCs were isolated from rabbit bone marrow aspirates. Briefly, marrow aspirates (20-ml volume) were harvested and immediately transferred into plastic tubes. Isolated rMSCs were expanded in minimum essential medium containing fetal bovine serum (10%), d-glucose (4.5 mg/ml), nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), Hepes buffer (100 mM), penicillin (100 Ul/ml), streptomycin (100 g/ml), and l-glutamate (0.29 mg/ml). Medium was changed twice a week, and rMSCs were used at passage 2 for the following experiments. TGF3 (10 ng/ml) was added in the medium for 2 weeks, and then TGF3 was replaced with BMP4 (50 ng/ml) in some of the cultures for another 4 weeks. Medium was also changed twice a week. Immunofluorescence staining of chondrogenic markers (Col1A1, Col2A1, Aggrecan, and Col10A1) was conducted to compare the generated chondrocyte phenotype and observed under confocal microscopy (Leica, Japan). The expression of chondrogenesis markers (SOX9, Col1A1, and Col2A1), superficial zone chondrocyte markers (ACAN, PRG4, CILP2, GDF5, and Col22A1), and deep zone chondrocyte markers(Col10A1, RUNX2, and ALP) after TGF3 or BMP4 incubation for 6 weeks was analyzed by real-time polymerase chain reaction (RT-PCR) using an ABI 7300 RT-PCR system (Applied Biosystems, USA). Six-week-old tissues generated under both conditions were stained with toluidine blue and alcian blue for proteoglycan production. The stained images were taken using a light microscope (Leica Microsystems, Germany).

Different joint tissue constructs for joint reconstruction were fabricated using 3D bioprinting with OPUS (Novaprint). 3D bioprinting cell-laden hydrogels together with biodegradable polymers was conducted for specific articular joint. The motion program and alignment of cell-laden hydrogel and PCL fibers were demonstrated in the printing process of anisotropic cartilage tissues in movie S1. Bioprinting rabbit-derived MSC-laden hydrogels together with physically and chemically gradient biodegradable polymers was conducted for knee cartilage repair using OPUS. The rMSCs suspension (a total of 1 107 cells) was loaded into the composite hydrogel (table S1). The printing chamber was kept at a constant 17C. The native cartilage structure inspired us to produce four-layer 3D structures by placing together cell-laden hydrogel and PCL (~100-m diameter for hydrogel and ~200-m diameter for PCL) to construct a composite cartilage scaffold (17). Needle sizes for the hydrogel and PCL were 100 and 200 m, respectively. Briefly, PCL was molten (~60C) to fabricate the physically gradient supporting structure for the scaffold, while MSC-laden hydrogel (~37C) encapsulating PLGA microparticles carrying TGF3 or BMP4 in different layers was bioprinted into the microchannels between PCL fibers from different syringes (movie S1). During plotting, the needle diameter, layer thickness, and speed for PCL printing were kept constant at 200 m, 200 m, and 180 mm/min, respectively, as previously reported (15). The extrusion pressure for PCL and hydrogel was 1.2 to 1.8 kPa and 0.5 to 0.8 kPa, respectively. The fiber spacing was kept constant at 150 or 750 m for NG scaffolds and varied gradually from 150 to 750 m throughout the gradient scaffolds. The gradient microchannels between PCL range gradually from 150 m wide from the superficial zone of the cartilage to 750 m wide in the deep zone of the cartilage construct. The fiber spacing was changed every millimeter. The scaffolds were plotted in blocks of 4 4 4 mm for rabbit cartilage construct and 14 14 14 mm for human cartilage construct.

rhTGF3 and rhBMP4 were microencapsulated in PLGA (50:50 PLA/PGA) S to deliver TGF3 (20 ng/ml) and BMP4 (100 ng/ml) in hydrogel as previously described (15, 17). TGF3 and BMP4 S were mixed in the cell-laden hydrogel (table S1), respectively, and printed into the microchannels between PCL fibers with different syringes. To chemically simulate the deep layer in native cartilage, PLGABMP4-encapsulated MSC-laden hydrogel was used in the deepest layer with a 750-m PCL fiber spacing, while PLGATGF3 was used for the other three layers of the cartilage construct. Generated PLGA S was shown with SEM. Printability was also shown with a test run for the PLGA-encapsulated MSC-laden hydrogel. Release kinetics of TGF3 and BMP4 from PLGA S were measured by incubating S (10 mg/ml) encapsulating TGF3 (0.1% bovine serum albumin) or BMP4 [in phosphate-buffered saline (PBS)] at 37C with mild agitation for up to 60 days. Upon centrifugation at 2500 revolutions per minute for 5 min, supernatant of the PLGA S incubation solution was collected. Released TGF3 and BMP4 concentration was measured using enzyme-linked immunosorbent assay kits following the manufacturers protocols (15). To validate S distribution in MSC-laden hydrogel, fluorophore-conjugated rhodamine was encapsulated into PLGA S and delivered to the hydrogel. At day 7, PLGA rhodamine S and cell viability (live/dead assay) in the hydrogel was observed under a confocal microscope.

To validate the cartilage-generating capability of the composite scaffold, the protein-carrying scaffolds were incubated and observed for 12 weeks in vitro. Photographs of cartilage-like tissue development surrounding the scaffolds were taken to show the cartilage-generating potential in vitro of the scaffolds. Mechanical measurements on scaffolds and native cartilage were carried out with a single-column static instrument (Instron 5843, USA) equipped with two flat compression stages and a 10-N load cell.

To see the differences within the rMSCs cultured in the different areas of the gradient scaffolds, after 6 weeks under differentiation conditions, the constructs were collected, washed three times with PBS, and cut in four portions of 1 mm in height. The images of each layer were taken using a light microscope. The viability of the BMSCs on the scaffolds were analyzed with live/dead assay and observed under confocal microscopy for 3, 7, and 21 days, while the morphology of cells was observed under confocal microscopy at end point (21 days). Briefly, The MSCs in the scaffold were fixed with 4% paraformaldehyde and treated with rhodamine phalloidin (Thermo Fisher Scientific, USA) to stain the F-actin for 1 hour and incubated with DAPI (Thermo Fisher Scientific, USA) to stain the nucleus for 5 min in turn. Cell proliferation in the constructs was assessed with alamarBlue assay kit (DAL1100; Life Technologies) according to the manufacturers instruction as previously described (12).

Biochemical studies were performed to the full and partitioned scaffolds. Toluidine blue and alcian blue staining were applied to determine the production of GAGs in each layer of the gradient scaffold. The sections for the different layers were prepared and then treated with Safranin O and toluidine blue staining to identify GAG formation in each layer. Immunofluorescence staining of chondrocyte markers (PRG4, Col2A1, aggrecan, and Col10A1) was conducted for layer-specific chondrogenesis and observed under confocal microscopy.

Different groups of scaffolds were transplanted under the dorsal skin of nude mice in vivo subcutaneously for 12 weeks. The cartilage scaffolds were retrieved after 12 weeks in vivo, and zone-specific expressions of PRG4, aggrecan, and type II and X collagens were assayed with immunofluorescence. GAG production was determined with toluidine blue and alcian blue staining.

Adult male New Zealand white rabbits weighing 3.0 to 3.5 kg were used for the study in vivo. Rabbits were randomized into three groups (two knees of each rabbit were used): NG-750 (BCS group), NG-150 scaffold (BMS group), and the gradient scaffold (DS group). After anesthesia, the knee joint of the rabbits was exposed after dislocating the patella. A cylindrical defect (4-mm diameter, 4-mm depth) on the trochlear groove of the distal femur was created using corneal trephine. Then, suited 3D bioprinted BCS, BMS, or DS scaffolds were implanted matching with the defect. Forced flexion and extension were conducted for the operated knee to confirm the localization of the implanted scaffolds in the defect. Last, the operated knee joint was closed with suture (4-0 thread), and antibiotics were given intramuscularly for prophylactic infection. After the operation, rabbits were allowed to move freely in their single cages and fed with standard food and water. Eight, 12, and 24 weeks later, rabbits were euthanized for further study. The protocol was approved by the local Institutional Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals, revised in 2010 and published by the National Academy of Sciences.

Serial sections (4 mm thick) were cut sagittally through the center of the operative site and stained with H&E, toluidine blue, Safranin O and fast green, toluidine blue, alcian blue, and picrosirius red according to standard protocols. Immunohistochemical staining of markers (PRG4, RUNX2, and collagens II and X) for chondrocyte phenotype and microvessel ingrowth (CD31 and smooth muscle actin) was conducted according to standard protocols in the generated cartilage tissue sections in different groups compared with the native cartilage. The stained images were taken, and regenerated cartilage thickness (n = 6 for each) was calculated for different bioprinted scaffolds using a light microscope. A modified method was used to evaluate the histological repair of articular cartilage defects (18).

Acknowledgments: Funding: This work was funded by the National Key R&D Program of China (nos. 2018YFB1105600 and 2018YFA0703000), the China National Natural Science Funds (nos. 51631009 and 81802122), the Chinese postdoctoral funding (no. 2019M661559), and the Funds from Shanghai Jiao Tong University for the Clinical and Translational Research Center for 3D Printing Technology. Author contributions: Y.S. and Y.Y. contributed equally to conceiving the study and designing the experiments. W.J. helped design the 3D bioprinted scaffolds. B.W. helped synthesize the growth factorencapsulated microspheres. Y.S. and Q.W. conducted the animal experiment. Y.S. and Y.Y. analyzed the data and wrote the manuscript. K.D. helped edit the manuscript and provided oversight. All authors read and approved the final manuscript. Competing interests: The authors declare that they have no competing interests. Data materials and availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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3D bioprinting dual-factor releasing and gradient-structured constructs ready to implant for anisotropic cartilage regeneration - Science Advances

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Father of Three with Incurable Cancer is Helping Researchers Get One Step Closer to a Cure – The Suburban Newspaper

By daniellenierenberg

In 2016, Danny Wade, a successful marketing professional and an active and doting father to his three young children, aged 11, 8 and 6, checked himself into the emergency department when he began experiencing severe, inexplicable bone pain and unusual fatigue.

Two days later, after undergoing a battery of tests, Danny was diagnosed with multiple myeloma, a little-known and incurable cancer of the plasma cells. He was just 42 years old.

"I was shocked when I got the news, Danny recalls. What upset me most was when the doctor told me that the average life expectancy for myeloma patients was only five to seven years. The thought that I would not see my children grow up was devastating. I knew I had to fight for my life.

Thats exactly what Danny has been doing. Within six months of being diagnosed, Danny went through a difficult high-dose chemotherapy regimen to prepare for an autologous stem cell transplant using his own stem cells. Then in 2017 after further tests, Dannys doctors recommended that his best option for survival was to undergo another transplant with stem cells from a healthy donor. He took his doctors advice and underwent the procedure. Fortunately, he was eligible to participate in a clinical trial at Maisonneuve-Rosemont Hospital where he received a breakthrough therapy involving bi-weekly injections that he will continue to take for a year.

Dannys condition is relatively stable at this time, and he extremely thankful to still be alive. He is thrilled to watch his children grow and to resume being an active part of their lives. He credits his survival to the life-saving treatments that he has access to and the love and support he receives from his partner, Anik. With my beloved Anik by my side, Ive had the courage to get through this nightmare and to have faith that I can get through whatever else the future holds.

Danny is eager to do what he can to help others living with myeloma. I made a promise that once I was doing well, I would do everything in my power to help find a cure so that other patients dont have to live through the horrors I have," says Danny. Danny is a member of the organizing committee of the Montreal Support Group, and recently co-founded the South Shore Myeloma Support Group.

Over the past four years, Danny has seen, first-hand, the life-changing impact that advances in myeloma research are having on the lives of those living with this incurable cancer. Thats why he and his family are more intent than ever to raise as much awareness and funds for myeloma as they can, and will be participating in Myeloma Canadas 12th annual Montreal Multiple Myeloma March on Sunday, September 20, at 10 am.

This years Montreal March has been modified to help stop the spread of COVID-19. In compliance with physical distancing measures, participants are encouraged to hold their own walk in their neighbourhood at the same time as the regularly scheduled March on September 20. Danny and his fellow Montreal Marchers have set their fundraising goal at $60,000 to help further crucial research for this deadly blood cancer that affects nine new Canadians every day.

Myeloma research has produced extremely promising results over the past two decades. In fact, for the first time, theres a cure in sight, says Dr Richard LeBlanc, Medical Hematologist and Oncologist, and holder of the Myeloma Canada Chair in Multiple Myeloma Research at the Universit de Montral. We cant afford to let the current situation stop the progress weve made and put vulnerable people living with myeloma at risk, which is why its more crucial than ever to invest in research and find a cure.

The Multiple Myeloma March, Myeloma Canadas flagship fundraiser is now in its 12th year. The annual five-kilometer event brings Canadian communities together to raise essential funds for research and to help improve the lives of all Canadians impacted by myeloma. Montreal is one of a record 33 communities across the country to be included in this years Multiple Myeloma March. The national fundraising goal is set at $650,000. To learn more about how this event will be working, please click here.

While this years March will undoubtedly be different because of the pandemic, its crucial to stay positive, says Martine Elias, Executive Director of Myeloma Canada. Fundraising has taken a huge hit for many organizations. We need to do all we can to increase awareness and raise essential funds for research that will improve the lives of Canadians impacted by myeloma, and bring us closer to a cure, Martine added. As we mark Myeloma Canadas 15th anniversary, we celebrate the strength of our incredible community. More than ever, were counting on our supporters to help us achieve our goal of $650,000. Canadians impacted by this incurable cancer are depending on us.

This year, a minimum of 50% of funds raised by the Multiple Myeloma March will go directly to support Myeloma Canadas Myeloma Research Priority Setting Partnership (PSP), the first program of its kind in myeloma. The PSP will use input provided by the Canadian myeloma community to identify and define investments in myeloma research over the next 18 months. The balance raised will go toward supporting various myeloma research projects and initiatives that are pivotal for improving quality of life and moving the needle toward a cure.

Multiple myeloma, also known as myeloma, is the second most common form of blood cancer. Myeloma affects a type of immune cell called the plasma cell, found in the bone marrow. Every day, nine Canadians are diagnosed, yet in spite of its growing prevalence, the disease remains relatively unknown. While there is no cure, people with myeloma are living longer and better lives, thanks to recent breakthroughs in treatment. To find the cure, more funding and research are required. To learn more, or to donate, please visit http://www.myeloma.ca

Myeloma Canada

http://www.myeloma.ca

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Father of Three with Incurable Cancer is Helping Researchers Get One Step Closer to a Cure - The Suburban Newspaper

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Dior Skincare Ambassador Joanna Czech On Her Self-Care Routine And How To Prevent Maskne – Tatler Malaysia

By daniellenierenberg

Image: Steve Wrubel/Parfums Christian Dior By Chloe Pek August 14, 2020

The beauty expert counts Bella Hadid and Kim Kardashian amongst her clientele

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When it comes to facial massages, celebrity facialist Joanna Czech has that magic touch. Counting Hollywood and runway bigwigs like Bella Hadid, Kim Kardashian, Jennifer Aniston, Cate Blanchett, Kate Winslet, Liam Neeson and more amongst her star-studded list of clients, Joannas skincare treatments are highly coveted internationally.

Now, the skincare expert is adding another credential to her portfoliojoining the House of Dior as a skincare ambassador and lending her expertise in developing the Dior skincare techniques international training.

In an interview via Zoom, Czech told us she had her reservations about joining Dior Skincare at first. I mean, theyre famous for make-up and fashion. I couldnt put my name next to fashion skincareIm very particular about that. Then, I heard about how the Capture Totale range stimulates cell energy, so that changed everything and my skin as well.

Czech, who originally planned to go to medical school in her schooling years, fell in love with skincare when she enrolled in the Aesthetics Institute and never looked back. However, she has remained inquisitive and fascinated with science. This is evident from her holistic approach to beauty, which combines both traditional techniques and cutting-edge technology.

See also: The Best Beauty Launches In August 2020

Adenosine triphosphate (ATP, the main carrier of energy for cellular activities) is responsible for the very first mitosis of cells. It is human physiology that production of ATP drastically drops at around seven years old, and the energy keeps slowing down. So any treatment or product that would stimulate cell energy is fascinating to me.

Culminated from Diors decades-long research into stem cells, the Capture Totale line is infused with a regenerative floral complex of Madagascan longozo, Chinese peony, white lily, and Chinese jasmine, which help to re-energise stem cells rather than replenish them.

Dont believe a product that says it contains stem cells because the stem cells are not alive within the product. Only stem cells that are directly re-injectedand most likely come from your bone marrowworks, Joanna explained, debunking one of the most popular beauty fads in recent years.

With the expert on the line, we took the opportunity to ask our burning questions about maskneand skincare misconceptions.

What is a skincare philosophy that you live by?

Respect, support and protect. This goes for skincare, how we treat ourselves and others.

Your all-time favourite Dior Skincare product and why?

The Capture Totale C.E.L.L. Energy Super Potent Serum because it contains the most concentrated version of the cell energising complex and acetylated hyaluronic acid. It creates hydrated, plump and radiant skin. If you are consistent, you see results in days. My skin has never looked better.

A common skincare mistake many people make?

In my opinion, its using toner. Thats a misconception because still, many people use a toner as the second step of cleansing as opposed to the first step of treating the skin, and this is from my experience of talking with clients.

They put toner or micellar water on a cotton pad and they keep wiping and seeing more make-up. If you see more make-up on your cotton pad, that means you need to go back into washing.

Toner is very often misunderstood or skipped, and it shouldnt be. I cant imagine, for instance, applying a serum on my face without applying toner first. There is no way the efficacy of the product will be the same if you have not applied a toner. Depending on the toner, they offer hydration and sometimes micro-exfoliation, but mainly they are used to maintain the pH of the skin. The optimal pH for our skin is 5.5, and many factors from our diet to lifestyle, and even washing our face can throw the skins pH off the scale, so it's very important to balance it back.

See also: Lancme's Celebrity Make-Up Artist Lisa Eldridge & Neelofa Share 5 Beauty Tips

With face masks becoming part of everyday life, maskne has become a real problem. How can we prevent these breakouts?

When you wear a mask, it creates a micro-climate and we keep breathing carbon dioxide back and forth, so there is not enough of anti-bacterial oxygen getting into the skin. There is sometimes too much moisture happening, so we will get super hydrated initially, and then get quite dehydrated right after. Thats when you will experience eczema and redness.

What I recommend is keeping the skin as clean as possible before wearing the mask, with just a balancing toner, and protecting balm or healing ointments to lubricate areas where the mask could potentially irritate the skin. Very often, its on the nose bridge, as well as on the side and behind the ears.

For less reactivity, I wouldnt go through with the whole routine, but if you have to, I would advise starting your routine earlier so the products are on your skin for at least 30-40 minutes. If you will be stepping out shortly, reduce the routine and skip some steps. But no matter what, never forget about your SPF because the friction of the mask could also get rid of our stratum corneum and create little scabs, causing discolourations.

Then, as soon as you arrive home, take the mask off, wash your face, and again balance your skin with toner and use your serums.

What are your tips for soothing breakouts or eczema caused by wearing masks?

Even with microscopic breakouts, I would continue using any product that is hydrating because sometimes we misunderstand we have a breakout and then we try to use benzoyl peroxide, or everything that is dehydrating. No, your skin would be producing even more sebum. So keep hydrating your skin with soothing ingredients like colostrum and hyaluronic acid.

Your skincare routine?

My morning routine is very brief: cleanser, toner, a serum and then there is moisturiser. For my night time routine, my very first step is getting into the shower when I get home. I begin with massaging my body with my shower gel and silicone gloves under the shower, then I apply products like multi-vitamin oils and sometimes micro-exfoliating toners all over my body.

Then, I go to my face. I usually dont wear any make-up, so I start right away with my cleanser with some massaging movements and I remove it with a linen washcloth, followed by a toner. My favourite way of applying toner is the sponge techniqueinitially, you spread the product on your face, and then you press and release. When you press, your skin microscopically opens and when you release, the skin grasps whatever is on the surface.

After the toner, I use my serum. Ive been using the Capture Totale C.E.L.L. Energy Super Potent Serum since September. If you have weaker areas like forehead lines and nasolabial folds, these are the areas I would concentrate longer on, followed by an eye cream and moisturiser. Thats usually my five-step basic routine.

About twice a week, I do facial masks, one of those quick ones because Im the kind of New Yorker who only has five seconds for myself. But no matter how busy or tired I am at night, I would never forget about my skincare routine. Your skin is 60 per cent more potent to absorb everything during relaxation and rejuvenation time. So if you dont take care of your skin at night, you might as well forget doing anything in the morning. Twenty-five per cent of our immune system is within our skin, and we can improve that percentage of our health with products chosen for your skin condition and consistent skincare.

See also: Sulwhasoo's Ginseng To Achieve Skin As Flawless As South Korean Superstar Song Hye Kyo's

You work with many notable clientswhats the most common skincare problem celebrities deal with and treatments that they request for?

Celebrities have exactly the same problems as we do. The only one little difference is that celebrities tend to wear more make-up and more often than some of us, usually under the heat of theatrical or film lights, so they need a lot of hydration and rebalancing. Ive been called to the movie set many times to help soothe their skin with algae masks, or any cooling and hydrating treatments.

