Bellin, M., Marchetto, M. C., Gage, F. H. & Mummery, C. L. Induced pluripotent stem cells: the new patient? Nat. Rev. Mol. Cell Biol. 13, 713726 (2012).

Matsa, E., Burridge, P. W. & Wu, J. C. Human stem cells for modeling heart disease and for drug discovery. Sci. Transl. Med. 6, 239 (2014).

Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616623 (2014).

Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230234 (2011).

Yang, X., Pabon, L. & Murry, C. E. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ. Res. 114, 511523 (2014).

Feric, N. T. & Radisic, M. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv. Drug Deliv. Rev. 96, 110134 (2016).

Domian, I. J. et al. Generation of functional ventricular heart muscle from mouse ventricular progenitor cells. Science 326, 426429 (2009).

Lundy, S. D., Zhu, W. Z., Regnier, M. & Laflamme, M. A. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev. 22, 19912002 (2013).

Nunes, S. S. et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat. Methods 10, 781787 (2013).

Mannhardt, I. et al. Human engineered heart tissue: analysis of contractile force. Stem Cell Reports 7, 2942 (2016).

Ribeiro, M. C. et al. Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitrocorrelation between contraction force and electrophysiology. Biomaterials 51, 138150 (2015).

Shadrin, I. Y. et al. Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nat. Commun. 8, 1825 (2017).

Brette, F. & Orchard, C. T-tubule function in mammalian cardiac myocytes. Circ. Res. 92, 11821192 (2003).

Wiegerinck, R. F. et al. Force frequency relationship of the human ventricle increases during early postnatal development. Pediatr. Res. 65, 414419 (2009).

Lopaschuk, G. D. & Jaswal, J. S. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. J. Cardiovasc. Pharmacol. 56, 130140 (2010).

Jackman, C. P., Carlson, A. L. & Bursac, N. Dynamic culture yields engineered myocardium with near-adult functional output. Biomaterials 111, 6679 (2016).

Radisic, M. et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc. Natl Acad. Sci. USA 101, 1812918134 (2004).

Eng, G. et al. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes. Nat. Commun. 7, 10312 (2016).

Hasenfuss, G. et al. Energetics of isometric force development in control and volume-overload human myocardium. Comparison with animal species. Circ. Res. 68, 836846 (1991).

Chung, S. et al. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat. Clin. Pract. Cardiovasc. Med. 4, S60S67 (2007).

Gong, G. et al. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice. Science 350, aad2459 (2015).

Porter, G. A. Jr et al. Bioenergetics, mitochondria, and cardiac myocyte differentiation. Prog. Pediatr. Cardiol. 31, 7581 (2011).

Vega, R. B., Horton, J. L. & Kelly, D. P. Maintaining ancient organelles: mitochondrial biogenesis and maturation. Circ. Res. 116, 18201834 (2015).

Gottlieb, R. A. & Bernstein, D. Metabolism. Mitochondria shape cardiac metabolism. Science 350, 11621163 (2015).

Sun, R., Bouchard, M. B. & Hillman, E. M. C. SPLASSH: Open source software for camera-based high-speed, multispectral in-vivo optical image acquisition. Biomed. Opt. Express 1, 385397 (2010).

Hong, T. et al. Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia. Nat. Med. 20, 624632 (2014).

Bers, D. M. Cardiac excitationcontraction coupling. Nature 415, 198205 (2002).

Huebsch, N. et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Sci. Rep. 6, 24726 (2016).

Tulloch, N. L. et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109, 4759 (2011).

Ma, J. et al. High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. Am. J. Physiol. Heart Circ. Physiol. 301, H2006H2017 (2011).

Morikawa, K., Song, L., Ronaldson-Bouchard, K., Vunjak-Novakovic, G. & Yazawa, M. Electrophysiological recordings of cardiomyocytes isolated from engineered human cardiac tissues derived from pluripotent stem cells.Protoc. Exch. https://doi.org/10.1038/protex.2018.030 (2018).

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Advanced maturation of human cardiac tissue grown from ...

Cardiac Stem Cells Comments Off on Advanced maturation of human cardiac tissue grown from … | April 4th, 2019

At the turn of the century, prevailing dogma stated that the adult mammalian heart was incapable of self-repair. Postnatal growth reflected increases in cardiomyocyte size alone rather than through increases in cell number. This dogma was shaken by the demonstration that bone marrow cells could be used to regenerate heart muscle. The subsequent discovery that adult hearts contained cells that expressed the haematological stem cell marker c-Kit led to a large body of literature, mostly from Piero Aversas laboratory, which advanced the premise that cardiac c-Kit+ cells were clonogenic, multipotent, and capable of self-renewal (i.e. genuine heart stem cells). While this hypothesis was popularized and espoused by many, the validity of Anversas findings were questioned early on by several investigators who failed to reproduce key findings.1,2

On 14 October 2018, the Harvard Medical School and Brigham and Womens Hospital brought an end to this chapter as 31 papers from the lab pioneering heart c-Kit+ cells were recommended for retraction because the validity of the scientific data was uncertain. While the full identity of the papers affected is still unknown, the New England Journal of Medicine promptly issued an expression of concern that the data presented in two (heretofore) landmark papers in cardiac regeneration may not be reliable3 and outright retracted a 2011 paper demonstrating evidence for human lung c-Kit+ stem cells.4

On the heels of multiple corrections,511 institutional settlements,12 lawsuits,13 and prior retractions,14 it appears much of the literature supporting resident (in situ) c-Kit+ cells having any role in cardiac repair is open to question. The impact of this verdict is only now starting to be understood and has led many to question the concept of heart stem cells in the post-Anversa era.

Yes. Archaeological carbon-14 dating conclusively established that half of all cardiomyocytes are renewed over an individual lifespan.15 This repopulation decreases with advanced years. For example, at 25years old almost 1% of cardiomyocytes turn-over every year compared with only 0.5% turnover after 75years. Such numberslow but definitely not zerohave been confirmed by others using complementary methods in experimental animals.16,17

No. Reports began to emerge 10years ago questioning the cardiomyogenic potential of c-Kit+ cells.1820 Recent lineage tracking from multiple labs using complimentary techniques has established that endogenous cardiac c-Kit+ cells do not generate cardiomyocytes.2123

Probably not. Early reports panned through tissue lysate and heart sections for cells expressing embryonic or haematological stem markers in hopes of identifying cells that could be enticed to express cardiac markers in culture. In the absence of lineage tracking, the origin of the cells discovered is uncertain and very well may represent extra-cardiac contamination. It follows that cardio myogenesis seen before or after injury likely arises from myocardial de-differentiation only.24 Although cardiosphere-derived cells (CDCs) are clonogenic and multipotent in vitro,25 they have long been recognized not to function as cardiac progenitors after transplantation in vivo.26

In 2004, Messina et al. demonstrated a mixed population of CD105+ CD45-cells, explant-derived cells that spontaneously emigrate from heart tissue plated in culture.27 Forensic analysis showed these cells are intrinsically cardiac with no detectable seeding from extra-cardiac organs.28 To enable cell expansion to clinical doses, explant-derived cells have been antigenically selected or sphere cultured to generate c-Kit+ cells or CDCs, respectively (see Figure1). Independent labs have shown that both c-Kit+ cells (6 labs) or CDCs (45+ labs) improve heart function when delivered after injury. Unfortunately, studies providing direct comparisons between either cell type are often difficult to interpret as divergent cell culture methods or patient comorbidities influence cell potency; however, within CDCs, the small c-Kit+ cell fraction does not contribute to and is not necessary for, the observed gains in function.29

Figure 1

Schematic outline of heart-derived cell therapeutic manufacturing and identity. Explant-derived cells are cultured from myocardial tissue for antigenic selection (c-Kit+ cells, left panels) or sphere culture (CDCs, right panels) prior to expansion. Representative c-Kit+ cell images demonstrate freshly isolated human c-Kit+ cells (left panel, black dots, beads from magnetic-activated cell sorting) and during cell expansion (right panel, low confluence to highlight cell morphology). Representative images of CDCs cultured from transgenic mouse tissue expressing the c-Kit reporter (green fluorescent protein)18 highlighting the proportion of c-Kit+ cells within. Also shown is flow cytometry characterization from the SCIPIO (c-Kit+ cell trial, left panel)35 and CADUCEUS (CDC trial, right panel)41 trials contrasting the antigenic identity of each heart-derived cell therapeutic used in clinical trials.

Figure 1

Schematic outline of heart-derived cell therapeutic manufacturing and identity. Explant-derived cells are cultured from myocardial tissue for antigenic selection (c-Kit+ cells, left panels) or sphere culture (CDCs, right panels) prior to expansion. Representative c-Kit+ cell images demonstrate freshly isolated human c-Kit+ cells (left panel, black dots, beads from magnetic-activated cell sorting) and during cell expansion (right panel, low confluence to highlight cell morphology). Representative images of CDCs cultured from transgenic mouse tissue expressing the c-Kit reporter (green fluorescent protein)18 highlighting the proportion of c-Kit+ cells within. Also shown is flow cytometry characterization from the SCIPIO (c-Kit+ cell trial, left panel)35 and CADUCEUS (CDC trial, right panel)41 trials contrasting the antigenic identity of each heart-derived cell therapeutic used in clinical trials.

Not as much as we thought! Ex vivo expanded c-Kit+ cells were inspired by the Anversa literature and it was thought, until recently, that robust cell numbers persisted for many years after intramyocardial injection.30 The in situ c-Kit+ cell findings, which largely emanated from the well-funded Anversa lab, were directly extended to ex vivo expanded c-Kit+ cells. Since then, it has been concretely established that few transplanted cells engraft beyond a few days.31 This surprising observation revealed that c-Kit+ cells were evanescent, and thus not functioning as stem cells.

This realization came very late for c-Kit+ cells, unlike CDCs, which have been known for >10years to be effective despite little persistence of injected cells beyond 4weeks (i.e. 23% of the initial injectate).32,33 Fortunately, the CDC literature provides a clear template for these investigations with several articles listing comprehensive proteomic analysis, cytokine over-expression/subtraction data supporting causation, exosome profiling data and microRNA addition/subtraction data supporting a causative role in post infarct repair.34

Although very late in the game, a great deal of the basic phenotyping work is not yet known about c-Kit+ cells; including the fundamental differences between heart-derived and extra-cardiac c-Kit+ cells. It may be that c-Kit+ cells stimulate many of the immunomodulatory (macrophage polarization) and trophic (angiogenic, anti-apoptotic, mitotic and anti-scarring) endogenous repair mechanisms already identified in the CDC literature but much waits to be uncovered.

Reports of their death have been greatly exaggerated. The 2011 Phase 1 SCIPIO Trial demonstrated intra-coronary injection of c-Kit+ cells was safe and provided encouraging hints of efficacy as shown by increases in cardiac ejection fraction, New York Heart Association (NYHA) class and viable myocardium.35 But the subsequent 2014 expression of concern by The Lancet36 reflected cell product characterization, identity and manufacturing which were both done in Boston by Dr Anversas team.37 The impact of recent events on interpretation of the SCIPIO Trial is still not known but may emerge as the journals affected by the list of articles recommended for retraction receive more information.

