In 1996, Dolly the sheep made headlines around the world after becoming the first mammal to be successfully cloned from an adult cell. Many commentators thought this would catalyze a golden age of cloning, with numerous voices speculating that the first human clone must surely be just a few years away.

Some people suggested that human clones could play a role in eradicating genetic diseases, while others considered that the cloning process could, eventually, eliminate birth defects (despite research by a group of French scientists in 1999 finding that cloning may actually increase the risk of birth defects).

There have been various claims all unfounded, it is important to add of successful human cloning progams since the success of Dolly. In 2002, Brigitte Boisselier, a French chemist and devout supporter of Ralism a UFO religion based on the idea that aliens created humanity claimed that she and a team of scientists had successfully delivered the first cloned human, whom she named Eve.

However, Boisselier was unwilling or indeed unable to provide any evidence, and so it is widely believed to be a hoax.

So why, almost 30 years on from Dolly, haven't humans been cloned yet? Is it primarily for ethical reasons, are there technological barriers, or is it simply not worth doing?

Related: What are the alternatives to animal testing?

"Cloning" is a broad term, given it can be used to describe a range of processes and approaches, but the aim is always to produce "genetically identical copies of a biological entity," according to the National Human Genome Research Institute (NHGRI).

Any attempted human cloning would most likely utilize "reproductive cloning" techniques an approach in which a "mature somatic cell," most probably a skin cell, would be used, according to NHGRI. The DNA extracted from this cell would be placed into the egg cell of a donor that has "had its own DNA-containing nucleus removed."

The egg would then begin to develop in a test tube before being "implanted into the womb of an adult female," according to NHGRI.

However, while scientists have cloned many mammals, including cattle, goats, rabbits and cats, humans have not made the list.

"I think there is no good reason to make [human] clones," Hank Greely, a professor of law and genetics at Stanford University who specializes in ethical, legal and social issues arising from advances in the biosciences, told Live Science in an email.

"Human cloning is a particularly dramatic action, and was one of the topics that helped launch American bioethics," Greely added.

The ethical concerns around human cloning are many and varied. According to Britannica, the potential issues encompass "psychological, social and physiological risks." These include the idea that cloning could lead to a "very high likelihood" of loss of life, as well as concerns around cloning being used by supporters of eugenics. Furthermore, according to Britannica, cloning could be deemed to violate "principles of human dignity, freedom and equality."

In addition, the cloning of mammals has historically resulted in extremely high rates of death and developmental abnormalities in the clones, Live Science previously reported.

Another core issue with human cloning is that, rather than creating a carbon copy of the original person, it would produce an individual with their own thoughts and opinions.

"We've all known clones identical twins are clones of each other and thus we all know that clones aren't the same person," Greely explained.

A human clone, Greely continued, would only have the same genetic makeup as someone else they would not share other things such as personality, morals or sense of humor: these would be unique to both parties.

People are, as we well know, far more than simply a product of their DNA. While it is possible to reproduce genetic material, it is not possible to exactly replicate living environments, create an identical upbringing, or have two people encounter the same life experiences.

So, if scientists were to clone a human, would there be any benefits, scientific or otherwise?

"There are none that we should be willing to consider," Greely said, emphasizing that the ethical concerns would be impossible to overlook.

However, if moral considerations were removed entirely from the equation, then "one theoretical benefit would be to create genetically identical humans for research purposes," Greely said, though he was keen to reaffirm his view that this should be thought of as "an ethical non-starter."

Greely also stated that, regardless of his own personal opinion, some of the potential benefits associated with cloning humans have, to a certain degree, been made redundant by other scientific developments.

"The idea of using cloned embryos for purposes other than making babies, for example producing human embryonic stem cells identical to a donor's cells, was widely discussed in the early 2000s," he said, but this line of research became irrelevant and has subsequently not been expanded upon post-2006, the year so-called induced pluripotent stem cells (iPSCs) were discovered. These are "adult" cells that have been reprogrammed to resemble cells in early development.

Shinya Yamanaka, a Japanese stem cell researcher and 2012 Nobel Prize winner, made the discovery when he "worked out how to return adult mouse cells to an embryonic-like state using just four genetic factors," according to an article in Nature. The following year, Yamanaka, alongside renowned American biologist James Thompson, managed to do the same with human cells.

When iPSCs are "reprogrammed back into an embryonic-like pluripotent state," they enable the "development of an unlimited source of any type of human cell needed for therapeutic purposes," according to the Center of Regenerative Medicine and Stem Cell Research at the University of California, Los Angeles.

Therefore, instead of using embryos, "we can effectively do the same thing with skin cells," Greely said.

This development in iPSC technology essentially rendered the concept of using cloned embryos both unnecessary and scientifically inferior.

Related: What is the most genetically diverse species?

Nowadays, iPSCs can be used for research in disease modeling, medicinal drug discovery and regenerative medicine, according to a 2015 paper published in the journal Frontiers in Cell and Developmental Biology.

Additionally, Greely also suggested that human cloning may simply no longer be a "sexy" area of scientific study, which could also explain why it has seen very little development in recent years.

He pointed out that human germline genome editing is now a more interesting topic in the public's mind, with many curious about the concept of creating "super babies," for example. Germline editing, or germline engineering, is a process, or series of processes, that create permanent changes to an individuals genome. These alterations, when introduced effectively, become heritable, meaning they will be handed down from parent to child.

Such editing is controversial and yet to be fully understood. In 2018, the Council of Europe Committee on Bioethics, which represents 47 European states, released a statement saying that "ethics and human rights must guide any use of genome editing technologies in human beings," adding that "the application of genome editing technologies to human embryos raises many ethical, social and safety issues, particularly from any modification of the human genome which could be passed on to future generations."

However, the council also noted that there is "strong support" for using such engineering and editing technologies to better understand "the causes of diseases and their future treatment," noting that they offer "considerable potential for research in this field and to improve human health."

George Church, a geneticist and molecular engineer at Harvard University, supports Greely's assertion that germline editing is likely to garner more scientific interest in the future, especially when compared with "conventional" cloning.

"Cloning-based germline editing is typically more precise, can involve more genes, and has more efficient delivery to all cells than somatic genome editing," he told Live Science.

However, Church was keen to urge caution, and admitted that such editing has not yet been mastered.

"Potential drawbacks to address include safety, efficacy and equitable access for all," he concluded.

Originally published on Live Science.

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Why haven't we cloned a human yet? - Livescience.com

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image:A team at Scripps Research demonstrated how protein-sugar clusters called proteoglycans can guide processes like cell maturation and neuronal synapse formation, among other functions. As one example, pictured, semi-synthetic syndecan-1 proteoglycan rescues the maturation of mouse embryonic stem cells into neural precursor cells (red and green). view more

Credit: Meg Critcher, Scripps Research

LA JOLLA, CAScientists at Scripps Research have developed a set of methods for the closer study of one of the least-accessible, least-understood players in biology: protein-sugar conjugates called proteoglycans.

These molecules are often thickly present on the surfaces of cells and are known to have a broad set of functions in the body, though how they work and how their dysfunctions contribute to diseases are largely mysteries.

The scientists, who report their work in Nature Chemical Biology on May 12, 2022, devised synthetic proteoglycans that closely mimic real ones but have convenient chemical handles for modifying them. These and other aspects of their research platform enable the systematic study of how proteoglycans structure affects their functions in health and disease. The scientists demonstrated the effectiveness of their platform by using it to make new discoveries about proteoglycans roles in early cell development and in cancer cell spreading.

Were essentially unpacking the complexity of these molecules by constructing them in a modular way ourselves, and studying them in a tightly controlled environment, says study senior author Mia Huang, PhD, associate professor in the Department of Molecular Medicine at Scripps Research.

A proteoglycan starts as just a proteinthe so-called core proteinbut this protein contains special sites where any of a variety of sugar-related molecular chains called glycosaminoglycans (GAGs) can be linked. Within the cell where the protein originates, enzymes catalyze the attachment of GAGs to it, and this newborn proteoglycan normally is further decorated with clusters of sulfur and oxygen atoms called sulfates. The finished proteoglycan may be anchored into the cell membrane, its GAG chains waving in the extracellular fluid like seagrass, or it may be secreted from the cell to perform other functions.

With such complexity, it is no surprise that proteoglycans have versatile functionsthey are present in virtually all tissues, including cartilage, collagen, bone, skin, blood vessels, brain cells and mucosal surfaces. They help steer processes such as cell maturation, cell adhesion, cell migration, and neuronal synapse formation; serve as receptors for protein signaling partners; and are even used by some viruses and bacteria to latch onto cells. But proteoglycans complexity also means that how they do what they do, and with what partners, remains largely undiscovered. Scientists arent even certain how many proteoglycans there are in human and other mammalian cellsalthough there are at least dozens.

Huang and her team, including first authors Timothy OLeary, PhD and Meg Critcher, respectively a postdoctoral researcher and doctoral candidate in the Huang Lab during the study, constructed proteoglycan core proteins that are almost identical to known core proteins, but contain special molecular handles enabling the researchers to change the numbers and locations and types of GAG chains that bind to them. This allows the researchers to study systematically how the function of a proteoglycan changes as its GAG arrangement changes.

The researchers also developed techniques allowing them to anchor their proteoglycans in cell membranes or to let them float freely, to see how this affects proteoglycans functions in different circumstances.

Using their synthetic versions of common proteoglycans called syndecans, the scientists were able to study the respective contributions of GAG chains and core proteins. Specifically, they looked at two key biological processes mediated by syndecans: the maturing of stem cells, and the spreading of breast cancer cells on an extracellular matrix.

We learned from these experiments that not only the GAG chains but also the core proteins contribute to proteoglycan function, says Critcher. Notably, we also found that proteoglycans role in cancer cell spreading depends heavily on whether they are anchored to the cell membrane or free-floating.

The team also incorporated a method called proximity tagging to help them identify proteoglycans interaction partners. Huang and colleagues are now using this, and their modular construction technique, to study the interactions of syndecans and other proteoglycans in different contextsand with different GAG arrangementsand otherwise to explore their structures and functions.

Chemical editing of proteoglycan architecture was co-authored by Timothy OLeary, Meg Critcher, Tesia Stephenson, Xueyi Yang, Abdullah Hassan, Noah Bartfield, Richard Hawkins, and Mia Huang.

Funding for the research was provided by the National Institutes of Health (R00HD090292, R35GM142462).

About Scripps Research

Scripps Research is an independent, nonprofit biomedical institute ranked the most influential in the world for its impact on innovation by Nature Index. We are advancing human health through profound discoveries that address pressing medical concerns around the globe. Our drug discovery and development division, Calibr, works hand-in-hand with scientists across disciplines to bring new medicines to patients as quickly and efficiently as possible, while teams at Scripps Research Translational Institute harness genomics, digital medicine and cutting-edge informatics to understand individual health and render more effective healthcare. Scripps Research also trains the next generation of leading scientists at our Skaggs Graduate School, consistently named among the top 10 US programs for chemistry and biological sciences. Learn more atwww.scripps.edu.

Nature Chemical Biology

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Mucosal barrier injury (MBI) in the gastrointestinal tract remains a major clinical obstacle in the effective treatment of hematological malignancies, driving local and systemic complications that negatively impact treatment outcomes. Here, we provide the first evidence of hyper-activation of the IL-1/CXCL1/neutrophil axis as a major driver of MBI (induced by melphalan), which supports evaluating the IL-1RA anakinra, both preclinically and clinically. Our data reinforce that strengthening the mucosal barrier with anakinra is safe and effective in controlling MBI which in turn, stabilises the host microbiota and minimises febrile events. Together, these findings represent a significant advance in prompting new therapeutic initiatives that prioritise maintenance of the gut microenvironment.

The IL-1/CXCL1/neutrophil axis is documented to drive intestinal mucosal inflammation, activated by ligation of intestinal pattern recognition receptors, including toll-like receptors (TLRs)31. In the context of MBI, TLR4 activation is known to drive intestinal toxicity32, 33, however targeting TLR4 directly is challenging due to emerging regulation of tumour response34,35,36,37. As such, we selected anakinra as our intervention to inhibit inflammatory mechanisms downstream of TLR4. While anakinra was able to minimise the intensity and duration of MBI, it did not completely prevent it with comparable citrulline dynamics across animal groups in the first 48h after melphalan treatment. This reflects the core pathobiological understanding of MBI which is initiated by direct cytotoxic events which activate a cascade of inflammatory signalling that serve to exacerbate mucosal injury and the subsequent breakdown of the mucosal barrier33. By preventing this self-perpetuating circle of injury with anakinra, we were able to effectively minimise the duration of MBI and thus have a profound impact on the clinical symptomology associated with MBI including weight loss and anorexia. These findings firstly highlight the cluster of (pre-)clinical symptoms related to MBI (malnutrition, anorexia, diarrhea)38 and suggest that the mucoprotective properties of anakinra will provide broader benefits to the host, mitigating the need for intensive supportive care interventions (e.g. parenteral nutrition).

In line with our hypothesised approach, minimising the duration of MBI reduced secondary events including enteric pathobiont expansion and fever. This again reiterates that changes in the host microbiome and associated complications can be controlled by strengthening the mucosal barrier39. It can be postulated that by minimising the intensity of mucosal injury, the hostility of the microbial environment is reduced ensuring populations of commensal microbes to be maintained. This is supported by our results with the abundance of Faecalibaculum maintained throughout the time course of MBI. Faecalibaculum is a potent butyrate-producing bacterial genus documented to control pathogen expansion by acidification of the luminal environment. Administration of Faecalibacteria prausnitzii has been shown to reduce infection load in a model of antibiotic-induced Clostridioides difficile infection, whilst also showing mucoprotective benefits in models of MBI40, 41. Furthermore, it is documented to cross feed other commensal microbes increasing colonization resistance. Together, these underscore the luminal benefits of strengthening the mucosal barrier and suggest that maintenance of commensal microbes is central to minimizing translocation events and subsequent BSI.

In our clinical Phase IIA study with 3+3 design, we have shown that treatment with anakinra, up until a dose of 300mg, appears to be safe, feasible, and tolerated well. Of course, the sample size of this study was relatively small. However, anakinra was previously evaluated for its efficacy in the treatment of acute and chronic GvHD in patients allogeneic HSCT. In these studies, patients were treated for a similar time period (with higher doses of anakinra). No differences were seen between the anakinra and placebo group regarding (S)AEs, including infections and time to neutrophil recovery. There were no significant changes in our exploratory analyses, however, it was of note to see marked increase in IL-10 in patients that received 300mg anakinra. This may reflect anakinras capacity to promote anti-inflammatory signaling as observed in COVID-19 related respiratory events42. However, with our sample size it is not possible to make any conclusions on this mechanism. Our conclusion is that the recommended dose (RP2D) for anakinra is 300mg QD, which will be investigated in Phase IIB trial (AFFECT-2 study: Anakinra: Efficacy in the Management of Fever During Neutropenia and Mucositis in ASCT; clinicaltrials.gov identifier NCT04099901)43.

While encouraging, our data must be viewed in light of some limitations. Most importantly, our animal model purposely did not include any antimicrobials as we aimed to dissect the true contribution of MBI in pathogen expansion and subsequent febrility. While it is unclear if melphalan has a direct cytotoxic effect on the microbiota, it is likely that MBI drives dysbiosis with antibiotics serving to exacerbate these changes, with previous data demonstrating no direct impact of specific chemotherapeutic agents on microbial viability44. As such, assuming dysbiosis is secondary to mucosal injury as recently demonstrated45, we anticipate that anakinra will still have an appreciable impact on the severity of dysbiosis and may even prompt more protocolised/limited antibiotic use. Similarly, while we used body temperature as an indicator of BSI, we did not culture peripheral blood or mesenteric lymph nodes as was performed in our animal model development. The ability of anakinra to prevent BSI and thus minimise antibiotic use will be best evaluated in AFFECT-2 where routine blood culture is performed. It is also important to consider that we detected episodes of bacteremia in our participants that were likely caused by skin colonizing organisms; a mechanism anakinra will not influence. While these are expected in HSCT recipients, the majority of infectious cases originate from the gut, and we therefore anticipate anakinras capacity to strengthen the mucosal barrier will be clinically impactful in our next study. It must also be acknowledged that limited mechanistic investigations were conducted to identify the way in which anakinra provided mucoprotection. It is well documented that MBI is highly multifactorial, involving mucosal, microbial and metabolic dysfunction33, 46; each of which is mediated through aberrant cytokine production. It is therefore unlikely that anakinra will affect distinct pathways, instead dampening multiple mechanisms. In translating this evidence to the clinic, the impact of anakinra on symptom control is of greater significance than mechanistic insight.