Their needs are equal to ours. They want to work on their facial contours and ensure their skin is as evenly textured as possible so their makeup looks perfect, so I would offer some mild exfoliation, perhaps micro-currents for targeted muscle stimulation, and maybe manual massaging to stimulate blood flow to create the sort of rosy healthy oxygenated looking skin. Many people call it a red carpet facial but I call them ordinary facial because every woman wants the samesmooth hydrated skin with nice cheekbones, beautiful jawlines, and thats what really works.

See also: Beauty Talk With Aznita Azman, Founder Of Nita Cosmetics

The rest is here:
Dior Skincare Ambassador Joanna Czech On Her Self-Care Routine And How To Prevent Maskne - Tatler Malaysia

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How Adam Smith Might Have Valued Amazon, Netflix, Tesla, And Tiny Biotechs – Seeking Alpha

By daniellenierenberg

Adam Smith (1723-1790) was not who you think he was. I'm talking about the original Adam Smith who wrote The Wealth of Nations (1776) and spent most of his life in Edinburgh, Scotland. The more recent "Adam Smith" - nom de plume of the late George Goodman who wrote The Money Game (1967) - bears much more resemblance to the Adam Smith you think you know.

The first Adam Smith would have had little interest in stock market wisdom because he regarded himself as a moral philosopher rather than an analyst of markets. In fact, he was not even a capitalist. His works do not include the words "capitalist" or "capitalism" because neither came into use in his lifetime. The first mention of "capitalism" in print was in the 1854 novel The Newcomes by William Makepeace Thackeray, whose father had been involved with the East India Company. Karl Marx, oddly enough, helped popularize the term in his classic Das Kapital (1867). The irony is that if Marx did not quite invent the concept of capitalism, he certainly made the term popular in the process of opposing and bashing it.

No one can know what Adam Smith would have thought about free market capitalism as presently practiced, nor can we guess what he would have thought about the aftermarket in shares which we call "the stock market." The first stock exchanges came into being a couple of years after his death and shares were traded in only a small handful of companies including the still extant Bank of New York (NYSE:BK). Security trading over Smith's lifetime was concerned primarily with credit instruments, the exceptions being one-off exchanges organized by and for the British and Dutch East India Companies. So no capitalism, no market opinions from Adam Smith. Sorry to have to tell you.

The primary interest of Adam Smith was the goal which gave his book its full title - an inquiry into the nature and causes of the wealth of nations, in short, the well-being of the general populace. Counterintuitively paired with this was the self-interest which led tradesmen and the early industrialists to seek profit. He used the term "invisible hand" only three times in his writing and just once in The Wealth of Nations, to wit:

The rich consume little more than the poor, and in spite of their natural selfishness and rapacity, though they mean only their own conveniency, though the sole end which they propose from the labours of all the thousands whom they employ be the gratification of their own vain and insatiable desires, they divide with the poor the produce of all their improvements. They are led by an invisible hand to make nearly the same distribution of the necessaries of life which would have been made, had the earth been divided into equal portions among all its inhabitants, and thus without intending it, without knowing it, advance the interest of the society, and afford means to the multiplication of the species...the beggar, who suns himself by the side of the highway, possesses that security which kings are fighting for."

This is the central core of Adam Smith's thinking. It has always interested me that the ultimate goals of Adam Smith and Karl Marx did not differ greatly. The important difference is that Smith believed in freedom of the market while Marx believed that the solution was the top-down mandate of a command economy. We are familiar at this point with the general course of events in top down economies. The 20th Century resolved that question definitively in favor of Smith's view, which we now call capitalism.

Smith, however, never imagined a world with an after-market of securities measured by such things as price earnings ratios and discounted free cash flow. He would have been astonished at the use of these and other forms of analysis central to modern markets including shares of corporations with thousands of shareholders and many millions of shares. The few larger businesses in his day - a few early industrialists and the enormous East India Companies - did not lend themselves to that kind of analysis.

Does that mean that the thinking of Adam Smith is useless in trying to understand value in the modern financial markets? Not at all. Smith's model of the invisible hand contains a clue as to the way he might have valued companies and their shares. In fact, the view of Adam Smith may take us back to the primary purpose of capital markets which focus on start-ups, IPOs, unicorns, perhaps even SPACs, and all companies in their early stages. Such companies seek capital with which they aspire to bring innovations. They hope to profit by serving the unmet and often unrecognized needs of a body of potential customers.

What Smith saw was the intricate interplay between the needs and desires of customers and the self-interest of a risk-taking capitalist. That is the core transaction of the capitalist system. Without so much as a glance at discounted future cash flow, Smith implicitly understood that for a business the important thing was the population for which a business might add value. The issues for the entrepreneur involve the accuracy of their estimate of that market, the share of that market they might expect to win, the revenues they might expect to receive, and the profit margin they might expect to realize on those revenues.

In short, Adam Smith's thinking may not ordinarily be very helpful in the after-market we call "the stock market" but is central to the universe of young and innovative companies. It is directly connected with the way businesses and customers are conjoined. What a business does for its customers, he implies, provides an outline of its ultimate value. For this reason, I see the conjunction of businesses and customers as potentially useful in thinking about leading companies in the current market, especially for those companies which cannot be analyzed usefully by the standard market metrics of sales, margins, earnings, PE, and discounted cash flow.

In Adam Smith terms, a company should be worth a reasonable return for what it contributes to the greater good of the general populace. This single sentence is at the heart of what I am calling the "Adam Smith Model" of valuation. Does it actually work when trying to value innovative companies? Can one make decisions based on this model? To a large degree I think it is the only really helpful approach in valuing companies driven by new products and concepts.

To show how this sort of analysis might work, I will start with my daughter's portfolio of innovative biotech companies, which she put together in the early days of the pandemic. It is a pretty good model of the kind of thing I have always kept a careful distance from. Her surprising success with this portfolio prompted my own internal debate.

My daughter is a bright young woman who will soon turn 50. She has a doctorate in art history from Penn but retrained as a nurse in order to live in the woods in western Massachusetts and raise her children as a single mom close to nature and away from urban centers. Her life is modeled more on Thoreau's Walden than on Ben Graham's The Intelligent Investor. Despite sitting at my dinner table for seventeen years she remained almost entirely ignorant about financial markets until recently. The after-market in stocks seemed to her insufficiently serious to deserve her attention, which might well have been Adam Smith's view had he lived to see it. I confess to having had similar thoughts myself at times but have suppressed them.

In recent years, however, prompted by the realization that she may one day retire and need an income, she has begun to take an interest in markets. Around the beginning of the COVID-19 crisis (on which she had early insight and much sound advice), she put together without telling me a portfolio of biotech companies. She did this on a very small scale. Over four or five months she is up well over 200%, an amount I have never made in anything like that period. Here's an excerpt from an email she sent me on her portfolio:

Yes, that's why I like leronlimab - CytoDyn (OTCQB:CYDY). It has many uses, a high safety profile (I don't give a second glance to drugs with a low safety profile-anyone could have seen that with hydroxychloroquine, and now dexamethasone-which is a broad-target immunosuppressant, hence will never be a commonly used drug for Covid). Leronlimab has a great safety profile and works with a known mechanism vs. the cytokine storm. Anything good for Covid (or the other viruses that are still around: SARS, MERS and Ebola) must not suppress the immune system as a whole (as do all steroids such as dexamethasone). Leronlimab is targeted at the CCR5 receptor-which makes it effective for coronaviruses as well as cancers and autoimmune disease. Amazing for metastatic cancer, including prostate (though the recent studies are on a hard to treat breast cancer), and probably other untreatable but common cancers. It's going to be great for HIV. It's going to work for host vs graft disease (post-transplants, when we go back to doing them). It also appears to work for NASH (non-alchoholic fatty liver disease, which has increased dramatically in numbers, but is silent in most people until it is at a late stage.) It is the next diabetes.

Mesoblast (MESO):

The next wave of medical advances are going to come through better understanding of immunomodulation. Most if not all diseases-including cardiac disease and diabetes--will come to be understood as inflammatory diseases to be manipulated at the cellular level. We will see more and more of these diseases due to our inflammatory (sedentary, antioxidant-deprived) lifestyle and toxic environment. In any case, I'm interested in the companies who are leading the way in specialized research in immunomodulation. Mesoblast is using stem cell technologies to repair the immune system, and applying that technology to many untreatable diseases.

Avalon GloboCare (AVCO):

Same argument as Mesoblast: multiple technologies, targeted immunotherapy. I'm not so interested in any single technology, but they are partnering on several important technologies (stem cells, diagnostic technologies), with broad implications and clinical uses. They are partnering to develop a nasal vaccine for Covid, but again, I'm not as interested in that particular product, but the broader technology. Nasal vaccines are going to be a winner for many reasons-ease of use, global application, and the fact that we will run short on syringes for other vaccines).

Altimmune (ALT):

Same as above: leader in NASH (non-alcoholic fatty liver disease), nasal vaccine technology

Okay, those are my four picks. Amazing for metastatic cancer, including prostate (though the recent studies are on a hard to treat breast cancer). The others, JNJ, Becton Dickinson, and DaVita, you know."

I love the fact that my daughter comes at investing from an angle so different from mine and with a skill set that does not overlap mine at all. I also love that its method combines brains and a good heart - the assumption that a company is worth the sum of what it contributes to human well being. What I find most intriguing is that her natural way of coming at things aligns so closely with the Adam Smith view. Can growth investing possibly have such a simple foundation?

You will probably have guessed that I have never bought anything like these biotech companies nor used anything resembling this kind of analysis. I do not, and could not possibly, recommend any or all of them. They are well outside my areas of knowledge and expertise. The only counsel I was able to give my daughter included the fact that when buying companies like this you should probably buy a basket of them - something which she had already done, intuitively.

By early July she had tripled her money and was beginning to be worried about what felt to her like an overhyped sector of an overpriced market. This was where she thought my advice might be useful. I laughed and said that she should be giving me financial advice instead of vice versa, but if she was nervous she should probably sell down to her comfort level (she's in a low tax bracket so cap gains aren't a problem). Perhaps she should at least sell down to the point at which she was investing with house money. I added that it was okay to leave a few chips on the table and let her long term bet ride. She agreed and did something close to that.

Her insight had been pretty simple. The value of a company should correspond to the amount of value added via the "invisible hand" to the health, happiness, and well-being of its customers - perhaps even to the general populace. You would start by estimating the size of the market for which it provided a product or service. You would then adjust to take into account the competition for that market and finally the probability of your particular company capturing a major part of that market. Then, and only then, you might begin to make rough estimates as to potential revenues and profit margin. The key correlation is not revenue and profit margin, which are well out in the future, but the value the company is likely to add to society. The payoff in small biotechs like these, if it comes at all, is likely to come in a rush when a large pharma company sees the potential and buys them out, fulfilling the Adam Smith projection of appropriate reward for a large service.

When I started to formulate it this way, I realized that I have missed quite a lot in never owning stocks which might be best measured in this way. This includes not just small biotechs and niche technology startups but also giant current market leaders such as Amazon (AMZN), Netflix (NFLX), and Tesla (TSLA). At every point in the lives of these three companies, I have found that the methods by which I have always valued stocks - things like discounted earnings, dividends, and cash flow - made me unable to put together any reasonable argument for owning them.

Had I finally stumbled upon a valuation model that might provide a rationale for buying them? Up to this point, I have not seen a persuasive methodology for thinking about the value of these companies. Could this simple approach account for their unusually high valuations?

Adam Smith implied that the relationship between a business and the population it served was the invisible force behind what we call capitalism. It takes only a small further step to propose that the population served by a business can also be described as an "asset" owned by that business. In some cases, especially young or innovative companies, it is customers acquired that is the central asset. The idea of a business "owning" its customers is not new. I first read about it in a novel at least fifty years ago when a literary agent retires by essentially selling his customers to a rival - a practice that was apparently commonplace even then.

This customer-based approach seems to be the way the founders of these three market leaders looked at the opportunity. Customers weren't just part of the picture. They were the whole thing. Acquiring customers is what these companies set out to do. Everything else could come later. They were determined to do everything it takes to own the largest number of customers, including running their businesses with negative earnings and free cash flow for a long time. The market caught on to their goals and their prices shot up to the stratosphere.

Amazon, Netflix, and Tesla have always sold at ridiculous multiples of earnings and cash flow, if any, and are ridiculously expensive by pretty much every other traditional measure. When you look at them the way my daughter looks at biotechs, however, the picture changes. You set the standard ratios aside and instead ask: what is the value of these companies if measured by the sum of value they provide in service to their actual and potential customers? The transmission of that value to shareholders is initially as invisible as the invisible hand by which value is distributed to the populace. It is nevertheless reflected in the stock price.

Here's how one might do a broad estimate of value for the three companies:

Today, online commerce saves customers money and precious time," writes Bezos. "Tomorrow, through personalization, online commerce will accelerate the very process of discovery. Amazon.com uses the internet to create real value for its customers and, by doing so, hopes to create an enduring franchise, even in established and large markets.

We believe that a fundamental measure of our success will be the shareholder value we create over the long-term. This value will be a direct result of our ability to extend and solidify our current market leadership position. The stronger our market leadership, the more powerful our economic model.

Because of our emphasis on the long-term, we may make decisions and weigh tradeoffs differently than some companies... We will continue to make investment decisions in light of long-term market leadership considerations rather than short-term profitability considerations or short-term Wall Street reactions...We aren't so bold as to claim that the above is the 'right' investment philosophy, but it's ours, and we would be remiss if we weren't clear in the approach we have taken and will continue to take.

From the beginning, our focus has been on offering our customers compelling value," explained Bezos. "We brought [customers] much more selection than was possible in a physical store (our store would now occupy six football fields), and presented it in a useful, easy-to-search, and easy-to-browse format in a store open 365 days a year, 24 hours a day."

That's Amazon's mission statement summed up in a few paragraphs. The guiding purpose to this business model is positioning yourself to "own" more and more customers. This customer-obsession of Bezos amounts to is a manifesto for innovative companies. The second paragraph flows directly from the core principle of Adam Smith. Get first things first, Bezos is saying, meaning understanding the potential market and seizing it. Profitability and measurements commonly used by Wall Street come later.

Amazon is no longer a young company in chronological age, but the vision embedded in its mission statement is to remain a young company forever. A Day 1 company, as Bezos calls it, is always visionary and entrepreneurial in its thinking. What Bezos is saying to investors is: disregard the numbers used by Wall Street analysts. They are important measures only for Day 2 companies (slow-moving, mature companies in stasis, for which the next stage is death). Keep your eyes on the main thing - the growth of your customer base and a high level of customer satisfaction. Facebook (FB) and Alphabet (GOOG)(GOOGL) were like that in early stages but moved fairly quickly to address the question of how to monetize their users, eventually succeeding and becoming measurable by ordinary metrics. They are now ordinary growth companies with moderately high PEs, at least in context of the current market. Bezos rejected early monetization. Have faith, he said. We will monetize our customer base when we get around to it.

The greatest single risk for Amazon is its increasing size, which makes it difficult to remain nimble and full of energy. At some point, it will face the horror which confronts history's great empires - running out of worlds to conquer. Political constraints may have something to do with that, but pure size is the major burden. Summing it up, I would buy Amazon at something like 50-60% of its present price if nothing had gone wrong in the business in the meantime.

2. Elon Musk somehow manages to top Bezos. His manifesto, much of which comes out in random statements and tweets, is that Tesla will one day produce pretty much every car sold in the US, maybe even the world. At the very least it will be the driving force in a new industry. His business has a powerful technological core, but the rational for it is the prospect of capturing much of the total customer base for vehicles. It currently appears to be priced on the assumption that Musk will succeed in this ambition to a large degree.

Musk is confident that Tesla's technology will become the universal standard and squeeze most of the current auto industry into terminal decline. Its panache stems from great aesthetics and the promise of enlisting his customers in the project of slowing climate change and helping save the world. Tesla, he implies, will almost incidentally become highly profitable, an outcome to which Musk himself seems to be personally indifferent but in which his investors might have some interest. If he is right, Tesla will probably look cheap if bought today or tomorrow at 160 times its current (and first annual) positive earnings.

Like Bezos, Musk would have us remember: we don't care about all that. That's the old valuation model. What we care about is a market of 17 million vehicles sold annually in the US and a number around five times that in the world. That's the scale of customers Elon wants to own. Once that happens, he will bite the bullet and monetize.

To own Tesla at anything like the current price you have to make a few audacious assumptions. You have to believe that vehicles will continue to be bought on very large scale and that the overwhelming number of vehicles sold will become electric within a short period of time. You then have to believe that Tesla will become the company that owns most of the customers and sells most of the vehicles. It's not impossible, but there are obstacles to overcome.

If Ford, GM, Toyota, Honda and others launch a modestly successful counterattack, or the whole market shrinks, you will see the earnings and cash flow multiples of Tesla shares contract in the general direction of the valuations of those "Day 2" companies. In other words, if you are an investor, you don't want Tesla to become just another car company, nor do you want it to be the last giant in an industry that is contracting and possibly dying. If one of those things happens, Tesla, as measured by the Adam Smith premise, is likely to be a disappointing investment. This is very broad brush analysis, but that's the only way to really deal with Tesla, a company quite similar to my daughter's biotechs. The risks for Tesla seem high and hard to calculate. These are the problems routinely faced by innovative companies in their early stages, and you must also pay attention to the risk that Tesla could run out of time to overthrow the industry while the industry still exists in its present form.

3. Netflix is a company I have looked at only recently. Until a few months ago I had never used their product - not once. Entertainment is OK - I'm being entertained by writing this, and I dare to hope that you readers are both entertained and stimulated to further thought by it - but I didn't experience Netflix until a millennial step child and her husband spent some time with us and promptly realized that they couldn't live without it. They put it on a couple of our TVs so that they would have some kiddie movies to bribe their 3-year-old to eat dinner plus an hour of decompressing entertainment for themselves before sleep.

A few months ago my wife and discovered that we still had it, linked somehow to their home two thousand miles away, and it turns out that the shows are pretty good. They turned out to be especially valuable during the lockdown. We had run out of old movies, so we started over with Netflix. I started paying attention to articles on Netflix and ultimately took a look at their numbers.

Egads! They have been unprofitable from day one and their negative cash flow has done nothing but increase. Their costs for content are going up and their competition is mounting. On the other hand, Stranger Things is the kind of nitwit escapism that I found that I like after a long hot day teaching tennis (my wife not so much).

How do I put the two views of Netflix together. In this case, the risks and uncertainties make the stock uninvestable for me. For one thing, I am used to having entertainment piped into me for free (I automatically tune out all ads.) The numbers needed are just too daunting for Netflix, the rising costs for content are worrisome, and ultimate limits in a market now sliced several ways implies limits to growth. I am doubtful that Netflix will ever morph into a company I can measure more conventionally. I'm pretty sure I wouldn't renew if our faraway relatives stopped providing it for free. That's the core of it: I'm a customer of sorts, but they don't really own me. I don't own them either, and am not likely to any time soon.

The outperformance of high growth companies over the last decade and most spectacularly over recent months has naturally invited vigorous debate. The catastrophic dot.com crackup exactly two decades ago has receded sufficiently that alt explanations of market behavior are once again beginning to be proposed in earnest. This article is perhaps one of them but exists within the frame of traditional methods.

The dot.com era which reached its peak in 2000 crashed amidst assertions that eyeballs and clicks were better measures of value than earnings or cash flow. I lived through it as a bystander, listening to fellow fitness enthusiasts in the workout room at my tennis club boast about their portfolios, then noticing their absences one by one as the crisis unfolded. I didn't feel schadenfreude, far from it, only relief that I myself had not been ruined.

Valuations are once again at a point which calls ordinary prudence into question. Are the traditional models of valuation no longer worth using? This was suggested recently by BlackRock quant Jeff Shen who argued here that traditional efforts to solve the "mystery" of value are worthless. The Shen view, by the way, derives from this article by another BlackRock analyst, Gerald T Garvey, published in the prestigious Journal of Portfolio Management. The Garvey article comes down firmly on the growth side of the growth/value debate arguing that "elevated percentage value spreads predict higher risk, not higher returns."

In more down to earth terms, Shen and Garvey are saying that companies whose shares haven't been able to grow in this environment are losing ground and possibly dying, and should be avoided. If a stock goes up a lot it is probably safe because the wisdom of crowds is behind its rise. This is the kind of statement that is true until it isn't. Shen goes on to argue that contemporary investors should look for alt indicators and models such as the happiness of a company's employees. That particular idea didn't exactly blow me away, and neither Bezos nor Musk seem to be proponents of using that principle to focus or drive their businesses.

On the other hand, an effort to measure a company's success in terms of the overall value it provides to its customers does seem to me an interesting way to think about growth companies. Most companies trading in the aftermarket for stocks - by now you know that when I use this awkward but accurate phrase I am referring to the "stock market" - are not high growth companies and are probably best analyzed by traditional measures. Ultimately some form of traditional value measurement must appear within the life-cycle of a successful company.

To Jeff Bezos, the moment when traditional cash measures become important to a company is the day that it wakes up as a Day 2 company, a company that does not attempt to reinvent the world afresh every morning. While such a company may still turn out to be a decent investment, it's important for value investors to pay careful attention to their risk of having their business disrupted by new technologies and methods. This is a fairly straightforward way of thinking about the world we now live in, and I have learned to ask the hard questions about everything I own - even companies with seemingly strong moats.