The CONCERT HF Trial (ClinicalTrials.gov Identifier: NCT02501811) began in 2015 to explore the effects of combining heart-derived c-Kit+ cells with blood mesenchymal stem cells on post infarct repair.38 This trial was based upon two preclinical studies suggesting combined therapy increases transplanted cell engraftment to enhance cell treatment outcomes.39,40 With the Harvard c-Kit+ cell retractions, the NIHBLI paused the trial on 29 October 2018 to provide the Data and Safety Monitoring Board (DSMB) an opportunity to review the literature supporting the scientific foundations of the trial. Given the invasive nature of the trial (and the observation that a patient died during endomyocardial biopsy), this caution is appreciated to ensure that sufficient pre-clinical insight and clinical equipoise still exist in the new post-Anversa era.

At best, the future of heart c-Kit+ cells is uncertain. With the astounding number of key publications likely to be retracted, it may very well be that adult c-Kit+ cells are not fundamentally different enough from other heart-derived cells to warrant efforts exploring clinical efficacy beyond the multiple clinical trials completed or underway using CDCs or the CDC secretome.

Conflict of interest: none declared.

References are available as supplementary material at European Heart Journal online.

Published by Oxford University Press on behalf of the European Society of Cardiology 2019.

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Cardiac stem cells in the post-Anversa era | European ...

Cardiac Stem Cells Comments Off on Cardiac stem cells in the post-Anversa era | European … | April 3rd, 2019

University of Arizona researchers Churko and colleagues describe new findings of gene expression patterns in cardiac stem cells, which could be used to create heart regeneration therapies.

Heart disease affects millions of people each year and has the potential to impair heart function by damaging heart muscle. Although many preventative therapies are available, once damage has occurred to the cells of the heart, there are not many treatment options available. The heart has a limited capacity to heal itself, but one option that could result from new research into stem cell therapies is regenerative therapy, leading to cardiac regeneration. However, the processes involved in stem cell differentiation into various heart muscle tissues are not well understood.

In a new study published in Nature Communications, University of Arizona researchers Churko and colleagues investigate the gene expression patterns that are responsible for the differentiation of heart cells. This will clarify how heart cells develop and respond to drugs or other factors.

The researchers found that heart muscle cells vary in gene expression as they mature, between days 14 and 45. Younger cells have gene expression profiles more like those of cells of the heart atrium, whereas more mature cells have gene expression profiles more like those of the heart ventricle. Churko and colleagues also identified one gene, NR2F2, that, when overexpressed blunted expression of the specific genes that are expressed within muscle cells and heart cells, and led to increased expression of the genes associated with pluripotent stem cells and neuronal cells.

Churko and colleagues findings will help other researchers working on heart stem cells and regenerative therapy. By understanding the genetic expression patterns that lead to or characterize the differentiation of stem cells into heart muscle cells, researchers will be able to guide pluripotent stem cells into becoming cardiac cells. Ultimately, this will lead to better treatment for patients with heart disease and a damaged heart.

Written by C.I. Villamil

Reference: Churko et al. 2018. Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis. Nature communications 9:4906.

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New insights into cardiac stem cells could lead to heart ...

Cardiac Stem Cells Comments Off on New insights into cardiac stem cells could lead to heart … | March 8th, 2019

Theranostics 2018; 8(3):815-829. doi:10.7150/thno.19577

Review

Dong Wang1*, LeeAnn K. Li1,2*, Tiffany Dai3, Aijun Wang4, Song Li1,5

1. Department of Bioengineering, University of California, Los Angeles, CA 90095, USA;2. David Geffen School of Medicine, University of California, Los Angeles, CA 90024, USA;3. Department of Bioengineering, University of California, Berkeley, CA 94720, USA;4. Surgical Bioengineering Laboratory, Department of Surgery, School of Medicine, University of California, Davis, Sacramento, CA 95817, USA;5. Department of Medicine, University of California, Los Angeles, CA 90095, USA.* Equal contribution

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Understanding the contribution of vascular cells to blood vessel remodeling is critical for the development of new therapeutic approaches to cure cardiovascular diseases (CVDs) and regenerate blood vessels. Recent findings suggest that neointimal formation and atherosclerotic lesions involve not only inflammatory cells, endothelial cells, and smooth muscle cells, but also several types of stem cells or progenitors in arterial walls and the circulation. Some of these stem cells also participate in the remodeling of vascular grafts, microvessel regeneration, and formation of fibrotic tissue around biomaterial implants. Here we review the recent findings on how adult stem cells participate in CVD development and regeneration as well as the current state of clinical trials in the field, which may lead to new approaches for cardiovascular therapies and tissue engineering.

Keywords: Cardiovascular disease, Stem cell, atherosclerosis, vascular grafts, vascular smooth muscle cell.

Cardiovascular diseases (CVDs) such as ischemic heart disease, stroke, and peripheral artery disease are the leading cause of mortality and morbidity around the world: about 30% of global deaths and 10% of global disease burden a year are due to CVDs [1, 2]. In the past three decades, these diseases have been increasing in underdeveloped and developing countries. Although deaths from CVDs have declined in some developed countries with better healthcare interventions and systems and primary prevention, population growth and aging will drive up global CVDs in coming decades [1, 2].

Atherosclerosis is a chronic inflammatory disease resulting in clogged arteries or unstable plaque rupture [3, 4]. Currently, treatment of atherosclerosis includes reducing risk factors such as treatment of hypercholesterolemia and hypertension [1, 2] and, for advanced disease, surgery such as stent implantation and bypass surgery using autologous vessels or tissue-engineered vascular grafts [5]. However, thrombosis and secondary atherosclerosis are common complications following stent and graft implantation, particularly in small-diameter arteries and grafts [6]. New therapies are thus urgently needed for better prevention and treatment of atherosclerosis.

It is widely accepted that endothelial cell (EC) dysfunction, inflammatory cell recruitment, and vascular smooth muscle cell (SMC) de-differentiation contribute to atherogenesis [3, 4, 7]. In the past two decades, several types of vascular stem cells (VSCs), in addition to circulating progenitors, have been identified and characterized, with evidence that they are not only involved, but also play pivotal roles in blood vessel remodeling and disease development. VSCs or similar stem cells in mesenchymal tissues, for instance, also participate in the regeneration of blood vessels following the implantation of vascular grafts. Elucidating the regulatory mechanisms of these VSCs is fundamental to understanding vascular remodeling and may pave the way to developing novel, successful therapies for atherosclerosis. In this review, we analyze vascular remodeling through the lens of stem cells, and discuss the challenges we face in developing improved therapies for vascular diseases and regeneration.

Large and medium size blood vessels have three distinct layers: 1) the tunica intima, an inner lining of ECs, which may contain a small number of endothelial progenitor cells (EPCs) [8, 9]; 2) the tunica media, a thick middle layer composed of smooth muscle cells (SMCs) and a small number of stem cells; and 3) the tunica adventitia, an outer layer of connective tissue containing a heterogeneous population of cells, including fibroblasts, resident inflammatory cells (including macrophages, dendritic cells, T cells and B cells), microvascular (vasa vasorum) ECs around which pericytes reside, adrenergic nerves, and also stem cells (including multipotent mesenchymal stem cells, or MSCs) and progenitor cells (including those with macrophage, endothelial, smooth muscle, and hematopoietic potential) [10-18]. All these cells contribute, to varying extents, to the pathogenesis of atherosclerosis and vascular remodeling.

Atherosclerosis is thought to be initiated by dysfunctional or activated ECs [3, 7]. Various risk factors include genetic defects and environmental risks, behaviors like cigarette smoking and harmful use of alcohol, as well as disturbed blood flow, hypertension, hypercholesterolemia, infections, and other chronic conditions such as diabetes, obesity, autoimmune diseases, and aging [1, 2]. The injured endothelial area may be repaired by adjacent EC proliferation or EPCs from bone marrow or resident endothelium [19]. Disease begins when such endothelial repair does not occur properly.

Malfunctioning ECs secrete cytokines and upregulate expression of surface adhesive molecules to recruit circulating platelets, monocytes, T cells, neutrophils, dendritic cells, and mast cells to adhere to the site of endothelial injury and infiltrate into the subendothelial space. Within this space, monocytes differentiate into macrophages and scavenge lipid deposited from the circulation, becoming foam cells in the process [3, 20-22]. Notably, most of these foam cells are initially derived from preexisting intimal-resident myeloid progenitors rather than recently recruited blood monocytes [23]. In addition, the inflammatory cells activate medial SMCs and stem cells, prompting adventitial stem cells to proliferate and migrate into the intima, where they may differentiate and also obtain some properties of myofibroblasts and macrophages [3, 20-22, 24, 25]. Disease proceeds as the abnormal vascular wall processes prompt macrophages, together with leukocytes, activated ECs, and SMCs, to secrete increasing amounts of inflammatory cytokines to recruit more inflammatory cells from the circulation and resident adventitial tissues. This forms a cycle of inflammatory responses in local atherosclerotic lesions [3, 4, 26-28]. All these events lead to the development of fatty streaks, formation of neointima, and thickening of arterial walls seen at the early stages of atherosclerosis [3, 26]. The extracellular matrix, too, may play a role in lipid retention [29]. As these atherosclerotic lesions continue to grow and narrow the lumen, arteries may attempt to compensate by gradual dilation; however, this compensation reaches its limit beyond a certain size of atherosclerotic lesion.

Advanced atherosclerotic plaques have developed a fibrous cap that sequesters the underlying inflammatory mixture, which includes foam cells and extracellular lipid droplets, infiltrated T cells, macrophages, and mast cells, and necrotic tissue [3, 26]. The cap itself is mainly comprised of SMCs and collagen matrix, which can be degraded and ruptured by metalloproteases released by macrophages and mast cells. Stability of plaques is thus defined by thickness of the fibrous cap. Severe thrombosis may occur upon fibrous cap rupture, leading to acute coronary artery disease (myocardial infarction) and stroke [3, 26].

Several groups provide direct evidence that smooth muscle myosin heavy chain (SM-MHC)+ SMCs are a major contributor to neointimal thickening and atherosclerotic lesions, using transgenic mice with tamoxifen-regulated CreER under the control of a SM-MHC promoter (SM-MHC-CreER) [22, 30-33]. Interestingly, some studies suggest that SMCs in human atherosclerotic lesions are monoclonal [34, 35], implying heterogeneity of the SMC population. By using multi-colored lineage tracing in ApoE-/-/SM-MHC-CreER/Rosa26-Confetti transgenic mice, a recent study demonstrates that only a small number of SMCs proliferate and contribute to atherosclerotic plaques [36]. This is consistent with our single-cell analysis of SMCs showing that only a small subpopulation of SMCs is capable of proliferation and differentiation (unpublished data). However it is worth noting that, in addition to medial SMCs, other non-SMCs such as stem cells and ECs also contribute to the SMCs of neointima and atherosclerotic lesions [22, 33, 37, 38], while lesional macrophage-like cells can also be derived from SMCs [39], suggesting alternative mechanisms may also account for vascular disease development.