In conclusion, we have demonstrated that not only is anakinra safe in HSCT recipients treated with HDM, but may also be an effective strategy to prevent acute MBI. Our data are critical in supporting new antibiotic stewardship efforts directed at mitigating the emerging consequences of antibiotic use. We suggest that minimizing the severity and duration of MBI is an important aspect of infection control that may optimize the efficacy of anti-cancer treatment, decreasing its impact on antibiotic resistance and the long-term complications associated with microbial disruption.

This study is reported using the ARRIVE guidelines for the accurate and reproducible reporting of animal research.

All animal studies were approved by the Dutch Centrale Commissie Dierproeven (CCD) and the Institutional Animal Care and Use Committee of the University Medical Centre Groningen, University of Groningen (RUG), under the license number 171325-01(-002). The procedures were carried out in accordance with the Dutch Experiments on Animals (Wet op de Dierproeven) and the EU Directive 2010/63/EU. All animals were individually housed in conventional, open cages at the Centrale Dienst Proefdieren (CDP; Central Animal Facility) at the University Medical Centre Groningen. Rats (single housed) were housed under 12h light/dark cycles with ad libitum access to autoclaved AIN93G rodent chow and sterile water. All rats acclimatised for 10days and randomised to their treatment groups via a random number sequence generated in Excel. Small adjustments were made to ensure comparable body weight at the time of treatment and cages were equally distributed across racks to minimise confounding factors. HRW was responsible for animal allocation and assessments while RH/ARDSF performed treatments. Softened chow and subcutaneous saline were provided to rats to reduce suffering/distress and were humanely euthanised if a clinical toxicity score>/=12 was observed. This score was calculated based on weight loss, diarrhea, reluctance to move, coat condition and food intake; each of which were assessed 03. At completion of the study, rats were anaesthetised with 5% isoflurane in an induction chamber, followed by cardiac puncture and cervical dislocation (isoflurane provided by a facemask).

We have previously reported on the development and validation of our HDM model of MBI, which exhibits both clinical and molecular consistency with patients undergoing HDM treatment21. During model development, plasma (isolated from whole blood) was collected and stored for cytokine analysis to inform the selection of our intervention. Repeated whole blood samples (75l) were collected from the tail vein into EDTA-treated haematocrit capillary tubes on day 0, 4, 7 and 10.

Cytokines (IFN-, IL-1, IL-4, IL-5, IL-6, IL-10, IL-13, KC/GRO and TNF-) using the Meso Scale Discovery V-Plex Proinflammatory Panel Rat 2 following manufacturers guidelines. On the day of analysis, all reagents were brought to room temperature, samples were centrifuged to remove any particulate matter and diluted 1:4. Data analysis was performed using the Meso Scale Discovery Workbench.

Male albino Wistar rats (150180g) were randomized (Excel number generator) to one of four experimental groups (N=16/group): (1) controls (phosphate buffered saline (PBS)+0.9% NaCl), (2) anakinra+0.9% NaCl, (3) PBS+melphalan, and (4) anakinra+melphalan. Melphalan was administered as a single, intravenous dose on day 0 (5mg/kg, 10mg/ml) via the penile vein under 3% isoflurane anaesthetic. Anakinra was administered subcutaneously (100mg/kg, 150mg/ml) twice daily from day 1 to+4 (8 am and 5pm). N=4 rats per group were terminated at the exploratory time points (day 4, and 7) and N=8 on day 10 (recovery phase) by isoflurane inhalation (3%) and cervical dislocation. The primary endpoint for the intervention study was plasma citrulline, a validated biomarker of MBI19, 47, which was used for all power calculations (N=8 required, alpha=0.05, beta=0.8).

Clinical manifestations of MBI were assessed using validated parameters of body weight, food intake and water intake, as well as routine welfare indicators (movement, posture, coat condition). Rats were weighed daily, and water/food intake monitored by manual weighing of chow and water bottles.

Plasma citrulline is an indicator of intestinal enterocyte mass48, and a validated biomarker of intestinal MBI. Repeated blood samples (75l) were collected from the tail vein into EDTA-treated haematocrit capillary tubes on day 0, 2, 4, 6, 7, 8 and 10. Citrulline was determined in 30l of plasma (isolated from whole blood via centrifugation at 4000g for 10min) using automated ion exchange column chromatography as previously described49.

Whole blood samples (200l) were collected from the tail vein into MiniCollect EDTA tubes on day 0, 4, 7 and 10 for differential morphological analysis which included: white blood cell count (WBC, 109/L), red blood cell count (RBC, 109/L), haemoglobin (HGB, mmol/L), haematocrit (HCT, L/L), mean corpuscular volume (MCV, fL), mean corpuscular haemoglobin (MCH, amol), mean corpuscular hemoglobin concentration (MCHC, mmol/L), platelet count (PLT, 109/L), red blood cell distribution width (RDW-SD/-CV, fL/%), mean platelet volume (fL), mean platelet volume (MPV, fL), platelet large cell ratio (P-LCR, %), procalcitonin (PCT, %), nucleated red blood cell (NRBC, 109/L and %), neutrophils (109/L and %), lymphocytes (109/L and %), monocytes (109/L and %), eosinophils (109/L and %), basophils (109/L and %) and immunoglobulins (IG, 109/L and %). For the purpose of the current study only neutrophils, lymphocytes and monocytes were evaluated.

Core body temperature was used as an indicator of fever. Body temperature was assessed daily using the Plexx B.V. DAS-7007R handheld reader and IPT programmable transponders. Transponders were inserted subcutaneously under mild 2% isoflurane anaesthesia on day 4. Average values from day 4 to 1 were considered as baseline body temperature.

The microbiota composition was assessed using 16S rRNA sequencing in N=8 rats/group. Repeated faecal samples were collected on day 0, 4, 7 and 10 and stored at 80C until analysis. Sample preparation (including DNA extraction, PCR amplification, library preparation), quality control, sequencing and analyses were all performed by Novogene (please see supplementary methods for full description).

All data (excluding 16S data) were analysed in GraphPad Prism (v8.0. Repeated measures across multiple groups were assessed by mixed-effect models with appropriate post-hoc analyses. Terminal data analyses were assessed by one-way ANOVA. Statistical analyses are outlined in figure legends and P<0.05 was considered significant.

This Phase IIA trial (AFFECT-1: NCT03233776, 17/6/2017) aimed to i) assess the safety of anakinra in autologous HSCT recipients undergoing conditioning with HDM, and ii) determine the maximum tolerated dose of anakina (100, 200 or 300mg).

This study was approved by the ethical committee Nijmegen-Arnhem (NL59679.091.16; EudraCT 2016-004,419-11) and performed in accordance with (a) theDeclaration of Helsinki (1964, amended October 2013), (b) Medical Research Involving Human Subjects Act and c) Good Clinical Practice guidelines.We enrolled patients from Radboud University Medical Centre who were at least 18years of age and were scheduled to undergo an autologous HSCT after receiving conditioning with HDM (200mg/m2) for multiple myeloma. All participants provided informed consent. Important exclusion criteria were active infections, a history of tuberculosis or positive Quantiferon, glomular filtration rate<40ml/min, and colonization with highly resistant micro-organisms or with gram-negative bacteria resistant to ciprofloxacin.

Patients were involved in the design of the AFFECT trials, through involvement of Hematon, a patient organization for patients with hemato-oncological diseases in the Netherlands. The project plan, including trial materials, have been presented to patient experts from Hematon. They have given their advice on the project, and provided input on the design of the study as well as on patient information. Patients will also be involved in the dissemination of the results of the AFFECT trials. Information on both the design as well as the outcome of the AFFECT trials is and/or will be available on websites specifically aimed at patients, such as the Dutch website kanker.nl.

Conforming with routine clinical practice and care, study participants were admitted at day 3, treated with melphalan 200mg/m2 at day 2, and received their autologous HSCT at day 0. They were treated with IL-1RA anakinra (Kineret, SOBI) intravenously once daily from day 2 up until day+12.

A traditional 3+3 design was used (Fig. S1), in which the first cohort of patients was treated with 100mg, the next cohort with 200mg and the third cohort with 300mg of anakinra. In this study design, the cohort is expanded when dose limiting toxicities (DLTs) occur. The primary study endpoint was safety, using the common toxicity criteria (CTCAE) version 4.050, as well as the maximum tolerated dose of anakinra (MTD; 100, 200 or 300mg). DLTs were defined as the occurrence of (1) an infection due to an opportunistic pathogen (including Pneumocystis jirovecii pneumonia, mycobacterial infections and invasive mould disease), (2) a suspected unexpected serious adverse reaction (SUSAR), (3) severe non-hematological toxicity grade 34 (meaning toxicity that does not commonly occur in the treatment with HDM and HSCT, or that is more severe than is to be expected with standard treatment) and (4) primary graft failure or prolonged neutropenia (neutrophils have not been>0.5109/l on one single day, assessed on day+21, and counting from day 0).

Secondary endpoints included: incidence of fever during neutropenia (defined as a tympanic temperature38.5C and an absolute neutrophil count (ANC)<0.5109/l, or expected to fall below 0.5109/l in the next 48h), CRP levels, intestinal mucositis as measured by (the AUC of) citrulline, clinical mucositis as determined by daily mouth and gut scores, incidence and type of BSI, short term overall survival (100days and 1year after HSCT), length of hospital stay in days and use of systemic antimicrobial agents, analgesic drugs and total parenteral nutrition (incidence and duration).

Patients received standard antimicrobial prophylaxis including ciprofloxacin and valacyclovir, as well as antifungal prophylaxis (fluconazole) on indication; i.e. established mucosal colonization. Upon occurrence of fever during neutropenia, empirical treatment with ceftazidime was started. The use of therapies to prevent or treat mucositis (i.e. oral cryotherapy) was prohibited. Also, treatment with acetaminophen or non-steroidal anti-inflammatory drugs was not allowed during hospital admission. All other supportive care treatments (i.e. morphine, antiemetics, transfusions, TPN) were allowed.

Laboratory analysis was performed three times a week, which included hematological and chemistry panels and plasma collection for citrulline analysis. Blood cultures were drawn daily from day+4 up until day+12, which was halted upon occurrence of fever. Outside this period, conforming to standard of care, blood cultures were drawn twice weekly and in occurrence of fever. Conforming standard of care, surveillance cultures of mucosal barriers were obtained twice weekly.

Plasma was longitudinally collected from participants throughout the study period for the evaluation of cytokines using the Meso Scale Discovery Customised U-Plex 9-analyte panel following manufacturers guidelines (IL-1/, IL-1RA, CXCL1, TNF, IL-10, IL-17, IL-6, GM-CSF). 16S sequencing was performed by Novogene (as per preclinical analysis methodology).

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7. Litchi & Titch Mini Aromatherapy Serum

Made with essential oils and botanical extracts of the highest quality, this blend is made with calmness and serenity in mind. Neroli, Grapefruit and Chamomile are heroed, and the formula is suitable for all skin types.

Tip: Apply 5 to 7 drops of the serum and press onto the skin after misting or dampening skin slightly, then cup your hands over your nose and breathe in to set a calming mood.

This brand is proudly South African, free from nasties, and cruelty-free.

Explore more onwww.litchiandtitchnaturals.comor@litchiandtitchnaturals

8. NUXE rve de miel Face & Body Gel 100ml

An ultra-rich cleansing gel using natural active ingredients such as honey, coconut and sunflower. The formula is gentle, yet effective enough to be used on your face and body, and is especially suitable for dry and sensitive skin.

Tip: Thanks to the deliciously rich and creamy texture, it works wonderfully for shaving.

Explore more onwww.nuxe.comor@nuxe_sa

9. Skin Republic Wrinkle Smooth Complex Sheet Mask

A biodegradable sheet mask packed with Adenosine, Green Tea and Plant Stem Cells was chosen for their skin-firming and texture-smoothing powers.

Explore more onwww.theskinrepublic.co.zaor@skinrepublic

To enter, fill in your details and answer the question below:

*Winners will be announced Monday 15 May 2022.

*Hint: All entrants can expect some exciting news on Monday 15 May 2022.

Question: Which item in the Beauty Box are you most excited to try?

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WIN: A beauty box of dreams valued at R3000 - CapeTown ETC

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Worlds 1st Focused Ultrasound Cancer Immunotherapy Center LaunchedUVA Health and the Charlottesville-based Focused Ultrasound Center today announced the launch of theFocused Ultrasound Cancer Immunotherapy Center, the worlds first center dedicated specifically to advancing a focused ultrasound and cancer immunotherapy treatment approach that could revolutionize 21st-century cancer care.

A Study by the Gwangju Institute of Science and Technology Investigates Mercury Contamination in Freshwater Lakes in KoreaDuring the 1950s and 1960s, Minamata Bay in Japan was the site of widespread mercury poisoning caused by the consumption of fish containing methylmercurya toxic form of mercury that is synthesized when bacteria react with mercury released in water.

Researchers identify possible new target to treat newborns suffering from lack of oxygen or blood flow in the brainThe condition, known as hypoxic-ischemic encephalopathy (HIE), can result in severe brain damage, which is why researchers at theCase Western Reserve University School of Medicineand UH Rainbow Babies & Childrens Hospital (UH Rainbow) are studying the condition to evaluate how HIE is treated and develop new, more effective options.

Should You Give Your Child Opioids for Post-Operative Pain Management?Routine head and neck procedures, such as removal of tonsils and adenoids and the placement of ear tubes, may cause moderate to severe pain in pediatric patients.

Two birds with one stone: a refined bioinformatic analysis can estimate gene copy-number variations from epigenetic dataA team led by Dr. Manel Esteller, Director of the Josep Carreras Leukaemia Research Institute, has improved the computational identification of potentially druggable gene amplifications in tumors, from epigenetic data.

Some Shunts Used After Epilepsy Surgery May Risk Chronic HeadachesSurgeons who observe persistent fluid buildup after disconnecting epileptic and healthy brain areas should think twice before installing low-pressure nonprogrammable drainage shunts, according to a study coauthored by Rutgers pediatric and epilepsy neurosurgeonYasunori Nagahamathat found chronic headaches could result from these procedures.

Re-defining the selection of surgical procedure in sufferers with tuberous sclerosis complicatedBy illustrating a number of instances of tuberous sclerosis in sufferers whove undergone surgical resection with seizure-free outcomes, researchers have recognized components that decide choice of sufferers for profitable surgical procedure.

Scientists study links between obesity, age and body chemistryA team of Clemson University scientists is making inroads in understanding the relationship between certain enzymes that are normally produced in the body and their role in regulating obesity and controlling liver diseases.

Clemson scientists discover new tools to fight potentially deadly protozoa that has pregnant women avoiding cat litter boxesNow, a group of researchers from Clemson University have discovered a promising therapy for those who suffer from toxoplasmosis, a disease caused by the microscopic protozoa Toxoplasma gondii.

Rising income inequality linked to Americans declining healthRising levels of income inequality in the United States may be one reason that the health of Americans has been declining in recent decades, new research suggests.

New research to understand how the brain handles optical illusions and makes predictionsNew research projects are underway at the Allen Institute to address these questions through OpenScope, the shared neuroscience observatory that allows scientists around the world to propose and direct experiments conducted on one of the Institutes high-throughput experimental platforms.

Robotic therapy: A new effective treatment for chronic stroke rehabilitationA study led by Dr. Takashi Takebayashi and published in the journal Stroke suggests continuing therapy for chronic stroke patients is still beneficial while suggesting a radical alternative.

Children with history of maltreatment could undergo an early maturation of the immune systemThe acute psychosocial stress states stimulate the secretion of an antibody type protein which is decisive in the first immune defence against infection, but only after puberty.

Toxoplasmosis: propagation of parasite in host cell stoppedA new method blocks the protein regulation of the parasite Toxoplasma gondii and causes it to die off inside the host cell.

Research shows the role empathy may play in musicCan people who understand the emotions of others better interpret emotions conveyed through music? A new study by an international team of researchers suggests the abilities are linked.

Effects of stress on adolescent brains triple networkA new studyinBiological Psychiatry: Cognitive Neuroscience and Neuroimaging, published by Elsevier, has used functional magnetic resonance imaging (fMRI) to examine the effects of acute stress and polyvicitimization, or repeated traumas, on three brain networks in adolescents.

Reform to Mental Health Act must prompt change in support for familiesFamily members of people with severe mental health challenges need greater support to navigate the UKs care system following changes announced in yesterdays Queens Speech, say the authors of a new study published in theBritish Journal of Social Work.

New knowledge about airborne virus particles could help hospitalsMeasurements taken by researchers at Lund University in Sweden of airborne virus in hospitals provide new knowledge about how best to adapt healthcare to reduce the risk of spread of infection.