Disruption is a major theme of the contemporary world, and every thoughtful person would do well to put the world together afresh every morning. Even with an open mind, it's hard to anticipate what hidden risks might cause a company's current defenses to collapse. Because of the incredible speed of change and the prevalence of unsuspected collateral effects, this questioning is important in a way that it has never been in the past. That was the important lesson number two from the dot.com event. Buying the disruptors rarely made fortunes, but not being sufficiently cautious about potential disruptees was a good way to lose a fortune.

For these businesses the Adam Smith Model needs to be turned upside down so that it becomes a story about loss of customers. One of the great anecdotal examples was Bill Gates stunning a 1990s gathering of Buffett's value investor pals by using his knowledge of the digital world to inform them that Eastman Kodak (KODK), then a market stalwart, was "toast." The customer criterion proves its importance when inverted. I was unable to estimate the outcome for Amazon - haven't made a nickel directly by buying it - but it was obvious to me instantly that it was going to be the end of the road for many other retailers, as well as many malls and REITs. The history of Sears Roebuck and Walmart were powerful precedents. The only thing not entirely clear was the time frame, which is proving to be much faster than most people expected.

The astonishing thing was how eagerly investors jumped on the Amazon bandwagon, which has many uncertainties, and how slowly the investor mind adjusted to the knock-on effects, which were far more certain. The key to grasping this quickly, is to focus on customers "owned" but sure to slip away, as in the case of Kodak.

The Adam Smith Model is simply one of the ways of making an estimate concerning what the cumulative value should be somewhere down the road at whatever time the company decides to monetize the cash value of owning its customer base. At that point, it will begin to report profits and cash flow, pay dividends, and buy back shares. Apple (AAPL) may be the best current example of this model. It started paying dividends and buying back shares about a year before its growth began to level off. As the dream of perpetual growth disappeared, investors were rewarded by the cold cash that abundantly flowed.

This is the distant event that Bezos' mission statement grudgingly projects. For Bezos, earnings, dividends, and buybacks are Day 2 concerns, and you get the feeling that he would just as soon not live to see them. Being a Day 2 company, is like living a comfortable and happy life: the great second-best award for those who have given up their aspirations to greatness. So Apple was once an innovative company priced on the basis of the Adam Smith Model and has now normalized into a Day 2 company which can be valued by the traditional tools. Who knows, maybe it has a few positive tricks up its sleeve but relentless regular growth is a thing of the past.

Amazon seems to be on the same general course as Apple, but with ordinary shareholder gratification deferred into a less well defined and more distant future. You just have to wait for it, and at an incredibly low discount rate such as the current Treasury rates you are willing to pay up for the ultimate awards now and wait a long time. This is part of the current market infatuation with rapid and persistent growth. If you project very far into the future, the value may approach infinity, or since that concept no longer exists even in physics, you could approximate it by the difficulty Amazon would have if Amazon's business became the major part of the gross product of the planet.

High valuations in the current market can be partially explained by a number of factors including historically low interest rates and the appeal of the tech leaders during a broad public lockdown. It also true, however, that the most optimistic thinking stems from a gambling mentality which is supported by the famous Petersburg Paradox which has come to bear in their valuations. There are a number of recent articles with varied approaches to this subject, and you can sample them by googling Petersburg Paradox.

The Petersburg Paradox is generally credited to Daniel Bernoulli, who published an article on it in 1738, but is sometimes credited to his cousin Nicolaus Bernoulli who talked about it in a letter written in 1713. It is a simple gambling game that doubles your winnings with each successive throw of tails. Its expected return generates an infinite series of events the probability of which decline by the exponential 1/2 to the N power exactly offsetting the exponential increase in winnings (2 to the N power).

Each successive term is exactly 1. The mathematically expected return is the sum of that infinite number of ones. I suppose that this means you max out when the number in dollars is equal to the number of bits (or Planck units) in the universe.

This series, therefore, produces quite large expectation of winnings despite the fact that the probability of large winnings at any particular future point obviously diminishes enormously and becomes very slight after a few coin tosses. It is famous for the contradiction of the expected total return and the relatively small amount that any reasonable person would be willing to wager on that return. A number of mathematicians have attempted to resolve this contradiction - economist and quant Paul Samuelson having been one of them - but their efforts at refutation have been unsatisfying.

Recent articles have related the Petersburg Paradox to investor expectations for stocks with high and persistent earnings growth. An extremely smart and interesting article was published way back in 1957 by David Durand (The Journal of Finance, Vol 12, No 3, Sep 1957, pp 348-363). Durand explored the problem of valuation for growth stocks including the then relatively new approach of using multiple discount rates at various break points in time. The growth numbers are quaint - annual growth at numbers like 5 and 6.5% - chickenfeed compared to growth rates of modern high tech companies.

Durand related the question of pricing long growth periods to the Petersburg Paradox, addressing the infinity problem and the need to truncate the infinite series at some point. This has a parallel to the problem of valuing current growth companies where it is necessary to consider not only forecasts for future earnings growth rates but also the length of waiting time before cash flows and dividends appear. There's also the question of the interest rate used for discounting, which is now virtually nil but has been very significant at times in the past.

The Adam Smith Model happens to dovetail nicely with the distant outcomes of the Petersburg Paradox coin flip game. The further away the payout is from the present the larger the rewards become when you finally throw heads. It's just that in the case of fast growing but not yet profitable companies, you more or less defer the chance of hitting heads early in order to let the reward build exponentially and have the promise of hitting a very large summative outcome in the future. That's where the thinking of investors in Amazon, Tesla, and Netflix must come from, and it's more or less rational if their estimate of the payoff and the time necessary to achieve it are reasonably accurate. It has been pretty accurate in the case of Apple.

There's just one more thing, of course. What if the estimate of Adam Smith value proves to be outright wrong? What if a tough new competitor with a better technology or improved business model appears? What if competition already in place proves to be more formidable than assumed? Even with Amazon these risks must be taken into account, but with Tesla they should be major concerns, and with Netflix they should be very major concerns.

There could also be exogenous risks such as a major rise in interest rates which would wreck the denominator and greatly reduce the value of a distant payoff. That high denominator, by the way, was what drove price earnings ratios in the 1970s down to the single digits. Returns even a few out years were so heavily discounted that no one wanted to look that far into the future. This sort of thinking served to greatly diminish the appeal of growth stocks.

Innovative companies don't always work out. I thought about that a lot around the year 2000, when I attached a 95% probability to my belief that the investing world had lost its collective mind but reserved a 5% probability I was the one who just didn't get it. The odd thing is that some of the new model dot.coms did, in fact, contribute quite a bit to the general welfare. They made all sorts of businesses more efficient, and at the same time made basic communication for everyone cheaper, faster, and better. This is presumably a good thing. In the end, however, it didn't work out well for shareholders who held on long term.

But there's another question. Did their temporarily outrageous valuations represent a magical mechanism for pulling forward a proper reward for founders and the most nimble shareholders despite the fact that the companies themselves were destined to never ultimately earn any money? In a just version of Adam Smith's invisible handsome reward was certainly owed to the founders and early owners who contributed so much to human well being but the mechanism by which they received it is somewhat murky,

So what if that deferred payoff never comes?

Schopenhauer asked a similar question in his Studies in Pessimism, except that he asked it about death, not a sudden gush of cash flow. Calm down, Schopenhauer argued. Why fear death? If you knew it wasn't the end of things, that you would wake up tomorrow morning feeling fine, you wouldn't worry about it much. What about waking up next week? What about next year? What about five or ten years out? What about a thousand years before you wake up? Ten thousand? What about... never? Would it make a difference?

Schopenhauer's courage in the face of non-being is reflected in the large number of investors who seem unworried about the possible absence of cash returns many years into the future. Once you take the Schopenhauer premise, it doesn't matter if the payoff never arrives. The stock is going up today in anticipation of it. That's all that really matters. Shareholders are happy. You can always cash out at your convenience. What is the future anyway but a dwindling infinite series?

It's really more like heaven than death. Both of my grandmothers believed strongly in its existence, although I can't imagine what they really thought it would be like. It probably didn't matter. In both cases, it sustained them over the course of long and productive lives which they lived with great confidence of a wonderful if not precisely defined eternity. Ignorance was bliss.

A business is its customers. Is it as simple as that? The value of a business is the value of the services provided to its present and prospective customers discounted for the distance in time to monetization of those customers but discounted to reflect the possibility of various things that could happen to reduce or wipe out that future payoff. Both new and rapid growth businesses generally defer that payout further into the future than most businesses, especially if the discounting factor is relatively low.

This model produces huge winners and abject losers. We marvel at the winners when we see them without considering survivor bias. We discard the losers even if they have played a major part in the evolution of the economic world and traded at high prices in optimistic moments along the way. In retrospect we wonder why they once traded at such high prices.

In very young industries such as biotechs, the outcome often leaves losers by the way side and rewards just one or two competitors. One way of thinking about this is that at the outset many such companies own a similar probability of surviving but one or two end up "owning" most of the customers and the cash flow bonanza that will eventually come with them. The probability of winning gradually shrinks for most but rises for the winners. For that reason, my daughter's approach of buying a basket of these companies is probably the best way for investors to participate.

This approach may also be very helpful in evaluating growth companies which are not new but remain at some distance from giving investors serious cash rewards. Here the method for selecting a basket of winners draws upon the kind of broad-brush estimates and calculations used in selecting a basket of small biotechs. If looking closely at Amazon, Tesla, and Netflix doesn't help much, it's important to make and constantly update estimates bearing upon the scale and strength of their "ownership" of customers as well as rough estimates of risks. This broad and approximate approach is how Adam Smith would probably have looked at valuation of companies if he was as interested in profits as we sometimes assume him to have been.

Disclosure: I am/we are long JNJ, BDX. I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it (other than from Seeking Alpha). I have no business relationship with any company whose stock is mentioned in this article.

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How Adam Smith Might Have Valued Amazon, Netflix, Tesla, And Tiny Biotechs - Seeking Alpha

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Studies uncover new approaches to combat hair loss in men and women – Business MattersBusiness Matters

By daniellenierenberg

The studies show that JAK inhibitors and other small molecules are capable of reawakening dormant hair follicles and stem cell therapies that can develop new follicles.

The first study was led by Angela Christiano, whos a professor of Dermatology at Columbia University Vagelos College of Physicians and Surgeons. The researchers discovered some previously unknown cells that ensure that mouse hair follicles dont leave a resting state. If the activities of these cells are inhibited, dormant follicles can be reawakened.

In the second research, the team of Christiano found a method for growing human hair in a dish. This method could make more men and women start exploring the idea of hair restoration surgery. It could also enhance the method that pharmaceutical companies consider when looking for new hair-growth medications.

In pattern baldness, lots of hair follicles are still in existence. However, they are dormant. The focus of researchers on getting drugs that function effectively in the same pathways as minoxidil and finasteride has affected their quest to find new drugs that can reawaken follicles and inhibit hair growth. Notably, minoxidil and finasteride are the only two medications that can be used by men suffering from male pattern baldness.

The researchers had previously found a new pathway, known as JAK-STAT, which is active in the stem cells of resting hair follicles and makes them remain in a state of dormancy. Christiano and other researchers in his team showed that JAK inhibitors used on mouse skin are good for reawakening resting follicles in mice.

Their second study was targeted at knowing more about the natural processes of making sure that the follicles remain dormant. Therefore, the researchers searched for factors that managed the activity of the JAK pathway in the hair follicle.

During the search, the Colombian researchers found a formerly unknown immune-related cell type that is capable of creating a substance called Oncostatin M. This substance makes sure that the follicle doesnt leave a dormant state. One of the authors of the study, Etienne Wang, Ph.D., notes Rare subsets of immune cells were previously difficult to identify in a whole skin, but this work was facilitated by our ability to sequence individual cells and pinpoint the ones making Oncostatin M.

There are some resemblances between these cells and macrophages, which are regarded as the immune systems scavenger cells. The researchers discovered that these cells are close to resting hair follicles.

These cells have been named trichophages. It should be noted that this name is taken from tricho, which is a Greek word for hair.

Besides, the hair cycle could be turned on when the trichophages are being targeted. By utilizing antibodies and small molecular inhibitors for inhibiting Csf1R, which is a receptor that is found on the trichophages, the flow of Oncostatin M could be blocked so that the hair cycle can start once again.

The second study involved the creation of a means of growing human hair in a dish. This method could lead to hair restoration surgery for an increased number of individuals including women. This new development could also enhance the method through which pharmaceutical companies find new hair growth medications.

It is worthwhile to note that this study brings a novel idea of growing human hair follicles in a dish without using any implantation in the skin.

Notably, researchers have been generating new rat or mouse hairs by culturing cells that were extracted from the end of existing follicles.

To find a way around human hair cells resistance, the Colombian researchers have been looking for ways to build conditions that look like the 3D environment that is the habitat of human hair cells. Although they failed at the beginning, they are making progress now as they have found a way to grow new human hair follicles in a dish in a lab.

In a nutshell, these new approaches can bring a significant change to the process of dealing with hair loss in both men and women. While studies are still going on, you can cope with your hair loss by picking the right products at Lilyhair.co.uk to improve your overall look.

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Studies uncover new approaches to combat hair loss in men and women - Business MattersBusiness Matters

categoriaSkin Stem Cells commentoComments Off on Studies uncover new approaches to combat hair loss in men and women – Business MattersBusiness Matters dataJuly 8th, 2020
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Research and therapy with induced pluripotent stem cells …

By daniellenierenberg

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Research and therapy with induced pluripotent stem cells ...

categoriaIPS Cell Therapy commentoComments Off on Research and therapy with induced pluripotent stem cells … dataJuly 8th, 2020
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Tyson, Ronaldo, and more sports stars who use stem cell treatment costing up to 15,000 to speed up healing – The Sun

By daniellenierenberg

THE world's top sports stars are preferring to use stem cell treatment, instead of undergoing major surgery that could leave them out for months.

Cristiano Ronaldo, Rafael Nadal, and most recently Mike Tyson have all tried the therapy, which can cost anywhere from 4,000 to 15,000, when they've suffered injury.

Ailments that can be treated, include tendon inflammation, muscle strain, arthritis, degenerative disc disease, and even bone fractures.

And sportsman who have undergone stem cell therapy are benefitting from improved results, as well as a faster recovery time.

Collected from the blood from a newborn babys umbilical cord, the bone marrow or from body fat, stem cells are injected into an athletes' affected area.

They get to work by replenishing damaged cells from an injury or through wear and tear.

Stem cells also help reduce pain and inflammation, increase blood flow, and promote soft-tissue growth.

It helps the body to heal naturally, and means sports stars can potentially avoid going under the surgeon's knife.

When you're a top sports star, if you get injured the first thing you want to do is get back into the thick of action as quick as possible.

Unfortunately, many injuries can take a long time to heal, and will never allow the sportsman in question to return to the same level he/she was at before the injury.

That's where stem cell treatment is a game-changer.

Forget surgery, steroid injections, and lengthy physiotherapy, which don't always repair the issue at hand.

Stem cell treatments offer an alternative, albeit at a price, to have a non-surgical therapy that's non-evasive and, more importantly, heals the problem fast cutting out the need of rehabilitation.

Better still, some patients have reported that the therapy has not only reversed existing damage, but has strengthened cells against further damage.

Juventus star Ronaldo and Spanish tennis hero Nadal are all fans of stem cell treatment.

Back in 2016, when the Portuguese forward was playing for Real Madrid, he suffered a hamstring tear that threatened to keep him out of action of an important Champions League game against Manchester City.

Although he missed the first leg, he was back for the second - less than three weeks after suffering the problem.

That same year, Ronaldo tore a collateral ligament in his knee during Portugals Euro 2016 final against France.

Again, he turned to stem cell treatment and was back in training with Los Blancos just a month after his knee complaint.

Nadal's chronic knee problems forced him to take seven months off from tennis in 2013.

But stem cell treatment allowed the cartilage to repair. In the seven years since he's won six Grand Slams, there's been no setbacks from his troublesome knee and he appears as mobile as ever.

The Spaniard also cured a long-standing back problem with the therapy.

The former heavyweight champion, who is considering making a comeback, is the latest name to have tried stem cell treatment.

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It is not known what Tyson, 53, was suffering from - but he was happy to reveal all in an Instagram chat with basketball legend Shaquille O'Neal.

Iron Mike said: "You know what I had done? I had stem-cell research therapy.

"I feel like a different person but I can't comprehend why I feel this way. It's really wild what scientists can do."

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Tyson, Ronaldo, and more sports stars who use stem cell treatment costing up to 15,000 to speed up healing - The Sun

categoriaBone Marrow Stem Cells commentoComments Off on Tyson, Ronaldo, and more sports stars who use stem cell treatment costing up to 15,000 to speed up healing – The Sun dataMay 22nd, 2020
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Epidermal Stem Cells of the Skin – PubMed Central (PMC)

By daniellenierenberg

Abstract

The skin constantly renews itself throughout adult life, and the hair follicle undergoes a perpetual cycle of growth and degeneration. Stem cells (SCs) residing in the epidermis and hair follicle ensure the maintenance of adult skin homeostasis and hair regeneration, but they also participate in the repair of the epidermis after injuries. We summarize here the current knowledge of epidermal SCs of the adult skin. We discuss their fundamental characteristics, the methods recently designed to isolate these cells, the genes preferentially expressed in the multipotent SC niche, and the signaling pathways involved in SC niche formation, SC maintenance, and activation. Finally, we speculate on how the deregulation of these pathways may lead to cancer formation.

Keywords: hair follicle, multipotency, self-renewal, cell fate determination, Wnt signaling, Bmp, cancer

Skin and its appendages ensure a number of critical functions necessary for animal survival. Skin protects animals from water loss, temperature change, radiation, trauma, and infections, and it allows animals to perceive their environment through tactile sense. Through camouflage, the skin provides protection against predators, and it also serves as decoration for social and reproductive behavior.

Adult skin is composed of a diverse organized array of cells emanating from different embryonic origins. In mammals, shortly after gastrulation, the neurectoderm cells that remain at the embryo surface become the epidermis, which begins as a single layer of unspecified progenitor cells. During development, this layer of cells forms a stratified epidermis (sometimes called interfollicular epidermis), the hair follicles (HRs), sebaceous glands, and, in nonhaired skin, the apocrine (sweat) glands. Mesoderm-derived cells contribute to the collagen-secreting fibroblasts of the underlying dermis, the dermovasculature that supplies nutrients to skin, arrector pili muscles that attach to each hair follicle (HF), the subcutaneous fat cells, and the immune cells that infiltrate and reside in the skin. Neural crestderived cells contribute to melanocytes, sensory nerve endings of the skin, and the dermis of the head. Overall, approximately 20 different cell types reside within the skin.

In the adult, many different types of stem cells (SCs) function to replenish these various cell types in skin as it undergoes normal homeostasis or wound repair. Some SCs (e.g., those that replenish lymphocytes) reside elsewhere in the body. Others (e.g., melanoblasts and epidermal SCs) reside within the skin itself. This review concentrates primarily on epidermal SCs, which possess two essential features common to all SCs: They are able to self-renew for extended periods of time, and they differentiate into multiple lineages derived from their tissue origin (Weissman et al. 2001).

Mature epidermis is a stratified squamous epithelium whose outermost layer is the skin surface. Only the innermost (basal) layer is mitotically active. The basal layer produces, secretes, and assembles an extracellular matrix (ECM), which constitutes much of the underlying basement membrane that separates the epidermis from the dermis. The most prominent basal ECM is laminin5, which utilizes 31-integrin for its assembly. As cells leave the basal layer and move outward toward the skin surface, they withdraw from the cell cycle, switch off integrin and laminin expression, and execute a terminal differentiation program. In the early stages of producing spinous and granular layers, the program remains transcriptionally active. However, it culminates in the production of dead flattened cells of the cornified layer (squames) that are sloughed from the skin surface, continually being replaced by inner cells moving outward ().

Epidermal development and hair follicle morphogenesis. The surface of the early embryo is covered by a single layer of ectodermal cells that adheres to an underlying basement membrane of extracellular matrix. As development proceeds, the epidermis progressively stratifies and acquires layers of terminally differentiating cells that are required to establish a functional barrier. During embryonic development, some of the undifferentiated basal cells are instructed by the underlying dermis (signal 1) to adopt a hair follicular fate. Subsequently, the epidermis sends a message to the dermis (signal 2) to make the dermal papilla (DP). Finally, the DP sends a message to the developing follicle (signal 3), allowing its growth and differentiation to form the discrete lineages of the hair follicle and its hair. Encased by a basement membrane, the basal layer of the follicle is referred to as the outer root sheath (ORS). At the base of the mature follicle is the highly proliferative compartment called the matrix (Mx). Matrix cells differentiate to form the concentric rings of differentiating cells that give rise to the hair shaft, its channel (the inner root sheath, IRS), and the companion layer. Hair follicles also contain sebaceous glands to ensure the water impermeability of the hair and lubricate the hair channel and skin surface.