Endothelial to mesenchymal transition (EndoMT) is one possible mechanism. Some studies utilized Tie2-Cre mice for lineage tracing ECs and found that ECs contribute to pulmonary artery neointimal formation by differentiating into cells positive for smooth muscle -actin (-SMA) [40, 41]. However, other researchers found a very low frequency, in contrast, of EndoMT in the neointima [38]. Similarly, using Tie2-Cre mice to trace ECs in carotid artery neointimal formation, we found that although ECs contributed to neointimal formation, they still maintained endothelial identities and expressed CD31 but no or low -SMA expression [37]. This discrepancy requires further investigation with different animal models and tissue locations, and still leaves open the possibility of additional mechanisms for neointimal pathogenesis.

In addition to vascular SMCs and ECs, vascular stem and progenitor cells have been isolated from the circulation and from different layers of the artery wall, and have been implicated in vascular disease development. Key examples found in or around the vasculature are summarized in Table 1. The list is organized based on differentiation potential and tissue(s) of origin, and is discussed in detail below.

Vascular stem cells and progenitors

Bone marrow cells were reported to differentiate into SMCs in neointima and atherosclerotic lesions in the early 2000s [42-45]. These findings, however, remain controversial, as later studies in vascular transplant and injury models countered by arguing that bone marrow-derived cells did not in fact differentiate into neointimal SMCs, although they did participate in the inflammatory response [46-48]. A mouse wire injury model, for instance, found that some bone marrow cells were recruited to the neointima and expressed -SMA, but never became positive for mature SMC marker SM-MHC. Rather, these bone marrow cells expressed markers of monocytes and macrophages [48].

Other bone marrow-derived cells - specifically, certain EPCs - have also been identified as important for endothelial regeneration. It should be noted that the term endothelial progenitor cell has been applied to many different cell types, and defining what precisely it means to be an EPC is a source of controversy. Classification traditionally is divided into two methods: antigen classification, and culture-based classification. Both have been used to identify vascular-relevant EPCs.

Using the first method, cell-surface antigens are examined typically with flow cytometry to quantify relevant populations. Putative EPCs were first isolated by Asahara et al. (1997) from human peripheral blood by flow cytometry using surface markers CD34 and vascular endothelial growth factor receptor 2 (VEGFR-2, also known as kinase insert domain receptor, KDR, or fetal liver kinase 1, Flk1), both of which are characteristically expressed by ECs [49]. These circulating cells could contribute to neoangiogenesis postnatally by homing to angiogenic sites and acquiring characteristics of endothelium. Thereafter, other groups reported that EPCs contribute to endothelial regeneration in rodent models after various arterial injuries including vein graft atherosclerosis and mechanical injury [50-52], as well as in human diabetic wound healing [53].

Studies further showed that EPCs are in fact a heterogeneous population comprised of different subpopulations with different cell surface markers. In addition to CD34 and VEGFR-2, in an attempt to distinguish between immature and mature endothelial cells, investigators also commonly use markers like CD133 (also known as AC133), which is lost during endothelial maturation [54]. For example, Peichev et al. (2000) identified a unique subpopulation of EPCs (CD34+/VEGFR-2+/AC133+) in human fetal liver and peripheral blood [55]; another subpopulation of Flk1+/AC133+/CD34-/VE-cadherin- cells were also identified as EPCs in human bone marrow [56]. Despite the advantages of having specific markers for lineage tracing and drawing ties between disparate populations, one can see here too how antigen-based definitions may still be somewhat nonspecific in phenotype. The more antigen markers utilized, the more specific the definition, but also the fewer the cells identified - particularly considering the inherently probabilistic nature of antigen carriage for given cell types.

In the second method of classification, cells are isolated based on in vitro culture. Given the difficulties of finding specific surface markers for EPCs, some research groups isolated EPCs by single-cell colony-formation assay (SCCFA) based on the high self-renewal and proliferation potential of stem cells. Some studies subdivided EPCs based on their time of appearance in culture into populations which, interestingly, have different differentiation potential: early EPCs cannot differentiate into ECs, but only differentiate into macrophages and contribute to angiogenesis through paracrine factors, and thus were named as myeloid angiogenic cells (MACs); and late EPCs can differentiate into ECs and contribute to de novo blood vessel formation, and were dubbed endothelial colony forming cells (ECFCs) [57-61].

In addition to circulation-derived EPCs, EPCs with similar properties have been derived based on colony-formation assay from the vascular endothelium of large human blood vessels, placenta, and adipose tissue [62-64]. Mouse ECFCs have also been isolated from endothelial culture by surface markers lin-CD31+CD105+Sca1+c-Kit+, with c-Kit expression found to be critical for the clonal expansion of these ECFCs [65].

Beyond the nature of EPC classification, their functions, too, remain controversial. The concept of bone marrow-derived EPCs playing a fundamental role in the mechanism of vascular repair and regeneration has acquired many proponents as we described, though it remains hotly debated [66]. Pre-clinical animal studies showed that transplanted human EPCs formed microvessels and promoted vascular regeneration in vivo [49, 55, 56, 67, 68]. In mouse models of vascular graft transplantation, for instance, bone marrow cells contributed to the regenerated ECs of the grafts [50, 69, 70]. Nevertheless, another study countered that bone marrow-derived EPCs do not contribute to vascular endothelium in mouse models of bone marrow transplantation, tumor formation, and a parabiotic system [71].

A role for bone marrow-derived EPCs in atherogenesis similarly has been inferred, but accumulation of solid evidence in this role is mixed and still work in progress [52]. In an ApoE-/- mouse model, bone marrow-derived Sca-1+/CD34+/Flk-1+/CD133+ EPCs were found in the lesion-prone area of endothelium, possibly for repairing the injured endothelium [72]. However, other studies have said that, although there may exist a population of bone marrow-derived EPCs, ECs derived from the vascular bed are instead responsible for the EC replacement and regeneration seen in transplant arteriosclerosis [73].

In the clinical context, the role of EPCs remains unclear. Large-scale clinical studies suggested that high levels of EPCs were associated with reduced risk of cardiovascular diseases [74, 75] and improved outcomes after acute ischemic stroke [76-78] (versus poorer stroke outcomes if blood EPCs failed to increase [79]), and that vascular trauma, acute coronary diseases, and stroke induced elevated level of EPCs [76, 80, 81], presumably for purposes of vascular repair and maintenance. However, some also found no clear correlation between EPC level and endothelial function [82].

To date, much ambiguity and controversy remains in regards to the existence of true EPCs that can differentiate into ECs, their marker expression, location, and contribution to endothelial regeneration. It is possible that EPCs are a rare but dynamic population that respond to specific stimuli such as severe endothelial injury of large arteries or vascular transplantation [50, 69, 70], but not to tumor growth, which involves microvessels [71].

Stem and progenitor cells resident to vasculature have been identified across the different vessel wall layers. Similar to the bone marrow-derived progenitor cells, isolation has relied on antigen selection or culture-based characterization. Although those derived from the adventitia are better characterized and supported - evidence which will be elaborated momentarily - a few groups of stem cells have also been characterized in the media.

A population of calcifying vascular cells (CVCs) was first isolated from human atherosclerotic lesions in the arterial medial layer by Bostrm et al. (1993) and Tintut et al. (2003) and found to differentiate into SMC, osteogenic, and chondrogenic lineages [83, 84]. CVCs were harvested by tissue explant culture and were identified as expressing CD29 and CD44, two non-specific mesenchymal cell markers (adhesion receptors). However, no specific transcriptional markers were identified.

Later, in 2006, Sainz et al. isolated a small population of Sca-1+, c-kit (-/low), Lin-, CD34-/low cells from the media layer (around 60.8% prevalence in tunica media) of healthy murine thoracic and abdominal aortas [85]. They used a Hoechst DNA binding dye method to identify non-tissue-specific stem/progenitor cells based on their ability to expel the dye via the transmembrane transporter ATP-binding cassette transporter subfamily G member 2 (ABCG2). These cells gave rise to ECs (as determined by VE-cadherin, CD31, and von Willebrand factor expression) and SMCs (determined by -SMA, calponin, and SM-MHC expression) when cultured with vascular endothelial growth factor (VEGF) and transforming growth factor 1 (TGF-1)/platelet-derived growth factor BB (PDGF-BB) respectively, similar to Flk-1+ mesoangioblasts found in the embryonic dorsal aorta, and also produced (VE-cadherin+ and -SMA+) vascular-like branching structures of cells [85, 86].

Another population of vascular progenitors were isolated by Zaniboni, et al. from the media by internal digestion of porcine aortas with collagenase [87]. These cells were described as similar to both MSCs and pericytes. Like MSCs, they had elongated, spindle-shaped, fibroblast-like morphology, and met minimum MSC criteria [88] for CD90 and CD105 positivity while lacking expression of CD34 and CD45. They also expressed additional MSC markers CD44 and CD56 and displayed classic MSC differentiation potential into adipocytes, chondrocytes, and osteocytes. At the same time, in behavior considered distinctive of pericytes, in coculture with human umbilical vein endothelial cells they were able to form network-like structures [87].

MSCs themselves have also been implicated in atherosclerosis [89]. MSCs expressing Oct-4, Stro-1, Sca-1, and Notch-1, for instance, were identified in the wall of a range of vessel segments such as the aortic arch, and thoracic and femoral arteries. These multipotent cells exhibited adipogenic, chondrogenic, and leiomyogenic potential [14, 15].

Our group, too, has identified a population of multipotent vascular stem cells (MVSCs) in the arterial medial and adventitial layers that could significantly contribute to the population of traditionally defined proliferative and synthetic SMCs in SMC culture and in neointima [25, 37]. Upon vascular injury (e.g., denudation injury), Sox10+ MVSCs are activated, become proliferative, and migrated from both medial and adventitial layers to contribute to neointima formation [25, 37]. In addition, some Sox10- cells became Sox10+, suggesting Sox10 may be a marker of activated cells (Fig. 1). In wound healing and scar formation, MVSC-like Sox10+ cells (which are also found in soft tissues around blood vessels and throughout the body) can differentiate into both myofibroblasts and SMCs [24]. Following the implantation of polymer vascular grafts for instance, these cells, rather than SMCs, are recruited to the outer surface of the grafts and gradually differentiate into SMCs [70], recapitulating some aspect of vascular development.