Guidance developed for rare dancing eyes syndromeExperts from Evelina London Childrens Hospital developed the guidance in collaboration with a worldwide panel of experts and families of children with the condition.

Genetic study identifies migraine causes and promising therapeutic targetsQUT genetic researchers have found blood proteins that cause migraine and have a shared link with Alzheimers disease that could potentially be prevented by repurposing existing therapeutics.

How do genomes evolve between species? The key role of 3D structure in male germ cellsA study led by scientists at the UAB and University of Kent uncovers how the genome three-dimensional structure of male germ cells determines how genomes evolve over time.

Novel Supramolecular CRISPRCas9 Carrier Enables More Efficient Genome EditingRecently, a research team from Kumamoto University, Japan, have constructed a highly flexible CRISPR-Cas9 carrier using aminated polyrotaxane (PRX) that can not only bind with the unusual structure of Cas9 and carry it into cells, but can also protect it from intracellular degradation by endosomes.

Obesity, diabetes and high blood pressure increase mortality from COVID-19 especially among young and middle-aged peopleObesity, impaired blood glucose metabolism, and high blood pressure increase the risk of dying from COVID-19 in young and middle-aged people to a level mostly observed in people of advanced age.

Are most ORR electrocatalysts promising nanocatalytic medicines for tumor therapy?The current searches for medical catalysts mainly rely on trial-and-error protocols, due to the lack of theoretical guidance.

The combination makes the difference: New therapeutic approach against breast cancerResearchers at the University of Basel have now discovered an approach that involves a toxic combination with a second target gene in order to kill the abnormal cells.

Glatiramer acetate compatible with breastfeedingA study conducted by the neurology department of Ruhr-Universitt Bochum (RUB) at St. Josef Hospital on the drug glatiramer acetate can relieve mothers of this concern during the breastfeeding period.

A*STAR, NHCS, NUS And Novo Nordisk To Collaborate On Cardiovascular Disease ResearchThe Agency for Science, Technology and Researchs (A*STAR) Genome Institute of Singapore (GIS) and Bioinformatics Institute (BII), as well as the National Heart Centre Singapore (NHCS), National University of Singapore (NUS), and pharmaceutical company Novo Nordisk have signed an agreement to study the mechanisms underlying cardiovascular disease progressionespecially the condition called heart failure with preserved ejection fraction (HFpEF).

Taking ownership of your healthA study published this month inAge and Ageing by The Japan Collaborate Cohort (JACC) Study group at Osaka University assessed the impact of modifying lifestyle behaviors on life expectancy from middle age onwards.

Experimental evolution illustrates gene bypass process for mitosisResearchers from Nagoya University demonstrated gene bypass events for mitosis using evolutionary repair experiments.

Temporomandibular Disorder-Induced Pain Likely to Worsen in Late Menopause TransitionNew study evaluates the influence of menopause symptoms on the intensity of temporomandibular disorder-induced pain throughout the full menopause transition.

Breathtaking solution for a breathless problemA drop in oxygen levels, even when temporary, can be critical to brain cells. This explains why the brain is equipped with oxygen sensors. Researchers from Japan and the United States report finding a new oxygen sensor in the mouse brain.

How calming our spinal cords could provide relief from muscle spasmsAn Edith Cowan University (ECU) studyinvestigating motoneurons in the spine has revealed two methods can make our spinal cords less excitable and could potentially be usedto treat muscle spasms.

Analysis Finds Government Websites Downplay PFAS Health RisksState and federal public health agencies often understate the scientific evidence surrounding the toxicity of per- and polyfluoroalkyl substances (PFAS) in their public communications, according toan analysispublished today in the journalEnvironmental Health.

Multiple diagnoses are the norm with mental illness; new genetic study explains whyThe study, published this weekin the journalNature Genetics, found that while there is no gene or set of genes underlying risk for all of them, subsets of disordersincluding bipolar disorder and schizophrenia; anorexia nervosa and obsessive-compulsive disorder; and major depression and anxietydo share a common genetic architecture.

Drinkers sex plus brewing method may be key to coffees link to raised cholesterolThe sex of the drinker as well as the brewing method may be key to coffees link with raised cholesterol, a known risk factor for heart disease, suggests research published in the open access journalOpen Heart.

Artificial cell membrane channels composed of DNA can be opened and locked with a keyIn new research, Arizona State University professorHao Yan, along with ASU colleagues and international collaborators from University College London describe the design and construction of artificial membrane channels, engineered using short segments of DNA.

Single cell RNA sequencing uncovers new mechanisms of heart diseaseResearchers at the Hubrecht Institute have now successfully applied a new revolutionary technology (scRNA-seq) to uncover underlying disease mechanisms, including specifically those causing the swelling.

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Other Notable Health Studies & Research From May 11, 2022 - Study Finds

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In a new set of firsts for Barbie, the latest in its most diverse and inclusive doll line yet will include a Barbie with hearing aids and a Ken doll with vitiligo, a condition where skin loses its pigment cells.

Mattels global head of Barbie Dolls, Lisa McKnight, said in a statement: Its important for kids to see themselves reflected in product and to encourage play with dolls that dont resemble them, to help them understand and celebrate the importance of inclusion.

Mattel said it worked alongside Dr Jen Richardson, a practitioner in educational audiology, to accurately model behind-the-ear devices for the doll.

Im honored to have worked with Barbie to create an accurate reflection of a doll with behind-the-ear hearing aids, Richardson said. As an educational audiologist with over 18 years of experience working in hearing loss advocacy, its inspiring to see those who experience hearing loss reflected in a doll.

Im beyond thrilled for my young patients to see and play with a doll who looks like them.

The 2022 Fashionistas line, out next month, will also feature a Barbie with a prosthetic leg, a Barbie who uses a wheelchair, and male dolls that are thinner and less muscular.

Barbie has described its upcoming 175-look collection as its most diverse and inclusive doll line, offering a variety of skin tones, eye colors, hair colors and textures, body types, disabilities, and fashions, to inspire even more stories.

In recent years, Barbie has launched more inclusive doll lines including one inspired by real-life women who have upended societal norms. In 2017, Barbie released a doll wearing a hijab, modelled after Ibtihaj Muhammad, a fencer who became the first American to compete and win an Olympic medal wearing the garment.

Last year, Barbie launched a set of dolls that honoured Covid workers including vaccinologists, nurses and paramedics.

One of the dolls is modelled after Dame Sarah Gilbert, the co-creator of the Oxford/AstraZeneca vaccine.

I am passionate about inspiring the next generation of girls into Stem careers and hope that children who see my Barbie will realise how vital careers in science are to help the world around us, Gilbert told the Guardian.

My wish is that my doll will show children careers they may not be aware of, like a vaccinologist.

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Barbie unveils first doll with hearing aid as part of inclusivity push - The Guardian US

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The new multi-organ chip has the size of a glass microscope slide and allows the culture of up to four human engineered tissues, whose location and number can be tailored to the question being asked. These tissues are connected by vascular flow, but the presence of a selectively permeable endothelial barrier maintains their tissue-specific niche. Credit: Kacey Ronaldson-Bouchard/Columbia Engineering

Major advance from Columbia Engineering team demonstrates the first multi-organ chip made of engineered human tissues linked by vascular flow for improved modeling of systemic diseases like cancer.

Engineered tissues have become an essential component for modeling diseases and testing the efficacy and safety of drugs in a human context. A key hurdle for researchers has been figuring how to model body functions and systemic diseases with multiple engineered tissues that can physiologically communicate just like they do in the body. However, it is essential to provide each engineered tissue with its own environment so that the specific tissue phenotypes can be maintained for weeks to months, as required for biological and biomedical studies. Making the challenge even more complex is the necessity of linking the tissue modules together to facilitate their physiological communication, which is required for modeling conditions that involve more than one organ system, without sacrificing the individual engineered tissue environments.

Up to now, no one has been able to meet both conditions. Today, a team of researchers from Columbia Engineering and Columbia University Irving Medical Center reports that they have developed a model of human physiology in the form of a multi-organ chip consisting of engineered human heart, bone, liver, and skin that are linked by vascular flow with circulating immune cells, to allow recapitulation of interdependent organ functions. The researchers have essentially created a plug-and-play multi-organ chip, which is the size of a microscope slide, that can be customized to the patient. Because disease progression and responses to treatment vary greatly from one person to another, such a chip will eventually enable personalized optimization of therapy for each patient. The study is the cover story of the April 2022 issue of the journal Nature Biomedical Engineering.

In our study, we cultured liver, heart, bone, and skin, connected by vascular flow for four weeks. These tissues can be generated from a single human induced pluripotent stem cell, generating a patient-specific chip, a great model for individualized studies of human disease and drug testing. Credit: Keith Yeager/Columbia Engineering

This is a huge achievement for usweve spent ten years running hundreds of experiments, exploring innumerable great ideas, and building many prototypes, and now at last weve developed this platform that successfully captures the biology of organ interactions in the body, said the project leader Gordana Vunjak-Novakovic, University Professor and the Mikati Foundation Professor of Biomedical Engineering, Medical Sciences, and Dental Medicine.

Taking inspiration from how the human body works, the team has built a human tissue-chip system in which they linked matured heart, liver, bone, and skin tissue modules by recirculating vascular flow, allowing for interdependent organs to communicate just as they do in the human body. The researchers chose these tissues because they have distinctly different embryonic origins, structural and functional properties, and are adversely affected by cancer treatment drugs, presenting a rigorous test of the proposed approach.

The tissues cultured in the multi-organ chip (skin, heart, bone, liver, and endothelial barrier from left to right) maintained their tissue-specific structure and function after being linked by vascular flow. Credit: Kacey Ronaldson-Bouchard/Columbia Engineering

Providing communication between tissues while preserving their individual phenotypes has been a major challenge, said Kacey Ronaldson-Bouchard, the studys lead author and an associate research scientist in Vunjak-Novakovics Laboratory for Stem Cells and Tissue Engineering. Because we focus on using patient-derived tissue models we must individually mature each tissue so that it functions in a way that mimics responses you would see in the patient, and we dont want to sacrifice this advanced functionality when connecting multiple tissues. In the body, each organ maintains its own environment, while interacting with other organs by vascular flow carrying circulating cells and bioactive factors. So we chose to connect the tissues by vascular circulation, while preserving each individual tissue niche that is necessary to maintain its biological fidelity, mimicking the way that our organs are connected within the body.

The group created tissue modules, each within its optimized environment and separated them from the common vascular flow by a selectively permeable endothelial barrier. The individual tissue environments were able to communicate across the endothelial barriers and via vascular circulation. The researchers also introduced into the vascular circulation the monocytes giving rise to macrophages, because of their important roles in directing tissue responses to injury, disease, and therapeutic outcomes.

All tissues were derived from the same line of human induced pluripotent stem cells (iPSC), obtained from a small sample of blood, in order to demonstrate the ability for individualized, patient-specific studies. And, to prove the model can be used for long-term studies, the team maintained the tissues, which had already been grown and matured for four to six weeks, for an additional four weeks, after they were linked by vascular perfusion.

The researchers also wanted to demonstrate how the model could be used for studies of an important systemic condition in a human context and chose to examine the adverse effects of anticancer drugs. They investigated the effects of doxorubicin a broadly used anticancer drug on heart, liver, bone, skin, and vasculature. They showed that the measured effects recapitulated those reported from clinical studies of cancer therapy using the same drug.

The team developed in parallel a novel computational model of the multi-organ chip for mathematical simulations of drugs absorption, distribution, metabolism, and secretion. This model correctly predicted doxorubicins metabolism into doxorubicinol and its diffusion into the chip. The combination of the multi-organ chip with computational methodology in future studies of pharmacokinetics and pharmacodynamics of other drugs provides an improved basis for preclinical to clinical extrapolation, with improvements in the drug development pipeline.

While doing that, we were also able to identify some early molecular markers of cardiotoxicity, the main side-effect that limits the broad use of the drug. Most notably, the multi-organ chip predicted precisely the cardiotoxicity and cardiomyopathy that often require clinicians to decrease therapeutic dosages of doxorubicin or even to stop the therapy, said Vunjak-Novakovic.

The development of the multi-organ chip began from a platform with the heart, liver, and vasculature, nicknamed the HeLiVa platform. As is always the case with Vunjak-Novakovics biomedical research, collaborations were critical for completing the work. These include the collective talent of her laboratory, Andrea Califano and his systems biology team (Columbia University), Christopher S. Chen (Boston University) and Karen K. Hirschi (University of Virginia) with their expertise in vascular biology and engineering, Angela M. Christiano and her skin research team (Columbia University), Rajesh K. Soni of the Proteomics Core at Columbia University, and the computational modeling support of the team at CFD Research Corporation.

The research team is currently using variations of this chip to study, all in individualized patient-specific contexts: breast cancer metastasis; prostate cancer metastasis; leukemia; effects of radiation on human tissues; the effects of SARS-CoV-2 on heart, lung, and vasculature; the effects of ischemia on the heart and brain; and the safety and effectiveness of drugs. The group is also developing a user-friendly standardized chip for both academic and clinical laboratories, to help utilize its full potential for advancing biological and medical studies.

Vunjak-Novakovic added, After ten years of research on organs-on-chips, we still find it amazing that we can model a patients physiology by connecting millimeter sized tissues the beating heart muscle, the metabolizing liver, and the functioning skin and bone that are grown from the patients cells. We are excited about the potential of this approach. Its uniquely designed for studies of systemic conditions associated with injury or disease, and will enable us to maintain the biological properties of engineered human tissues along with their communication. One patient at a time, from inflammation to cancer!

Reference: A multi-organ chip with matured tissue niches linked by vascular flow by Kacey Ronaldson-Bouchard, Diogo Teles, Keith Yeager, Daniel Naveed Tavakol, Yimu Zhao, Alan Chramiec, Somnath Tagore, Max Summers, Sophia Stylianos, Manuel Tamargo, Busub Marcus Lee, Susan P. Halligan, Erbil Hasan Abaci, Zongyou Guo, Joanna Jackw, Alberto Pappalardo, Jerry Shih, Rajesh K. Soni, Shivam Sonar, Carrie German, Angela M. Christiano, Andrea Califano, Karen K. Hirschi, Christopher S. Chen, Andrzej Przekwas and Gordana Vunjak-Novakovic, 27 April 2022, Nature Biomedical Engineering.DOI: 10.1038/s41551-022-00882-6

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Plug-and-Play Human Organ-on-a-Chip Can Be Customized to the Patient - SciTechDaily

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All thalassemia patients need timely supply of safe blood for regular blood transfusion

There are 270 million Thalassemia patients in the world, yet India is called the Thalassemia capital because the country has the largest number of children suffering from this inherited blood disorder that causes the body to have less hemoglobin than normal.

More than 10,000 children are born in India with Thalassemia every year. According to World Health Organization, four million Indians are thalassemia carriers, while more than 1,00,000 are the actual patients battling the disease. Parents, who are usually asymptomatic, are the carriers of Thalassemia, and have a 25% chance of passing the disease to their children.

Even though thalassemia affects millions of people in the world, not many are aware about this condition. Early symptoms include fatigue, weakness, bone deformities (especially in the face), pale appearance or yellowish skin tone, slow growth rate, lowered immunity levels, and iron overload.

Due to financial barriers and lack of access to treatment, many young patients in India do not survive into adulthood. One of the major challenges is the lack of adequate pre-natal diagnosis facilities for Thalassemia, especially in rural areas. To manage the disease, patients need to take regular blood transfusions lifelong, along with iron chelation to treat iron overload in the body. These are very taxing procedures for young children that strain their health.

All Thalassemia patients need timely supply of safe blood for regular blood transfusion. It is not a permanent solution and is quite a painful process for a child. The annual requirement of packed red cells for Thalassemia patients is about 2 million units in India. Although patient organizations and regional blood banks have been working relentlessly towards fulfilling this requirement, the demand is significantly more than the current supply. Lot of families have to struggle month after month in making packed cells available for their children.

Repeated packed red cell transfusions lead to iron overload in patients. Excess iron gets deposited in organs like liver, heart and endocrine glands. This iron overload is usually the cause of death in the second or third decade of life. These patients need to be on medications to remove the extra iron from their bodies to ensure a normal life span for them. Yet, in spite of freely available drugs, less than 10% of Thalassemia patients are adequately chelated in India. Factors like ignorance, poor compliance and, more importantly, unaffordability are major reasons for poor chelation in Thalassemia patients.

Thalassemia is a multi-system problem. In addition to regular blood transfusion and iron chelation, the disease needs to be managed by a multidisciplinary team that should have hematologist, endocrinologist, cardiologist, nutritionist, nurse practitioner, etc. to provide holistic care. There are hardly any comprehensive centers for thalassemia care in India.