The major structural proteins of the epidermis are keratins, which assemble as obligate heterodimers into a network of 10-nm keratin intermediate filaments (IFs) that connect to 64-integrin-containing hemidesmosomes that anchor the base of the epidermis to the laminin5-rich, assembled ECM. Keratin IFs also connect to intercellular junctions called desmosomes, composed of a core of desmosomal cadherins. Together, these connections to keratin IFs provide an extensive mechanical framework to the epithelium (reviewed in Omary et al. 2004). The basal layer is typified by the expression of keratins K5 and K14 (also K15 in the embryo), whereas the intermediate suprabasal (spinous) layers express K1 and K10. Desmosomes connected to K1/K10 IFs are especially abundant in suprabasal cells, whereas basal cells possess a less robust network of desmosomes and K5/K14. Rather, basal cells utilize a more dynamic cytoskeletal network of microtubules and actin filaments that interface through -and -catenins to E-cadherin-mediated cell-cell (adherens) junctions, in addition to the 1-integrin-mediated cell-ECM junctions (reviewed in Green et al. 2005, Perez-Moreno et al. 2003). Filaggrin and loricrin are produced in the granular layer. The cornified envelope seals the epidermal squames and provides the barrier that keeps microbes out and essential fluids in (Candi et al. 2005, Fuchs 1995) (). The program of terminal differentiation in the epidermis is governed by a number of transcription factor families, including AP2, AP1, C/EBPs, Klfs, PPARs, and Notch (reviewed in Dai & Segre 2004).

Although the molecular mechanisms underlying the process of epidermal stratification are still unfolding, several studies have recently provided clues as to how this might happen. Increasing evidence suggests the transcription factor p63 might be involved. Mice null for the gene encoding p63 present an early block in the program of epidermal stratification (Mills et al. 1999, Yang et al. 1999).

There are several possible mechanisms by which stratification could be achieved with an inner layer of mitotically active cells and suprabasal differentiating layers. In the first mechanism, a proliferating basal cell progressively weakens its attachment to the basement membrane and to its neighbors and is pushed off the basal layer and up into the spinous layer. In vitro studies demonstrated that this process, referred to as delamination, effectively allows stratification (Vaezi et al. 2002, Watt & Green 1982). A possible alternative to delamination is that basal cells in a stratifying tissue might orient their mitotic plane of division perpendicular to the underlying basement membrane, which would consequently place one of the two daughter cells in the suprabasal layer.

Recent studies in mice suggest that during embryonic development in skin, the majority of mitotic cells within the epidermis go from having their spindle plane parallel to the basement membrane to a perpendicular orientation (Lechler & Fuchs 2005, Smart 1970). In these perpendicular orientations, the apical centriole associates with a complex containing Nuclear Mitotic Apparatus protein, partitioning-defective protein 3, atypical protein kinase C, Inscuteable, and partner of inscuteable. The association with this cortical complex is intriguing because most of these evolutionarily conserved proteins have been shown genetically to be essential for the asymmetric cell divisions that occur in Drosophila neuroblasts and in Caenorhabditis elegans embryos (Cowan & Hyman 2004, Wodarz 2005). Although many features of the underlying mechanism remain to be addressed, proper spindle orientation appears to require 1-integrin and -catenin, further underscoring the importance of basement membrane and adherens junctions in the establishment of epidermal polarity and tissue architecture (Lechler & Fuchs 2005). More studies are now needed to determine the respective role of asymmetrical cell division and delamination during development, skin homeostasis, and pathological conditions such as wound healing.

The development of HFs involves a temporal series of epithelial-mesenchymal interactions (reviewed in Hardy 1992) (). First, the dermis signals to the overlying epidermis to make an appendage. In response, the epidermis then transmits a signal to instruct the underlying dermal cells to condense and form the dermal papilla (DP). Another signal is then sent from the DP to promote the proliferation and elaborate differentiation required to form the epidermal appendage.

The process of HF development has been divided into discrete stages distinguished by their morphological and biochemical differences (Paus et al. 1999). The first morphological sign of HF development is the formation of a hair placode, in which the basal epithelium becomes elongated and invaginates at sites where dermal condensates form. As the developing follicle extends downward and en-wraps the DP, the cells at the base maintain a highly proliferative state. During follicle maturation, these proliferating (matrix) cells begin to differentiate into the inner root sheath (IRS), which is the envelope for the future hair shaft and is marked by the expression of the transcription factor GATA3 and the structural protein trichohyalin (Kaufman et al. 2003, O'Guin et al. 1992). The outer layer of cells becomes the outer root sheath (ORS), which is contiguous with the epidermis and is surrounded externally by the basement membrane. The ORS expresses K5 and K14, similar to the interfollicular epidermis. As the follicle continues to widen, a new inner core of cells appears and begins to express the hair keratin genes of the hair shaft (reviewed in Omary et al. 2004). By postnatal day 8 in mice, follicle downgrowth is complete, and for the next 7 days, matrix cells proliferate and differentiate into the six concentric layers of the IRS and hair shaft ().

At postnatal day 16, proliferation in the matrix ceases, and the lower two-thirds of the HF rapidly degenerates by a process involving apoptosis (catagen stage). An epithelial strand surrounded by the retracting basement membrane draws the DP upward, where in backskin it comes to rest just below the base of this permanent segment of the HF called the bulge. This resting stage is referred to as telogen. In the first hair cycle, telogen lasts approximately one day, but in subsequent cycles, this phase becomes increasingly extended, suggesting the need to reach a biochemical threshold before the next hair cycle can be activated. The new cycle of hair regeneration (anagen) begins with the emergence of a proliferating hair germ, and the progression to form the mature follicle bears a significant resemblance to embryonic folliculogenesis (Muller-Rover et al. 2001) (). The periodic cycling of hair growth and degeneration persists throughout the life of the animal and implicates the existence of SCs to fuel the regenerative process.

The hair follicle cycle. When matrix cells exhaust their proliferative capacity or the stimulus required for it, hair growth stops. At this time, the follicle enters a destructive phase (catagen), leading to the degeneration of the lower two-thirds of the follicle. The upper third of the follicle remains intact as a pocket of cells surrounding the old hair shaft (club hair). The base of this pocket is known as the bulge, which is the natural reservoir of hair follicle stem cells (SCs) necessary to form a new hair follicle. After catagen, the bulge cells enter a quiescent stage (telogen), in which the DP is now in close contact with bulge SCs. In the mouse, the first telogen lasts approximately one day, after which all the hair follicles synchronously enter a new cycle of regeneration and hair growth (anagen stage). The bulge as a structure develops when the new hair must emerge from the original orifice, which is often shared by the old club hair. Subsequent hair cycles involve increasingly longer telogen phases, resulting in considerably less synchronous hair cycles.

The molecular mechanisms that govern HF morphogenesis and cycling are still poorly understood, but genetic studies in mice reveal the importance of Wnt/-catenin, bone morphogenetic protein (Bmp), sonic hedgehog (Shh), fibroblast growth factor (Fgf), epidermal growth factor receptor (Egf), NFkB, and Notch signaling pathways (reviewed in Millar 2002, Schmidt-Ullrich & Paus 2005).

The adult skin epithelium is composed of molecular building blocks, each of which consists of a pilosebaceous unit (HF and sebaceous gland) and its surrounding interfollicular epidermis (IFE). The IFE contains its own progenitor cells to ensure tissue renewal in the absence of injury, and HFs contain multipotent SCs that are activated at the start of a new hair cycle and upon wounding to provide cells for HF regeneration and repair of the epidermis.

The IFE, which generates the lipid barrier of adult skin, constantly renews its surface throughout the entire life of the animal and also undergoes reepithelialization after wound injuries. These renewing and repairing activities of the skin epidermis imply the existence of SCs to ensure these critical functions. Histological analysis has shown that mouse epidermis is organized in stacks of cells with a hexagonal surface area lying on a bed of ten basal cells (Mackenzie 1970; Potten 1974, 1981). This structure was hypothesized to function as an epidermal proliferative unit (EPU) with one putative SC per unit. Researchers tested experimentally the existence of EPUs using lineage-tracing analyses. The first type of lineage tracing was performed by infecting cultured mouse and human keratinocytes with a retrovirus expressing LacZ and grafting these marked keratinocytes onto immunodeficient mice. Alternatively, mice were directly infected with LacZ-virus in skin, following dermabrasion (Ghazizadeh & Taichman 2001, Kolodka et al. 1998, Mackenzie 1997). Analysis of the chimeric skin revealed the presence of discrete columns of blue cells from the basal cells to the most differentiated uppermost layer of cells. These findings demonstrate that EPUs exist in the basal IFE and can be maintained individually as a separate unit for extended periods of time. Such domains can be explained by a mechanism whereby basal cells divide asymmetrically relative to the basement membrane to maintain a proliferative daughter and give rise to a differentiating daughter cell overlying it (Lechler & Fuchs 2005).

Self-renewal within the epidermis has also been studied using genetic fate mapping, which circumvents the wound response generated in transplantation experiments (Ro & Rannala 2004). In this case, transgenic mice were engineered to express a mutant form of green fluorescent protein (GFP) that cannot be translated owing to the presence of a stop codon in the EGFP-coding sequence. Subsequently, the mice received topical application of a mutagen to induce mutations that can remove the stop codon and restore expression of a functional GFP protein. These sporadic mutations resulted in patches of GFP-positive cells within the IFE, allowing the visualization of EPU columns. Although elegant, these experiments did not address how many SCs are present in each EPU and where the SCs reside within the unit.

In human skin, the epidermis is thicker and undulates to form deep epidermal ridges (rete ridges) that extend downward in the epidermis and help to anchor the epidermis to the dermis. Used only sparingly, SCs have been proposed to cycle less frequently. The infrequently cycling cells within the IFE are located at the base of these ridges, which is conveniently in a more protected site than elsewhere within the IFE (Lavker & Sun 1982).

To identify characteristics of IFE SCs, researchers have turned toward in vitro experiments. Cultured human IFE keratinocytes expressing the highest level of 1-integrin have the highest proliferative potential in vitro (Jones & Watt 1993). Other genes have also been shown to be preferentially expressed in 1-enriched human keratinocytes, underscoring the biochemical distinctions of this population of basal cells (Legg et al. 2003). As would be expected, the 1-bright cells are found in the basal layer, but interestingly, they reside in clusters (Jones et al. 1995). Additionally, the 1-bright cells do seem to reside at the base of the deepest epidermal ridges of palmoplantar skin, consistent with the location of slow-cycling SCs observed by Lavker & Sun (1982). Elsewhere, however, the 1-bright clusters reside outside these zones, in a seemingly more compromised position for SCs. Hence, the extent to which 1-integrin levels define the distinguishing features of IFE SCs must await further studies. In this effort, additional markers are needed to enrich the purification and analyses of IFE cells with high proliferative potential. Such markers should also help in defining the location and the number of IFE SCs within their functional EPU columns and in discerning the extent to which less frequent cycling is a measure of stemness within the IFE population. A final issue to be resolved is the extent to which cells with high proliferative potential in the basal layer of the IFE are able to contribute to other cell lineages, i.e., those of the sebaceous gland and HF.

In the mid-1970s, Rheinwald & Green (1975) defined culture conditions allowing the growth of human IFE SCs in vitro. This seminal discovery allowed the propagation of keratinocytes from severely burned patients and their subsequent grafting as sheets of autologous cultured cells that were functional in reepithelializing the damaged skin (Gallico et al. 1984, O'Connor et al. 1981, Pellegrini et al. 1999, Ronfard et al. 2000). In the past 25 years, this technology has saved many lives. Although the patient's repaired skin epithelium does not regenerate sweat glands or HFs, it does have a normal epidermis, which can undergo wound repair.

When plated at low cell density, cultured human keratinocytes can form three types of colonies: (a) highly proliferative colonies (holoclones) of small round cells that present an undifferentiated morphology and that can be passaged long-term, (b) aborted colonies (paraclones) displaying large flat morphology typical of terminally differentiated cells, and (c) relatively small heterogeneous colonies (meroclones) of limited proliferative potential that become senescent after a few rounds of passaging (Barrandon & Green 1987). Although the term holoclone refers only to the proliferative capacity of the colony, the progeny of a single epidermal holoclone in vitro can re-form a functional and renewable epidermis in vivo (Rochat et al. 1994). This implies that at least some cells within holoclones possess the fundamental characteristics of a SC in that they can self-renew and differentiate into a functional tissue. By contrast, meroclones have been likened to so-called transit-amplifying cells, i.e., cells with a limited number of cell divisions before they commit to terminally differentiate. Although the precise physiological relevance of these cultured populations of cells remains to be determined, the in vitro description of their clonal properties has served as a useful foundation for the analyses of SCs in vivo.

In the hair follicle, SCs reside in a discrete microenvironment called the bulge, located at the base of the part of the follicle that is established during morphogenesis but does not degenerate during the hair cycle. Bulge SCs are more quiescent than other cells within the follicle. However, during the hair cycle, bulge SCs are stimulated to exit the SC niche, proliferate, and differentiate to form the various cell types of mature HFs. In addition, to provide cells during HF regeneration, the bulge SC is a reservoir of multipotent SCs that can be recruited during wound healing to help the repair of the epidermis. We summarize here the recent progress in the functional and molecular characterization of bulge SCs.

For many years, it was thought that the SCs that regenerate HFs during the hair cycle are the highly proliferative matrix cells (Kligman 1959). This model was later challenged when Montagna & Chase (1956) observed that X-ray irradiation kills the matrix cells, but hairs can still re-form from cells within the ORS. The ability of the upper ORS to act in concert with the DP to make HFs was further substantiated by dissection and transplantation experiments (Jahoda et al. 1984; Oliver 1966, 1967).

Mathematical modeling has supported the notion that SCs may be used sparingly and hence divide less frequently than their progeny (Potten et al. 1982). This notion was bolstered by administering repeated doses of marked nucleotide analogs such as BrdU or 3[H]-thymidine to label the S-phase cycling cells of the skin (pulse period) and then following the fate of the incorporated label over time (chase period). The differentiating cells are sloughed from the skin surface, and the more proliferative cells dilute their label as they divide, marking the least proliferative cells as label-retaining cells (LRCs) (Bickenbach 1981).

To locate HF LRCs, Lavker and colleagues (Cotsarelis et al. 1990) administrated BrdU for a week in newborn mice and then analyzed label retention in the skin after four weeks of chase. The majority of LRCs in the skin resided in a specialized region at the base of the permanent segment of the HF. Known as the bulge, this region was described more than a century ago by histologists (Stohr 1903). Within the ORS, the bulge resides just below the sebaceous gland at a site where the arrector pili muscle attaches to the follicle (). Although its origins are likely to be traced to the early stages of HF embryogenesis, the bulge acquires its distinctive appearance when the first postnatal hair germ emerges before the prior club hair has been shed (). During the first telogen phase, a single layer of quiescent cells surround the old club hair; as the new hair cycle initiates, the bulge acquires a second layer of cells (Blanpain et al. 2004).

Although generally quiescent, bulge cells can be prompted to proliferate artificially in response to mitogenic stimuli such as phorbol esters (TPA) or naturally at the start of each hair cycle. In an elegant double-label study to demonstrate a precursor-product relation, Taylor et al. (2000) showed that when BrdU-labeled LRCs in the bulge are exposed to a brief pulse of a second nucleotide label, they incorporate 3[H]-thymidine as they exit and proliferate to develop the new hair germ. To directly determine whether the bulge region contains SCs, Barrandon and coworkers (Kobayashi et al. 1993) dissected rat and human HFs and assessed the growth potential of different HF segments in vitro. In rat-whisker follicles, 95% of the derived holoclones came from cells of the bulge segments, whereas less than 5% of the growing colonies could be derived from the matrix region. In adult human skin, keratinocytes with high proliferative potential were also found within bulge segments, but the zone of clonogenic cells was broader, extending from the bulge to the lower ORS (Rochat et al. 1994). In this regard, in adult human skin, the bulge is notably a less distinctive structure than it is in rodents.

Early studies involving reepithelialization during wound repair led researchers to posit that HFs may have the capacity to regenerate epidermis upon injury (Argyris 1976). To evaluate whether bulge LRCs have this capacity, Taylor et al. (2000) extended their double-labeling techniques to wound-healing experiments. Indeed, following a wound, BrdU-labeled cells derived from the bulge could be found proliferating within the epidermis near the HF orifice (infundibulum).

Fuchs and coworkers (Tumbar et al. 2004) recently adapted the nucleotide pulse-chase experiments to the protein level by engineering mice expressing a tetracycline-regulated histone H2B-GFP protein in their skin epithelium. In the absence of tetracycline, all the skin epithelial nuclei were green with H2B-GFP expression, but when tetracycline was administered, the gene was shut off, and after four weeks, only the bulge cells still labeled brightly with H2B-GFP protein (). Upon wounding, H2B-GFP-positive cells were detected in the epidermis and infundibulum, confirming the ability of bulge LRCs to reepithelialize the epidermis in response to injury (Tumbar et al. 2004). Upon activation of the hair cycle, the emerging hair germ displayed H2B-GFP-positive cells with much weaker fluorescence than the bulge, suggesting that they were derived from the bulge LRCs. These findings support the studies of Barrandon, demonstrating the ability of bulge cells to regenerate the HF during the normal hair cycle.

The bulge stem cells (SCs). Bulge (Bu) SCs are more quiescent than are other keratinocytes with proliferative potential in the skin. Tumbar et al. (2004) developed a strategy for conducting fluorescent pulse-chase experiments in mice engineered to express a tetracycline-regulatable H2B-GFP transgene. After labeling all the skin epithelial cells with H2B-GFP, a four-week chase resulted in significant H2B-GFP-label retention only in the bulge (a). Label-retaining cells (LRCs) could be found along the basal layer of cells that express 64-integrins, as well as in a suprabasal location within the bulge (b). Bulge SCs express a high level of the cell surface protein CD34, which has been used with 6-integrin to isolate basal and suprabasal bulge cells, using flow cytometry (Blanpain et al. 2004, Trempus et al. 2003). [The approximate fluorescence of the outer root sheath (ORS) and interfollicular epidermis (IFE) cells is also indicated on the FACS profile.] Tissues were counterstained with Dapi (blue) to mark the nuclei. Abbreviations used: Cb, club hair; HF, hair follicle; SG, sebaceous gland.

Several lines of evidence suggest that there is a continuous flux of bulge cells throughout the growing stage of the hair cycle. During the anagen phase of the backskin hair cycle, Tumbar et al. (2004) detected a trail of H2B-GFP-positive cells along the lower ORS. Although these cells were less bright than their bulge LRC counterparts, the results were intriguing in light of rat-whisker bulge transplantation and clonogenic experiments performed by Barrandon and colleagues (Oshima et al. 2001). Based on these seminal studies, researchers proposed that SCs migrate from the bulge along the basal layer of the ORS to the matrix, where they proliferate and differentiate to produce the hair and IRS. Although the hair cycle of whisker follicles differs from those in the backskin in that the growing stage is longer and follicles transit from mid-catagen directly to anagen, a common theme for SC movement and activation likely applies for HFs, irrespective of whether they are whisker or pelage follicles.

In the past ten years, researchers have made considerable strides in isolating and purifying cells from the HF bulge. Given the complexity of the skin, purification of bulge cells using flow cytometry (FACS) has focused on isolating bulge cells in the simpler, telogen-phase follicles, where the quiescent bulge marks the base. Kaur and colleagues (Li et al. 1998) have employed conjugated antibodies against 6-integrin and anti-CD71 (antitransferin Ab or 10G7) to show that 6-bright, CD71-dim cells from skin possess similar colony-forming efficiency but higher long-term growth potential than the rest of the population. Bulge LRCs share this expression pattern and are enriched in the 6-bright, CD71-dim population by approximately twofold (Tani et al. 2000). Other markers such as S100A4 and S100A6 proteins (Ito & Kizawa 2001), K19 (Michel et al. 1996), K15 (Lyle et al. 1998), and CD34 (Trempus et al. 2003) have also been reported to exhibit preferential expression in the bulge. Although most of these antibodies have not proven useful for isolating living bulge cells by FACS, CD34 is an exception. CD34-positive cells are enriched tenfold for LRCs, and they form larger colonies than unfractionated epidermis (Trempus et al. 2003).

When transgenic expression of a basal epidermal marker (K14-GFP) is used in conjunction with antibodies against 6-integrin and CD34, purification of bulge cells is enhanced substantially (Blanpain et al. 2004). On the basis of differential 6 expression, the CD34/K14-GFP-positive cells from the inner and outer layers of the mature bulge can also be fractionated (). Bulge cells have also been purified from K15-GFP-transgenic skin in conjunction with 6-integrin antibodies (Morris et al. 2004), and when tetracycline-regulatable H2B-GFP mice are employed for bulge purification, a 70-fold enrichment of bulge LRCs can be achieved over unfractionated skin epithelial cells (Tumbar et al. 2004). In all three of these methods for obtaining bulge cells with high purity, bulge cells form large colonies that can be passaged in vitro (Morris et al. 2004, Tumbar et al. 2004). This is true for both the inner and the outer layer of the bulge (Blanpain et al. 2004) (). Clonogenicity studies further demonstrate that a large colony derived from a single bulge cell can give rise to multiple large colonies upon passaging, implying the occurrence of SC self-renewal in vitro (Blanpain et al. 2004, Claudinot et al. 2005).

The two major properties of SCs are their abilities to self-renew and to differentiate along multiple lineages. To address the differentiation potential of bulge SCs, researchers have used a variety of methods, including (a) transplantation studies of microdissected HF segments, (b) direct transplantation and clonal analysis of isolated bulge cells, and (c) genetic fate mapping in mice.

In pioneering studies, Oshima et al. (2001) generated chimeric rodent-whisker follicles by removing the unlabeled bulge of a wild-type vibrissae follicle, replacing it with a lacZ-expressing bulge microdissected from a transgenic mouse-whisker follicle and transplanting the chimeric follicle into the kidney capsule and/or embryonic backskin from immunodeficient mice. Thirty days after transplantation, lacZ-marked cells were detected in the epidermis, sebaceous gland, and HFs (Oshima et al. 2001). Morris et al. (2004) have obtained similar results using 105-FACS-isolated K15-GFP-tagged bulge cells transplanted into immunodeficient mice.