Of special note is that vessel-derived stem/progenitor cells as well as MSCs isolated from ApoE-/- mice respond to the inflammatory environment and undergo calcification in the form of significantly greater osteogenesis and chondrogenesis [90]. MVSCs can also differentiate mesenchymally into osteogenic, chondrogenic, and adipogenic cells in vitro [25] and in vivo (unpublished observation), suggesting a possible role for them in vascular fat accumulation and calcification. As CVCs, in contrast, can differentiate into osteogenic and chondrogenic cells but not adipogenic cells in vitro, it is possible that CVCs are derived from MVSCs that have partially differentiated. Because almost all VSCs share some characteristics of MSCs, it is also possible that MSCs are derived from one or multiple subpopulations of VSCs.

The adventitia is the outermost layer of a blood vessel and is composed of a collagen-rich extracellular matrix embedded with a mixture of cells. The complexity of cellular composition reflects the pivotal role of the adventitia in vascular remodeling. Indeed, of the three blood vessel layers, evidence for vascular stem/progenitor cell enrichment in the adventitia, specifically along its border with the media, is the most abundant and robust. Its significance makes physiological and anatomical sense. Proximity to the vasa vasorum, which connect to the peripheral circulation, enable vessel wall communication with otherwise removed stem cell niches including the aforementioned bone marrow [14, 15], and the pivotal role of vasa vasorum density, structural integrity, and expansion in atheroma development and complications is well documented [91].

In human arteries, in addition to the Sox10+ MVSCs we described in the previous section [25], a population of vascular wall-resident multipotent stem cells (VW-MPSCs) were isolated from the adventitia by Klein, et al. [92]. They expressed certain MSC surface markers (including Stro1, CD105, CD73, CD44, CD90 and CD29) and positivity for stem cell-associated transcription factors Oct4 and Sox2, and demonstrated lack of contaminating mature EC or EPCs and hematopoietic stem cells (HPCs) by negativity for CD31, CD34, CD45, CD68, CD11b, and CD19. These VW-MPSCs also demonstrated adipocyte, chondrocyte, and osteocyte differentiation in culture conditions. In vivo transplantation with human umbilical vein endothelial cells (HUVECs) into immunodeficient mice via Matrigel resulted in new vessel formation covered with VW-MPSC-derived pericyte- and smooth muscle-like cells, an effect enhanced by VEGF, FGF-2, and TGF1 stimulation [92]. These authors more recently identified that HOX genes may epigenetically regulate VW-MPSC differentiation into SMCs, potentially contributing to neointimal formation and tumor vascularization [93].

Sox10+ MVSCs in aorta ring ex vivo culture. Aorta rings of Sox10-Cre/Rosa-RFP mice were cultured ex vivo, and imaged by two-photon microscopy. Arrows indicate the emerging Sox10+ cells. Scale bar, 100 m.

Progenitors have also been derived from human veins, dubbed saphenous vein-derived progenitor cells (SVPs) for their specific location of origin. Assessing endothelial markers CD34, CD31, and von Willebrand factor (vWF) in these cells showed CD34+, CD31-, vWF-. These highly proliferative cells were found to be localized around adventitial vasa vasorum, and expressed pericyte/mesenchymal antigens as well as stem cell marker Sox2. In an ischemic hindlimb model in immunodeficient mice, intramuscular injection of SVPs improved neovascularization and blood flow recovery, and the cells established N-cadherin-mediated physical contact with the capillary endothelium by day 14 post-transplantation [94]. These therapeutic benefits of vein-derived adventitial stem cells have been replicated in other studies using mouse models of ischemia, with one beginning to look towards manufacturing these cells for human angina therapy [95-97]. Spindle shaped MSCs (CD13+, CD29+, CD44+, CD54+) have also been isolated from human varicose saphenous vein intima. Displaying a similar gene expression profile to bone marrow-derived MSCs, these could differentiate into osteoblasts, chondrocytes, and adipocytes [98].

In rodents, another important progenitor population, Sca-1+ stem cells, has been described in the adventitia along the medial border. This population also expresses other stem cell markers including c-kit, CD34, and Flk1 and was first identified by Hu et al. in the aortic roots of ApoE-/- mice [99]. They had demonstrated capacity to differentiate into SMCs in vivo, with LacZ-labeled Sca-1+ cells found in vein graft atherosclerotic lesions after transplantation in the adventitial space, implying the migration of Sca-1+ cells from the adventitia to the neointima [99]. Years later, the same group illustrated the multipotency of the cells by demonstrating in a decellularized vessel graft mouse model the cells' in vitro differentiation into SMCs (with PDGF) and ECs (with VEGF) [100]. Implications to reduce neointimal thickness by applying VEGF to the adventitial layer, promoting stem cell differentiation into ECs rather than SMCs, were made clear as well [100].

Other studies have since further implicated Sca-1+ stem cells in atherosclerosis and adventitial remodeling [28, 101, 102]. The later stages of atherosclerosis, for instance, mainly involve resident proliferating macrophages rather than those differentiated from bone marrow monocytes [27]. These local resident proliferating macrophages were found to be derived from a subpopulation of Sca-1+ stem cells, resident macrophage progenitors, that also expressed CD45 [28]. In aging, Sca1+ adventitial cells enriched for monocyte/macrophage markers and CD45 were shown to be depleted by 3-fold in mature versus young mice, raising the question of whether age-related vascular degeneration may be due to such effects on progenitors in the vascular wall [103].

Recently, Majesky et al. used two in vivo SMC lineage-tracing approaches and showed that some Sca1+ vascular adventitial progenitors (CD34+) are derived from differentiated SMCs, potentially thereby contributing to maintenance of the resident vascular progenitor cell population [33]. In an earlier study, Shankman et al. had suggested that SMCs could de-differentiate into progenitor-like cells capable of differentiating into MSC- and macrophage-like cells [32]. Interestingly, in both cases, KLF4 was identified as a key modulator of cell phenotypic changes. This intriguing relationship between SMCs and VSCs (or VSC-like cells) warrants further investigation.

Overall, although a human ortholog of Sca-1 has yet to be identified, study of pathways and mechanisms surrounding these cells have been of great value, and results suggest that locally manipulating microenvironment is a possible angle for treating atherosclerotic disease [104].

Pericytes play important roles in regulating microvascular stability and dynamics [105]. They were first described over a century ago, and defined as another type of vascular mural cell that surround microvessels, forming an incomplete envelope around ECs and found within the microvascular basement membrane [106]. Pericyte-like cells have also been reported in the inner intima (mostly subendothelium) in human arteries of all sizes [107]. Several markers have been used to identify pericytes, including NG2 [108], CD146 [109, 110], PDGFR, and -SMA [111].

In recent years, accumulating studies have discovered important roles for pericytes in development and diseases. Pericyte-like cells were identified in atherosclerotic lesions and thought to be one of the sources of atherosclerotic cells [83, 112], which may come from the vasa vasorum, a specialized microvessel inside large vessel walls [91]. Cells histologically characterized as true pericytes were also found to comprise a second net-like subendothelial tissue layer, which combines with the endothelium to form the intimal barrier in healthy human and bovine microvasculature. In contrast with the endothelium, these pericytes were highly prothrombotic when exposed to serum and display overshooting growth behavior in endothelium-denuded vascular areas, making them potential key players in atherosclerosis, thrombosis, and thrombotic side-effects of venous coronary bypass grafting [92].

In the porcine aortic media, novel vascular progenitor cells with pericyte- and MSC-like properties were also found capable of differentiating into osteocytes, chondrocytes, and adipocytes [87]. Pericytes around microvessels in skeletal muscle are another type of myogenic progenitor cell distinct from satellite cells [113, 114].

Pericytes in multiple organs have been reported to have properties of MSCs [111]. Moreover, pericytes can differentiate into myofibroblasts and are another important cellular source of organ fibrosis [115-117]. It is likely that pericytes include subpopulations of stem cells or progenitors. In our recent work, we found Sox10+ stem cells in the stroma of subcutaneous connective tissues which had the same properties as MVSCs in large vessels [24, 25]. These Sox10+ stem cells are precursors of pericytes and fibroblasts, as described in the previous section, and contribute to both fibrosis and microvessel formation during tissue repair and regeneration [24]. Gli1+ stem cells had similarly wide distribution as the Sox10+ stem cells and were found in the perivascular space and also adventitial layer of large arteries. They could differentiate into myofibroblasts contributing to organ fibrosis, and neointimal SMCs contributing to atherosclerotic lesions and arterial calcification [115, 118].

Therapeutically, two separate studies examined the benefit of pericyte transplantation in mouse models of myocardial infarction. They found that pericytes from both saphenous vein [119] and skeletal muscle [120] attenuated left ventricular dilation, improved cardiac contractility and ejection fraction, reduced myocardial fibrosis and scarring, and improved neovascularization and angiogenesis. Saphenous vein-derived pericytes also reduced cardiomyocyte apoptosis, attenuated vascular permeability, and improved myocardial blood flow [119], while the skeletal muscle-derived pericytes significantly diminished host inflammatory cell infiltration at the infarct site as well [120]. Both studies attributed benefits to cellular interactions and paracrine effects [119, 120].

Dellavalle, et al. demonstrated the skeletal muscle-regenerating properties of both normal human pericytes and dystrophin-reprogrammed human Duchenne patient pericytes when transplanted into mouse models of muscular dystrophy [113]. In small-diameter tissue-engineered vascular grafts (TEVGs), exogenously seeded pericytes improved maintenance of patency after TEVG implantation into the aorta of rats (100% at 8 weeks, versus 38% unseeded controls) [121]. An endogenous approach has met with similar success, where promoting the differentiation of Sca-1+ stem/progenitor cells into the endothelial lineage has reduced neointimal thickness by up to 80% [100]. Altogether, these findings highlight stem cells as important players and potentially significant therapeutic targets in vascular remodeling, and underscore the multifactorial complexity of vascular disease pathogenesis.

The microenvironment plays important roles in regulating vascular cell function and the stem cell renewal and fate decision, and includes both biochemical factors (e.g., growth factors, cytokines) and biophysical factors (e.g., extracellular matrix, stiffness, flow shear stress and mechanical stretch).

Inflammatory cytokines, in addition to adhesion molecules, govern recruitment of relevant immune cells to the arterial wall in atherosclerosis. Beyond these traditional roles in regulating cell function and homeostasis, though, and notably for our discussion here, in recent years cytokines have also been found to regulate stem cell recruitment and activation during vascular remodeling [122, 123]. Cytokines like stromal cell-derived factor 1 (SDF-1), for example, has been shown to recruit bone marrow EPCs to form microvessels in hindlimb ischemic angiogenesis [124, 125] and to promote adventitial Sca1+ stem cells to migrate through vein graft walls and differentiate into neointimal SMCs [126]. In advanced atherosclerotic plaques, it is also believed that SDF-1 recruits SMC progenitor cells from bone marrow to the fibrotic cap [127]. Another cytokine, tumor necrosis factor- (TNF-), induces adventitial Sca1+ stem cells to differentiate into ECs, while suppressing SMC gene activation [128]. Growth factors like VEGF and PDGF-BB/TGF-1 can stimulate adventitial and medial stem cells to differentiate into ECs and SMCs, respectively [85, 100].