However, there is some good news too. With advances in the medical field, thalassemia major, once considered a cumbersome disease with lifelong blood transfusions, iron overload and limited lifespan, has seen a shift in the last decade with blood stem cell transplant, the only curative treatment option available for thalassemia.

Recent data shows 85-90% success rate of stem cell marrow transplantation in patients who have HLA matched stem cell donor. In a blood stem cell transplant, stem cells are collected from blood of the donors and transplanted into the thalassemia patient after their bone marrow has been destroyed by radiation or chemotherapy. Only 30% patients who need transplants have a fully HLA (Human Leukocyte Antigen) matched donor in their family, rest of them depend on an unrelated donor.

Patients and donors of Indian origin have unique HLA characteristics that are severely under-represented in the global database, which makes the probability of finding a suitable donor even more difficult. Indian patients mainly require an Indian tissue match. This calls for increased awareness and need to encourage people in India to register as a potential blood stem cell donor.

Registering as a potential donor is an easy process which can be done through online portal of a stem cell registry such as DKMS BMST Foundation India. Once you sign-up, you will receive a DIY home swab kit to take your cheek swab samples and send it back to the registry! Once an individual comes up as a match for a blood cancer patient, blood stem cells from the individual are obtained from the bloodstream using a procedure called peripheral blood stem cell collection, which is similar to a blood platelet donation..

Source: WHO, GLOBOCAN and Ministry of Health

(Dr. Nitin Agarwal, MD, Transfusion Medicine, HOD, Donor Request Management, DKMS BMST Foundation India)

Disclaimer: This content including advice provides generic information only. It is in no way a substitute for qualified medical opinion. Always consult a specialist or your own doctor for more information. NDTV does not claim responsibility for this information.

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World Thalassemia Day 2022: Why India Is The Thalassemia Capital Of The World - NDTV Doctor

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One is that there are some people that are naturally resistant to heart attack and have lifelong, low levels of LDL, the cardiologist says. Second, there are some genes that can be switched off that lead to very low LDL cholesterol, and individuals with those genes switched off are resistant to heart attacks.

Kathiresan and his team formed a hypothesis in 2016 that if they could develop a medicine that mimics the natural protection that some people enjoy, then they might identify a powerful new way to treat and ultimately prevent heart attacks. They launched Verve in 2018 with the goal of creating a one-time therapy that would permanently lower LDL and eliminate heart attacks caused by high LDL.

The medication is targeted specifically for patients who have a genetic form of high cholesterol known as heterozygous familial hypercholesterolemia, or FH, caused by expression of a gene called PCSK9. Verve also plans to develop a program to silence a gene called ANGPTL3 for patients with FH and possibly those with or at risk of atherosclerotic cardiovascular disease.

FH causes cholesterol to be high from birth, reaching levels of 200 to 300 milligrams per deciliter. Suggested normal levels are around 100 to 129 mg/dl, and anything above 130 mg/dl is considered high. Patients with cardiovascular disease usually are asked to aim for under 70 mg/dl, but many still have unacceptably high LDL despite taking oral medications such as statins. They are more likely to have heart attacks in their 30s, 40s and 50s, and require lifelong LDL control.

The goal for drug treatments for high LDL, Kathiresan says, is to reduce LDL as low as possible for as long as possible. Physicians and researchers also know that a sizeable portion of these patients eventually start to lose their commitment to taking their statins and other LDL-controlling medications regularly.

If you ask 100 patients one year after their heart attack what fraction are still taking their cholesterol-lowering medications, its less than half, says Kathiresan. So imagine a future where somebody gets a one-time treatment at the time of their heart attack or before as a preventive measure. Its right in front of us, and its something that Verve is looking to do.

In late 2020, Verve completed primate testing with monkeys that had genetically high cholesterol, using a one-time intravenous injection of VERVE-101. It reduced the monkeys LDL by 60 percent and, 18 months later, remains at that level. Kathiresan expects the LDL to stay low for the rest of their lives.

Verves gene editing medication is packaged in a lipid nanoparticle to serve as the delivery mechanism into the liver when infused intravenously. The drug is absorbed and makes its way into the nucleus of the liver cells.

Verves program targeting PCSK9 uses precise, single base, pair base editing, Kathiresan says, meaning it doesn't cut DNA like CRISPR gene editing systems do. Instead, it changes one base, or letter, in the genome to a different one without affecting the letters around it. Comparing it to a pencil and eraser, he explains that the medication erases out a letter A and makes it a letter G in the A, C, G and T code in DNA.

By making that simple change from A to G, the medication switches off the PCSK9 gene, automatically lowering LDL cholesterol.

Once the DNA change is made, all the cells in the liver will have that single A to G change made, Kathiresan says. Then the liver cells divide and give rise to future liver cells, but every time the cell divides that change, the new G is carried forward.

Additionally, Verve is pursuing its second gene editing program to eliminate ANGPTL3, a gene that raises both LDL and blood triglycerides. In 2010, Kathiresan's research team learned that people who had that gene completely switched off had LDL and triglyceride levels of about 20 and were very healthy with no heart attacks. The goal of Verves medication will be to switch off that gene, too, as an option for additional LDL or triglyceride lowering.

Success with our first drug, VERVE-101, will give us more confidence to move forward with our second drug, Kathiresan says. And it opens up this general idea of making [genomic] spelling changes in the liver to treat other diseases.

The approach is less ethically concerning than other gene editing technologies because it applies somatic editing that affects only the individual patient, whereas germline editing in the patients sperm or egg, or in an embryo, gets passed on to children. Additionally, gene editing therapies receive the same comprehensive amount of testing for side effects as any other medicine.

We need to continue to advance our approach and tools to make sure that we have the absolute maximum ability to detect off-target effects, says Euan Ashley, professor of medicine and genetics at Stanford University and founding director of its Center for Inherited Cardiovascular Disease. Ashley and his colleagues at Stanfords Clinical Genomics Program and beyond are increasingly excited about the promise of gene editing.

We can offer precision diagnostics, so increasingly were able to define the disease at a much deeper level using molecular tools and sequencing, he continues. We also have this immense power of reading the genome, but were really on the verge of taking advantage of the power that we now have to potentially correct some of the variants that we find on a genome that contribute to disease.

He adds that while the gene editing medicines in development to correct genomes are ahead of the delivery mechanisms needed to get them into the body, particularly the heart and brain, hes optimistic that those arent too far behind.

It will probably take a few more years before those next generation tools start to get into clinical trials, says Ashley, whose book, The Genome Odyssey, was published last year. The medications might be the sexier part of the research, but if you cant get it into the right place at the right time in the right dose and not get it to the places you dont want it to go, then that tool is not of much use.

Medical experts consider knocking out the PCSK9 gene in patients with the fairly common genetic disorder of familial hypercholesterolemia roughly one in 250 people a potentially safe approach to gene editing and an effective means of significantly lowering their LDL cholesterol.

Nurse Erin McGlennon has an Implantable Cardioverter Defibrillator and takes medications, but she is also hopeful that a gene editing medication will be developed in the near future.

Erin McGlennon

Mary McGowan, MD, chief medical officer for The Family Heart Foundation in Pasadena, CA, sees the tremendous potential for VERVE-101 and believes patients should be encouraged by the fact that this kind of research is occurring and how much Verve has accomplished in a relatively short time. However, she offers one caveat, since even a 60 percent reduction in LDL wont completely eliminate the need to reduce the remaining amount of LDL.

This technology is very exciting, she said, but we want to stress to our patients with familial hypercholesterolemia that we know from our published research that most people require several therapies to get their LDL down., whether that be in primary prevention less than 100 mg/dl or secondary prevention less than 70 mg/dl, So Verves medication would be an add-on therapy for most patients.

Dr. Kathiresan concurs: We expect our medicine to lower LDL cholesterol by about 60 percent and that our patients will be on background oral medications, including statins that lower LDL cholesterol.

Several leading research centers are investigating gene editing treatments for other types of cardiovascular diseases. Elizabeth McNally, Elizabeth Ward Professor and Director at the Center for Genetic Medicine at Northwestern Universitys Feinberg School of Medicine, pursues advanced genetic correction in neuromuscular diseases such as Duchenne muscular dystrophy and spinal muscular atrophy. A cardiologist, she and her colleagues know these diseases frequently have cardiac complications.

Even though the field is driven by neuromuscular specialists, its the first therapies in patients with neuromuscular diseases that are also expected to make genetic corrections in the heart, she says. Its almost like an afterthought that were potentially fixing the heart, too.

Another limitation McGowan sees is that too many healthcare providers are not yet familiar with how to test patients to determine whether or not they carry genetic mutations that need to be corrected. We need to get more genetic testing done, she says. For example, thats the case with hypertrophic cardiomyopathy, where a lot of the people who probably carry that diagnosis and have never been genetically identified at a time when genetic testing has never been easier.

One patient who has been diagnosed with hypertrophic cardiomyopathy also happens to be a nurse working in research at Genentech Pharmaceutical, now a member of the Roche Group, in South San Francisco. To treat the disease, Erin McGlennon, RN, has an Implantable Cardioverter Defibrillator and takes medications, but she is also hopeful that a gene editing medication will be developed in the near future.

With my condition, the septum muscles are just growing thicker, so Im on medicine to keep my heart from having dangerous rhythms, says McGlennon of the disease that carries a low risk of sudden cardiac death. So, the possibility of having a treatment option that can significantly improve my day-to-day functioning would be a major breakthrough.

McGlennon has some control over cardiovascular destiny through at least one currently available technology: in vitro fertilization. Shes going through it to ensure that her children won't express the gene for hypertrophic cardiomyopathy.

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Acta Naturae. 2010 Jul; 2(2): 1828.

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cellsthat can be obtained by reprogramming animal and human differentiated cells. In this review,issues related to the nature of iPSCs are discussed and different methods ofiPSC production are described. We particularly focused on methods of iPSC production withoutthe genetic modification of the cell genome and with means for increasing the iPSC productionefficiency. The possibility and issues related to the safety of iPSC use in cell replacementtherapy of human diseases and a study of new medicines are considered.

Keywords: induced pluripotent stem cells, directed stem cell differentiation, cell replacement therapy

Pluripotent stem cells are a unique model for studying a variety of processes that occur inthe early development of mammals and a promising tool in cell therapy of human diseases. Theunique nature of these cells lies in their capability, when cultured, for unlimitedselfrenewal and reproduction of all adult cell types in the course of theirdifferentiation [1]. Pluripotency is supported by acomplex system of signaling molecules and gene network that is specific for pluripotent cells.The pivotal position in the hierarchy of genes implicated in the maintenance of pluripotency isoccupied by Oct4, Sox2 , and Nanog genes encodingtranscription factors [2, 3]. The mutual effect of outer signaling molecules and inner factors leads tothe formation of a specific expression pattern, as well as to the epigenome statecharacteristic of stem cells. Both spontaneous and directed differentiations are associatedwith changes in the expression pattern and massive epigenetic transformations, leading totranscriptome and epigenome adjustment to a distinct cell type.

Until recently, embryonic stem cells (ESCs) were the only wellstudied source ofpluripotent stem cells. ESCs are obtained from either the inner cell mass or epiblast ofblastocysts [46]. A series of protocols has been developed for the preparation of variouscell derivatives from human ESCs. However, there are constraints for ESC usein cell replacement therapy. The first constraint is the immune incompatibility between thedonor cells and the recipient, which can result in the rejection of transplanted cells. Thesecond constraint is ethical, because the embryo dies during the isolation of ESCs. The firstproblem can be solved by the somatic cell nuclear transfer into the egg cell and then obtainingthe embryo and ESCs. The nuclear transfer leads to genome reprogramming, in which ovariancytoplasmic factors are implicated. This way of preparing pluripotent cells from certainindividuals was called therapeutic cloning. However, this method is technologyintensive,and the reprogramming yield is very low. Moreover, this approach encounters theabovementioned ethic problem that, in this case, is associated with the generation ofmany human ovarian cells [7].

In 2006, the preparation of pluripotent cells by the ectopic expression of four genes Oct4 , Sox2 , Klf4 , and cMyc in both embryonic and adult murine fibroblasts was first reported[8]. The pluripotent cells derived from somatic ones werecalled induced pluripotent stem cells (iPSCs). Using this set of factors(Oct4, Sox2, Klf4, and cMyc), iPSCs were prepared later from variousdifferentiated mouse [914] and human [1517] cell types. Human iPSCs were obtainedwith a somewhat altered gene set: Oct4 , Sox2 , Nanog , and Lin28 [18].Induced PSCs closely resemble ESCs in a broad spectrum of features. They possess similarmorphologies and growth manners and are equally sensitive to growth factors and signalingmolecules. Like ESCs, iPSCs can differentiate in vitro intoderivatives of all three primary germ layers (ectoderm, mesoderm, and endoderm) and formteratomas following their subcutaneous injection into immunodeficient mice. MurineiPSCs injected into blastocysts are normally included in the development toyield animals with a high degree of chimerism. Moreover, murine iPSCs, wheninjected into tetraploid blastocycts, can develop into a whole organism [19, 20]. Thus, an excellent method thatallows the preparation of pluripotent stem cells from various somatic cell types whilebypassing ethical problems has been uncovered by researchers.

In the first works on murine and human iPSC production, either retro or lentiviralvectors were used for the delivery of Oct4 , Sox2 , Klf4 , and cMyc genes into somatic cells. Theefficiency of transduction with retroviruses is high enough, although it is not the same fordifferent cell types. Retroviral integration into the host genome requires a comparatively highdivision rate, which is characteristic of the relatively narrow spectrum of cultured cells.Moreover, the transcription of retroviral construct under the control of a promoter localizedin 5LTR (long terminal repeat) is terminated when the somatic celltransform switches to the pluripotent state [21]. Thisfeature makes retroviruses attractive in iPSC production. Nevertheless, retroviruses possesssome properties that make iPSCs that are produced using them improper for celltherapy of human diseases. First, retroviral DNA is integrated into the host cell genome. Theintegration occurs randomly; i.e., there are no specific sequences or apparent logic forretroviral integration. The copy number of the exogenous retroviral DNA that is integrated intoa genome may vary to a great extent [15]. Retrovirusesbeing integrated into the cell genome can introduce promoter elements and polyadenylationsignals; they can also interpose coding sequences, thus affecting transcription. Second, sincethe transcription level of exogenous Oct4 , Sox2 , Klf4 , and cMyc in the retroviral constructdecreases with cell transition into the pluripotent state, this can result in a decrease in theefficiency of the stable iPSC line production, because the switch from the exogenous expressionof pluripotency genes to their endogenous expression may not occur. Third, some studies showthat the transcription of transgenes can resume in the cells derived fromiPSCs [22]. The high probability thatthe ectopic Oct4 , Sox2 , Klf4 , and cMyc gene expression will resume makes it impossible to applyiPSCs produced with the use of retroviruses in clinical trials; moreover,these iPSCs are hardly applicable even for fundamental studies onreprogramming and pluripotency principles. Lentiviruses used for iPSC production can also beintegrated into the genome and maintain their transcriptional activity in pluripotent cells.One way to avoid this situation is to use promoters controlled by exogenous substances added tothe culture medium, such as tetracycline and doxycycline, which allows the transgenetranscription to be regulated. iPSCs are already being produced using suchsystems [23].

Another serious problem is the gene set itself that is used for the induction of pluripotency[22]. The ectopic transcription of Oct4 , Sox2 , Klf4 , and cMyc can lead to neoplastic development from cells derived from iPSCs,because the expression of Oct4 , Sox2 , Klf4, and cMyc genes is associated with the development ofmultiple tumors known in oncogenetics [22, 24]. In particular, the overexpression of Oct4 causes murine epithelial cell dysplasia [25],the aberrant expression of Sox2 causes the development of serrated polypsand mucinous colon carcinomas [26], breast tumors arecharacterized by elevated expression of Klf4 [27] , and the improper expression of cMyc is observed in 70% of human cancers [28].Tumor development is oberved in ~50% of murine chimeras obtained through the injection ofretroviral iPSCs into blastocysts, which is very likely associated with thereactivation of exogenous cMyc [29, 30].

Several possible strategies exist for resolving the above-mentioned problems:

The search for a less carcinogenic gene set that is necessary and sufficient for reprogramming;

The minimization of the number of genes required for reprogramming and searching for the nongenetic factors facilitating it;

The search for systems allowing the elimination of the exogenous DNA from the host cell genome after the reprogramming;

The development of delivery protocols for nonintegrated genetic constructs;

The search for ways to reprogram somatic cells using recombinant proteins.