In the experiments of Barrandon and coworkers, temporal analysis of anagen-phase chimeric whisker follicles revealed a downward flux of lacZ-positive cells originating from the transplanted bulge, migrating to the matrix and subsequently differentiating into one of the six concentric rings of IRS and hair shaft lineages. Although at reduced frequency, cells residing in the lower HF were also able to differentiate into multiple skin cell lineages (Oshima et al. 2001). These findings support the view that SCs migrate from the bulge to the base of the follicle before they differentiate and lose their potential. As outlined above, it still remains to be resolved as to whether a continuous downward flux of bulge cells occurs only in whiskers or human HFs, in which the hair cycle displays a prolonged anagen phase, or whether it is a feature common to all HFs.

The studies above beautifully underscore the potential of cells within the bulge region to differentiate along the three different lineages afforded to the skin keratinocyte. However, they do not address whether the bulge consists of multiple types of unipotent progenitors, each of which are able to differentiate along one lineage, or whether individual bulge cells possess multipotency, the ability to differentiate along any of the three lineages. To date, technical hurdles have precluded testing for multipotency using in vivo clonal analyses. However, in the past few years, researchers have employed clonal analyses in vitro to demonstrate definitively the multipotency of bulge cells when passaged in vitro (Blanpain et al. 2004, Claudinot et al. 2005).

In the first study, Fuchs and coworkers (Blanpain et al. 2004) placed isolated K14-GFP-tagged bulge cells in culture to obtain individual holoclones. After short-term expansion, the descendents from a single bulge cell were then transplanted onto the backs of nude mice. The progeny of single bulgederived holoclones each gave rise to GFP-positive HFs, IFE, sebaceous gland, and even bulge SCs (Blanpain et al. 2004). Similar results were obtained by Barrandon and coworkers (Claudinot et al. 2005), who were able to generate thousands of HFs from the progeny of a single cultivated rat-whisker SC. These experiments provide compelling evidence in support of the notion that cells within the adult follicle bulge possessing the classical criteria of bona fide multipotent SCs. That the inner bulge layer also has this capacity further suggests that even when bulge cells detach from the basal lamina and appear to undergo early commitment to the HF lineage, the process is reversible, at least after in vitro culture (Blanpain et al. 2004).

Under normal circumstances, the bulge acts as a reservoir of follicle SCs, and only in response to injury has it been shown to mobilize and function as a multipotent SC reservoir. Whether there are other multipotent SCs in adult skin remains to be demonstrated. However, there is substantial evidence that unipotent SCs exist in other locations in the skin. Fate-mapping experiments using a Cre recombinase that permanently marks bulge cells reveal that under physiological conditions, the IFE contains only rare patches of -galactosidase-positive cells derived from bulge cells. These data reinforce the notion postulated above on the basis of EPU columns: Normal IFE homeostasis is controlled by the presence of unipotent progenitors that reside within the IFE (Ito et al. 2005, Levy et al. 2005, Morris et al. 2004). That bulge SCs are not necessary for epidermal homeostasis is perhaps best exemplified by the fact that palmoplantar skin lacks HFs altogether, as do a number of genetic hair disorders, yet epidermal homeostasis and wound repair can still take place (Montagna et al. 1954).

To determine which genes and signaling pathways operate within the bulge SCs, researchers have performed transcriptional profiling on isolated telogen-phase bulge cells (Blanpain et al. 2004, Morris et al. 2004, Tumbar et al. 2004). In most cases, these profiles have been compared with those of basal epidermal cells, which have proliferative capacity but are thought to contain few if any multipotent SCs. Notably, most of the transcripts upregulated in either the Tumbar or Morris arrays were upregulated in the Blanpain array, which encompassed a considerably larger gene set compared with the two earlier studies. Blanpain et al. (2004) list 56 transcripts that scored as upregulated in bulge cells irrespective of the isolation method, hair cycle stage, or attachment to the basal lamina and that can be viewed as a molecular signature of bulge cells.

Interestingly, 14% of genes found to be upregulated in other types of SCs (hematopoeitic SC, neuronal SC, and embryonic SC) (Ivanova et al. 2002, Ramalho-Santos et al. 2002) were also found to be a part of the bulge signature (Blanpain et al. 2004), suggesting that certain genes within this list are likely involved in the unique properties common to many if not all SCs. Related to this issue are the important similarities recently uncovered between these mouse bulge SC profiles and those of human bulge SCs (Ohyama et al. 2006). Although some differences were noted (CD34, for example, extends to the lower ORS in human follicles), this similarity bodes well for future clinical studies aimed at improving the potential of skin SCs for therapeutic purposes.

The bulge signature now provides a constellation of markers that should enable researchers to examine the extent to which bulge cells retain their program of gene expression as they respond to natural stimuli, e.g., during the hair cycle and upon injury, and as they exit the niche to migrate and/or differentiate along particular lineages. The list should also be useful in examining how the bulge cells change their properties in response to various genetic manipulations. Through such future examinations, scientists should begin to uncover the extent to which the bulge signature is a reflection of the quiescence of these SCs and identify the subset of these genes involved in self-renewal and in suppression of lineage determination irrespective of whether a skin SC is quiescent or proliferative.

Although these studies are in their infancy, a few important lessons are already emerging. One intriguing aspect of the transcriptional profiling conducted on the bulge to date is the high degree to which the bulge signature is maintained in both anagen and telogen stages of the hair cycle and in basal and suprabasal bulge layers (Blanpain et al. 2004). These findings underscore the powerful influence that the microenvironment of the bulge niche has on its residents. In turn, for a bulge SC to become mobilized and exit the niche, this dominance must be overcome.

Although researchers are conducting additional experiments to dissect the molecular significance of the bulge signature, it is tempting to speculate on the roles of various transcripts that are either up- or downregulated preferentially in the bulge. To this end, a number of bulge signature genes encode cell adhesion, cytoskeleton, and ECM components. We posit that these genes may reflect the specialized microenvironment that must be suitable not only for maintaining their SC characteristics within the niche, but also for allowing bulge SCs to exit their niche and migrate during wound repair and/or in hair regrowth.

The bulge signature also provides a battery of candidate genes likely to play a role in SC quiescence. Most notable are the many upregulated genes encoding cell-cycle inhibitory factors, such as Cdkn1b (p27), Cdkn1c (p57), and Cdkn2b (p15), and the numerous downregulated genes encoding cell-cycle-promoting factors, such as Ki67, proliferating cell nuclear antigen, cyclins (Cyclin D1, D2, A2, B1) and cyclin-dependent kinases, and cell-division-cycle-related genes (Cdc2a, 2b, 6, 7, 25c) (Blanpain et al. 2004, Morris et al. 2004, Tumbar et al. 2004). Although the cell cycle is typically thought to be regulated largely at the posttranslational level, the transcriptional regulation of these cell-cycle genes suggests that the quiescent nature of the bulge is governed by unique operational control mechanisms.

Finally, another interesting set of bulge signature genes contains those that are likely involved in maintaining the SCs in an undifferentiated, growth-inhibited state. Of these genes, it is particularly interesting that many components of the Wnt/-catenin signaling pathway (Tcf3; Tcf4; Dkk-3; sFRP1; Fzd 2, 3, 7; Dab2; Ctbp2) and the TGF-/Bmp signaling pathways (Ltbp1, 2, 3; Tgf-2; Gremlin) are upregulated in the bulge. These pathways are discussed individually in the sections below.

The Wnt/-catenin signaling pathway is conserved throughout the eukaryotic kingdom, where it controls a myriad of different cellular decisions during embryonic and postnatal development (). Wnt deregulation leads to an imbalance of proliferation and differentiation, often resulting in cancers (Reya et al. 2001).

The Wnt/-catenin signaling pathway during hair follicle (HF) morphogenesis and regeneration. (a) Schematic of the canonical Wnt pathway (for more details, see http://www.stanford.edu/%7Ernusse/). In the absence of a Wnt signal, the excess of cytoplasmic -catenin is targeted for degradation through its association with a multiprotein complex. Upon binding Wnt, its activated receptor complex recruits certain key components of the -catenin degradation targeting machinery. Stabilized free cytoplasmic -catenin is now translocated to the nucleus, where it can associate with transcription factors of the LEF/TCF family to transactivate the expression of their target genes. (b) Loss- and gain-of-function studies in mice have highlighted the different functions of Wnt/-catenin signaling during morphogenesis and adult skin homeostasis. During HF morphogenesis, Wnt/-catenin is required to specify the HF (placode) fate in the undifferentiated basal epidermis. During the adult hair cycle, Wnt/-catenin is required to maintain HF stem cell (SC) identity. As judged by a Wnt reporter transgene, an increase in Wnt signaling promotes SC activation to initiate the growth of a new hair during the telogen-to-anagen transition. An even stronger signal appears to be involved later at the transition of matrix cells to commit to terminally differentiate specifically along the hair shaft lineage. (c) When a constitutively active form of -catenin is expressed for sustained periods in skin epidermis, mice develop de novo HFs from the interfollicular epidermis (IFE), outer root sheath (ORS), and sebaceous glands (SGs). Eventually, these mice develop HF tumors called pilomatricoma, which consist of immortalized matrix-like cells at the periphery, and pure hair cells in the centers (no inner root sheath or companion layer cells). Visualization was enhanced by breeding the K14-N mice on a background of K14-GFP mice. (d) The different signal strengths of Wnt reporter gene activity, combined with the -catenin dosage dependency associated with these different outcomes in mice, can be explained by a model whereby the effective strength of Wnt signaling controls the behavior and fate of the follicle SC. Note: The so-called gradient of Wnt activity refers to the status of Tcf/Lef/-catenin transcriptional activity within the cell, which in fact could be achieved as a gradient, without even involving a Wnt per se. DP, dermal papilla.

Wnts compose a large family of cysteine-rich secreted glycoproteins that activate Frizzled receptors, which in turn stimulate a cascade of events culminating in the stabilization and accumulation of cytoplasmic -catenin. Normally, cellular -catenin is complexed with E-cadherin and -catenin at adherens junctions, and free cytoplasmic -catenin is degraded by the proteasome. Upon Wnt signaling, excess -catenin is no longer degraded, and it is free to complex with and activate members of the Tcf/Lef1 family of transcription factors (Logan & Nusse 2004) ().

The sonic hedgehog (Shh) signaling pathway during hair follicle morphogenesis and adult hair cycle. (a) Schematic of the Shh pathway. In the absence of Shh, its receptor Patched (Ptch) inhibits Smoothened (Smo) activity. Upon Shh binding, Ptch can no longer repress Smo, which activates the translocation of Gli into the nucleus, allowing it to transactivate its target genes. (b) The role of Shh in the hair follicle. Loss-of-function studies in mice have revealed the importance of Shh in sustaining proliferation in the embryonic and adult hair germ. Gain-of-function studies underscore the striking relation between basal cell carcinomas and deregulation of the Shh pathway. (c) Shh is not expressed in the quiescent bulge stem cells. During hair regeneration, there is a lag before Shh is strongly activated in the developing hair germ. Sustained expression of Shh seems to rely on close association with the dermal papilla (DP). Both in embryonic development and the adult, Shh appears to act downstream of the Wnt/-catenin signaling pathway. Bu, bulge; HG, hair germ.

In the skin, Wnt and -catenin play diverse roles in HF morphogenesis, SC maintenance and/or activation, hair shaft differentiation, and also pilomatricoma tumor formation in mice and humans (Alonso & Fuchs 2003). Activation of Wnt/-catenin signaling is critical during the first stage of HF morphogenesis, as evidenced by the absence of placode formation on conditional ablation of -catenin (Huelsken et al. 2001) or constitutive expression of a soluble Wnt inhibitor (Dkk1) (Andl et al. 2002). Although the source and identity of the putative Wnt signal required to induce placode formation remain elusive, it may be the first dermal signal to instruct epidermal cells to make hair. Consistent with this notion is the activation in both the placode and the postnatal hair germ of a Wnt reporter gene driving lacZ under the control of an enhancer composed of multimerized binding sites for the Lef1/Tcf DNA-binding proteins that interact with and are activated by association with -catenin (DasGupta & Fuchs 1999, Reya & Clevers 2005) (). Nuclear -catenin and Lef1 expression are also seen in embryonic placodes and postnatal hair germs at this time (Merrill et al. 2004, van Genderen et al. 1994, Zhou et al. 1995). Noggin, a soluble inhibitor of Bmps, is expressed by the mesenchymal condensate and is required in the early stage of HF morphogenesis and cycling. It appears to act at least in part by promoting expression of Lef1 (Botchkarev et al. 2001, Jamora et al. 2003).

Transgenic mouse studies support a role for Wnt signaling in the specification of HF development. Mice expressing a constitutively stabilized -catenin (>N-catenin) display de novo HFs (Gat et al. 1998) (), whereas mice lacking Lef1 (van Genderen et al. 1994) or -catenin (Huelsken et al. 2001) or overexpressing the Wnt inhibitor Dkk1 exhibit a paucity of follicles (Andl et al. 2002).

Postnatally, the strongest Wnt signal is associated with the terminally differentiating cortical cells of the hair shaft (DasGupta & Fuchs 1999) (). The hair keratin genes possess Lef1/Tcf DNA-binding domains and are bona fide targets for Wnt-mediated gene expression (Merrill et al. 2001, Zhou et al. 1995). This lineage of the matrix cells appears to be particularly singled out for robust Wnt signaling, as K14-N-catenin transgenic mice develop pilomatricomas, which are pure tumor masses of cortical cells (Gat et al. 1998). Similarly, the majority of human pilomatricomas possess N-terminal stabilizing mutations in the coding sequence of the -catenin gene (Chan et al. 1999, Xia et al. 2006).

In contrast to the cortical cells, the bulge is largely silent for Wnt reporter activity (DasGupta & Fuchs 1999). Microarray data suggest that the bulge is normally in a Wnt-inhibited environment, showing an upregulation of genes encoding putative Wnt-inhibitory factors (sFRP1, Dkk3, Wif) and a downregulation of genes encoding Wnt-promoting factors in the bulge (Wnt3, Wnt3a). However, bulge SCs express a number of frizzled surface receptors (Fz2, 3, and 7) to enable them to receive Wnt signals as well as Wnt-signaling-related transcription factors (Tcf3, Tcf4, Tle1, Ctbp2) to enable them to transmit a Wnt signal (see Tumbar et al. 2004). In this regard, Tcf3 is intriguing, as it has been shown to act as a repressor in the absence of Wnt signaling (Merrill et al. 2001, 2004). Taken together, these findings suggest that bulge SCs are in a quiescent, Wnt-inhibited state and that Wnt signaling plays a key role in driving these cells along at least one hair differentiation lineage ().

Several studies suggest that the role of Wnt signaling in the postnatal HF may be even broader. The involvement of Wnts in HF morphogenesis suggests that Wnt signaling may be important for activating bulge SCs. Consistent with this notion is the presence of a few Wnt-reporter-driven, LacZ-positive bulge cells at the beginning of the hair cycle (DasGupta & Fuchs 1999). The number of activated bulge cells can be considerably enhanced by breeding the Wnt-reporter mice on the background of K14-N-catenin mice; at most stages of the hair cycle, however, the bulge remains silent for Wnt-reporter activity (DasGupta & Fuchs 1999, Merrill et al. 2001).

By inducing the expression of stabilized -catenin in telogen-phase follicles, several groups have observed precocious activation of hair regeneration (Lo Celso et al. 2004, Lowry et al. 2005, Van Mater et al. 2003), in a fashion reminiscent of the de novo follicle morphogenesis that occurs in the IFE (Gat et al. 1998). Despite the premature transition from telogen to anagen, the K14-N-catenin bulge reenters its relatively quiescent state once the follicle has grown downward (Lowry et al. 2005). These findings imply that some additional factor(s) is required in addition to elevated Wnt signaling to change the status of Lef1/Tcf-regulated genes (including TopGal) and activate bulge SCs. It is tempting to speculate that this signal emanates from the DP, given the close proximity of the DP to the bulge prior to the start of the hair cycle. One candidate may be the Bmp-inhibitor Noggin, produced by the DP and shown to be required for Lef1 expression in the embryonic hair placode and in the matrix cells as well (Andl et al. 2004, Botchkarev et al. 1999, Jamora et al. 2003, Kobielak et al. 2003). Fgf7 and Fgf10 are additional candidates known to be expressed in the bulge and to have an impact on follicle morphogenesis (Guo et al. 1993, Petiot et al. 2003).

Despite the continuous presence of an elevated level of stabilized -catenin, the size of the SC niche does not change over time (Lowry et al. 2005). This means that if elevated -catenin promote the self-renewal of bulge SCs, it must be accompanied by an increase in the rate at which SCs exit the niche. Two factors consistent with this notion are that the rate of BrdU-label retention is reduced and the level of BrdU-label incorporation is enhanced in the K14-N-catenin bulge. That said, this increased proliferation appears to be manifested in precocious SC activation, as it was not accompanied by a noticeable increase in the length of the hair or the cellularity of HFs.

To understand how -catenin elevation can promote SC activation in the bulge, Lowry et al. (2005) conducted microarray analyses on telogen- or anagen-phase SCs isolated from N-catenin or wild-type follicles. Intriguingly, some telogen-phase bulge genes affected by N-catenin were similarly affected in the normal anagen-phase bulge, suggesting the transgene-induced changes may reflect natural changes that occur in the telogen-to-anagen transition of the hair cycle. Although further studies are needed to assess the extent to which this is the case, genes that surfaced in these arrays and that may play a role in Wnt-mediated bulge SC activation include Cyclin D2 (Ccnd2), Sox4, and Biglycan (Lowry et al. 2005). Another protein upregulated in the early anagen bulge appears to be the transcriptional corepressor Hairless, which has been proposed to function by blocking the expression of the soluble Wnt inhibitor Wise, which in turn may lead to Wnt-mediated SC activation (Beaudoin et al. 2005). An additional interesting twist is the recent study reporting that Shh is a downstream target of Wnt-mediated activation of follicle SCs (Silva-Vargas et al. 2005). Shh is particularly intriguing as a Wnt candidate, as it would integrate these two key signaling pathways essential for HF morphogenesis. That said, on the basis of the differential expression of direct Wnt target genes and Shh, it seems unlikely that Shh is a direct target for Wnt signaling in bulge cells (Lowry et al. 2005). We discuss the Shh pathway in greater depth below.

In summary, these findings delineate sequential roles for Wnt signaling in temporally regulating follicle SC lineages, perhaps in a fashion that depends on the level of the signal: (a) -catenin stabilization promotes bulge SC activation, proliferation, and induction of follicle regeneration; (b) -catenin stabilization promotes the specification of matrix cells to terminally differentiate along the hair (cortical) cell lineage; (c) -catenin stabilization promotes de novo HF morphogenesis; and (d) constitutively active -catenin expression results in pilomatricoma hair tumors. The particular fate selected by a follicle cell appears to depend on a constellation of intrinsic and extrinsic factors, which together influence the status of Tcf/Lef1-regulated genes. At the Wnt-inhibited end of the spectrum is SC quiescence, and at the constitutive Wnt end is tumorigenesis ().

Similar to Wnt/-catenin, Shh is an ancient signaling pathway involved in cell fate specification and proliferation during animal development (Taipale & Beachy 2001). The Shh transmembrane receptor is Patched (Ptch), which is active in the absence of Shh (). Ptch functions by inhibiting Smoothened (Smo), which is essential to transduce the Shh signal through the Gli family of transcription factors to induce target gene expression. Ptch itself is a Shh target gene, resulting in the localized sequestration of Shh and the restriction of long-range signaling (Casali & Struhl 2004).

Given the prominence of the Shh pathway in development and proliferation, it is not surprising to find that when deregulated, this pathway leads to tumorigenesis. Rubin et al. (2005) illuminated its importance in skin with the finding that Ptch1 gene mutations cause basal cell nevus syndrome, a hereditary predisposition to basal cell carcinomas (BCCs), the most common type of skin cancer in humans. In the skin, Ptch acts as a tumor suppressor gene, as loss of heterozygosity at the Ptch locus (chromosome 9q22.3) has been observed in sporadic BCC and BCCs isolated from patients with basal cell nevus syndrome (Gailani et al. 1996, Hahn et al. 1996, Johnson et al. 1996, Unden et al. 1996). Activating mutations in Smo have also been detected in sporadic BCCs (Xie et al. 1998), and overexpression of Shh, Smo, Gli1, or Gli2 leads to BCCs in mice (Dahmane et al. 1997, Grachtchouk et al. 2000, 2003; Hutchin et al. 2005; Oro et al. 1997; Xie et al. 1998). Recently, Vidal et al. (2005) demonstrated that an HMG transcription box factor, Sox9, is also upregulated in BCC, and epistasis experiments suggest that Sox9 is downstream of the Shh signaling pathway in skin.

BCCs are thought to be derived from HFs, and consistent with this notion, Shh is expressed in the hair placodes of embryonic skin (St-Jacques et al. 1998) (). As revealed by Ptch expression, Shh is likely to signal in both the epithelial hair germ and its underlying mesenchymal condensate, suggesting its potential role in the epithelial-mesenchymal cross talk essential for follicle formation (Oro & Higgins 2003, Oro et al. 1997). Loss-of-function mutations in Shh are still permissive for hair germ formation, placing Shh genetically downstream of Wnt and Noggin signaling (). However, placodes fail to develop further, thus positioning Shh upstream from the proliferative cascade essential for HF morphogenesis (Chiang et al. 1999, St-Jacques et al. 1998, Wang et al. 2000). Mice deficient in Gli2 present a phenotype similar to Shh-null mice, suggesting that Shh acts mainly through Gli2 in HF (Mill et al. 2003).