Among the biophysical factors found important for vascular cells, local disturbed flow is a major factor that induces EC dysfunction in the branches and curvatures of the arterial tree [129]. Disturbed flow shear stress can induce a series of intracellular signaling pathways in ECs and activate proliferative and inflammatory gene expression, initiating neointimal formation and atherosclerosis even in newborns [129, 130].

The extracellular matrix (ECM) is also important in regulating vascular dynamics. Subendothelial matrix proteoglycans are thought to contribute to lipid retention in the early stages of atherosclerosis [29]. ECM stiffness and embedded growth factors are critical in regulating cell functions. Our previous work has showed that stiff surfaces, together with TGF, promoted MSC differentiation into SMCs in vitro [131]. Collagen IV, too, has been reported to be critical in promoting embryonic stem cell differentiation into Sca-1+ stem cells, and to act together with aforementioned cytokines and growth factors to promote differentiation [132, 133]. Mechanical stretch and microtopography can regulate SMC differentiation and function as well [134, 135].

To date, the niche of VSCs has not been well defined. Although we know connection to the peripheral circulation via the vasa vasorum enables vessel niche communication with other stem cell niches like the bone marrow, how VSCs are activated by such communication, inflammatory signals, and local microenvironmental changes remains to be investigated.

As our understanding of the importance and mechanism of stem and progenitor cell involvement in human vascular remodeling has evolved, two therapeutic angles have arisen: 1) influencing endogenous VSC behavior to prevent initiation and progression of disease, and 2) exogenous stem cell delivery to promote disease reversal and healing of tissue injury. The application of more immature stem cells with greater differentiation potential such as embryonic and induced pluripotent stem cells to cardiovascular disease (including myocardial infarction, vascular regeneration in coronary and peripheral artery disease) has been reviewed elsewhere [136-138]. Adult stem cells such as those we have discussed pose multiple advantages in their accessibility (e.g., the stromal vascular fraction of adipose aspirates contain human blood vessel fragments; coronary bypass surgery makes pieces of aorta or segments of internal thoracic artery, radial artery, and saphenous vein readily available), decreased risk of uncontrolled differentiation (e.g., teratomas), and immune-privileged nature (in the case of MSCs and pericytes) that enables allogeneic use as well [139].

That said, clinical trials and therapies utilizing such VSCs are still sadly lacking. No human clinical trials to date have examined application of pericytes or resident VSCs for vascular disease. MSCs and EPCs, perhaps because of the broadness of their definition, have accumulated a more substantial body of clinically relevant evidence. The majority of clinical trials for atherosclerosis and diseases for which it is the primary cause - such as angina, myocardial ischemia, and ischemic stroke, all diseases primarily of the macrovasculature - utilize MSCs and EPCs instead. These trials focus, too, more on stem cell/progenitors for disease treatment rather than disease prevention. Limited evidence for underlying mechanisms suggests stem cell angiogenic roles play a large part in measurable therapeutic benefit; evidence for a therapeutic role in neointimal regression, in contrast, is lacking [140, 141]. It should be noted that MSCs and EPCs have also been utilized therapeutically to promote angiogenesis in diseases of the microvasculature such as diabetic ischemia-induced chronic wounds [53, 142] and peripheral occlusive disease [140, 141, 143], but we focus on macrovascular plaque-related diseases here instead.

In 2013, a phase III trial for refractory angina locally transplanted (G-CSF-stimulated) autologous blood cells positive for the EPC marker CD34 via percutaneous intramyocardial injection. The trial showed preliminary results consistent with those of earlier phase studies [144], although with higher placebo effects than previously detected, and animal studies lead us to believe benefit is derived from cell contribution to myocardial neoangiogenesis, and possible differentiation into cardiomyocytes and ECs [145-147]. If completed, it would have provided the requisite information for regulatory approval of the first cellular therapeutic for a cardiovascular indication [148]. Results may merit an expanded examination of therapeutic EPC transplantation, perhaps in combination with other vasculogenic mediators and scaffolds to improve EPC survival and function.

Other clinical trials have also attempted direct exogenous transplantation of adult bone-marrow-derived stem cells, but for myocardial ischemia (MI) and ischemic stroke patients. Several have found such intracoronary transplantation improves regional systolic function recovery and infarct size reduction in MI patients [149, 150], and a number of recent meta-analyses have confirmed improvements in not only left ventricular contractility after therapy [151-153] but also decreased mortality, acute MI recurrence, and readmission for heart failure [150, 152]. Still, effects of transplantation on infarct volume and remodeling are contradictory and inconclusive [150, 152-156]. BM cells, rather than incorporating, may prompt ischemic tissues to secrete paracrine signals (e.g., angiogenic factors); these signals in conjunction with transdifferentiation potential may underlie functional recovery [149, 156-158].

In stroke, promising results in experimental models [159] prompted clinical trials of intra-arterial or intravenous transplantation of autologous bone marrow mononuclear cells (including CD34+ progenitors). A phase I/II clinical trial in middle cerebral artery stroke patients transplanted 5-9 days after stroke found that changes in serum levels of GM-CSF, PDGF-BB, and MMP-2 associated with better functional outcomes were induced; however, varied impact on functional outcomes themselves was not measured [160]. Another phase II randomized control trial (RCT) found that cell therapy was safe, but had no beneficial effect on stroke outcome [161]. The first trial to explore dose-dependent efficacy of intra-arterial transplantation of bone marrow mononuclear cells in moderate-to-severe acute ischemic stroke patients is currently ongoing (IBIS trial, prospective phase II RCT) [162]. Despite promising animal studies, which suggest BM cell-based treatments can benefit endogenous neurorestoration by promoting contralesional pyramidal axon sprouting and preservation, increasing neurotrophic factor secretion, and possible synergistic effects between microvascular angiogenesis and neurogenesis, demonstrable long-term clinical therapeutic benefit of cell therapies for stroke is still being determined [141].

Secondary stimulation of endogenous progenitors has also been attempted. Granulocyte colony-stimulating factor (G-CSF) is one agent that can stimulate the bone marrow to release EPCs, in addition to release of granulocytes and hematopoietic stem cells [18]. Multiple clinical trials, encouraged by prior positive results in various animals [163], sought to assess its utility in upregulating endogenous EPC release in patients with ischemic heart disease. Results, however, have been mixed: although one study found an improvement of severe ischemia in severe MI patients [164] and a meta-analysis of seven RCTs including 364 acute MI patients found improvement of left ventricular ejection fraction (LVEF) [165], others (including an RCT and a meta-analysis of ten clinical trials including 445 patients) concluded no impact on infarct size, LV function, or coronary restenosis [166-168]. Interestingly, physical exercise, strongly established by many large-scale epidemiological studies as being robustly associated with decreased cardiovascular mortality and potent primary and secondary CVD prevention [169-173], has been found to mobilize EPCs from the bone marrow and is thought to exert its benefits mechanistically via the maintenance of an intact endothelial layer [174].

Using G-CSF in stroke patients has been less studied. A phase IIb RCT concluded in 2012 that G-CSF successfully and safely increased CD34+ cells by 9.5-fold relative to placebo, with a trend of reducing ischemic lesion volume [175]. Further study, though, is necessary.

The majority of completed clinical trials (as reported on clinicaltrials.gov) involving MSC transplantation for vascular disease focuses on treatment of myocardial ischemia, finding that treatment is tolerable and safe with improvements seen in metrics such as LVEF [176-178] and global EF [179], LV end-systolic [176, 178, 179] and diastolic volumes [178], and functional walk and cardiac tests [176] and global symptom scores [177]. A phase I/II clinical trial for patients with severe stable coronary artery disease and refractory angina transplanted autologous bone marrow-derived MSCs into their viable myocardium, and found similarly promising results. The trial showed sustained safety three years post-transplantation, significant clinical improvements in symptomatic and functional metrics, as well as reduced hospital admissions for CV disease [180].

Delivery route of MSCs, furthermore, was found by meta-analysis of six clinical trials involving 334 MI patients to shape efficacy of treatment. Greatest improvement in LVEF was seen if transendocardial injection and intravenous infusion, rather than intracoronary infusion, were used to deliver MSCs [181].

In 2015 an observational clinical study for coronary atherosclerosis examined outcomes of plasmonic resonance therapy using silica-gold nanoparticles that had been incubated with allogeneic mesenchymal CD73+ CD105+ stem-progenitor cells. Results showed highly safe, significant plaque regression relative to stenting controls (reduction of total atheroma volume up to 60mm3, or 37.8% of plaque burden, relative to current maximal success of conventional drugs of 6-14mm3) and late lumen enlargement without arterial remodeling [182].

Overall, although animal and preliminary clinical studies have revealed much promise, there remains much to be done in understanding the mechanism of VSC therapeutic benefits in order to appropriately target them for effective therapy.

Strong evidence has accumulated to demonstrate the involvement of various stem and progenitor cells in vascular regeneration and disease, including atherosclerotic neointimal formation. These stem cells display a nonuniform distribution both across the vessel wall as well as across different vascular territories, a distribution perhaps contributing to explanations of why different vascular segments may have variable susceptibility to vascular disease despite similar hemodynamics and environment [183]. Different populations of vascular cells, including SMCs, ECs, inflammatory cells (including macrophage and dendritic cell progenitors), and stem cells, may interact with and be subject to regulation by each other and by the local microenvironment during neointimal thickening. Recent studies show exosomes, nanometer lipid bilayer signaling particles secreted by cells with important roles in many physiological and pathological processes [184-187], have a hand in this regulation by mediating vascular calcification as found in atherosclerosis [188], atheroprotective communication between ECs and SMCs [189], and anti-inflammatory effects of MSCs [187]. Exosomes could thus be therapeutic targets of interest as well [190].

Identifying proper cellular targets (e.g., using screening methods such as RNA-sequencing and epigenetic profiling to characterize VSCs, along with other techniques such as laser microdissection and immunofluorescence to identify key VSC markers) and understanding the underlying regulatory mechanisms will facilitate the development of successful therapies for vascular disease. Given their differentiation potential into SMCs and ECs, these stem cells could also be good cellular sources for fabricating vascular grafts or otherwise promoting vascular regeneration.

Far as the field has come, several critical questions remain to be addressed. First, given the diversity of stem cells discovered by different research groups, confirming whether these cells are distinct populations and determining their relationship with proliferative/synthetic SMCs will be necessary. It will be helpful to obtain consensus on specific panels of markers to define different stem cell populations. Examining to what degree the difference in their marker expression profiles may be a result of different culture conditions in vitro, too, will be of importance.

Second, the niche of VSCs needs to be further characterized to define the macro and microenvironmental factors that maintain VSCs in a quiescent versus activated state, and how such factors promote healthy survival.