The ectopic expression of cMyc and Klf4 genes isthe most dangerous because of the high probability that malignant tumors will develop [22]. Hence the necessity to find other genes that couldsubstitute cMyc and Klf4 in iPSC production. Ithas been reported that these genes can be successfully substituted by Nanog and Lin28 for reprogramming human somatic cells [18;] . iPSCs were prepared from murine embryonic fibroblastsby the overexpression of Oct4 and Sox2 , as well as the Esrrb gene encoding the murine orphan nuclear receptor beta. It has alreadybeen shown that Esrrb , which acts as a transcription activator of Oct4 , Sox2 , and Nanog , is necessary for theselfrenewal and maintenance of the pluripotency of murine ESCs. Moreover, Esrrb can exert a positive control over Klf4 . Thus, the genes causingelevated carcinogenicity of both iPSCs and their derivatives can besuccessfully replaced with less dangerous ones [31].

The Most Effectively Reprogrammed Cell Lines . Murine and humaniPSCs can be obtained from fibroblasts using the factors Oct4, Sox2, and Klf4,but without cMyc . However, in this case, reprogramming deceleratesand an essential shortcoming of stable iPSC clones is observed [32, 33]. The reduction of a number ofnecessary factors without any decrease in efficiency is possible when iPSCsare produced from murine and human neural stem cells (NSCs) [12, 34, 35]. For instance, iPSCs were produced fromNSCs isolated from adult murine brain using two factors, Oct4 and Klf4, aswell as even Oct4 by itself [12, 34]. Later, human iPSCs were produced by the reprogramming offetal NSCs transduced with a retroviral vector only carrying Oct4 [35] . It is most likely that the irrelevanceof Sox2, Klf4, and cMyc is due to the high endogenous expression level of these genes inNSCs.

Successful reprogramming was also achieved in experiments withother cell lines, in particular, melanocytes of neuroectodermal genesis [36]. Both murine and human melanocytes are characterized by a considerableexpression level of the Sox2 gene, especially at early passages.iPSCs from murine and human melanocytes were produced without the use of Sox2or cMyc. However, the yield of iPSC clones produced from murine melanocytes was lower(0.03% without Sox2 and 0.02% without cMyc) in comparison with that achieved when allfour factors were applied to melanocytes (0.19%) and fibroblasts (0.056%). A decreasedefficiency without Sox2 or cMyc was observed in human melanocyte reprogramming (0.05%with all four factors and 0.01% without either Sox2 or cMyc ). All attempts to obtain stable iPSC clones in the absence of both Sox2 andcMyc were unsuccessful [36]. Thus, theminimization of the number of factors required for iPSC preparation can be achieved by choosingthe proper somatic cell type that most effectively undergoes reprogramming under the action offewer factors, for example, due to the endogenous expression of pluripotencygenes. However, if human iPSCs are necessary, these somatic cellsshould be easily accessible and wellcultured and their method of isolation should be asnoninvasive as possible.

One of these cell types can be adipose stem cells (ASCs). This is aheterogeneous group of multipotent cells which can be relatively easily isolated in largeamounts from adipose tissue following liposuction. Human iPSCs weresuccessfully produced from ASCs with a twofold reprogramming rate and20fold efficiency (0.2%), exceeding those of fibroblasts [37].

However, more accessible resources for the effective production of humaniPSCs are keratinocytes. When compared with fibroblasts, human iPSC productionfrom keratinocytes demonstrated a 100fold greater efficiency and a twofold higherreprogramming rate [38].

It has recently been found that the reprogramming of murine papillary dermal fibroblasts(PDFs) into iPSCs can be highly effective with theoverexpression of only two genes, Oct4 and Klf4 ,inserted into retroviral vectors [39;].PDFs are specialized cells of mesodermal genesis surrounding the stem cells ofhair follicles . One characteristic feature of these cells is the endogenous expression of Sox2 , Klf4 , and cMyc genes,as well as the geneencoding alkaline phosphatase, one of the murine and humanESC markers. PDFs can be easily separated from other celltypes by FACS (fluorescenceactivated cell sorting) using life staining with antibodiesagainst the surface antigens characteristic of one or another cell type. The PDF reprogrammingefficiency with the use of four factors (Oct4, Sox2, Klf4, and cMyc) retroviral vectorsis 1.38%, which is 1,000fold higher than the skin fibroblast reprogramming efficiency inthe same system. Reprogramming PDFs with two factors, Oct4 and Klf4 , yields 0.024%, which is comparable to the efficiency of skinfibroblast reprogramming using all four factors. The efficiency of PDF reprogramming iscomparable with that of NSCs, but PDF isolation is steady and far lessinvasive [39]. It seems likely that human PDF lines arealso usable, and this cell type may appear to be one of the most promising for human iPSCproduction in terms of pharmacological studies and cell replacement therapy. The use of suchcell types undergoing more effective reprogramming, together with methods providing thedelivery of pluripotency genes without the integration of foreign DNA into thehost genome and chemical compounds increasing the reprogramming efficiency and substitutingsome factors required for reprogramming, is particularly relevant.

Chemical Compounds Increasing Cell Reprogramming Efficiency. As was noted above,the minimization of the factors used for reprogramming decreases the efficiency of iPSCproduction. Nonetheless, several recent studies have shown that the use of genetic mechanisms,namely, the initiation of ectopic gene expression, can be substituted by chemical compounds,most of them operating at the epigenetic level. For instance, BIX01294 inhibitinghistone methyltransferase G9a allows murine fibroblast reprogramming using only two factors,Oct4 and Klf4, with a fivefold increased yield of iPSC clones in comparison with the controlexperiment without BIX01294 [40]. BIX01294taken in combination with another compound can increase the reprogramming efficiency even more.In particular, BIX01294 plus BayK8644 elevated the yield of iPCSs 15 times, andBIX01294 plus RG108 elevated it 30 times when only two reprogramming factors, Oct4 andKlf4, were used. RG108 is an inhibitor of DNA methyltransferases, and its role in reprogrammingis apparently in initiating the more rapid and effective demethylation of promoters ofpluripotent cellspecific genes, whereas BayK8644 is an antagonist of Ltypecalcium channels, and its role in reprogramming is not understood very well [40]. However, more considerable results were obtained inreprogramming murine NSCs. The use of BIX01294 allowed a 1.5foldincrease in iPSC production efficiency with two factors, Oct4 and Klf4, in comparison withreprogramming with all four factors. Moreover, BIX01294 can even substitute Oct4 in thereprogramming of NSCs, although the yield is very low [41]. Valproic (2propylvaleric) acid inhibiting histone deacetylases canalso substitute cMyc in reprogramming murine and human fibroblasts. Valproic acid (VPA)increases the reprogramming efficiency of murine fibroblasts 50 times, and human fibroblastsincreases it 1020 times when three factors are used [42, 43]. Other deacetylase inhibitors,such as TSA (trichostatin A) and SAHA (suberoylanilide hyroxamic acid), also increase thereprogramming efficiency. TSA increases the murine fibroblast reprogramming efficiency 15times, and SAHA doubles it when all four factors are used [42]. Besides epigenetic regulators, the substances inhibiting the proteincomponents of signaling pathways implicated in the differentiation of pluripotent cells arealso applicable in the substitution of reprogramming factors. In particular, inhibitors of MEKand GSK3 kinases (PD0325901 and CHIR99021, respectively) benefit the establishment of thecomplete and stable pluripotency of iPSCs produced from murineNSCs using two factors, Oct4 and Klf4 [41, 44].

It has recently been shown that antioxidants can considerably increase the efficiency ofsomatic cell reprogramming. Ascorbic acid (vitamin C) can essentially influence the efficiencyof iPSC production from various murine and human somatic cell types [45]. The transduction of murine embryonic fibroblasts (mEFs) with retrovirusescarrying the Oct4 , Sox2 , and Klf4 genes results in a significant increase in the production level of reactive oxygen species(ROS) compared with that of both control and Efs tranduced with Oct4 , Sox2 , cMyc , and Klf4 . Inturn, the increase in the ROS level causes accelerated aging and apoptosis of the cell, whichshould influence the efficiency of cell reprogramming. By testing several substances possessingantioxidant activity such as vitamin B1, sodium selenite, reduced glutathione, and ascorbicacid, the authors have found that combining these substances increases the yield ofGFPpositive cells in EF reprogramming (the Gfp genewas under the control of the Oct4 gene promoter). The use of individualsubstances has shown that only ascorbate possesses a pronounced capability to increase thelevel of GFPpositive cells, although other substances keep theirROSdecreasing ability. In all likelihood, this feature of ascorbates is not directlyassociated with its antioxidant activity [45]. The scoreof GFPpositive iPSC colonies expressing an alkaline phosphatase hasshown that the efficiency of iPSC production from mEFs with three factors (Oct4, Sox2, andKlf4) can reach 3.8% in the presence of ascorbate. When all four factors (Oct4, Sox2, Klf4, andcMyc) are used together with ascorbate, the efficiency of iPSC production may reach8.75%. A similar increase in the iPSC yield was also observed in the reprogramming of murinebreast fibroblasts; i.e., the effect of vitamin C is not limited by one cell type. Moreover,the effect of vitamin C on the reprogramming efficiency is more profound than that of thedeacetylase inhibitor valproic (2propylvaleric) acid. The mutual effect of ascorbate andvalproate is additive; i.e., these substances have different action mechanisms. Moreover,vitamin C facilitates the transition from preiPSCs to stablepluripotent cells. This feature is akin to the effects of PD0325901 and CHIR99021, which areinhibitors of MEK and GSK3 kinases, respectively. This effect of vitamin C expands to humancells as well [45]. Following the transduction of humanfibroblasts with retroviruses carrying Oct4 , Sox2 , Klf4 , and cMyc and treatment with ascorbate, theauthors prepared iPSCs with efficiencies reaching 6.2%. The reprogrammingefficiency of ASCs under the same conditions reached 7.06%. The mechanism ofthe effect that vitamin C has on the reprogramming efficiency is not known in detail.Nevertheless, the acceleration of cell proliferation was observed at the transitional stage ofreprogramming. The levels of the p53 and p21 proteins decreased in cells treated withascorbate, whereas the DNA repair machinery worked properly [45]. It is interesting that an essential decrease in the efficiency of iPSCproduction has been shown under the action of processes initiated by p53 and p21 [4650].

As was mentioned above, for murine and human iPSC production, both retro andlentiviruses were initially used as delivery vectors for the genes required for cellreprogramming. The main drawback of this method is the uncontrolled integration of viral DNAinto the host cells genome. Several research groups have introduced methods fordelivering pluripotency genes into the recipient cell which either do notintegrate allogenic DNA into the host genome or eliminate exogenous genetic constructs from thegenome.

CreloxP Mediated Recombination. To prepareiPSCs from patients with Parkinsons disease, lentiviruses were used,the proviruses of which can be removed from the genome by Cre recombinase. To do this, the loxP site was introduced into thelentiviral 3LTRregions containing separate reprogramming genesunder the control of the doxycyclineinducible promoter. During viral replication, loxP was duplicated in the 5LTR of the vector. As aresult, the provirus integrated into the genome was flanked with two loxP sites. The inserts were eliminated using the temporary transfection ofiPSCs with a vector expressing Cre recombinase[51].

In another study, murine iPSCs were produced using a plasmid carrying the Oct4 , Sox2 , Klf4I, and cMyc genes in the same reading frame in which individual cDNAs were separatedby sequences encoding 2 peptides, and practically the whole construct was flanked with loxP sites [52]. The use ofthis vector allowed a notable decrease in the number of exogenous DNA inserts in the hostcells genome and, hence, the simplification of their following excision [52]. It has been shown using lentiviruses carrying similarpolycistronic constructs that one copy of transgene providing a high expression level of theexogenous factors Oct4, Sox2, Klf4, and cMyc is sufficient for the reprogramming ofdifferentiated cells into the pluripotent state [53,54].

The drawback of the CreloxP system is the incomplete excisionof integrated sequences; at least the loxP site remains in thegenome, so the risk of insertion mutations remains.

Plasmid Vectors . The application of lentiviruses and plasmids carrying the loxP sites required for the elimination of transgene constructsmodifies, although insignificantly, the host cells genome. One way to avoid this is touse vector systems that generally do not provide for the integration of the whole vector orparts of it into the cells genome. One such system providing a temporary transfectionwith polycistronic plasmid vectors was used for iPSC production from mEFs [29]. A polycistronic plasmid carrying the Oct4 , Sox2 , and Klf4 gene cDNAs, as well as aplasmid expressing cMyc , was transfected into mEFs one, three, five,and seven days after their primary seeding. Fibroblasts were passaged on the ninth day, and theiPSC colonies were selected on the 25th day. Seven out of ten experiments succeeded inproducing GFPpositive colonies (the Gfp gene wasunder the control of the Nanog gene promoter). The iPSCsthat were obtained were similar in their features to murine ESCs and did not contain inserts ofthe used DNA constructs in their genomes. Therefore, it was shown that wholesome murineiPSCs that do not carry transgenes can be reproducibly produced, and that thetemporary overexpression of Oct4 , Sox2 , Klf4 , and cMyc is sufficient for reprogramming. The maindrawback of this method is its low yield. In ten experiments the yield varied from 1 to 29 iPSCcolonies per ten million fibroblasts, whereas up to 1,000 colonies per ten millions wereobtained in the same study using retroviral constructs [29].

Episomal Vectors . Human iPSCs were successfully produced fromskin fibroblasts using single transfection with polycistronic episomal constructs carryingvarious combinations of Oct4 , Sox2 , Nanog , Klf4 , cMyc , Lin28 , and SV40LT genes. These constructs were designed on the basis of theoriP/EBNA1 (EpsteinBarr nuclear antigen1) vector [55]. The oriP/EBNA1 vector contains the IRES2 linker sequence allowing theexpression of several individual cDNAs (encoding the genes required for successfulreprogramming in this case) into one polycistronic mRNA from which several proteins aretranslated. The oriP/EBNA1 vector is also characterized by lowcopy representation in thecells of primates and can be replicated once per cell cycle (hence, it is not rapidlyeliminated, the way common plasmids are). Under nonselective conditions, the plasmid iseliminated at a rate of about 5% per cell cycle [56]. Inthis work, the broad spectrum of the reprogramming factor combinations was tested, resulting inthe best reprogramming efficiency with cotransfection with three episomes containing thefollowing gene sets: Oct4 + Sox2 + Nanog + Klf4 , Oct4 + Sox2 + SV40LT + Klf4 , and cMyc + Lin28 . SV40LT ( SV40 large T gene )neutralizes the possible toxic effect of overexpression [57]. The authors have shown thatwholesome iPSCs possessing all features of pluripotent cells can be producedfollowing the temporary expression of a certain gene combination in human somatic cells withoutthe integration of episomal DNA into the genome. However, as in the case when plasmid vectorsare being used, this way of reprogramming is characterized by low efficiency. In separateexperiments the authors obtained from 3 to 6 stable iPSC colonies per 106transfected fibroblasts [55]. Despite the fact that skinfibroblasts are wellcultured and accessible, the search for other cell types which arerelatively better cultured and more effectively subject themselves to reprogramming throughthis method is very likely required. Another drawback of the given system is that this type ofepisome is unequally maintained in different cell types.

PiggyBacTransposition . One promising system used foriPSC production without any modification of the host genome is based on DNA transposons.Socalled PiggyBac transposons containing2linkered reprogramming genes localized between the 5 and3terminal repeats were used for iPSC production from fibroblasts. The integrationof the given constructs into the genome occurs due to mutual transfection with a plasmidencoding transposase. Following reprogramming due to the temporary expression of transposase,the elimination of inserts from the genome took place [58, 59]. One advantage of the PiggyBac system on CreloxP is that the exogenous DNA iscompletely removed [60].

However, despite the relatively high efficiency of exogenous DNA excision from the genome by PiggyBac transposition, the removal of a large number of transposoncopies is hardly achievable.

Nonintegrating Viral Vectors . Murine iPSCs were successfullyproduced from hepatocytes and fibroblasts using four adenoviral vectors nonintegrating into thegenome and carrying the Oct4 , Sox2 , Klf4 , and cMyc genes. An analysis of the obtainediPSCs has shown that they are similar to murine ESCs in their properties(teratoma formation, gene promoter DNA methylation, and the expression of pluripotent markers),but they do not carry insertions of viral DNA in their genomes [61]. Later, human fibroblastderived iPSCs wereproduced using this method [62].

The authors of this paper cited the postulate that the use of adenoviral vectors allows theproduction of iPSCs, which are suitable for use without the risk of viral oroncogenic activity. Its very low yield (0.00010.001%), the deceleration ofreprogramming, and the probability of tetraploid cell formation are the drawbacks of themethod. Not all cell types are equally sensitive to transduction with adenoviruses.