Shh signaling is also important for follicle regeneration during the adult hair cycle. Although not expressed in the bulge, Shh is expressed in the matrix and in the developing germ, where it becomes polarized to one side during anagen progression (). The mechanisms underlying this exquisite restriction in expression are not understood, but Shh signaling is likely to span the matrix, as evidenced by Ptch expression (Gat et al. 1998, Oro & Higgins 2003). As would be predicted from the relative roles of Shh and Wnt signaling in embryonic skin, anti-Shh antibodies delivered to postnatal follicles block anagen progression (Wang et al. 2000), and similarly the Shh inhibitor cyclopamine blocks hair regeneration (Silva-Vargas et al. 2005). Conversely, Shh or small-molecule Shh agonists accelerate the progression from telogen to anagen (Paladini et al. 2005, Sato et al. 1999).

Whereas Shh plays a role in matrix cell proliferation in the hair cycle, Indian hedgehog (Ihh) is expressed in the sebaceous gland. Additionally, both human and mouse sebaceous tumors overexpress Ihh but not Shh. In normal sebaceous glands, Ihh is expressed in differentiating sebocytes, and nuclear Gli1 is present in sebocyte progenitors (Niemann et al. 2003). In vitro inhibition of hedgehog signaling inhibits growth and stimulates differentiation of sebocytes, suggesting a paracrine mechanism by which Ihh secreted by differentiated sebocytes stimulates proliferation of sebocyte precursors (Niemann et al. 2003). Transgenic overexpression of the other members of Shh family shows that Desert hedgehog is a functional homolog to Shh in the skin (Adolphe et al. 2004).

Bmps are secreted proteins that activate signal transduction by binding to a transmembrane receptor complex composed of Bmpr1a and Bmpr1b receptors. Upon ligand binding, Bmpr1b phosphorylates the cytoplasmic tail of Bmpr1a, which in turns phosphorylates the R-Smad DNA-binding protein (Smad 1, 5, and 8), which in turn complexes with one of its partner Smads (typically Smad 4) to translocate to the nucleus and mediate target gene expression (Shi & Massague 2003) ().

Bone morphogenetic protein (BMP) signaling pathway during hair follicle morphogenesis and differentiation. (a) Schematic of the BMP pathway. The extracellular availability of BMP proteins is tightly regulated by soluble BMP inhibitors such as Noggin. BMP dimers bind a heterodimeric receptor complex (BMPR-I and BMPR-II) that phosphorylates and activates R-Smad (Smads 1, 5, and 8), which then associates with its co-Smad (Smad 4) partner. Once activated, the R-Smad/co-Smad complex is translocated into the nucleus, where it transactivates its target genes. (b) Role of BMPs in hair follicle morphogenesis. BMP signals are transmitted to and from the overlying epidermis to underlying dermal condensates. Although the role these BMP signals play is not fully understood, this exchange of signaling is thought to play a role in the early specification of sites of hair follicle morphogenesis. As dermal condensates form, they express the BMP-inhibitor Noggin, which is required for normal follicle development and permissive for Lef1 expression and Wnt signaling. Later, as follicle maturation has progressed, the activation of BMP receptor signaling is essential for the matrix cells to differentiate to form the hair shaft and its inner root sheath (IRS) channel. BMP signaling also regulates epidermal proliferation in the skin. DP, dermal papilla.

Bmpr1a is expressed throughout most of the developing skin epithelium. The pattern of Bmp expression is particularly elaborate in the HF. In early skin development, Bmp2 is expressed in the placode epithelium, whereas Bmp4 is expressed by the underlying mesenchyme (Kratochwil et al. 1996; Lyons et al. 1989, 1990; Wilson et al. 1999). In adult HFs, Bmps also appear to function in epithelial-mesenchymal interactions. In the DP, Bmp4, -6, and -7 are expressed (Kratochwil et al. 1996; Lyons et al. 1989, 1990; Rendl et al. 2005; Wilson et al. 1999), although Bmp6 may also function in bulge SC quiescence and/or maintenance (Blanpain et al. 2004). In addition, Bmps are differentially expressed in the various lineages of the HF, with Bmp7 and -8 in the IRS and Bmp2 and -4 in the hair shaft precursors.

The role for Bmp signaling in skin development begins in the neuroepithelium, when Bmp signaling specifies uncommitted ectodermal cells to become epidermis (Nikaido et al. 1999). Once the embryonic skin SC progenitor cells have been specified, the next crossroads for signaling appears to be at the juncture of hair placode formation. In a process bearing a certain resemblance to the formation of the neural tube, placode formation is dependent on Noggin, a soluble inhibitor of Bmp signaling (Botchkarev et al. 1999, Jamora et al. 2003). Conditional ablation of the Bmpr1a gene also results in the accumulation of large masses of undifferentiated, Lef1-expressing, placode-like cells, further emphasizing a role for Bmp inhibition in the early stages of HF morphogenesis (Andl et al. 2004, Kobielak et al. 2003).

The conditional targeting of the Bmpr1a gene also revealed a positive role for Bmp signaling in the differentiation of matrix cells into IRS and hair shaft lineages (Andl et al. 2004, Kobielak et al. 2003, Ming Kwan et al. 2004, Yuhki et al. 2004). Several markers of matrix cell differentiation (FoxN1/nude, Hoxc13, Msx2, and GATA3) were strongly reduced or absent following the ablation of Bmpr1a. Notably and in striking contrast, Shh and Lef1 expression was expanded, as is also seen in transgenic mice expressing Noggin under the control of the Msx2 promoter (Kulessa et al. 2000). Nuclear -catenin was also decreased in the Bmpr1a-deficient matrix cells, demonstrating that Bmp signaling lies upstream of -catenin signaling during matrix cell differentiation. These findings strengthen the view that the inhibition of Bmp signaling is required for SC activation toward the HF cell fate, whereas Bmp signaling is required for the differentiation of activated SCs to adopt one or more of the six different lineages that compose the mature HF (Kobielak et al. 2003).

Several other lines of evidence suggest that the inhibition of Bmp signaling promotes SC activation. At the conclusion of the normal hair cycle, proliferation ceases and the HF enters the destructive phase (catagen). By contrast, Bmpr1a-null ORS continues to proliferate and grow downward, leading to an accumulation of matrix cells and the formation of follicular tumors (Andl et al. 2004, Ming Kwan et al. 2004). Conversely, treatment of cultivated bulge SCs with BMP6 inhibits their proliferation and leads to a transient withdrawal from the cell cycle (Blanpain et al. 2004, Botchkarev et al. 1999).

Similar to other major signaling pathways in skin, Notch signaling is involved in a variety of cell fate decisions across the animal kingdom. Transmembrane Notch receptors (Notch14 in mammals) bind transmembrane ligands, either Jaggeds (2) or deltas (3). Upon ligand engagement, membrane Notch receptors are sequentially cleaved, first by a metalloproteinase and then by -secretase, which releases the active Notch intracellular domain (NICD), freeing it to translocate to the nucleus and associate with the DNA-binding protein RBP-J. Upon NICD binding, RBP-J is converted from a transcriptional repressor to an activator, leading to the induction of downstream Notch target genes (Artavanis-Tsakonas et al. 1999) ().

Notch signaling pathway during epidermal stratification and hair follicle differentiation. (a) Schematic of canonical Notch signaling. Upon ligand (Jagged or Delta) binding, the Notch transmembrane receptor is cleaved by proteases (ADAM protease and -secretase), releasing the Notch intracellular domain (NICD), which can then translocate into the nucleus and associate with the DNA-binding protein RBP-Jk to permit transcription of target genes. (b) Role of Notch1 in skin development. Notch1 is cleaved and generates its active form, NICD1, which controls epidermal stratification and differentiation. Early, NICD1 is present in basal cells but later it is found primarily in suprabasal cells. Loss-of-function studies suggest that Notch1 acts as a tumor suppressor in skin epidermis to restrict proliferation to the basal layer. Notch1 also plays a role in the hair follicle, where it has been demonstrated to play a critical role in the differentiation of the inner root sheath and the hair shaft.

Multiple components of the Notch signaling pathway are expressed in embryonic and adult epidermis. During the early stage of epidermal stratification, Notch1 is expressed and active in the basal and suprabasal cells of the epidermis and sebaceous glands (Okuyama et al. 2004, Rangarajan et al. 2001) (). In the latter stages of epidermal stratification, Notch1 activity decreases in the basal layer and becomes more restricted to the spinous layer (K1-positive cells) (Okuyama et al. 2004). Loss of Notch1 function results in a defect of IFE differentiation (Rangarajan et al. 2001).

In the HF, Notch13 are expressed in proliferative matrix cells and in differentiating HF cells (Kopan & Weintraub 1993, Pan et al. 2004) (). When both Notch 1 and Notch2 or PS1 and PS2 genes involved in Notch processing are conditionally ablated in the matrix, HFs are quantitatively lost and epidermal cysts arise, underscoring the role for Notch signaling in follicle maturation and differentiation (Pan et al. 2004). The consequences of Notch1 deletion are most directly deleterious to the sebaceous glands, which are reduced in the single conditional knockout animals; in the absence of both Notch1 and Notch2, sebaceous glands are missing altogether (Pan et al. 2004). Conditional gene targeting of RBP-J also results in hair loss and epidermal cyst formation (Yamamoto et al. 2003).

Related to the natural role of Notch signaling in skin, loss of Notch 1 potentiates skin tumor development upon chemically induced carcinogenesis (Nicolas et al. 2003). Conversely, NICD overexpression in cultured cells inhibits keratinocyte proliferation, in part by upregulating the p21 target gene, which possesses a functional RBP-J-binding site within its promoter (Rangarajan et al. 2001). Although these studies point to a role for Notch in hair differentiation and inhibition of proliferation, sustained activation of Notch signaling through NICD1 overexpression in hair shaft progenitors unexpectedly promotes matrix cell proliferation and impairs hair shaft differentiation (Lin & Kopan 2003, Lin et al. 2000). These findings raise the possibility that the roles for Notch signaling in the epidermis and HF may be distinct.

Further insights into Notch signaling in the skin come from studies on chicken feather formation. As in mice, Notch1 is expressed in chick epidermal placode, and delta is expressed in the underlying mesenchyme. Delta overexpression in a small epidermal patch leads to an acceleration of feather development, whereas massive overexpression in the epidermis leads to a decrease in feather development (Crowe et al. 1998). These findings suggest a model in which Notch signaling promotes HF development in the preexisting placode but restricts neighboring cells from adopting a similar fate. The generalization of this model for other appendage development in other species requires further investigation, but the model mirrors that of Notch signaling in epidermal and neural fate specification in Drosophila.

Although loss of Notch1 in the epidermis does not impair early follicle morphogenesis, it does progressively reduce the number of HFs over time (Vauclair et al. 2005). It is still unclear what the downstream genes regulated by Notch signaling in the epidermis are, and how these genes mediate their cellular function. It also remains to be determined how Notch signaling acts in the bulge SC niche, how Notch regulates hair cycle, and how the Notch signaling pathway is connected to the other signaling pathways known to influence SC maintenance and activation.

The ends of chromosomes are called telomeres, and they consist of short, tandem DNA sequence repeats that associate with specific proteins and protect chromosome ends from degradation and recombination. Telomerase is a reverse transcriptase that synthesizes the DNA repeats to circumvent telomere shortening during DNA replication. Telomerase reverse transcriptase (TERT) is the catalytic subunit of the protein complex that makes the telomerase. Telomerase is upregulated in many human cancers, and TERT cooperates with other oncogenes to transform normal cells into tumor cells (Blackburn 2001).

TERT has been postulated to extend the proliferative potential of cells, and hence it has been speculated to play a role in SC biology. When the K5 promoter is used to drive TERT overexpression in the basal epidermal layer of transgenic mice, animals are more susceptible to skin tumorigenesis when exposed to chemical carcinogens, and they repair their wounds more rapidly (Gonzalez-Suarez et al. 2001). Conversely, mice deficient for TERC, another key component of telomerase, are resistant to skin chemical carcinogenesis (Gonzalez-Suarez et al. 2000).

In bulge SCs, increased TERT activity results in proliferation and premature entry into the anagen stage (Flores et al. 2005, Sarin et al. 2005). Flores et al. (2005) assumed that the reduced epidermal proliferation seen in TERC-null mice reflected the importance of telomerase and telomere length in bulge SC behavior. In contrast, Sarin et al. (2005) discovered surprisingly that TERC affects the skin in a fashion independent of its role in telomerase function and telomere length. Both groups have posited that TERT and TERC exert their function on the quiescent SCs within the bulge. However, given the need to sustain proliferation in the matrix cells during the growth phase of the hair cycle, it seems more plausible that a need for enhancing conventional telomerase activity would be felt by the proliferating matrix compartment rather than the infrequently cycling cells of the bulge. Additional studies are needed to clarify these conflicting results and determine the mechanism by which telomerase overexpression allows or facilitates skin carcinogenesis and SC activation.

Bulge SCs display elevated levels of several cytoskeleton-related genes known to be regulated by small G proteins of the Rho family of small GTPases. Rac is a pleiotropic regulator of actin dynamics, intercellular adhesion, and cell migration, and as such, it is expressed broadly in proliferating cells. Conditional ablation of the Rac1 gene in the proliferating keratinocytes of skin rapidly depletes the proliferative compartments, leading to a mobilization and depletion of SCs (Benitah et al. 2005).

A priori, a similar outcome might be expected for the depletion of many different types of essential epidermal genes. However, Rac1 was of particular intrigue to Watt and her colleagues (Arnold & Watt 2001, Braun et al. 2003, Frye et al. 2003, Waikel et al. 2001) because it is a negative regulator of c-Myc, whose elevated expression has been reported to deplete the population of bulge LRCs. It will be interesting in the future to see the extent to which, as posited by the authors, Rac1 may act at the nexus of the transition between the SC and its committed progeny (Benitah et al. 2005).

Given the general consensus that overexpression of c-Myc depletes bulge SCs and drives them to differentiate along the epidermal lineage (Arnold & Watt 2001, Braun et al. 2003, Frye et al. 2003, Waikel et al. 2001), it came as a surprise that conditional loss of endogenous c-Myc also leads to a loss of bulge LRCs and precocious differentiation of basal epidermal cells (Zanet et al. 2005). Although the jury is still out, one possible explanation for the seemingly disparate results between gain and loss of c-Myc function is that c-Myc acts at multiple points along the bulge SC lineages, and a perturbation at one or more of these steps may indirectly impact the behavior of SCs. Consistent with this notion is that both gain- and loss-of-function studies point to a role for c-Myc in governing the sebaceous gland lineage, which is also thought to rely on bulge SCs.

The skin epithelium is a complex tissue containing three distinct lineages that form the IFE, the HF, and the sebaceous gland.

The IFE constantly self-renews to provide a new protective layer at the skin surface, and HFs undergo a perpetual cycle of growth and degeneration to ensure the renewal of the hair pelage.

Different populations of progenitor cells contribute to lineage homeostasis, but to date, only bulge SCs have been demonstrated clonally to be multipotent with the ability to differentiate into all three differentiation lineages.

Bulge SCs can be activated and mobilized, at each cycle of hair follicle regeneration and after wound healing, to provide cells for tissue repair.

Recent progress in the isolation and molecular characterization of bulge SCs has provided new insights into the various mechanisms implicated in SC maintenance and activation.

Conserved signaling pathways regulating developmental decisions throughout the animal kingdom are reutilized during adult life to regulate the functions of skin epithelial SCs, and deregulation of these signaling pathways leads to the development of cancer in various tissues.

In this review, we try to highlight some of the significant advances made recently in skin stem cell biology, and we place them within the context of the historical foundations that made this current research possible. In closing, we offer a few of the unanswered questions in the field of skin stem cells that we think are likely to capture the attention of skin biologists in the years to come.

Do common molecular mechanisms govern the fundamental characteristics shared by adult skin SCs and other SCs, namely self-renewal and maintenance of the undifferentiated state? Comparisons of the transcriptional profiles of different types of SCs isolated directly from their respective tissues should help to identify possible candidates, as will the profiling of SCs residing in and out of their niches, and in quiescent and activated states. As candidate genes are identified, functional analyses of putative self-renewal or differentiation inhibitory SC genes in skin should reveal their importance and begin to unravel the pathways involved.

What is the mechanism by which quiescent bulge SCs are activated? Little is known about the signals needed to mobilize bulge SCs to reepithelialize epidermal wounds and to replenish the sebaceous gland. Even for the better understood process of SC activation during the hair cycle, a number of key issues remain unsolved. At the crux of the problem is whether follicle SC activation involves an intrinsic clock mechanism and/or whether it involves a signal from the DP. Although a change in the status of Lef1/Tcf/-catenin-regulated genes has been implicated in follicle SC activation, it is still not clear where a Wnt signal is involved, where it comes from, how the pathway exerts its effects, how it converges with other key signaling pathways, and how the program of gene expression is established that leads to follicle formation.

What is the relationship between the bulge SCs and the proliferative compartments of the epidermis, sebaceous gland, and HF? Do proliferating skin keratinocytes retain unipotent or even multipotent SC properties, or are they committed to embark on a terminal differentiation program? The point of no return in the skin SC field is an important one. Lineage-tracing experiments and the recent studies on asymmetric cell divisions in the skin provide new insights into these issues, but additional studies are now needed to illuminate the molecular relations between these different proliferative populations within the skin.

What is the relationship between the multipotent progenitors of embryonic skin epidermis and the multipotent SCs of the bulge? Embryonic skin effectively begins as a single layer of multipotent progenitors, but they differ from bulge SCs in their proliferative status and their lack of an apparent niche. Are bulge SCs simple quiescent counterparts of their embryonic brethren, or are there intrinsic differences between them? As methods are honed to isolate and characterize the early embryonic SCs, this relationship should become clearer. Additionally, it will be helpful to trace the development of the bulge from its early origins to its site in the postnatal follicle.

Are SCs at the root of cancers in the skin? Cancer is the result of a multistep process requiring the accumulation of mutations in several genes. For most cancers, the target cells of oncogenic mutations are unknown. Adult SCs may be the initial target cells, as they self-renew for extended periods of time, providing increased opportunity to accumulate the mutations required for cancer formation. Certain cancers contain cells with SC characteristics with high self-renewal capacities and the ability to re-form the parental tumor on transplantation. However, whether the initial oncogenic mutations arise in normal SCs or in more differentiated cells that reacquire SC-like properties remains to be determined. The demonstrations that SCs are the target cells of the initial transforming events and that cancers contain cells with SC characteristics await the development of tools allowing for the isolation and characterization of normal adult SCs. For most epithelia in which cancer arises, such isolation techniques are not available. The new methods to isolate and specifically mark skin SCs make it now possible to test experimentally the cancer SC hypothesis in the skin.

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Epidermal Stem Cells of the Skin - PubMed Central (PMC)

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The London Patient, Cured of H.I.V., Reveals His Identity – The New York Times

By daniellenierenberg

A year after the London Patient was introduced to the world as only the second person to be cured of H.I.V., he is stepping out of the shadows to reveal his identity: He is Adam Castillejo.

Six feet tall and sturdy, with long, dark hair and an easy smile, Mr. Castillejo, 40, exudes good health and cheer. But his journey to the cure has been arduous and agonizing, involving nearly a decade of grueling treatments and moments of pure despair. He wrestled with whether and when to go public, given the attention and scrutiny that might follow. Ultimately, he said, he realized that his story carried a powerful message of optimism.

This is a unique position to be in, a unique and very humbling position, he said. I want to be an ambassador of hope.

Last March, scientists announced that Mr. Castillejo, then identified only as the London Patient, had been cured of H.I.V. after receiving a bone-marrow transplant for his lymphoma. The donor carried a mutation that impeded the ability of H.I.V. to enter cells, so the transplant essentially replaced Mr. Castillejos immune system with one resistant to the virus. The approach, though effective in his case, was intended to cure his cancer and is not a practical option for the widespread curing of H.I.V. because of the risks involved.

Only one other individual with H.I.V. Timothy Ray Brown, the so-called Berlin Patient, in 2008 has been successfully cured, and there have been many failed attempts. In fact, Mr. Castillejos doctors could not be sure last spring that he was truly rid of H.I.V., and they tiptoed around the word cure, instead referring to it as a remission.

Still, the news grabbed the worlds attention, even that of President Trump.

And by confirming that a cure is possible, it galvanized researchers.

Its really important that it wasnt a one-off, it wasnt a fluke, said Richard Jefferys, a director at Treatment Action Group, an advocacy organization. Thats been an important step for the field.

For Mr. Castillejo, the experience was surreal. He watched as millions of people reacted to the news of his cure and speculated about his identity. I was watching TV, and its, like, OK, theyre talking about me, he said. It was very strange, a very weird place to be. But he remained resolute in his decision to remain private until a few weeks ago.

For one, his doctors are more certain now that he is virus-free. We think this is a cure now, because its been another year and weve done a few more tests, said his virologist, Dr. Ravindra Gupta of the University of Cambridge.