Third, stem cell fate needs to be determined in long-term in vivo experiments. However, stem cells may become activated and differentiated quickly at the early phase of neointimal thickening in vivo, which makes capture of the phenotype by immunohistology difficult. Genetic lineage tracing techniques would address this problem, if obstacles of selection of good markers and of availability of transgenic animal models can be surmounted. Such techniques could also address the relative contributions of different cell types, and multi-color reporter mice could be used to investigate heterogeneity within the same population.

Fourth, the behavior of VSCs under various pathological conditions should be elucidated. Stem cell activation and differentiation are regulated by various microenvironmental factors. Changes in biochemical and biophysical factors in a disease state and the effects of these factors, individually or in combination, may have profound effects on stem cell functions. Conversely, taking creative inspiration from current successful therapies for atherosclerosis and brainstorming approaches for cellular therapies to target their same mechanisms could yield therapies with fewer side-effects and more targeted results. For instance, any conversation on atherosclerosis would be incomplete without mention of statins, the current mainstay of treatment [191, 192]. Research has shown that, independent of cholesterol reduction, statins may exert their beneficial effects via EPC mobilization. This may be a promising direction for future therapies [52]. Similarly, piggybacking on the putative plaque-stabilizing mechanism of statins by use of the chemokine SDF-1 to recruit bone marrow-derived SM progenitor cells to the fibrous cap has yielded increases in cap thickness without altering artery diameter in mice [127]. This finding may prove useful for unstable atherosclerosis if further studies in large animals and humans continue to yield promising results.

Fifth, especially with sourcing of vascular wall MSCs becoming increasingly feasible [17], there is great promise in cell therapies if details on differences in identity and manufacturing based on specific vascular and cell source can be fleshed out. Despite their mechanistic significance, EPCs and other progenitors without immune-privilege, in contrast with MSCs and pericytes which do, may pose a challenge in clinical application if the goal is exogenous transplantation [139]. Endogenous recruitment and processes may be more feasible for these other progenitors. Although stem cell transplantation has been proven to be safe and benefit tissue regeneration, the mechanisms of benefit, too, are unclear at present. Overall, clinical trials certainly remain of value - as phenomena in humans are ultimately distinct from those in animals - but it is clear that such applications are yet in the early stages. The mixed results clearly indicate that an improved understanding of underlying mechanisms is necessary not only for effective design of therapeutic translation and study, but also for interpretation of results. Ongoing risk and safety assessment will continue to be necessary in parallel.

Finally, besides delivery of exogenous stem cells for therapies, the potential of endogenous recruitment or of using stem cells as novel targets of therapies needs to be further investigated in vitro and in vivo. In vitro isolated VSCs can be used for drug screening. A well-defined culture model, such as co-culture with SMCs, mechanical loading, and 3D culture that mimics the in vivo microenvironment, would be valuable. Blood vessel tissue ex vivo culture is better than cell culture as it mimics the niche of cell-cell interactions and native extracellular matrix, which may be useful when combined with tissue clarity techniques and transgenic animal models. All these new tools and technologies will continue to facilitate further discoveries in vascular stem cell biology, enabling development of diagnostic and therapeutic strategies with unprecedented efficacy and capability to combat vascular disease and promote regeneration.

CVD: cardiovascular disease; EC: endothelial cell; EPC: endothelial progenitor cell; SMC: smooth muscle cell; VSC: vascular stem cell; MSC: mesenchymal stem cell; MVSC: multipotent vascular stem cell; CVC: calcifying vascular cells; -SMA: smooth muscle -actin; SM-MHC: smooth muscle myosin heavy chain.

This work was supported by grants from the National Institutes of Health (HL117213 and HL121450 to S.L.) and the Medical Scientist Training Program at UCLA (NIH T32 GM008042 to L.L.).

The authors have declared that no competing interest exists.

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Adult Stem Cells in Vascular Remodeling - Theranostics

Cardiac Stem Cells Comments Off on Adult Stem Cells in Vascular Remodeling – Theranostics | February 12th, 2019

Regenerative medicine may be defined as the process of replacing or "regenerating" human cells, tissues or organs to restore or establish normal function. This field holds the promise of regenerating damaged tissues and organs in the body by replacing damaged tissue or by stimulating the body's own repair mechanisms to heal tissues or organs. Regenerative medicine also may enable scientists to grow tissues and organs in the laboratory and safely implant them when the body is unable to heal itself. Current estimates indicate that approximately one in three Americans could potentially benefit from regenerative medicine.

Regenerative Medicine refers to a group of biomedical approaches to clinical therapies that may involve the use of stem cells. Examples include cell therapies (the injection of stem cells or progenitor cells); immunomodulation therapy (regeneration by biologically active molecules administered alone or as secretions by infused cells); and tissue engineering (transplantation of laboratory grown organs and tissues). While covering a broad range of applications, in practice the latter term is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, skin). Often, the tissues involved require certain mechanical and structural properties for proper functioning. The term has also been applied to efforts to perform specific biochemical functions using cells within an artificially-created support system (e.g., artificial pancreas or liver).

Cord blood stem cells are being explored in several applications including Type 1 diabetes to determine if the cells can slow the loss of insulin production in children; cardiovascular repair to observe whether cells selectively migrate to injured cardiac tissue, improve function and blood flow at the site of injury and improve overall heart function; and central nervous system applications to assess whether cells migrate to the area of brain injury alleviating mobility related symptoms, and repair damaged brain tissue (such as that experienced with cerebral palsy). Cord blood stem cells likely will be an important resource as medicine advances toward harnessing the body's own cells for treatment. Because a person's own (autologous) stem cells can be infused back into that individual without being rejected by the body's immune system, autologous cord blood stem cells have become an increasingly important focus of regenerative medicine research.

Regenerative medicine has made its way into clinical practice with the use of materials that are able to assist in the healing process by releasing growth factors and cytokines back into the damaged tissue (e.g., (chronic) wound healing). As additional applications are researched, the fields of regenerative medicine and cellular therapies will continue to merge and expand, potentially treating many disease conditions and improving health for a variety of diseases and health conditions.

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Adult stem cells are undifferentiated cells, found throughout the body after development, that multiply by cell division to replenish dying cells and regenerate damaged tissues. Also known as somatic stem cells (from Greek , meaning of the body), they can be found in juvenile as well as adult animals and humans, unlike embryonic stem cells.

Scientific interest in adult stem cells is centered on their ability to divide or self-renew indefinitely, and generate all the cell types of the organ from which they originate, potentially regenerating the entire organ from a few cells.[1] Unlike for embryonic stem cells, the use of human adult stem cells in research and therapy is not considered to be controversial, as they are derived from adult tissue samples rather than human embryos designated for scientific research. They have mainly been studied in humans and model organisms such as mice and rats.

A stem cell possesses two properties:

Hematopoietic stem cells are found in the bone marrow and umbilical cord blood and give rise to all the blood cell types.[3]

Mammary stem cells provide the source of cells for growth of the mammary gland during puberty and gestation and play an important role in carcinogenesis of the breast.[4] Mammary stem cells have been isolated from human and mouse tissue as well as from cell lines derived from the mammary gland. Single such cells can give rise to both the luminal and myoepithelial cell types of the gland, and have been shown to have the ability to regenerate the entire organ in mice.[4]

Intestinal stem cells divide continuously throughout life and use a complex genetic program to produce the cells lining the surface of the small and large intestines.[5] Intestinal stem cells reside near the base of the stem cell niche, called the crypts of Lieberkuhn. Intestinal stem cells are probably the source of most cancers of the small intestine and colon.[6]

Mesenchymal stem cells (MSCs) are of stromal origin and may differentiate into a variety of tissues. MSCs have been isolated from placenta, adipose tissue, lung, bone marrow and blood, Wharton's jelly from the umbilical cord,[7] and teeth (perivascular niche of dental pulp and periodontal ligament).[8] MSCs are attractive for clinical therapy due to their ability to differentiate, provide trophic support, and modulate innate immune response.[7] These cells have the ability to differentiate into various cell types such as osteoblasts, chondroblasts, adipocytes, neuroectodermal cells, and hepatocytes.[9] Bioactive mediators that favor local cell growth are also secreted by MSCs. Anti-inflammatory effects on the local microenvironment, which promote tissue healing, are also observed. The inflammatory response can be modulated by adipose-derived regenerative cells (ADRC) including mesenchymal stem cells and regulatory T-lymphocytes. The mesenchymal stem cells thus alter the outcome of the immune response by changing the cytokine secretion of dendritic and T-cell subsets. This results in a shift from a pro-inflammatory environment to an anti-inflammatory or tolerant cell environment.[10][11]

Endothelial stem cells are one of the three types of multipotent stem cells found in the bone marrow. They are a rare and controversial group with the ability to differentiate into endothelial cells, the cells that line blood vessels.

The existence of stem cells in the adult brain has been postulated following the discovery that the process of neurogenesis, the birth of new neurons, continues into adulthood in rats.[12] The presence of stem cells in the mature primate brain was first reported in 1967.[13] It has since been shown that new neurons are generated in adult mice, songbirds and primates, including humans. Normally, adult neurogenesis is restricted to two areas of the brain the subventricular zone, which lines the lateral ventricles, and the dentate gyrus of the hippocampal formation.[14] Although the generation of new neurons in the hippocampus is well established, the presence of true self-renewing stem cells there has been debated.[15] Under certain circumstances, such as following tissue damage in ischemia, neurogenesis can be induced in other brain regions, including the neocortex.

Neural stem cells are commonly cultured in vitro as so called neurospheres floating heterogeneous aggregates of cells, containing a large proportion of stem cells.[16] They can be propagated for extended periods of time and differentiated into both neuronal and glia cells, and therefore behave as stem cells. However, some recent studies suggest that this behaviour is induced by the culture conditions in progenitor cells, the progeny of stem cell division that normally undergo a strictly limited number of replication cycles in vivo.[17] Furthermore, neurosphere-derived cells do not behave as stem cells when transplanted back into the brain.[18]

Neural stem cells share many properties with haematopoietic stem cells (HSCs). Remarkably, when injected into the blood, neurosphere-derived cells differentiate into various cell types of the immune system.[19]

Olfactory adult stem cells have been successfully harvested from the human olfactory mucosa cells, which are found in the lining of the nose and are involved in the sense of smell.[20] If they are given the right chemical environment these cells have the same ability as embryonic stem cells to develop into many different cell types. Olfactory stem cells hold the potential for therapeutic applications and, in contrast to neural stem cells, can be harvested with ease without harm to the patient. This means they can be easily obtained from all individuals, including older patients who might be most in need of stem cell therapies.