Another method of gene delivery based on viral vectors was recently employed for theproduction of human iPSCs. The sendaivirus (SeV)based vector wasused in this case [63]. SeV is a singlestrandedRNA virus which does not modify the genome of recipient cells; it seems to be a good vector forthe expression of reprogramming factors. Vectors containing either all pluripotencyfactors or three of them (without ) were used for reprogramming the human fibroblast. The construct based on SeV is eliminatedlater in the course of cell proliferation. It is possible to remove cells with the integratedprovirus via negative selection against the surface HN antigen exposed on the infected cells.The authors postulate that reprogramming technology based on SeV will enable the production ofclinically applicable human iPSCs [63].

Cell Transduction with Recombinant Proteins . Although the methods for iPSCproduction without gene modification of the cells genome (adenoviral vectors, plasmidgene transfer, etc.) are elaborated, the theoretical possibility for exogenous DNA integrationinto the host cells genome still exists. The mutagenic potential of the substances usedpresently for enhancing iPSC production efficiency has not been studied in detail. Fullychecking iPSC genomes for exogenous DNA inserts and other mutations is a difficult task, whichbecomes impossible to solve in bulk culturing of multiple lines. The use of protein factorsdelivered into a differentiated cell instead of exogenous DNA may solve this problem. Tworeports have been published to date in which murine and human iPSCs wereproduced using the recombinant Oct4, Sox2, Klf4, and cMyc proteins [64, 65] . T he methodused to deliver the protein into the cell is based on the ability of peptides enriched withbasic residues (such as arginine and lysine) to penetrate the cells membrane. MurineiPSCs were produced using the recombinant Oct4, Sox2, Klf4, and cMycproteins containing eleven Cterminal arginine residues and expressed in E. coli . The authors succeeded in producing murine iPSCs during four roundsof protein transduction into embryonic fibroblasts [65].However, iPSCs were only produced when the cells were additionally treatedwith 2propylvalerate (the deacetylase inhibitor). The same principle was used for theproduction of human iPSCs, but protein expression was carried out in humanHEK293 cells, and the proteins were expressed with a fragment of nine arginins at the proteinCend. Researchers have succeeded in producing human iPSCs after sixtransduction rounds without any additional treatment [64]. The efficiency of producing human iPSC in this way was 0.001%, which isone order lower than the reprogramming efficiency with retroviruses. Despite some drawbacks,this method is very promising for the production of patientspecificiPSCs.

The first lines of human pluripotent ESCs were produced in 1998 [6]. In line with the obvious fundamental importance of embryonic stem cellstudies with regard to the multiple processes taking place in early embryogenesis, much of theinterest of investigators is associated with the possibility of using ESCs and theirderivatives as models for the pathogenesis of human diseases, new drugs testing, and cellreplacement therapy. Substantial progress is being achieved in studies on directed humanESC differentiation and the possibility of using them to correct degenerativedisorders. Functional cell types, such as motor dopaminergic neurons, cardiomyocytes, andhematopoietic cell progenitors, can be produced as a result of ESCdifferentiation. These cell derivatives, judging from their biochemical and physiologicalproperties, are potentially applicable for the therapy of cardiovascular disorders, nervoussystem diseases, and human hematological disorders [66].Moreover, derivatives produced from ESCs have been successfully used for treating diseasesmodeled on animals. Therefore, bloodcell progenitors produced from ESCs weresuccessfully used for correcting immune deficiency in mice. Visual functions were restored inblind mice using photoreceptors produced from human ESCs, and the normal functioning of thenervous system was restored in rats modeling Parkinsons disease using the dopaminergicneurons produced from human ESCs [6770]. Despite obvious success, the fullscale applicationof ESCs in therapy and the modeling of disorders still carry difficulties, because of thenecessity to create ESC banks corresponding to all HLAhaplotypes, whichis practically unrealistic and hindered by technical and ethical problems.

Induced pluripotent stem cells can become an alternative for ESCs in the area of clinicalapplication of cell replacement therapy and screening for new pharmaceuticals.iPSCs closely resemble ESCs and, at the same time, can be produced in almostunlimited amounts from the differentiated cells of each patient. Despite the fact that thefirst iPSCs were produced relatively recently, work on directed iPSCdifferentiation and the production of patientspecific iPSCs isintensive, and progress in this field is obvious.

Dopamine and motor neurons were produced from human iPSCs by directeddifferentiation in vitro [71, 72]. These types of neurons are damaged in many inherited oracquired human diseases, such as spinal cord injury, Parkinsons disease, spinal muscularatrophy, and amyotrophic lateral sclerosis. Some investigators have succeeded in producingvarious retinal cells from murine and human iPSCs [7375]. HumaniPSCs have been shown to be spontaneously differentiated in vitro into the cells of retinal pigment epithelium [76]. Another group of investigators has demonstrated that treating human andmurine iPSCs with Wnt and Nodal antagonists in a suspended culture induces theappearance of markers of cell progenitors and pigment epithelium cells. Further treating thecells with retinoic acid and taurine activates the appearance of cells expressing photoreceptormarkers [75].

Several research groups have produced functional cardiomyocytes (CMs) in vitro from murine and human iPSCs [7781]. Cardiomyocytes producedfrom iPSC are very similar in characteristics (morphology, marker expression,electrophysiological features, and sensitivity to chemicals) to the CMs ofcardiac muscle and to CMs produced from differentiated ESCs. Moreover, murineiPSCs, when injected, can repair muscle and endothelial cardiac tissuesdamaged by cardiac infarction [77].

Hepatocytelike cell derivatives, dendritic cells, macrophages, insulinproducingcell clusters similar to the duodenal islets of Langerhans, and hematopoietic and endothelialcells are currently produced from murine and human iPSCs, in addition to thealreadylisted types of differentiated cells [8285].

In addition to directed differentiation in vitro , investigators apply mucheffort at producing patientspecific iPSCs. The availability ofpluripotent cells from individual patients makes it possible to study pathogenesis and carryout experiments on the therapy of inherited diseases, the development of which is associatedwith distinct cell types that are hard to obtain by biopsy: so the use ofiPSCs provides almost an unlimited resource for these investigations.Recently, the possibility of treating diseases using iPSCs was successfullydemonstrated, and the design of the experiment is presented in the figure. A mutant allele wassubstituted with a normal allele via homologous recombination in murine fibroblastsrepresenting a model of human sickle cell anemia. iPSCs were produced fromrepaired fibroblasts and then differentiated into hematopoietic cell precursors.The hematopoietic precursors were then injected into a mouse from which the skin fibroblastswere initially isolated (). As a result, the initialpathological phenotype was substantially corrected [86].A similar approach was applied to the fibroblasts and keratinocytes of a patient withFanconis anemia. The normal allele of the mutant gene producing anemia was introducedinto a somatic cell genome using a lentivirus, and then iPSCs were obtainedfrom these cells. iPSCs carrying the normal allele were differentiated intohematopoietic cells maintaining a normal phenotype [87].The use of lentiviruses is unambiguously impossible when producing cells to be introduced intothe human body due to their oncogenic potential. However, new relatively safe methods of genomemanipulation are currently being developed; for instance, the use of synthetic nucleasescontaining zinc finger domains allowing the effective correction of genetic defects invitro [88].

Design of an experiment on repairing the mutant phenotype in mice modeling sickle cell anemia development [2]. Fibroblasts isolatedfrom the tail of a mouse (1) carrying a mutant allele of the gene encoding the human hemoglobin -chain (hs) were used for iPSCproduction (2). The mutation was then repaired in iPSCs by means of homological recombination (3) followed by cell differentiationvia the embryoid body formation (4). The directed differentiation of the embryoid body cells led to hematopoietic precursor cells (5)that were subsequently introduced into a mouse exposed to ionizing radiation (6).

The induced pluripotent stem cells are an excellent model for pathogenetic studies at the celllevel and testing compounds possessing a possible therapeutic effect.

The induced pluripotent stem cells were produced from the fibroblasts of a patient with spinalmuscular atrophy (SMA) (SMAiPSCs). SMA is an autosomalrecessive disease caused by a mutation in the SMN1 ( survival motorneuron 1 ) gene, which is manifested as the selective nonviability of lower motor neurons. Patients with this disorder usually die at the age of about two years.Existing experimental models of this disorder based on the use of flatworms, drosophila, andmice are not satisfactory. The available fibroblast lines from patients withSMA cannot provide the necessary data on the pathogenesis of this disordereither. It was shown that motor neurons produced from SMAiPSCs canretain the features of SMA development, selective neuronal death, and the lackof SMN1 transcription. Moreover, the authors succeeded in elevating the SMNprotein level and aggregation (encoded by the SMN2 gene, whose expressioncan compensate for the shortage in the SMN1 protein) in response to the treatment of motorneurons and astrocytes produced from SMAiPSCs with valproate andtorbomycin [89;]. iPSCs and theirderivatives can serve as objects for pharmacological studies, as has been demonstrated oniPSCs from patients with familial dysautonomia (FDA) [90]. FDA is an inherited autosomal recessive disorder manifested as thedegeneration of sensor and autonomous neurons. This is due to a mutation causing thetissuespecific splicing of the IKBKAP gene, resulting in a decreasein the level of the fulllength IKAP protein. iPSCs were produced fromfibroblasts of patients with FDA. They possessed all features of pluripotent cells. Neuralderivatives produced from these cells had signs of FDA pathogenesis and low levels of thefulllength IKBKAP transcript. The authors studied the effect of threesubstances, kinetin, epigallocatechin gallate, and tocotrienol, on the parameters associatedwith FDA pathogenesis. Only kinetin has been shown to induce an increase in the level offulllength IKBKAP transcript. Prolonged treatment with kinetininduces an increase in the level of neuronal differentiation and expression of peripheralneuronal markers.

Currently, a broad spectrum of iPSCs is produced from patients with variousinherited pathologies and multifactorial disorders, such as Parkinsons disease, Downsyndrome, type 1 diabetes, Duchenne muscular dystrophy, talassemia, etc., whichare often lethal and can scarcely be treated with routine therapy [51, 87, 89, 9194]. The data on iPSCs produced by reprogramming somaticcells from patients with various pathologies are given in the .

Functional categories of M. tuberculosis genes with changed expression level during transition to the NC state

One can confidently state that both iPSCs themselves and their derivativesare potent instruments applicable in biomedicine, cell replacement therapy, pharmacology, andtoxicology. However, the safe application of iPSCbased technologies requires the use ofmethods of iPSCs production and their directed differentiation which minimizeboth the possibility of mutations in cell genomes under in vitro culturingand the probability of malignant transformation of the injected cells. The development ofmethods for human iPSC culturing without the use of animal cells (for instance, the feederlayer of murine fibroblasts) is necessary; they make a viralorigin pathogen transferfrom animals to humans impossible. There is a need for the maximum standardization ofconditions for cell culturing and differentiation.

This study was supported by the Russian Academy of Sciences Presidium ProgramMolecular and Cell Biology.

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Induced Pluripotent Stem Cells: Problems and Advantages ...

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Somatic stem cells (also called adult stem cells) exist naturally in the body. They are important for growth, healing, and replacing cells that are lost through daily wear and tear.

Stem cells from the blood and bone marrow are routinely used as a treatment for blood-related diseases. However, under natural circumstances somatic stem cells can become only a subset of related cell types. Bone marrow stem cells, for example, differentiate primarily into blood cells. This partial differentiation can be an advantage when you want to produce blood cells; but it is a disadvantage if you're interested in producing an unrelated cell type.

Most types of somatic stem cells are present in low abundance and are difficult to isolate and grow in culture. Isolation of some types could cause considerable tissue or organ damage, as in the heart or brain. Somatic stem cells can be transplanted from donor to patient, but without drugs that suppress the immune system, a patient's immune system will recognize transplanted cells as foreign and attack them.

Therapy involving somatic stem cells is not controversial; however, it is subject to the same ethical considerations that apply to all medical procedures.

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Stem Cell Quick Reference - University of Utah

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Get the Highest Quality Multi Collagen Powder Highest Quality Multi Collagen Powder

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Collagen has become a popular hair growth supplement thanks to media attention and personal reports from consumers, despite a lack of scientific evidence linking collagen to hair growth.

Collagen is the most abundant protein in the human body, and it helps build and repair bones, tendons, ligaments, muscles, hair and skin, including skin on the scalp.

Some studies have shown that taking collagen improves hair growth, but most of these studies were funded by the cosmetic and supplement industries.According to the Harvard T.H. Chan School of Public Health, this presents a conflict of interest because most of the research on collagen supplements is funded by related industries that may benefit from positive results.

People have claimed that collagen supplementation can help regenerate hair follicles to regrow hair, thicken hair density, stop hair loss and possibly impact hair graying.

More objective and controlled studies are necessary to determine whether collagen supplementation may live up to these claims.

Collagen plays important roles in the health of the scalp and hair follicles, and it contains amino acids used to build keratin, which is the protein that hair is primarily made of.

Collagen is the primary component of the dermis, which is the connective tissue layer that contains hair follicles. Collagen plays a role repairing the dermis and the skin on the scalp at the base of the hair follicle, and it may impact age-related hair growth.

The following studies reported no conflicts of interest or suspicious funding sources.

An August 2021 study published in Aging investigated the role collagen may play in hair follicle regeneration. Tests showed that younger cells at the base of hair follicles upregulated (used more) collagen, while aged cells downregulated (used less) collagen. The young cells also aggregated together, which promotes hair follicle growth, while the aged cells had difficulty aggregating.

These results suggest that collagen helps younger people regenerate hair follicles, which lead to hair growth. As people age and collagen levels drop, there is less hair follicle regeneration and subsequent hair loss.

According to a 2018 study published in Experimental Dermatology, collagen type 17 is an important part of the microenvironment that promotes hair follicle stem cell health. Hair follicle stem cells become active during a new hair cycle, which is when new hair growth occurs.

The study also reported that people born with an inability to make collagen type 17 develop junctional epidermolysis bullosa, which is a genetic condition that causes skin to be fragile and blister. Patients with this condition develop serious hair loss and hair graying early in life.

Mice genetically altered to be deficient in type 17 collagen also develop hair loss and graying. Researchers reported that graying is believed to occur after collagen breakdown around hair follicle stem cells.

A 2016 study published in Science evaluated the effects of supplementing type 17 collagen in aged mice with hair loss. Results showed that maintaining levels of type 17 collagen helped aged hair follicles use collagen again to restore cell function and health.

Type 17 collagen is not available in supplement form but supplementing with different types of collagen may provide a wide range of amino acids the body needs to produce type 17 collagen.

Type I collagen is considered one of the best types for skin and hair, and it is found in both marine and bovine sources of collagen. This type of collagen is rich in hydroxyproline, which helps build keratin and is essential for skin health.

CB Supplements offers the highest quality multi collagen powder thats naturally formulated to keep your joints, skin, hair, nails and gut healthy.

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Taking collagen may improve the health of your scalp and hair, but there isnt much objective evidence that it will result in hair growth for everyone who takes it. Other potential collagen benefits include improvements in the health of skin, joints and bones.

Supplementing with collagen powder may promote the health and function of the scalp and hair follicles, but it must be taken on a long-term basis because the body produces an enzyme that constantly breaks down collagen.

Dietary supplements, including collagen supplements, are not reviewed by the U.S. Food and Drug Administration before they reach the U.S. market. It is important to look for a third-party verification seal on supplements before you buy them.

Examples of third-party verification organizations in the U.S. include the U.S. Pharmacopeia and NSF International. U.S. Pharmacopeia ensures that the ingredients have been verified, and NSF International certifies that a products ingredients match the label.

Make sure to discuss any new supplement with a doctor to review possible side effects, drug interactions and allergies. Collagen may be sourced from fish and eggs, which are common allergens.

Ask your doctor if any digestive problems could result from recommended dose and whether you should try a particular form of supplement first.

While there are no known drug interactions with collagen, some collagen supplements contain other ingredients, such as vitamin C, that may interact with drugs. Vitamin C should not be taken with bortezomib or deferoxamine.

Please seek the advice of a medical professional before making health care decisions.

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Collagen for Hair Growth | The Benefits It Has on Your Hair - DrugWatch.com

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Skin cells have been exposed to molecules that reverse their development but still retain their function, creating a kind of stem cell that keeps its original function in the body

By Chen Ly

A fluorescent light micrograph of fibroblast cells from human skin

VSHYUKOVA/SCIENCE PHOTO LIBRARY

Researchers have developed a method that can turn back the biological clock on skin cells by 30 years, creating stem cells from mature ones, which could be used to treat skin conditions in the future.

In 2007, Shinya Yamanaka at Kyoto University in Japan developed a technique that could transform adult skins cells into stem cells by inserting four specialist molecules, dubbed Yamanaka factors, that reverse cell development. It takes around 50 days of exposure to these molecules for normal cells to be reprogrammed into what are known as induced pluripotent stem cells (iPSCs).