Mr. Castillejo also tested his own readiness in small ways. He set up a separate email address and telephone number for his life as LP, as he refers to himself, and opened a Twitter account. He began talking weekly with Mr. Brown, the only other person who could truly understand what he had been through. In December, Mr. Castillejo prepared a statement to be read aloud by a producer on BBC Radio 4.

After talking through his decision with his doctors, friends and mother, he decided the time was right to tell his story.

I dont want people to think, Oh, youve been chosen, he said. No, it just happened. I was in the right place, probably at the right time, when it happened.

Mr. Castillejo grew up in Caracas, Venezuela. His father was of Spanish and Dutch descent which later turned out to be crucial and served as a pilot for an ecotourism company. Mr. Castillejo speaks reverently of his father, who died 20 years ago, and bears a strong resemblance to him. But his parents divorced when he was young, so he was primarily raised by his industrious mother, who now lives in London with him. She taught me to be the best I could be, no matter what, he said.

As a young man, Mr. Castillejo made his way first to Copenhagen and then to London in 2002. He was found to have H.I.V., the virus that causes AIDS, in 2003.

I do recall when the person told me and the panic set in, he said. At the time, an H.I.V. diagnosis was often seen as a death sentence, and Mr. Castillejo was only 23. It was a very terrifying and traumatic experience to go through.

With the support of his partner at the time, Mr. Castillejo persevered. He turned the passion for cooking he had inherited from his grandmother into a job as a sous chef at a fashionable fusion restaurant. He adopted an unfailingly healthy lifestyle: He ate well, exercised often, went cycling, running and swimming.

Then, in 2011, came the second blow. Mr. Castillejo was in New York City, visiting friends and brunching on the Upper East Side, when a nurse from the clinic where he went for regular checkups called him. Where are you? she asked. When Mr. Castillejo told her, she would say only that they had some concerns about his health and that he should come in for more tests when he returned to London.

He had been experiencing fevers, and the tests showed that they were the result of a Stage 4 lymphoma. I will never forget my reaction as once again my world changed forever, he said. Once again, another death sentence.

Years of harsh chemotherapy followed. Mr. Castillejos H.I.V. status complicated matters. Each time his oncologists adjusted his cancer treatment, the infectious-disease doctors had to recalibrate his H.I.V. medications, said Dr. Simon Edwards, who acted as a liaison between the two teams.

There is little information about how to treat people with both diseases, and H.I.V.-positive people are not allowed to enter clinical trials. So with each new chemotherapy combination, Mr. Castillejos doctors were venturing further into unchartered territory, Dr. Edwards said.

With each treatment that seemed to work and then didnt, Mr. Castillejo fell into a deeper low. He saw fellow patients at the clinic die and others get better, while he kept returning, his body weakening with each round.

I was struggling mentally, he said. I try to look at the bright side, but the brightness was fading.

In late 2014, the extreme physical and emotional toll of the past few years caught up to Mr. Castillejo, and two weeks before that Christmas he disappeared. His friends and family imagined the worst, and filed a missing persons report. Mr. Castillejo turned up four days later outside London, with no memory of how he had ended up there or what he had done in the interim. He described it as switching off from his life.

Around that same time, he said, he felt so defeated that he also contemplated going to Dignitas, the Swiss company that helps terminally ill people take their own lives: I felt powerless. I needed control, to end my life on my own terms. He made it through that dark period, and emerged with a determination to spend whatever was left of his life fighting.

Still, in the spring of 2015, his doctors told him he would not live to see Christmas. A bone-marrow transplant from a donor is sometimes offered to people with lymphoma who have exhausted their other options, but Mr. Castillejos doctors did not have the expertise to try that, especially for someone with H.I.V.

His close friend, Peter, was not ready to give up, and together they searched online for alternatives. (Peter declined to reveal his last name because of privacy concerns.) They discovered that at a hospital in London was Dr. Ian Gabriel, an expert in bone-marrow transplants for treating cancer, including in people with H.I.V. Because of their last-ditch effort, Mr. Castillejo said, Were here today. You never, never know.

Within a week, he met with Dr. Gabriel, who tried a third and final time to tap Mr. Castillejos own stem cells for a transplant. When that failed, Dr. Gabriel explained that Mr. Castillejos Latin background might complicate the search for a bone-marrow donor who matched the genetic profile of his immune system. To everyones surprise, however, Mr. Castillejo quickly matched with several donors, including a German one perhaps a legacy from his half-Dutch father who carried a crucial mutation called delta 32 that hinders H.I.V. infection. A transplant from this donor offered the tantalizing possibility of curing both Mr. Castillejos cancer and the H.I.V.

When Dr. Gabriel called with the news in the fall of 2015, Mr. Castillejo was on the top deck of one of Londons iconic red buses, on his way to see his general practitioner for a checkup. His thoughts raced alongside the scenery: He had only recently been told he was going to die, and now he was being told he might be cured of both cancer and H.I.V.

I was trying to digest what just happened, he recalled. But after that call, I had a big smile on my face. Thats where the journey began as LP.

With the possibility of an H.I.V. cure, the case immediately took on intense importance for everyone involved. Dr. Edwards, who had cared for Mr. Castillejo since 2012, had, as a young doctor in the early 1990s, seen many men his age die of H.I.V. What a privilege it would be to go from no therapy to a complete cure in my lifetime, he recalled telling Mr. Castillejo. So you have to get better no pressure.

Dr. Edwards involved Dr. Gupta, his former colleague and one of the few virologists in London he knew to be doing H.I.V. research. Dr. Gupta initially was skeptical; the approach had worked only once, 12 years earlier, with Mr. Brown. But Dr. Gupta also knew that the payoff could be huge. Antiretroviral drugs can suppress the virus to undetectable levels, but any interruption in the treatment can bring the virus roaring back, so a cure for H.I.V. is still the ultimate goal.

Dr. Gupta began carefully monitoring Mr. Castillejos H.I.V. status. In late 2015, Mr. Castillejo was preparing to receive the transplant when another major setback arose. His viral load shot back up with H.I.V. that appeared to be resistant to the drugs he had been taking.

This gave Dr. Gupta a rare glimpse at the typically suppressed virus, and allowed him to confirm that the viral strain was one that would be cleared by the transplant. But it also delayed the transplant by several months while the doctors adjusted Mr. Castillejos medications. He eventually received the transplant on May 13, 2016.

The next year was punishing. Mr. Castillejo spent months in the hospital. He lost nearly 70 pounds, contracted multiple infections and underwent several more operations. He had some hearing loss and began wearing a hearing aid. His doctors fretted over how to get his H.I.V. pills into his ulcer-filled mouth by crushing and dissolving them, or by feeding them to him through a tube. One of the doctors came to me and said to me, You must be very special, because I have more than 40 doctors and clinicians discussing your medication, Mr. Castillejo recalled.

Even after he left the hospital, the only exercise he initially was allowed to do was walking, so he walked for hours around the trendy Shoreditch neighborhood. He went to the flower market there every Sunday, treated himself to salted beef beigels to celebrate small successes and admired the colorful murals and vintage clothes.

A year on, as he became stronger, he slowly began thinking about forgoing the H.I.V. medications to see if he was rid of the virus. He took his last set of antiretroviral drugs in October 2017. Seventeen months later, in March 2019, Dr. Gupta announced the news of his cure.

Neither he nor Mr. Castillejo was prepared for what came next. Dr. Gupta found himself presenting the single case to a standing-room-only crowd at a conference, and shaking hands afterward with dozens of people. Mr. Castillejo was overwhelmed by the nearly 150 media requests to reveal his identity, and began to see a role he might play in raising awareness of cancer, bone-marrow transplants and H.I.V.

He has enrolled in several studies to help Dr. Gupta and others understand both diseases. So far, his body has shown no evidence of the virus apart from fragments the doctors call fossils and what seems to be a long-term biological memory of having once been infected.

Others in the H.I.V. community are reassured by this news, but expressed concern for Mr. Castillejos privacy and mental health.

It can be very important for people to have these kinds of beacons of hope, Mr. Jefferys, the Treatment Action Group director, said. At the same time, thats a lot of weight for someone to carry.

Mr. Castillejos friends have similar worries. But he is as ready as he will ever be, he said. He sees LP as his work identity and is determined to live his private life to its fullest. Having lost his lustrous dark hair several times over, he has now grown it to shoulder length. He has always enjoyed adventures, and with careful preparation he has begun traveling again, describing himself to fellow travelers only as a cancer survivor. He celebrated his 40th birthday with a trip to Machu Picchu, in Peru.

But in conversations about his status as the second person ever to be cured of H.I.V., Mr. Castillejo still adamantly refers to himself as LP, not Adam. When you call me LP, it calms me down, he said. LP to my name, that is kind of a big step.

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The London Patient, Cured of H.I.V., Reveals His Identity - The New York Times

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Are stem cells really the key to making humans live longer? – Wired.co.uk

By daniellenierenberg

To some longevity acolytes, stem cells promise the secret to eternal youth. For a hefty fee, you can pay a startup to extract your own stem cells and cryogenically freeze them, in the hope that they can one day be used in a treatment to help extend your life.

Other firms let you bank stem cells from your babys umbilical cord and placenta after childbirth, if youre convinced the high cost represents an insurance policy against future illness. Or you can follow the example of Sandra Bullock and Cate Blanchett and opt for an anti-ageing cream made with stem cells derived from the severed foreskins of newborn babies in South Korea.

Stem cells are the parent cells which give rise to other cells in our bodies. Since scientists first isolated human embryonic stem cells in a lab and grew them over 20 years ago, they have been mooted as a source of great hope for regenerative medical treatments, including for age-related degenerative conditions such as Parkinsons, Alzheimers, heart disease and stroke.

But apart from a few small-scale examples, the only stem cell-based medical treatment practised in clinics uses haematopoietic stem cells found in the blood and bone marrow which only produce blood cells for transplants in blood cancer patients. These cells are taken from a patients sibling or an unrelated donor, before being infused into a patients blood, or theyre taken from a patients own blood before being reinfused. The procedure has been used to treat blood malignancies for almost half a century, and recently multiple sclerosis too. So how likely is it that the predictions about stem cells' longevity-enhancing powers will become a reality?

In September 2019, Google banned ads for unproven or experimental medical techniques such as most stem cell therapy, citing a rise in bad actors attempting to take advantage of individuals by offering untested, deceptive treatments [that can often] lead to dangerous health outcomes. The decision was welcomed by the International Society for Stem Cell Research, which emphasised that most stem cell interventions remain experimental. Selling treatments before well-regulated clinical trials have been done, the body said, [threatens public] confidence in biomedical research and undermines the development of legitimate new therapies.

Its easy to see how less scrupulous companies can exploit the allure of stem cells, which seem to occupy a place in our collective consciousness as a kind of magical elixir. High hopes for stem cell-based therapies have grown since 2006, when the Japanese biologist Shinya Yamanaka created a new technology to reprogram adult cells, such as skin cells, into a similar state to embryonic stem cells, which are pluripotent, meaning they can develop into any tissue in the body. The Nobel prize-winning breakthrough was hailed as a major step in the study of stem cells without the need for controversial embryo research, and towards the use of these human induced pluripotent stem cells to regenerate damaged or diseased organs or effectively grow new spare parts which could treat the life-limiting and life-shortening illnesses associated with ageing.

Gerontologist Aubrey de Grey, whose Strategies for Engineered Negligible Senescence (SENS) research foundation aims to eliminate ageing-related diseases, thinks the chances well soon have stem cell based therapies are high. For anything that's in clinical trials, you're talking about maybe five years before it's available to the general public, he says, citing stem cell treatments for Parkinsons disease, currently being tested in phase two clinical trials, as one of the developments he thinks is likely to come soonest.

However, given that these trials involve a relatively small number of participants and most clinical trials ultimately fail, his predictions might be overly optimistic. Often described as a maverick, De Grey believes that humans can live forever and there is a 50 per cent chance medical advances of which stem cell therapies will play an important part will make this a reality within the next 17 years. Though living forever, he says, is not the ultimate goal but a rather large side effect of medicine which will successfully prevent or repair the damage that comes with ageing.

For New Jersey-based Robert Hariri, who co-founded Human Longevity Inc, which set its sights more modestly on making 100 the new 60, stem cells derived from placentas present especially exciting opportunities. A biomedical scientist, surgeon and entrepreneur, Hariri says his current venture Celularity which is focused on engineering placental cells, including stem cells, to create drugs for cancer and other conditions is not as concerned about the actual age number, but about preserving human performance as we age and treating the degenerative diseases that rob us of our quality of life.

Many of those working in the field, however, remain cautious in their optimism. Researchers have highlighted the potential risks of giving pluripotent cells to patients, whether they are induced or embryonic, as these cells can develop cancer-causing mutations as they grow.

Davide Danovi, a scientist at Kings College Londons Centre for Stem Cells & Regenerative Medicine, says the path to stem cell-based therapy is very long and full of hurdles. The supply chain involves challenges, he says. On the one hand, allogeneic treatments those with stem cells derived from one individual and expanded into big batches to create cells to treat many individuals have the advantage of being similar to the traditional pharmaceutical business models. The product is clear, its something that comes in a vial and can be scaled up and mass produced, Danovi says. But this treatment can present a greater risk of rejection from the patient, as opposed to the more bespoke autologous option which is more expensive and time-consuming as it involves extracting a patients own stem cells before reprogramming them.

Danovi is most excited by the potential of stem cells to treat age-related macular degeneration. In 2017 Japanese scientist Masayo Takahash led a team that administered transplants of artificially grown retinal cells created from induced pluripotent stem cells taken from donors to five patients with the eye condition, which can cause blindness, and theyre reported to be doing well. The eye, he says seems to be a place where immunity plays less of a role relative to other issues, so you can host cells which come from another individual with fewer problems [of rejection]. But, with other organs such as the liver, he says there are major conceptual problems with creating enough tissue. Its like the clean meat burger - you're talking about a production that is, in many cases, not easy to reach with the current technology.

Hariri believes placentas will solve some of the production challenges crucially, theyre an abundant commodity, with the vast majority thrown out after childbirth. His interest was sparked 20 years ago when his oldest daughter was in the womb: When I saw her first ultrasound in the first trimester, the placenta had already developed into a relatively sizable organ, even though she was just a peanut-sized embryo. Id been taught that the placenta was nothing more than an interface, but [if that was the case], you would expect that it would grow at the same rate as the embryo. His curiosity piqued, he began to see the placenta not as an interface but as a biological factory, where stem cells could be expanded and differentiated to participate in the development of that foetus. That intrigued me and I started to collect placentas and just, you know, basically disassemble them.

Placentas have numerous benefits, he says they dont carry the same ethical controversy as embryonic stem cells, for one thing. Scientists working on embryonic stem cells have to destroy an early embryo, and that option yields them a dozen cells, which have to be culture-expanded in the laboratory into billions of cells. In contrast, the placenta houses, billions and hundreds of billions of cells, which can be expanded as well, but you're starting out with a dramatically larger starting material.

Increasingly, scientists in the anti-ageing sphere are focusing on an approach that seems like the opposite of planting fresh stem cells into our bodies. Experts such as Ilaria Bellantuono at Sheffield Universitys Healthy Lifespan Institute are working towards creating senolytics medication that could kill off our senescent cells, the zombie cells that accumulate in tissues as we age and cause chronic inflammation. I think stem cells are very good for specific disease, where the environment is still young, Bellantuono says, but the data in animal models tells us that senolytics are actually able to delay the onset and reduce the severity of multiple diseases at the same time for example, there is evidence for osteoarthritis, osteoporosis, cardiovascular disease, Alzheimer's, Parkinson's, and diabetes. She explains that while human trials are still in their early stages, senolytics are likely to be more cost-effective than stem cell therapy and the status quo of older patients taking multiple pills for multiple diseases, which can interact with each other. Besides, she adds, they may actually work in tandem with stem-cell based therapies in the future, with senolytics creating a more hospitable environment in tissues to allow stem cells to do their work.

And as for the so-called penis facial? Its far from the only ultra-expensive stem cell skincare making bold anti-ageing claims but youre probably better off saving your money, as you are with the experimental medical treatments on offer. Stem cells are definitely exciting but theyre not the key to eternal youth. At least, not yet.

Robert Harari will be one of the speakers at WIRED Health in London on March 25, 2020. For more details, and to book your ticket, click here

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The 8 Most Iconic Beauty Products In The Black Friday Sales To Buy Now – elle.com

By daniellenierenberg

.

The Black Friday sales are almost among us, meaning the halls of your local department store are about to become a battleground. (The smartest of shoppers know the key to maintaining sanity is to do it all online.)

But when faced with deals and discounts from every angle, its crucial to keep your cool. You dont want to panic-buy all those as-seen-on-Instagram products before youre covered the essentials you know, the things you'll use day in, day out, until they're empty.

Resist those shiny, sparkling impulse buys (although yes, that glitter lipstick would look pretty good on NYE), and youll come out triumphant with a well-curated skincare edit that will keep your skin happy long after the last Quality Street has been polished off.

So, skip the scrolling and head straight for the good stuff this year. Here, discover the eight most unequivocally iconic beauty products we've spotted in the Black Friday sales. Trust us: you cannot go wrong with any (or all) of these heroes.

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1Soleil Tan De Chanel

Chanel

WAS 40 NOW 36

Soleil Tan de Chanel is the kind of product beauty dreams are made of. The weighty pot will last you all year, while the lacquered, double-C engraved pot will look incredibly bouji on your dressing table.

But of course,it's what's on the inside that really counts, and boy is this a brilliantly formulated bronzer. Unlike literally anything else on the market (many brands have tried and failed to 'dupe' it), this dense, mattecream can be picked up with a powder brush, dusted under or over foundation, and swiped strategically to carve out killer cheekbones it's revolutionary.

2Blanche Eau de Parfum

Byredo

WAS 110 NOW 93.50

Byredo's perfumes are coveted the world over, but with so many to choose from, you'll be hard-pressed to find a favourite without some kind of divine intervention.

Our advice? Head straight for Blanche, which is a byword for the crispest, cleanest scent you'll ever spritz. Forget cloying talc notes or blink-and-you'll-miss-it citrus: this is pure violet and musk-tinted freshness, like the cleanest cotton sheets that ever were.

And seeing as there's a very generous 15% discount (thank you, Liberty), maybe treat yourself to the body wash, too.

3The Rich Cream

205.00

WAS 205 NOW 174

The hype around Augustinus Bader's debut product was remarkable across the world, A-listers and beauty editors fawned. (Victoria Beckham even tapped him up to create her debut skincare product.)

Turns out, this clever cream really delivers. The secret is the Trigger Factor Complex, which works to kick-start the natural healing process of the body's stem cells. 30 years of research and development have clearly paid off.

4Bronzing Powder

Narsasos.com

WAS 31 NOW 24.50

The name of many a Nars product precedes its performance Orgasm, anyone? But not this one: the brand's Laguna and Casino bronzing powdersare famed for their supreme performance alone.

Both illuminate without relying on glitter (which always looks fake), and are warming without imparting those giveaway ruddy undertones.

Paler skins are destined for Laguna, while darker tones will love Casino. Your bank balance will love either.

5Diorshow Pump 'N' Volume HD Mascara

DIOR

WAS 28 NOW 25.20

Ask any beauty editor what their ride-or-die mascara is and you'll get...a lot of conflicting opinions.

Like the perfect shade of red lipstick, a favourite mascara is a subjective thing. After all,few can deliver perfection when it comes to length, volumeand colour. Enter Dior's Pump 'N' Volume: the mascara to unite us all. (And yes, it's the one with the no-wastesqueezy tube.)

6Do Son Eau De Parfum

Diptyque

WAS 120 NOW 96

Many of Diptyque's fragrances could be considered iconic, but Do Son is the one that'll see you being stopped by strangersin the street.

The tuberose trail is enticing enough, but it's the unusual addition of orange blossom and jasmine that take things to truly memorable heights. A spectacularode to its namesake beach, in Vietnam's Ha Long Bay.

7Ruby Woo Matte Lipstick

WAS 17.50 NOW 14

If you're a 'lipstick person', you already know about this one. You've likely got one stashed in your bag right now, as well as one on your dresser, and an 'emergency' onefloating around your bedroom somewhere.

So good it's never been successfully imitated, MAC's Ruby Woo is the ultimate lipstick. A true red, it's neither too orange or too blue, and there's no skin tone it won't look beautiful against.

8Glow Tonic

WAS 18 NOW 15.30

We all know that alpha-hydroxy-acids are the gold standard when it comes to resurfacing, but so many require a degree in dermatology to use correctly.

Forgo the faffing in favour of Pixis cult Glow Tonic: it might look cute, but its packed with 5% brightening glycolic acid alongside soothing aloe vera. Simply sweep it over cleansed skin nightly no brow-furrowing required.

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A Year Ago He Was Paralyzed From the Neck Down Then This Happened – Daily Reckoning

By Sykes24Tracey

You might feel a bit down if you watch the news. Who wouldnt?

Angry people might be grabbing headlines and making you wonder about the future, but the antidote is all around you.

Talk to some of your neighbors. Chances are, no matter what they look like or where theyre originally from, youll find theyre actually pretty decent people just like you.

The little improvements we all try to make may not register much, but the accumulation of them all eventually does.

And if theres one tangible piece of proof that the world is changing for the better, its Lucas Lindner.

2016 was not a kind year for 22-year-old Lucas.

Last May he lost control of his pickup truck when a deer ran out on the road. The front passenger tire blew out. The truck rolled, throwing him out of the window.

When he woke up in the hospital, he was paralyzed from the neck down. He was just heading to the grocery store on a Wisconsin Sunday morning.