Hair follicles contain two types of stem cells, one of which appears to represent a remnant of the stem cells of the embryonic neural crest. Similar cells have been found in the gastrointestinal tract, sciatic nerve, cardiac outflow tract and spinal and sympathetic ganglia. These cells can generate neurons, Schwann cells, myofibroblast, chondrocytes and melanocytes.[21][22]

Multipotent stem cells with a claimed equivalency to embryonic stem cells have been derived from spermatogonial progenitor cells found in the testicles of laboratory mice by scientists in Germany[23][24][25] and the United States,[26][27][28][29] and, a year later, researchers from Germany and the United Kingdom confirmed the same capability using cells from the testicles of humans.[30] The extracted stem cells are known as human adult germline stem cells (GSCs)[31]

Multipotent stem cells have also been derived from germ cells found in human testicles.[32]

To ensure self-renewal, stem cells undergo two types of cell division (see Stem cell division and differentiation diagram). Symmetric division gives rise to two identical daughter cells, both endowed with stem cell properties, whereas asymmetric division produces only one stem cell and a progenitor cell with limited self-renewal potential. Progenitors can go through several rounds of cell division before finally differentiating into a mature cell. It is believed that the molecular distinction between symmetric and asymmetric divisions lies in differential segregation of cell membrane proteins (such as receptors) between the daughter cells.

Discoveries in recent years have suggested that adult stem cells might have the ability to differentiate into cell types from different germ layers. For instance, neural stem cells from the brain, which are derived from ectoderm, can differentiate into ectoderm, mesoderm, and endoderm.[33] Stem cells from the bone marrow, which is derived from mesoderm, can differentiate into liver, lung, GI tract and skin, which are derived from endoderm and mesoderm.[34] This phenomenon is referred to as stem cell transdifferentiation or plasticity. It can be induced by modifying the growth medium when stem cells are cultured in vitro or transplanting them to an organ of the body different from the one they were originally isolated from. There is yet no consensus among biologists on the prevalence and physiological and therapeutic relevance of stem cell plasticity. More recent findings suggest that pluripotent stem cells may reside in blood and adult tissues in a dormant state.[35] These cells are referred to as "Blastomere Like Stem Cells"[36] and "very small embryonic like" "VSEL" stem cells, and display pluripotency in vitro.[35] As BLSC's and VSEL cells are present in virtually all adult tissues, including lung, brain, kidneys, muscles, and pancreas[37] Co-purification of BLSC's and VSEL cells with other populations of adult stem cells may explain the apparent pluripotency of adult stem cell populations. However, recent studies have shown that both human and murine VSEL cells lack stem cell characteristics and are not pluripotent.[38][39][40][41]

Stem cell function becomes impaired with age, and this contributes to progressive deterioration of tissue maintenance and repair.[42] A likely important cause of increasing stem cell dysfunction is age-dependent accumulation of DNA damage in both stem cells and the cells that comprise the stem cell environment.[42] (See also DNA damage theory of aging.)

Adult stem cells can, however, be artificially reverted to a state where they behave like embryonic stem cells (including the associated DNA repair mechanisms). This was done with mice as early as 2006[43] with future prospects to slow down human aging substantially. Such cells are one of the various classes of induced stem cells.

Adult stem cell research has been focused on uncovering the general molecular mechanisms that control their self-renewal and differentiation.

Adult stem cell treatments have been used for many years to successfully treat leukemia and related bone/blood cancers utilizing bone marrow transplants.[47] The use of adult stem cells in research and therapy is not considered as controversial as the use of embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo.

Early regenerative applications of adult stem cells has focused on intravenous delivery of blood progenitors known as Hematopetic Stem Cells (HSC's). CD34+ hematopoietic Stem Cells have been clinically applied to treat various diseases including spinal cord injury,[48] liver cirrhosis [49] and Peripheral Vascular disease.[50] Research has shown that CD34+ hematopoietic Stem Cells are relatively more numerous in men than in women of reproductive age group among spinal cord Injury victims.[51] Other early commercial applications have focused on Mesenchymal Stem Cells (MSCs). For both cell lines, direct injection or placement of cells into a site in need of repair may be the preferred method of treatment, as vascular delivery suffers from a "pulmonary first pass effect" where intravenous injected cells are sequestered in the lungs.[52] Clinical case reports in orthopedic applications have been published. Wakitani has published a small case series of nine defects in five knees involving surgical transplantation of mesenchymal stem cells with coverage of the treated chondral defects.[53] Centeno et al. have reported high field MRI evidence of increased cartilage and meniscus volume in individual human clinical subjects as well as a large n=227 safety study.[54][55][56][57] Many other stem cell based treatments are operating outside the US, with much controversy being reported regarding these treatments as some feel more regulation is needed as clinics tend to exaggerate claims of success and minimize or omit risks.[58]

The therapeutic potential of adult stem cells is the focus of much scientific research, due to their ability to be harvested from the parent body that is females during the delivery.[59][60][61] In common with embryonic stem cells, adult stem cells have the ability to differentiate into more than one cell type, but unlike the former they are often restricted to certain types or "lineages". The ability of a differentiated stem cell of one lineage to produce cells of a different lineage is called transdifferentiation. Some types of adult stem cells are more capable of transdifferentiation than others, but for many there is no evidence that such a transformation is possible. Consequently, adult stem therapies require a stem cell source of the specific lineage needed, and harvesting and/or culturing them up to the numbers required is a challenge.[62][63] Additionally, cues from the immediate environment (including how stiff or porous the surrounding structure/extracellular matrix is) can alter or enhance the fate and differentiation of the stem cells.[64]

Pluripotent stem cells, i.e. cells that can give rise to any fetal or adult cell type, can be found in a number of tissues, including umbilical cord blood.[65] Using genetic reprogramming, pluripotent stem cells equivalent to embryonic stem cells have been derived from human adult skin tissue.[66][67][68][69][70] Other adult stem cells are multipotent, meaning they are restricted in the types of cell they can become, and are generally referred to by their tissue origin (such as mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, etc.).[71][72] A great deal of adult stem cell research has focused on investigating their capacity to divide or self-renew indefinitely, and their potential for differentiation.[73] In mice, pluripotent stem cells can be directly generated from adult fibroblast cultures.[74]

In recent years, acceptance of the concept of adult stem cells has increased. There is now a hypothesis that stem cells reside in many adult tissues and that these unique reservoirs of cells not only are responsible for the normal reparative and regenerative processes but are also considered to be a prime target for genetic and epigenetic changes, culminating in many abnormal conditions including cancer.[75][76] (See cancer stem cell for more details.)

Adult stem cells express transporters of the ATP-binding cassette family that actively pump a diversity of organic molecules out of the cell.[77] Many pharmaceuticals are exported by these transporters conferring multidrug resistance onto the cell. This complicates the design of drugs, for instance neural stem cell targeted therapies for the treatment of clinical depression.

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All the organs and tissues of the body have adult stem cells for regenerating cells in case of injury or disease. As we age, adult stem cells gradually lose the ability to differentiate into functional tissue-specific cells. For example, cardiac muscle stem cells exist but elderly people have only one half the number of cardiac stem cells found in young people. Thus, adult stem cells become more dysfunctional as we age, causing progressively increased organ and tissue dysfunction.

An example of the aging role of adult stem cells is your skin continually losing dead cells, so that adult stem cells must continuously replenish the dying skin cells. With age, there are progressively fewer functional skin stem cells. Skin cell turnover slows, leading to thinner, dryer, less elastic skin that loses its youthful beauty. Hair thins and turns grey as functional hair follicle stem cells decline. Vision, hearing, smell, taste, and touch slowly decline with age, as the declining stem cell populations responsible for maintaining these functions are unable to fully rejuvenate.

Stimulating adult stem cell populations is not a simple task. If the proliferation of adult stem cells is over stimulated, then you may get overgrowth of tissues or a potential tumor. StemCell Maxum is a dietary supplement designed to improve the function of your existing stem cells. When an organ or tissue is damaged, it emits natural signals that new cells are needed to replace old or damaged cells. StemCell Maxum supports the adult stem cells that respond to provide new replacement cells.

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Theranostics 2017; 7(7):2067-2077. doi:10.7150/thno.19427

Review

Kiwon Ban1, Seongho Bae2, Young-sup Yoon2, 3

1. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong;2. Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia, USA;3. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea.

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.

Keywords: Cardiomyocytes, hPSCs

Heart failure is the leading cause of death worldwide [1]. Approximately 6 million people suffer from heart failure in the United States every year [1]. Despite this high incidence, existing surgical and pharmacological interventions for treating heart failure are limited because these approaches only delay the progression of the disease; they cannot directly repair the damaged hearts [2]. In the case of large myocardial infarction (MI), patients progress to heart failure and die within short time from the onset of symptoms [3].

The adult human heart has minimal regenerative capacity, because during mammalian development, the proliferative capacity of cardiomyocytes (CMs) progressively diminishes and becomes terminally differentiated shortly after birth [4].Therefore, once CMs are damaged, they are rarely restored [5]. When MI occurs, the infarcted area is easily converted to non-contractile scar tissue due to loss of CMs and replacement by fibrosis [6]. Development of a fibroblastic scar initiates a series of events that lead to adverse remodeling, hypertrophy, and eventual heart failure [2, 3, 7].

While heart transplantation is considered the most viable option for treating advanced heart failure, the number of available donor hearts is always less than needed [6]. Therefore, more realistic therapeutic options have been required [2]. Accordingly, over the past two decades, cell-based cardiac repair has been intensively pursued [2, 7]. Several different cell types have been tested and varied outcomes were obtained. Indeed, the key factor for successful cell-based cardiac repair is to find the optimal cell type that can restore normal heart function. Naturally, CMs have been considered the best cell type to repair a damaged heart [8]. In fact, many scientists hypothesized that implanted CMs would survive in damaged hearts and form junctions with host CMs and synchronously contract with the host myocardium [9]. In fact, animal studies with primary fetal or neonatal CMs demonstrated that transplanted CMs could survive in infarcted hearts [9-11]. These primary CMs reduced scar size, increased wall thickness, and improved cardiac contractile function with signs of electro-mechanical integration [9-11]. These studies strongly suggest that CMs can be a promising source to repair the heart. However, the short supply and ethical concerns disallow using primary human CMs. In a patient with ischemic cardiomyopathy, about 40-50% of the CMs are lost in 40 to 60 grams of heart tissue [7]. Even if we seek to regenerate a fairly small portion of the damaged myocardium, a large number of human primary CMs would be required, which is impossible.

Accordingly, CMs differentiated from human pluripotent stem cells (hPSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have emerged as a promising option for candidate CMs for cell therapy [12, 13]. hPSCs have many advantages as a source for CMs. First, hPSCs have obvious cardiomyogenic potential. hPSC derived-CMs (hPSC-CMs) possess a clear cardiac phenotype, displaying spontaneous contraction, cardiac excitation-contraction (EC) coupling, and expression of cardiac transcription factors, cardiac ion channels, and cardiac structural proteins [14, 15]. Second, undifferentiated hPSCs and their differentiated cardiac progeny display significant proliferation capacity, allowing generation of a large number of hPSC-CMs. Lastly, many pre-clinical studies demonstrated that implantation of hPSC-CMs can repair injured hearts and improve cardiac function [16-19]. Histologically, implanted hPSC-CMs are engrafted, aligned and coupled with the host CMs in a synchronized manner [16-19].