When you turn to a cell into an iPSC, you lose the original cell type and its functionality, says Diljeet Gill at the Babraham Institute in Cambridge, UK.

Gill and his colleagues have now devised a technique that uses Yamanaka factors to rejuvenate skin cells without losing their previous functionality.

The researchers collected skin cell samples from three human donors that had an average age of around 50, then exposed these to the Yamanaka factors for just 13 days to partially anti-age the cells. They then removed the Yamanaka factors and left the cells to grow.

As we age, our DNA gets tagged with chemicals, so tracking these markers can help us determine how old our bodies are. This is known as our epigenetic clock. Over time, some of our genes will either turn on or off, the collection of which is known as the transcriptome.

Gill and his team found that the epigenetic clock and transcriptome profiles of the partially reprogrammed cells matched the profiles of skin cells that belonged to people who were 30 years younger.

The rejuvenated cells also functioned like younger ones, too, creating more collagen than those that didnt undergo reprogramming. And when placed onto an artificial wound, the reprogrammed cells moved to close the gap much quicker than the older ones did.

In young people, if you cut yourself, itll take quicker to heal the wound, while it would take me longer to heal, says team member Wolf Reik, also at the Babraham Institute. Its very exciting not only the molecular read-outs that are younger, but the cell also functions more like young cells.

The key advance in this study is that we are now able to substantially rejuvenate cells without changing their identity or functionality, says Reik. In previous studies, you would end up with a stem cell, which is not what youd want for therapy.

The technique may one day be useful in treating skin conditions, such as burns and ulcers. There is also the added bonus that the cells wouldnt be rejected by an individuals body, because they would be their own cells, says Gill.

So far, weve only tested this technique in skin cells. Were excited to see if we can translate it across other cell types, says Gill.

Journal reference: eLife, DOI: 10.7554/eLife.71624

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Anti-ageing technique makes skin cells act 30 years younger - New Scientist

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It can be difficult to figure out what products are worth your money in such a saturated beauty space, but here are two telltale signs: If celebrities recommend the product without being paid and if beauty editors, who get sent free products to test on the regular, say theyd spend their own money on it, chances are the product is worth a place in your routine.

Augustinus Baders The Cream (for combination and oily skin) and The Rich Cream (for dry skin) check both these boxes. The Creams word-of-mouth testimonials have given the product cult-level status. In fact, its been recommended three separate times in I Swear By This, a franchise where creatives share their must-have beauty products.

I felt like this product came out of nowhere; like one day, it popped up and everyone was recommending it. I have really sensitive skin, so Im typically hesitant to try new products, but I gave this a go. When I woke up the next day, I was a believer. Everything that everyone says about this cream is true. The price point is definitely on the higher end, but its so worth it. Grace Pae, celebrity makeup artist

"This has become central to not just my routine, but the routine of many of our customers. We were the first platform to launch this product, and it was really excitingthe whole experience of discovering this beauty holy grail. It's been a best-seller since it launched." Cassandra Grey, founder of beauty e-commerce platform Violet Grey

If its good enough for burn victims, its good enough for me. It's expensive, but some stuff you can't really put a price on. Plus, Im not using that much. My girlfriend buys it, so its all over the house. Basically, when she's not around, I use it, so I dont get slapped on the wrist.

This is an interesting product because its become the hot chick thing. They sell this at The Row; its the one. Every two to three years, theres a product like that. I don't even know if it's celebrity-driven because, with anything that becomes as big and popular as this, its just because it's good and it works.

Skin care and beauty are so personal. You can't just have beautiful packaging and marketing. It really does have to work, and there has to be a word-of-mouth element. That criteria doesn't exist in other categories. Chris Black, writer, consultant, and co-host of the podcast How Long Gone

Given all the hype the product has received, I decided to try it out myself. Below, my review on Augustinus Bader The Cream.

The Formula

In 2008, Augustinus Bader, a professor of applied stem cell biology and cell technology at the University of Leipzig in Germany, formulated a wound gel that heals third-degree burns, without surgery. The eponymous skin-care line was born from this breakthrough. The ethos behind Baders work is the idea that your skin doesnt need a never-ending supply of new ingredients.

Our skin contains stem cells, which have healing and regenerative properties. However, according to research, in order to trigger the self-renewal process, the environment needs to give the stem cells a green light to do their job. If theyre not receiving signals to activate, the skin stem cells lay dormant.

Both creams are formulated with Trigger Factor Complex, which contains vitamins, peptides, amino acids, and lipidsingredients that nourish the environment and signal to the dormant stem cells to wake up. Once the bodys stem cells are activated, the healing process promotes skin-care benefits like reducing redness, fading hyperpigmentation, and minimizing the appearance of fine lines and wrinkles.

The Results

After years of hearing the buzz about the brand, I decided to give it a try to see if the cream was actually as good as people say. I have combination skin thats prone to dehydration and dark spots, so I chose The Cream over The Rich Cream. The brand recommends using the product for at least 27 days (the amount of time it takes for cell turnover to occur). They also suggest using it alone, with no other skin-care products aside from cleanser. However, I decided to work The Cream into my existing morning and evening routines. After cleansing, Id apply the product before layering my other serums on top.

Before

After

The Cream played well with the other products in my routine. Its lightweight texture glided smoothly across my skin and the formula didnt dry down stickythere was no greasy residue left behind. My skin felt (and looked) plump after applying and it absorbed quickly. After a couple weeks of using The Cream, I started to notice a difference. My skin, which usually looks dull and extra parched in the mornings, felt supple and looked dewy. My stubborn dark spots were beginning to fade. Overall, my complexion looked healthier.

I still have a couple of weeks to go before I hit the 27-day mark, but The Cream has quickly become a staple in my routine. Yes, its pricey, but because of its clinically backed formula and proven performance, I think its absolutely worth the money.

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Augustinus Bader The Cream Review - Coveteur

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ASCE+ HRLVis the second product of ExoCoBio's professional aesthetic brand "ASCE+" whose first product "ASCE+ SRLV" for skin care is now widely recognized in a global market. ASCE+ HRLV is targeted for scalp rejuvenation and hair loss market and expected to rapidly expand its market share by introducing four effects: (1) scalp rejuvenation, (2) hair loss care, (3) improvement of hair anti-aging and (4) trouble prevention. They are designed to take care of both of scalp and hair regardless of age and gender.

According to the research conducted by Grand View Research (global market and research company), "the global hair and scalp care market size is valued at US$ 80.81 billion in 2020 and is anticipated to grow at a CAGR of 6.6% from 2021 to 2028." In recent years, there are growing concerns about the harmful effects of pollution, excessive use of hair products with chemicals, and the demand for nature-friendly products, whose market needs led to the launch of ASCE+ HRLV.

Dr. Iigo de Felipe, a Dermatologist with clinics in Barcelona and London, completed forthe last 2 years a clinical study for patients with scalp rejuvenation & hair loss using ASCE+ HRLV. When asked about his experience with the product, he said it is "better than other traditional treatments such as oral dutasteride, minoxidil or PRP" and praised its effectiveness affirming that it is "especially good in the frontal area of the scalp, an area where many other treatments usually fail". Dr. De Felipe also emphasized that "even though treatment is very effective for men, it is equally and sometimes even more effective for women too."

Over the past five years, ExoCoBio has made utmost efforts for R&D relating to skin, by using its patented technology "ExoSCRT" that is designed to isolate quality exosomes with excellent efficacy and to mass-produce exosomes (For more information on ExoSCRT, visit the following link: https://www.youtube.com/watch?v=ZOGQI8VuNOU). As a result, ExoCoBio became a global leader in exosome industry by obtaining 38 patents that are considered to be the strongest patent portfolio for exosomes in the world. Recently ExoCoBio successfully registered the patent "Composition for rejuvenating hair &, preventing hair aging comprising an exosome derived from stem cells as an active ingredient (Patent No. KR 10-2265875-0000)," which is the world first and unique idea to rejuvenate the hair color (i.e., hair returns to its original color).

"It is anticipated that the launch of ASCE+ HRLV will lead to expansion of ExoCoBio's share in the scalp rejuvenation and hair loss market as well as skin care market. In addition to the aesthetic product, ExoCoBio plans to develop a bio-medicine for hair loss through its R&D based on its innovative technology," stated Byong Cho, CEO of ExoCoBio (For HRLV technology, visit the following: https://www.youtube.com/watch?v=vkRcNCvNKAI).

References:https://www.grandviewresearch.com/industry-analysis/hair-care-markethttps://www.imcas.com/en/academy/show/11792/exosomes-in-treatment-of-androgenetic-alopecia-aga?connected=1

About ExoCoBio Inc. (www.exocobio.com)

ExoCoBio Inc. is the global leader who specializes in next generation exosome-based regenerative aesthetics, regenerative medicine, & immunotherapy. Currently ExoCoBio offers a series of exosome-based aesthetic & cosmetic products which are innovative and brand-new items in the regenerative aesthetics industry. Among them, ASCE+, EXOMAGE, and CELLTWEET are leading brands for skin rejuvenation, skin immune-modulation, and scalp rejuvenation, which have shown dramatic sales increase worldwide.

SOURCE ExoCoBio Inc.

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ExoCoBio launches "ASCE+ HRLV" for Scalp Rejuvenation and Hair Loss - PR Newswire

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Few of us know what they are or exactly how they work. But many of us have heard about the healing powers of stem cells, as well as the controversy surrounding them. Stem cells are well-debated and highly complex with promises ranging from fixing damaged knees to regenerating receding hairlines.

Cleveland Clinic is a non-profit academic medical center. Advertising on our site helps support our mission. We do not endorse non-Cleveland Clinic products or services.Policy

But what are stem cells? And, whats all the fuss all about?

Director of the Center for Regenerative Medicine and Surgery, Amy Lightner, MD, shares the differences between stem cell types, how stem cells can be used and when to be cautious of claims that might be too good to be true.

When most of us think of stem cells, we probably recall images of Dolly the cloned sheep. While its true that Dolly was born of stem cells, her place in science history is just one of many advancements in the field.

In fact, there are many different types of stem cells, each of which has different responsibilities and abilities. What unifies them is their ability to regenerate into new cells.

Regenerative medicine is an emerging field that uses innovative treatments to help regenerate or heal cell function thats lost due to aging, disease or injury, Dr. Lightner explains. The way we achieve this is by using stem cells in large quantities, targeted to a certain area, that the body uses to promote healing.

Adult stem cells are the only type of stem cells that are currently approved for medical use in the United States by the U.S. Food and Drug Administration (FDA). The term adult stem cells is a little confusing because theyre actually found in infants, children and adults. These cells live in a variety of tissue in our bodies including bone marrow, muscles, your brain, your intestines and more.

Think of adult stem cells as a little army of cells that can regenerate themselves into new cells to maintain and repair the tissue or muscle where theyre found. The catch with adult stem cells is that they cant become different types of cells (for example, blood stem cells can only become new blood cells, not skin or brain cells).

Unlike adult stem cells, embryonic stem cells have many more possibilities. Harvested during an embryos blastocyst stage (about five or six days after an embryo has been fertilized in a lab), embryonic stem cells have the potential to become any type of cell (called pluripotent cells). For these reasons, embryonic stem cells are the type of stem cells that generate the controversy most people associate with the topic.

Stem cell therapy has been around since the 1970s, when the first adult bone marrow cells were used to treat blood disease. A bone marrow transplant allows a recipient whose bone marrow cells have been damaged by chemotherapy or disease to receive healthy bone marrow stem cells from a donor.

Those stem cells have the potential to mature within the blood system into different immune cells that recognize and fight off different types of blood cancer. And they also have the ability to heal, says Betty Hamilton, MD, Department of Hematology and Medical Oncology.

Bone marrow transplants are currently used to treat diseases including:

While you may have heard about the use of stem cell therapy for knees, back pain, arthritis, hair loss, diabetes and more, no other types of stem cell therapy beyond bone marrow transplants have yet been approved by the FDA. But thousands of clinical trials are available ranging from treatments for Crohns disease to multiple sclerosis and more. The common link between all these trials is the ability of the stem cells to reduce inflammation and repair damage to your body.

Dr. Hamilton and Dr. Lightner agree that were only just beginning to scratch the surface of stem cell therapy. In recent years, during the height of the COVID-19 pandemic, many clinical trials were underway to explore whether stem cells could be used to help treat the damaged lungs in people severely affected by the disease.

I think potential is the perfect word to describe stem cells, says Dr. Hamilton. We know they have these anti-inflammatory and regenerative properties where they can provide a significant improvement to someone suffering from a certain disease. There are so many diseases where inflammation happens, and something needs to be repaired, and so any help the immune system can get provides a lot of potential.

Scientists are also researching whether adult stem cells can turn into pluripotent stem cells, which would allow the cells to change into any cell type without involving the use of embryonic stem cells.

While the potential for stem cell therapy is great, doctors caution that were not quite there yet.

I always tell patients that ask about stem cell therapy clinics or traveling overseas for stem cell therapy treatment that if its not something that is a clinical trial with FDA oversight, then they have no real way of knowing whats being given to them, advises Dr. Lightner.

This means more harm can come than good if you dont know exactly whats being given to you. Or, in some cases, youre just spending thousands of dollars for what ends up being saline, Dr. Lightner says.

The best way to know that youre receiving sound medical treatment is to make sure the one youre considering is approved by the FDA on its Clinical Trials database.

Dr. Lightner cautions against treatments that sound too good to be true. While stem cell therapy has helped improve and save millions of lives, its best to know what exactly youre signing up for by seeking out a qualified medical provider offering an FDA-approved clinical trial.

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Is Stem Cell Therapy Right for You? - Health Essentials from Cleveland Clinic

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This spring, streamlineand save time and moneywith these seasonal tips and tricks

USE THE CODE 'SPRINGSKIN' FOR 10% OFF ATSCENTUALS.COM, ONLY UNTIL JUNE 21, 2022

The weather is getting warmer, the days a little longer, and flowers are starting to bloom. As the seasons change, so should our skin care routines. Vancouver-based Scentuals is celebrating the start of spring with seven easy ways to make the change.

Good skin starts from the inside out. Drink enough water, exercise regularly, eat healthy balanced meals, take your vitamins, and prioritize quality sleep.

Your skin care products have expiry dates too. Using products past their shelf life makes them less effectiveor entirely ineffectiveand can cause skin irritation.

ScentualsRefresh and gently balance your skin with an alcohol-free mist. Better yet, try one that smells like roses. Made with floral waters, witch hazel and hyaluronic acid, Scentuals Rose Facial Mist helps soothe and refine your skin.

Say goodbye to dull winter skin and hello to renewed healthy radiance. The Scentuals Radiance Facial Scrub gently exfoliates with ground apricot seeds, while cucumber extract calms skin irritation and reduces redness.

ScentualsSwap out thick cleansers and creams for lighter, fast-absorbing options. The Radiance Facial Cleanser and Cream by Scentuals have you covered.

ScentualsBrightening vitamin C leaves your skin with a healthy glow and promotes collagen production. Try the award-winning Scentuals Vitamin C Serum, effectively formulated with vitamin C, hyaluronic acid and plant stem cells.

Defend your skin against harmful UV rays by wearing sunscreen daily. Additional benefits to daily SPF include preventing sunburns, skin cancer and premature aging.

CREATED BY VANCOUVER MAGAZINE, IN PARTNERSHIP WITH SCENTUALS

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7 Easy Ways to Transition Your Skin Care Routine for Spring - Vancouver Magazine

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Cell and gene therapies are poised to have a major impact on the landscape of modern medicine, carrying the potential to treat an array of different diseases with unmet clinical need.

However, the number of approved, clinically adopted cell and gene therapies is mere compared to the amount that are currently in development. A major barrier for the translation of such therapies is the safe integration of therapeutic genes into the human genome. The insertion of therapeutic genes bears the risk of off target effects, or integration of the gene into an unintended location.

A number of different strategies have been proposed to mitigate this effect. The most recent body of work comes from a collaboration between Harvards Wyss Institute for Biologically Inspired Engineering, Harvard Medical School (HMS) and the ETH Zurich in Switzerland.

Published in Cell Report Methods, the research focused on identifying safe spots in the genome. These locations, known as genomic safe harbors (GSHs), are areas in the genome that meet the following criteria: they can be accessed easily by genome-editing strategies, are within a safe distance from genes that possess functional properties and permit expression of a therapeutic gene, only once it has landed in the harbor. A simple analogy is deciding which harbor to dock a boat there are many considerations, and these depend on the type of boat you are sailing, the weather conditions and ease of access.