It was an accident that could happen to anyone, to a friend or relative.

Normally, people like Lucas have no hope of restoring motor control of their bodies ever again.

In the United States, this awful story plays out 17,000 times every year. There are a quarter of a million people in the country with paralysis.

But Lucas story is working out a little bit differently.

Lucas was airlifted to Froedtert Hospital, a teaching hospital of the Medical College of Wisconsin.

There, Dr. Shekar N. Kurpad, professor of neurosurgery, applied 15 years of research into cell transplantation for spinal cord injury.

The procedure revolutionary and so were the cells Dr. Kurpad used.

The new procedure used cells that were developed over many years by researchers at a two companies leading the way in regenerative medicine.

Researchers at these companies have discovered how to grow stem cells and make them reliable for transplantation use.

On doctor, in fact, who Ive researched extensively, has been called the father of regenerative medicine.

Ive had the pleasure of meeting with him on a number of occasions.

Whenever I am in the San Francisco Bay Area, I try to visit him to learn whats going on in the field.

And from what Ive seen the therapeutic potential is hard to understate.

And were starting to see the results in people like Lucas Lindner.

Hes still wheelchair-bound we have a lot more to learn but he now has fine motor skills in his upper body. Thats extraordinary in cases like his.

Lucass miraculous improvement is due to newly designed pluripotent stem cells They are called pluripotent because they have the power to transform into any other cell type in the body.

And this Bay Area doctors company has accumulated the technology to make that happen.

Over the next few months, well get more clinical data from patients being treated with the full 20 million-cell dose and potentially more great news of restored motor function.

The recent headlines may have been about a few angry people rioting and hating each other, but the real important news is this

Recently, when the Cincinnati Reds played the Milwaukee Brewers, Lucas threw out the opening pitch.

Many U.S. presidents and other famous people have thrown pitches, but no pitch has been as historic as this one. And the advances I highlighted today are the reason why.

As this therapy matures and gets closer to market, I believe it will make a big impact on shares of companies in this space.

Which means the right-timed move in the upcoming months means a huge potential windfall of cash for you.

More to come soon.

For Tomorrows Trends Today,

Ray BlancoforThe Daily Reckoning

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Will putting leeches on his face help this blind man see? – USA TODAY

By daniellenierenberg

Most nights, Marcia Dunlap attaches seven or eight leeches around her husband John's eyes as part of an effort to restore some of his vision. Tom Bailey/The Commercial Appeal

With the help of his wife Marcia, John Dunlap receives his nightly leech treatment at his home in East Memphis. Marcia places several leeches on his face in an effort to increase pressure in his left eye. In conjunction with stem cell treatment, the Dunlaps hope that one day John may be a viable candidate for a procedure that could return some of his vision.(Photo: Jim Weber/The Commercial Appeal)

At home most evenings, Memphis, Tennessee, attorney John Dunlap, 80, unbuttons and removes his white dress shirt and counting his steps and remembering which way to turn carefullywalks with a tall white canefrom the living room to the dining table, where his wife Marcia has a plastic container of leeches.

Twenty-six months ago,the couple's schizophrenic sonAndrewattacked them in theirhome. The injuries blinded Dunlap. He's in total darkness.

After drapinga large, peach-colored towel around John's neck, Marcia reaches into the water for the skinniest leeches. Those are the hungriest and most likely to latchonto John's face.

One at a time, she gently presses four leeches to the skin around John's left eye and three around the right. She waits patiently wait for eachto bite and stay connected to John's skin.

"You can feel a bite,'' he says. "A little, stinging bite... And then after awhile you don't feel anything.''

The Dunlaps have carried out this unusualroutine60 or so times since December. It's a type of therapy prescribed by a Los Angeles doctor who offers experimental stem cell therapy designed to regenerate tissue.

"In the beginning he made it very clear he's not anophthalmologist and not an eye surgeon but he had had some success with stem cells in treating blindness. It's experimental,'' Dunlap said.

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The doctor prescribed the leech therapy as a preliminary step because, Dunlap said, the leech enzymesenhance the blood supply to the eye and nourishthe eye tissue.

The left eye had atrophied, or withered. The idea wasto restore health to the eyebefore the stem cell treatment. There is no right eye, but the hope is that the leech enzymes will help revive that optic nerve in case a transplant is ever possible.

Since the leech therapy,the pressure in the right eye has improved significantly, Dunlapsaid, referring to follow-upexams. The retina, which had folded into an ice-cream cone shape after the trauma, has begun returning to its normal shape, he said.

Even though he still cannot see out of the left eye and the optic nerve remains severed from the retina, Dunlap said, "I now have a live eye.''

The Dunlaps decline to identify the California doctor, describing him as a"humble'' person whodoes not seek the publicity.

With the help of his wife Marcia, John Dunlap receives his nightly leech treatment at his home in East Memphis. Marcia places several leeches on his face in an effort to increase pressure in his left eye. In conjunction with stem cell treatment, the Dunlaps hope that one day John may be a viable candidate for a procedure that could return some of his vision.(Photo: Jim Weber/The Commercial Appeal)

Andrew, the Dunlaps' mentally ill son, is charged with attempted murder and domestic assault, and remains in jail awaiting trial. Thecouplehave told authorities that they mainly want Andrew to receive mental health treatment.

The Dunlapshave experienced tragedy long before the 2015 assault.

Their son Jeff, one of four children, was a St. Jude Children's Research Hospital patient who died of cancer at age 10, in September 1974.

Dunlap recalls a return car tripfrom Knoxville, where he and Marcia had been visiting grandchildren shortly after he was released from rehab.

"As we were driving back I started thinking of all the things I won't get to do again. In my mind, I was going down the list,'' he said.

It would be a long list, including some leisure activities he loves. An avid Cubs fan, heenjoyed attending spring training games in Arizona. A passionate golfer, he enjoyedwatching how the ball flew when he struck it well.

But Dunlap stopped himself from completing the list of losses, telling himself, " 'You don't want to dwell on that'. . . It's as if the Lord sent me a message that hit me across my forehead, saying, 'John, get over it. It could be a whole lot worse.'

"Anytime I want to start thinking about the things I'm missing or not doing what I used to do, I think 'Get over it. Move on'.''

Sudden blindness is such a change in lifestyle. "I guess some people may feel the world has ended for them, but it hasn't,'' he said.

Marcia Dunlap gets special leeches for her husband John's nightly treatment from the laundry room where she keeps it out of sight. Marcia places several leeches on his face in an effort to increase pressure in his left eye. In conjunction with stem cell treatment, the Dunlaps hope that one day John may be a viable candidate for a procedure that could return some of his vision.(Photo: Jim Weber/The Commercial Appeal)

The stem cell and leech therapy is expensive and not covered by health insurance. Some have expressed their skepticism about the legitimacy of the experimental treatments.

"You have some people who are concerned for you, that your approach is not going to be effective,'' Dunlap said.

"Yet, several folks up herehave said, 'John, I'd take a shot at it. It is expensive but you're the one with the white cane and the one who is blind and has to live with it. You have everything to gain and nothing to lose.'''

While some might be concerned about the unusual treatments, many others are inspired by the Dunlaps,saidBlanche Tosh, a fellow church member and friend since high school.

"I have told them so many times, 'You just can't begin to know the lives you have affected,'' Tosh said.

"I know so many people who look at the way they are dealing with multiple things. How could anybody endure that and just go on and be pleasant and make it from day to day with the consistent attitude that the world sees.

"You are not going to find many people whoever see one of them without a smile,'' Tosh said.

She was inspired to start a gofundme account (gofundme.com/johndunlapvision) to help coverthe Dunlaps' expenses. As of midweek, $8,795 of the $100,000 goal had been raised.

Memphis lawyer John Dunlap and his wife Marcia continue to search for some medical procedure to restore at least partial vision after John was blinded a few years ago when their mentally ill son attacked him. (Photo: Jim Weber/The Commercial Appeal)

Since December, Dunlap has undergone two-and-a-half rounds of leech therapy and two series ofstem cell treatments. The couple traveled to California in June for the most recent stem cell procedures, and returned home with stem-cell eye drops and injections.

Nowthey are in the middle of the leech therapy they resumed this summer.

John has a follow-up exam next week, when he will learn if there's been continued progress from the stem cell and leech therapies.

The California doctor "indicated it would take two to three months to see if we were getting any results from stem cell therapy out there,'' Dunlap said. That time could come sometime this month or in September.

If the stem cell therapy has not worked by then, he said,"We'll just have to see what any third plan looks like, and the cost involved.''

Late in life, Dunlap has been forced to learn to type, work a computer, navigate with a cane, count the steps and memorize the turns from one spot to another, communicate with Siri, and smile as blood-sucking leeches dangle from his cheeks.

Asked about his sources of inner-strength, he responded, "I don't know I'd call it inner-strength.

"I can tell you I certainly believe in the Lord. We pray daily. I appreciate the prayers of others. I think it certainly is a faithissue.''

He also credits his late mother, Cora, a single parentwho managed a grocery. "She was a very optimistic, loving person,'' he recalled.

"And I've had Marcia's support. Marcia wasn't going to let me give up, just sit down and do nothing.''

The Dunlaps are starting to consider resuming their annual trips to Cubs spring training in Arizona. Maybe next spring.

"You may have your vision by then,'' Marcia told John.

"I might,'' he responded."We'll see.''

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SUPERHUMANS: Mars ‘will be colonised by genetically engineered Star Trek-style beings’ – Express.co.uk

By raymumme

GETTY STOCK IMAGE

The way to the Red Planet and other mysterious worlds is being inspired by the villainous Khan from the blockbuster films, according to new research.

The use of stem cell technology may mean the difference between life and death on any attempt to travel beyond Earth into the wilderness of space.

So the first person to walk on Mars is likely to be selected from the growing group of people whose parents took the step to store their child's stem cells at birth.

Stem cells are 'blank' cells that can be reprogrammed to turn into any other cell in the body, enabling the replacement of damaged cells.

More and more British parents, including TV presenter Natalie Pinkham and dancer Darcey Bussell, are paying more than 2,000 to freeze samples from their babies' umbilical cords at birth.

Stem cells are also found in bone marrow and some body tissue, but the procedure to harvest them from umbilical cords is less risky.

Adventurous Mars pioneers will have to be especially prepared for the dangerous trip, which could expose them to cancer and other diseases, through carefully researched gene therapy.

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We wince at the thought of genetically engineered humans

Mark Hall

Mark Hall, spokesperson for the UK's leading stem cell storage and diagnostics company StemProtect, said: "We wince at the thought of genetically engineered humans.

"And we are not going to create a Khan from Star Trek specifically to get to another planet. Getting humans to Mars and beyond will be both expensive and dangerous.

"But the scientific by-products - such as huge leaps in stem cell medicine - will benefit humanity for centuries to come."

Genetic engineering has featured in two Star Trek movies, and a number of TV episodes.

IG

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This still image strikes an uncanny resemblance to a figure of a woman

Khan, who appeared in Space Seed and Star Trek II: The Wrath of Khan, was modified to make him stronger and to give him greater stamina and intellectual capacity than a regular human.

Mr Hall said: "The first human to walk on Mars may not even be born yet - but that's an advantage."

StemProtect believes advanced medical techniques will be required to cope with the rigours of interplanetary space.

While a trip to Mars may appear "just around the corner" in galactic terms, it is highly possible exposure to radiation along the way could lead to the astronauts developing leukaemia and other cancers even before they arrived.

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This means future travellers will have to be 'immunised' before they leave Earth.

Mr Hall said: "There was an article in The Times suggesting elephants would make ideal Martian travellers because they'd be largely immune to the radiation.

"But those laughing at the ridiculous sounding headline completely missed the point - the fact is scientists are already working on ways of getting humans there and back alive."

Recent research has shown radiation in deep space increase the risk of leukaemia while long term exposure to micro gravity may leave astronauts open to infection.

The three year round trip to Mars would affect humans at the stem cell level, leaving them with a drastically lowered immune system, NASA funded scientists say.

And NASA's own findings say stem cells may be crucial to the future of space travel, particularly how they respond in a low gravity environment.

One study showed stem cells flown in space and then cultured back on Earth had greater ability to self renew and generate any cell type, changing more easily into specialised heart muscle cells, for instance.

Mr Hall said an astronaut will have to be prepared for the journey "quite literally at the stem cell level."

He explained: "That means working with the best and most effective stem cells available to the patient - those harvested from the umbilical cord at birth."

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The therapies required to 'immunise' humans to space travel are still being researched.

And with most space based science, it can only mean huge benefits to mankind back down on Earth when it comes to fighting otherwise deadly conditions and diseases.

Stem cells have the ability to treat a potentially infinite range of illnesses and diseases.

Stem cell therapy is already being used all over the world to treat some cancers and stroke victims - and there is fast progress being made in many other areas, including Parkinson's and Alzheimer's disease.

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SUPERHUMANS: Mars 'will be colonised by genetically engineered Star Trek-style beings' - Express.co.uk

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Neurotrophic factors in ALS: a winning combination? – ALS Research Forum

By LizaAVILA

Distinct growth factors promote the survival of specific types of motor neurons in the spinal cord, according to a study led by Georg Haase, of Aix-Marseille University in Marseille, France. The results suggest that these factors may work together to provide trophic support to motor neurons in the CNS and therefore, a combination of them may be needed to protect motor neurons damaged by disease.

Growth factors have always been tantalizingly attractive in ALS, said Nicholas Boulis of Emory University Medical School, who was not involved in the study. But the problem is, there has been a failure of growth factors to perform [in the clinic]. This study provides tangible evidence that you may be able to get a bigger effect by combining growth factors.

The study appeared on March 16 in the Proceedings of the National Academy of Sciences.

Neurotrophic Factors in ALS: The power of two+

Sorting out ALS. George Haases team at Aix-Marseille University in France used a FACS-based method to identify NTFs needed to protect distinct classes of motor neurons in the developing lumbar spinal cord. Now, the researchers are adapting this method to determine which of these substances may be needed to protect adult motor neurons, including those affected by ALS. The results may help clinicians develop neuroprotective treatment strategies tailored for the disease. [Courtesy of Schaller et al., 2017, PNAS]

Researchers first turned to neurotrophic factors (NTFs) in the early 1990s as a potential therapy for ALS in hopes to promote the survival of motor neurons damaged by the disease. But initial therapies proved ineffective in part due to delivery challenges (see Rogers, 2014).

In more recent years, neuroscientists discovered that many of these growth factors may work together to provide trophic support for motor neurons and promote their survival at least in the developing spinal cord (see Gould and Enomoto, 2009). But how these substances orchestrate this process remains an open question.

A growing number of researchers suspect that there may be distinct classes of motor neurons that are protected by distinct NTFs during development. To test this hypothesis, Haases team at Aix-Marseille University in France isolated motor neurons from the developing lumbar spinal cord in the mouse and determined which growth factors supported them.

To carry out this analysis, first author Sbastien Schaller and colleagues dissected out lumbar spinal cords at day E12 and suspended the tissue. Then, they used fluorescence-activated cell sorting (FACS) to isolate the motor neurons, cultured them and exposed them to combinations of neurotrophic substances.

The technique enabled motor neurons to be specifically captured from embryos by using Hb9:GFP mice, originally developed by Columbia Universitys Thomas Jessell in New York, which express GFP in motor neurons in the developing central nervous system.

100% of the cells expressed the motor neuronal markers ChAT and SMI 32, and none expressed interneuronal markers, indicating the exquisite purity of the isolated cells, said Haase. That, combined with the methods speed and degree of automation, make FACS-derived motor neurons a promising platform for future studies, he said, including screening for potential ALS therapies.

A combinatorial approach? Beginning in the early 1990s, researchers developed potential neuroprotective therapies for ALS that delivered single neurotrophic substances. But according to a new study, multiple NTFs may be needed to promote the survival of motor neurons affected by the disease. [Courtesy of Schaller et al., 2017, PNAS]

Next, the team exposed motor neurons to 12 different neurotrophic factors (BDNF, NT3, GDNF, neurturin, artemin, persephin, CNTF, CT1, LIF, HGF, IGF1, and VEGF), alone or in combination. Individually, all NTFs promoted neuronal survival after 3 days in culture, with GDNF being the most effective (43%). HGF, however, protected only about 20% of motor neurons in culture. But when HGF, CNTF and artemin were combined, motor neuron survival reached nearly 50%.

The effects were additive, explained Haase. That suggested to us that each [of these growth factors] were supporting a subset of motor neurons.

To test that hypothesis, the researchers used subtype cell surface-specific antibodies to label three major subsets of motor neurons from the lumbar spinal cordthe medial motor column, which innervate axial muscles, the lateral motor column, which innervate limb muscles, and preganglionic, which synapse with downstream neurons of the autonomic motor system. They then used FACS to separate each subtype, and exposed them to HGF, CNTF or artemin.

They found that each of these NTFs promoted the survival of distinct classes of motor neurons in the lumbar spinal cord. For example, HGF preferentially supported survival of motor neurons in the lateral motor column neurons, key motor neurons affected by ALS.

The effects were mediated by distinct neurotrophic factor receptors decorating the surface of each type of motor neuron, explained Haase. When we blocked the HGF receptor, we completely blocked the survival effect of HGF. That means these motor neurons depend on this particular factor for their survival.

Additional analysis indicated that CNTF and artemin protected other types of motor neurons located elsewhere in the spinal cord.

Lateral thinking. HGF promotes the survival of motor neurons that innervate the limbs through a c-Met-mediated mechanism at least in the developing spinal cord (Schaller et al., 2017). The neurotrophic substance is the basis of Viromeds VM202, a gene therapy-based strategy now being evaluated at the phase 1/2 stage (Sufit et al., 2017). [Image: Emw, Wikimedia Commons.]

Together, the findings suggest that these substances provide trophic support and promote the survival of specific types of motor neurons in the developing spinal cord.

This is a very high-quality paper that helps clarify the field, said Clive Svendsen of Cedars-Sinai in Los Angeles, California. Until now, it was not clear that distinct subsets of motor neurons may respond to their own subsets of growth factors.

Motor neurons that could potentially include those that descend from the brainstem, and those involved in breathing, also affected by the disease.

The results suggest that combining growth factors may offer more therapeutic benefit than single factors in ALS according to Nicholas Boulis.

Svendsen agreed. This is suggesting that for therapies, if you want to protect motor neurons, you may have to expand to include multiple growth factors, Svendsen said. However, he noted, and as confirmed in this study, GDNF by itself is still perhaps the most powerful all-around survival factor for motor neurons.

Svendsen is now developing a potential therapy for ALS that uses genetically engineered neural stem cells to deliver GDNF to the spinal cord. The Phase 1 clinical trial is soon to be launched (see October 2016 news).

Neuroprotective therapies: the next generation?

The next big question, which this paper leaves open, according to Svendsen is whether the growth factors identified in this study protect motor neurons in the adult nervous system.

Haase agreed. This is a critical question, and we are adapting our method to look at this now.

A stem cell-based approach? Haases team previously developed a FACS-based technique to isolate reprogrammed motor neurons generated from human iPS cells (Toli et al., 2015). The approach could be used to identify key neurotrophic substances that promote the survival of patient-derived motor neurons. [Image: Reprogrammed sALS motor neuron, Alves et al., 2015. CC BY 4.0].

Some neural circuits change drastically during adulthood, while others stay pretty much the same, so weve got to do the experiments to find out, explained Svendsen. But I will probably be trying HGF soon in my own experiments.

In the meantime, said Haase, it is important to keep in mind that the growth factors found to be less effective in this study should not be ruled out as potential therapies. They may act sequentially during development, or may require co-factors to exert their effect which were not present in our growth medium, he said.

It is also important to keep in mind that this study did not evaluate the ability of any of these substances to regenerate axons, a key goal in terms of developing therapies for ALS and other motor neuron diseases including SMA.

The challenges of delivery, which have stymied the field to date, remain paramount, Haase also noted. Gene delivery approaches with adeno-associated vectors have been studied for single growth factors, but if several are needed, a larger-capacity vector, such as lentivirus, may be required, according to Boulis. Multiple rounds of ex vivo gene therapy to equip stem cells with multiple growth factor genes, would be another option, followed by surgical implantation of the modified cells.

Further exploration in in vivo models and patient-derived iPS cells are an important next step to determine which combination of these substances could be of the most benefit, added Boulis.

But despite these challenges, Boulis agrees this approach is worth considering. As a surgeon who does translational work on the application of growth factors to ALS, this may be an Aha! moment.

Reference

Schaller S, Buttigieg D, Alory A, Jacquier A, Barad M, Merchant M, Gentien D, de la Grange P, Haase G. Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons. Proc Natl Acad Sci U S A. 2017 Mar 21;114(12):E2486-E2493. [PubMed].

Toli D, Buttigieg D, Blanchard S, Lemonnier T, Lamotte dIncamps B, Bellouze S, Baillat G, Bohl D, Haase G.Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique. Neurobiol Dis. 2015 Oct;82:269-80. [PubMed].

Further Reading

Rogers, ML. Neurotrophic Therapy for ALS/MND. New York: Springer New York; c2014. p. 1755-85. (Kostrzewa RM, editor. Handbook of Neurotoxicity.)

Gould TW, Enomoto H. Neurotrophic modulation of motor neuron development. Neuroscientist. 2009 Feb;15(1):105-16. [PubMed].

disease-als gdnf HGF neuroprotection neurotrophic factor topic-clinical topic-randd VEGF

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