The research team adopted computational strategies that enabled the identification of 2,000 predicted GSHs. From this initial identification, they successfully validated two of the sites both in vitro and in vivo using reporter proteins.

Technology Networks interviewed the studys first author, Dr. Erik Aznauryan, research fellow in the laboratory of Professor George Church at Harvard Medical School. Aznauryan dives into further detail on the history of GSH research, the methods adopted to validate the GSH sites and the potential applications of this research.

Molly Campbell (MC): Can you talk about the history of genomic safe harbor research, and how they were discovered?

Erik Aznauryan (EA): Three genomic sites were empirically identified in previous studies to support stable expression of genes of interest in human cells: AAVS1, CCR5 and hRosa26. All these examples were established without any a-priori safety assessment of the genomic loci they reside in.

Attempts have been made to identify human GSH sites that would satisfy various safety criteria, thus avoiding the disadvantages of existing sites. One approach developed by Sadelain and colleagues used lentiviral transduction of beta-globin and green fluorescence protein genes into induced pluripotent stem cells (iPSCs), followed by the assessment of the integration sites in terms of their linear distance from various coding and regulatory elements in the genome, such as cancer genes, miRNAs and ultraconserved regions.

They discovered one lentiviral integration site that satisfied all of the proposed criteria, demonstrating sustainable expression upon erythroid differentiation of iPSCs. However, global transcriptome profile alterations of cells with transgenes integrated into this site were not assessed. A similar approach by Weiss and colleagues used lentiviral integrations in Chinese hamster ovary (CHO) cells to identify sites supporting long-term protein expression for biotechnological applications (e.g., recombinant monoclonal antibody production). Although this study led to the evaluation of multiple sites for durable, high-level transgene expression in CHO cells, no extrapolation to human genomic sites was carried out.

Another study aimed at identifying GSHs through bioinformatic search of mCreI sites regions targeted by monomerized version of I-CreI homing endonuclease found and characterized in green algae as capable to make targeted staggered double-strand DNA breaks residing in loci that satisfy GSH criteria. Like previous work, several stably expressing sites were identified and proposed for synthetic biology applications in humans. However, local and global gene expression profiling following integration events in these sites have not been conducted.

All these potential GSH sites possess a shared limitation of being narrowed by lentiviral- or mCreI-based integration mechanisms. Additionally, safety assessments of some of these identified sites, as well as previously established AAVS1, CCR5 and Rosa26, were carried out by evaluating the differential gene expression of genes located solely in the vicinity of these integration sites, without observing global transcriptomic changes following integration.

A more comprehensive bioinformatic-guided and genome-wide search of GSH sites based on established criteria, followed by experimental assessment of transgene expression durability in various cell types and safety assessment using global transcriptome profiling would, thus, lead to the identification of a more reliable and clinically useful genomic region.

MC: If GSHs do not encode proteins, or RNAs with functions in gene expression, or other cellular processes what is their function in the genome?

EA: In addition to protein coding, functional RNA coding, regulatory and structural regions of the human genome, other less well understood and inactive DNA regions exist.

A large proportion of the human genome seems to have evolved in the presence of a variety of integrating viruses which, as they inserted their DNA into the eukaryotic genome over the course of million years, lead to an establishment of vast non-coding elements that we continue to carry to this day. Furthermore, partial duplications of functional human genes have resulted in the formation of inactive pseudogenes, which occupy space in the genome yet are not known to bear cellular functions.

Finally, functional roles of some non-coding portions of the human genome are not well understood yet. Our search of safe harbors was conducted using existing annotation of the human genome, and as more components of it are deciphered the identification of genomic regions safe for gene insertion will become more informed.

MC: Are you able to discuss why some regions of the genome were previously regarded as GSHs but are now recognized as non-GSHs?

EA: In the absence of other alternatives, AAVS1, CCR5 and hRosa26 sites were historically called GSHs, as they supported the expression of genes of interest in a variety of cell types and were suitable for use in a research setting.

Their caveats (mainly, location within introns of functional genes, closely surrounded by other known protein coding genes as well as oncogenes) however prevent them from being used for clinical applications. Therefore, in our paper we dont call them GSHs, and refer to our newly discovered sites as GSHs.

MC: You thoroughly scanned the genome to identify candidate loci for further study as potential GSHs. Can you discuss some of the technological methods you adopted here, and why?

EA: We used several publicly available databases to identify genomic coordinates of structural, regulatory and coding components of the human genome according to the GSH criteria we outlined in the beginning of our study (outside genes, oncogenes, lncRNAs etc.,). We used these coordinates and bioinformatic tools such as command lines bedtools to exclude these genomic elements as well as areas adjacent to them. This left us with genomic regions putative GSHs from which we could then experimentally validate by inserting reporter and therapeutic genes into them followed by transcriptomic analysis of GSH-integrated vs non-integrated cells.

MC: You narrowed down your search to test five, and then two GSHs. Can you expand on your choice of reporter gene when assessing two GSHs in cell lines?

EA: Oftentimes in research you go with what is available or what is of the most interest to the lab you are currently working in.

Our case was not an exception, and we initially (up until the T cell work) used the mRuby reporter gene as it was widely available and extensively utilized and validated in our lab at ETH Zurich back then.

When I moved to the Wyss Institute at Harvard, I began collaborating with Dr. Denitsa Milanova, who was interested in testing these sites in the context of skin gene therapy particularly the treatment of junctional epidermolysis bullosa caused by mutations in various anchor proteins connecting different layers of skin, among which is the LAMB3 gene. For this reason, we decided to express this gene in human dermal fibroblasts, together with green fluorescent protein to have a visualizable confirmation of expression. We hope we would be able to translate this study into clinics.

MC: Can you describe examples of how GSHs can be utilized in potential therapeutics?

EA: Current cell therapy approaches rely on random insertion of genes of interest into the human genome. This can be associated with potential side effects including cancerous transformation of therapeutic cells as well as eventual silencing of the inserted gene.

We hope that current cell therapies will eventually transition to therapeutic gene insertions precisely into our GSHs, which will alleviate both described concerns. Specific areas of implementation may involve safer engineering of T cells for cancer treatment: insertion of genes encoding receptors targeting tumor cells or cytokines capable of enhancing anti-tumor response.

Additionally, these sites can be used for the engineering of skin cells for therapeutic (as discussed earlier with the LAMB3 example) as well as anti-aging applications, such as expression of genes that result in youthful skin phenotype.

Finally, given the robustness of gene expression from our identified sites, they can be used for industry-scale bio-manufacturing: high-yield production of proteins of interest in human cell lines for subsequent extraction and therapeutic applications (e.g., production of clotting factors for patients with hemophilias).

MC: Are there any limitations to the research at this stage?

EA: A primary limitation to this study is the low frequency of genomic integration events using CRISPR-based knock-in tools. This means that cells in which the gene of interest successfully integrated into the GSH must be pulled out of the vastly larger population of cells without this integration.

These isolated cells would then be expanded to generate homogenous population of gene-bearing cells. Such pipeline is not ideal for a clinical setting and improvements in gene integration efficiencies are needed to help this technology easier translate into clinics.

Our lab is currently working on developing genome engineering tools which would eventually allow to integrate large genes into GSHs with high precision and efficiency.

MC: What impact might this study have on the cell and gene therapy development space?

EA: This study will hopefully lead to many researchers in the field testing our sites, validating them in other therapeutically relevant cell types and eventually using them in research as well as in clinics as more reliable, durable and safe alternatives to current viral based random gene insertion methods.

Additionally, since in our work we shared all putative GSHs identified by our computational pipeline, we hope researchers will attempt to test sites we havent validated yet by implementing the GSH evaluation pipeline that we outlined in the paper. This will lead to identification of more GSHs with perhaps even better properties for clinical translation or bio-manufacturing.

MC: What are your next steps in advancing this work?

We hope to one day translate our successful in vitro skin results and start using these GSHs in an in vivo context.

Additionally, we are looking forward to improving integration efficiencies into our GSHs, which would further support clinical transition of our sites.

Finally, we will evaluate the usability of our GSHs for large-scale production of therapeutically relevant proteins, thus ameliorating the pipeline of manufacturing of biologics.

Dr. Erik Aznauryan was speaking to Molly Campbell, Senior Science Writer for Technology Networks.

Link:
Sailing the Genome in Search of Safe Harbors - Technology Networks

Skin Stem Cells Comments Off on Sailing the Genome in Search of Safe Harbors – Technology Networks | April 3rd, 2022

image:zebrafish notochord nuclei at 15-somite stage. Grey: nuclear DNA (DAPI). Color: histone H3K9me3 view more

Credit: Phong Nguyen, Franka Rang & Kim de Luca. Copryight Hubrecht Institute.

How is the activity of genes regulated by the packaging of DNA? To answer this question, a technique to measure both gene expression and DNA packaging at the same time was developed by Franka Rang and Kim de Luca, researchers from the group of Jop Kind (group leader at the Hubrecht Institute and Oncode Investigator). This method, EpiDamID, determines the location of modified proteins around which the DNA is wrapped. It is important to gather information about these modifications, because they influence the accessibility of DNA, thereby affecting the gene activity. EpiDamID is therefore valuable for research into the early development of organisms. The results of the study are published in Molecular Cell on April 1st 2022.

In order to fit DNA into the nucleus of a cell, it is tightly packed around nuclear proteins: histones. Depending on the tightness of this winding, the DNA can be (in)accessible to other proteins. This therefore determines whether the process of gene expression, translation of DNA into RNA and eventually into proteins, can take place.

DNA packaging determine gene activity

The tightness of DNA winding around histones is regulated by the addition of molecular groups, so-called post-translational modifications (PTMs), to the histones. For example, if certain molecules are added to the histones, the DNA winding is loosened. This makes the DNA more accessible for certain proteins and causes the genes in this part of the DNA to become active, or expressed. Furthermore, proteins that are crucial for gene expression can directly recognize and bind the PTMs. This enables transcription: the process of DNA copying.

The regulation of gene expression, for instance through PTMs, is also known as epigenetic regulation. Since all cells in a body have the same DNA, regulation of gene expression is needed to (de)activate specific functions in individual cells. For instance, heart muscle cells have different functions than skin cells, thus require different genes to be expressed.

Analysis of single cells using EpiDamID

To understand how PTMs affect gene expression, first authors Franka Rang and Kim de Luca designed a new method to determine the location of the modifications. Using this approach, called EpiDamID, researchers can analyze single cells, whereas previous methods were only able to measure a large group of cells. Analysis on such a small scale results in knowledge on how DNA winding differs per cell, rather than information on the average DNA winding of many cells.

EpiDamID is based on DamID, a technique which is used to determine the binding location of certain DNA-binding proteins. Using EpiDamID, the binding location of specific PTMs on histone proteins can be detected in single cells. Compared to others, a great advantage of this technique is that researchers need very limited material. Furthermore, EpiDamID can be used in combination with other methods, such as microscopy, to study regulation of gene expression on different levels.

Future prospects

Following the development of this technique, the Kind group will focus on the role of PTMs from the point of view of developmental biology. Because single cells are analyzed using EpiDamID, only a limited amount of material is needed to generate enough data. This allows researchers to study the early development of organisms from its first cell divisions, when the embryo consists of only a few cells.

###

Publication

Rang, F. J.*, de Luca, K. L.*, de Vries, S. S., Valdes-Quezada, C., Boele, E., Nguyen, P. D., Guerreiro, I., Sato, Y., Kimura, H., Bakkers, J. & Kind, J. Single-cell profiling of transcriptome and histone modifications with EpiDamID. Molecular Cell, 2022.

*Authors contributed equally

Jop Kind is group leader at the Hubrecht Institute for Developmental Biology and Stem Cell Research and Oncode Investigator.

About the Hubrecht Institute

The Hubrecht Institute is a research institute focused on developmental and stem cell biology. It encompasses 21 research groups that perform fundamental and multidisciplinary research, both in healthy systems and disease models. The Hubrecht Institute is a research institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), situated on Utrecht Science Park. Since 2008, the institute is affiliated with the UMC Utrecht, advancing the translation of research to the clinic. The Hubrecht Institute has a partnership with the European Molecular Biology Laboratory (EMBL). For more information, visit http://www.hubrecht.eu.

Experimental study

Cells

Single-cell profiling of transcriptome and histone modifications with EpiDamID

1-Apr-2022

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Read the original:
Learning from the single cell: A new technique to unravel gene regulation - EurekAlert

Skin Stem Cells Comments Off on Learning from the single cell: A new technique to unravel gene regulation – EurekAlert | April 3rd, 2022

Transcript:

Nelson Chao, MD: Thank you for joining us at the Targeted Oncology Virtual Tumor Board, which is focused on practice updates in graft-versus-host disease [GVHD]. In todays presentation, my colleagues and I will review 3 clinical cases. We will discuss approaches to treating patients who are at risk or who develop graft-versus-host disease. We will share our perspectives on key clinical trial data that may impact our decisions. I am Dr Nelson Chao from Duke University [Durham, North Carolina]. Today, I am joined by Dr Corey Cutler from Dana-Farber Cancer Institute [Boston, Massachusetts] and Kerry [King] Minor from Duke University. Thank you for joining us.

I would like to start by giving a brief overview of the process for allogeneic transplant. We need to collect donor cells, which could be related or unrelated. The stem cells can be separated from the blood or given as a whole. There is a conditioning, or treatment of the recipient. Chemotherapy and/or radiation will kill the cancer and weaken the donor immune system. The cells are infused to the patient and then we wait for engraftment. Graft-versus-host disease is a leading cause of non-relapse mortality following allogeneic transplantation. In acute GVHD, the reaction of the donor immune cells against host tissues is the ideology of the disease. Three main tissues are affected: the skin, the liver, the gut. In chronic graft-versus-host disease, the syndrome is quite variable with features resembling autoimmune or other immunologic disorders. In chronic GVHD, you could have a single organ, or it might be widespread and have a significant impact on quality of life. The risk factors for acute and chronic are similar, although the greatest impact of chronic is having acute prior. With standard prophylaxis, 25% to 50% of patients receiving HLA [human leukocyte antigen]-matched transplant will develop acute GVHD. That may require high-dose systemic steroids, and up to 50% of patients will have inadequate response to steroid therapy, which is associated with poor prognosis.

So this is a sort of a classical slide down demonstrating the pathology where in number 1, the recipient, who received the conditioning regimen, ends up with tissue damage. Those tissues release inflammatory cytokines such as IL [interleukin]-1 and IL-6, and then there is damage to the small bowel, usually releasing LPS [lipopolysaccharides]. These go down through different pathways, one goes to host antigen-presenting cells, which will activate the donor cells that release IL-12, IFN [interferon-gamma]-, and IL-2. Both are within the small bowel because of the LPS, as well as the microbiota entry, and release of those pattern associated damage receptors. There is target cell apoptosis of the gut, which then amplifies the response from the cytotoxic lymphocytes and NK [natural killer] cells together with TNF [tumor necrosis factor]- and IL-1. So most commonly the GVHD prophylaxis includes calcineurin inhibitors; methotrexate; MMF [mycophenolate mofetil]; sirolimus [Rapamune]; T-cell antibody; Ex vivo T-cell depletion, such as CD34 selection; posttransplant cyclophosphamide; and then obviously the standard combination of tacrolimus and methotrexate. So Im going to stop here for a second and ask Dr Cutler to give us a sense of where these regimens fall within the Dana-Farber Cancer Institute.

Corey Cutler, MD, MPH, FRCPC: Sure. Thanks for the introduction, Nelson. At our center, the predominant regimen that we use in the mild of greater setting is still the classic combination of tacrolimus and methotrexate. In the reduced intensity setting, we often add in the M200 meter cells. We are using the posttransplant cyclophosphamide regimen whenever we do haploidentical transplants. At the moment, there isnt compelling data that suggests a superior regimen to tackling the methotrexate. So most of us dont use it as a matter of routine outside of either the clinical trials or the haploidentical setting.

Nelson Chao, MD: And Ms. Minor, what do you think are the major toxicities that you see with these standard regimens?

Kerry King Minor, MSN, ANP-BC: Well, we always educate the patients that are going to be receiving the methotrexate about the risk of mucositis. Thats probably 1 of the biggest things that we see in patient education with that prophylaxis agent and tacrolimus. We prepare them and monitor very closely for side effects, such as headaches, effects on their blood pressure, effects on their kidneys and their knees. Thats basically it. With cyclophosphamide, typically patients tolerate that fairly well.

Transcript edited for clarity.

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An Overview on Graft-Versus-Host Disease and Prophylaxis - Targeted Oncology

Skin Stem Cells Comments Off on An Overview on Graft-Versus-Host Disease and Prophylaxis – Targeted Oncology | April 3rd, 2022