In the last two decades, various protocols for differentiating hPSCs into CMs have been developed to improve the efficiency, purity and clinical compatibility [20] [18]. The reported differentiation methods include, but are not limited to: differentiation via embryoid body (EB) formation [20], co-culture with END-2 cells [18], and monolayer culture [15, 21, 22]. The EB-mediated CM differentiation protocol is one of the most widely employed methods due to its simple procedure and low cost. However, it often becomes labor-intensive to produce scalable EBs for further differentiation, which makes it difficult for therapeutic applications. EB-mediated differentiation also produces inconsistent results, showing beating CMs from 5% to 70% of EBs. Recently, researchers developed monolayer methods to complement the problems of EB-based methods [15, 21, 22]. In one representative protocol, hPSCs are cultured at a high density (up to 80%) and treated with a high concentration of Activin A (100 ng/ml) for 1 day and BMP4 (10 ng/ml) for 4 days followed by continuous culture on regular RPMI media with B27 [15]. This protocol induces spontaneous beating at approximately 12 days and produces approximately 40% CMs after 3 weeks. These hPSC-CMs can be further cultured in RPMI-B27 medium for another 2-3 weeks without significant cell damage [15]. However, these protocols use media with proprietary formulations, which complicates clinical application. As shown, most monolayer-based methods employ B27, which is a complex mix of 21 components. Some of the components of B27, including bovine serum albumin (BSA), are animal-derived products, and the effects of B27 components on differentiation, maturation or subtype specification processes are poorly defined. In 2014, Burridge and his colleagues developed an advanced protocol that is defined, cost-effective and efficient [22]. By subtracting one component from B27 at a time and proceeding with cardiac differentiation, the researchers reported that BSA and L-ascorbic acid 2-phosphate are essential components in cardiac differentiation. Subsequently, by replacing BSA with rice-derived recombinant human albumin, the chemically defined medium with 3 components (CDM3) was produced. The application of a GSK-inhibitor, CHIR99021, for the first 2 days followed by 2 days of the Wnt-inhibitor Wnt-59 to cells is an optimal culture condition in CDM3 resulting in similar levels of live-cell yields and CM differentiation [22].

Despite remarkable improvement in the generation of hPSC-CMs, obtaining pure populations of hPSC-CMs still remains challenging. Currently available methods can only generate a mixture of cells which include not only CMs but other cell types. This is one of the most critical barriers for applications of hPSC-CMs in regenerative therapy, drug discovery, and disease investigation. For Instance, cardiac transplantation of non-pure hPSC-CMs mixed with undifferentiated hPSCs or other cell types may produce tumors or unwanted cell types in hearts [23-28]. Accordingly, a pure or enriched population of hPSC-CMs would be required, particularly for cardiac cell therapy. Enriched hPSC-CMs would also be more beneficial for myocardial repair due to improved electric and mechanical properties [29]. A pure, homogeneous population of hPSC-CMs would pose less arrhythmic risk and have enhanced contractile performance, and would be more useful in disease modeling as they better reflect native CM physiology. Finally, purified hPSC-CMs would better serve for testing drug efficacy and toxicity. Therefore, many researchers have tried to develop methods to purify CMs from cardiomyogenically differentiated hPSCs.

There are three important topics that are not addressed in this review. First is the beneficial role of other cell types such as endothelial cells and fibroblasts in the integration, survival, and function of CMs [30-32]. We did not discuss this issue because it would need a separate review due to the volume of material. While the roles of such cells are important, the value of having purified hPSC-CMs is not diminished. Although cell mixtures or tissue engineered products can be used, unless purified CMs are employed, they would form tumors or other cells/tissues when implanted in vivo. Our point here is that even if cardiomyocytes are mixed with non-CMs, all cells should be clearly defined and purified as well. If the mixture is made in a non-purified or non-defined manner (for example, an unsophisticated top-down approach), there would be undefined cells that are neither CMs, ECs, nor fibroblasts and these unidentified cells will make aberrant tissues or tumors. Second, we did not deal with maturation of hPSC-CMs because of its broad scope and depth [33, 34]. Third is direct reprogramming or conversion of somatic cells into CMs. There has been another advancement in the generation of CMs by directly reprogramming or converting somatic cells into CM-like cells by introducing a combination of cardiac transcription factors (TFs) or muscle-specific microRNAs (miRNAs) both in vitro and in vivo [35-41]. These cells are referred to as induced CMs (iCMs) or cardiac-like myocytes (iCLMs). While this is an important advancement, we did not cover this topic either due to its size. Accordingly, this review will focus on the various strategies for purifying or enriching hPSC-CMs reported to date (Figure 1).

Early on, researchers isolated hPSC-CMs manually under microscopy by mechanically separating out the beating areas from myogenically differentiating hPSC cultures [18, 20, 42]. This method usually generates 5-70% hPSC-CMs. Although generally crude, it can enrich even higher percentages of CMs with further culture. This manual isolation method has the advantage of being easy, but while it can be useful for small-scale research, it is very labor intensive and not scalable, precluding large scale research or clinical application.

Currently available strategies for enriching cardiomyocytes derived from human pluripotent stem cells.

Xu et el. reported that hPSC-CMs, due to their physical and structural properties, can be enriched by Percoll density gradient centrifugation [43]. Percoll was first formulated by Pertoft et al [44] and it was originally developed for the isolation of cells, organelles, or viruses by density centrifugation. The Percoll-based method has several advantages. The procedure for Percoll-based separation is very simple and easy, it is inexpensive, and its low viscosity allows more rapid sedimentation and lower centrifugal forces compared to a sucrose density gradient. Lastly, it can be prepared and kept for a long time in an isotonic solution to maintain osmolarity. Although Percoll separation has resulted in major improvements in hPSC-CM isolation procedures, it has clear limitations with regard to purity and scalability. Previous studies found that Percoll separation is only able to enrich 40 -70% of hPSC-CMs. It is also not compatible with large-scale enrichment of hPSC-CMs.

Another traditional method for purifying hPSC-CMs is based on the expression of a drug resistant gene or a fluorescent reporter gene such as eGFP or DsRed, which is driven by a cardiac specific promoter in genetically modified hPSC lines [45, 46]. Here, enrichment of hPSC-CMs can be achieved by either drug treatment to eliminate cells that do not express the drug resistant gene or with FACS to isolate fluorescent cells [47, 48].

Briefly, enrichment of PSC-CMs by genetically based selection was first reported by Klug et al [49]. The authors generated murine ES cell lines via permanent gene transfection of the aminoglycoside phosphotransferase gene driven by the MHC (MYH7) promoter. With this approach, highly purified murine ESC-CMs up to 99% were achieved. Next, several studies reported the use of various CM-specific promoters to enrich ESC-CMs such as Mhc (Myh6), Myh7, Ncx (Sodium Calcium exchanger) and Mlc2v (Myl2) [46, 50, 51]. In the case of hESCs, MHC/EGFP hESCs were generated by permanent transfection of the EGFP-tagged MHC promoter [52]. Similarly, an NKX2.5/eGFP hESC line was generated to enrich GFP positive CMs [53]. However, since MHC and NKX2.5 are expressed in general CMs, the resulting CMs contain a mixture of the three subtypes of CMs, nodal-, atrial-, and ventricular-like CMs. To enrich only ventricular-like CMs, Huber et al. generated MLC2v/GFP ESCs to be able to isolate MLC2v/GFP positive ventricular-like cells by FACS [52] [54-57]. In addition, the cGATA6 gene was used to purify nodal-like hESC-CMs [58]. Future studies should focus on testing new types of cardiac specific promoters and devising advanced selection procedures to improve this strategy.

While fluorescence-based cell sorting is more widely used, the drug selection method may be a better approach to enrich high purity of hPSC-CMs during differentiation/culture as it does not require FACS. The advantage is its capability for high-purity cell enrichment due to specific gene-based cell sorting. These highly pure cells can allow more precise mechanistic studies and disease modeling. Despite its many advantages, the primary weakness of genetic selection is genetic manipulation, which disallows its use for therapeutic application. Insertion of reporter genes into the host genome requires viral or nonviral transfection/transduction methods, which can induce mutagenesis and tumor formation [50, 59-61].

Practically, antibody-based cell enrichment is the best method for cell purification to date. When cell type-specific surface proteins or marker proteins are known, one can tag cells with antibodies against the proteins and sort the target cells by FACS or magnetic-activated cell sorting (MACS). The main advantage is its specificity and sensitivity, and its utility is well demonstrated in research and even in clinical therapy with hematopoietic cells [62]. Another advantage is that multiple surface markers can be used at the same time to isolate target cells when one marker is not sufficient. However, no studies have reported surface markers that are specific for CMs, even after many years. Recently, though, several researchers demonstrated that certain proteins can be useful for isolating hPSC-CMs.

In earlier studies, KDR (FLK1 or VEGFR2) and PDGFR- were used to isolate cardiac progenitor cells [63]. However, since these markers are also expressed on hematopoietic cells, endothelial cells, and smooth muscle cells, they could not enrich only hPSC-CMs. Next, two independent studies reported two surface proteins, SIRPA [64] and VCAM-1 [65], which it was claimed could specifically identify hPSC-CMs. Dubois et al. screened a panel of 370 known antibodies against CMs differentiated from hESCs and identified SIRPA as a specific surface protein expressed on hPSC-CMs [64]. FACS with anti-SIRPA antibody enabled the purification of CMs and cardiac precursors from cardiomyogenically differentiating hPSC cultures, producing cardiac troponin T (TNNT2, also known as cTNT)-positive cells, which are generally considered hPSC-CMs, with up to 98% purity. In addition, a study performed by Elliot and colleagues identified another cell surface marker, VCAM1 [53]. In this study, the authors used NKX2.5/eGFP hESCs to generate hPSC-CMs, allowing the cells to be sorted by their NKX2.5 expression. NKX2.5 is a well-known cardiac transcription factor and a specific marker for cardiac progenitor cells [66, 67]. To identify CM-specific surface proteins, the authors performed expression profiling analyses and found that expression levels of both VCAM1 and SIRPA were significantly upregulated in NKX2.5/eGFP+ cells. Flow cytometry results showed that both proteins were expressed on the cell surface of NKX2.5/eGFP+ cells. Differentiation day 14 NKX2.5/eGFP+ cells expressed VCAM1 (71 %) or SIRPA (85%) or both VCAM1 and SIRPA (37%). When the FACS-sorted SIRPA-VCAM1-, SIRPA+ or SIRPA+VCAM1+ cells were further cultured, only SIRPA+ or SIRPA+VCAM1+ cells showed NKX2.5/eGFP+ contracting portion. Of note, NKX2.5/eGFP and SIRPA positive cells showed higher expression of smooth muscle cell and endothelial cell markers indicating that cells sorted solely based on SIRPA expression may not be of pure cardiac lineage. Hence, the authors concluded that a more purified population of hPSC-CMs could be isolated by sorting with both cell surface markers. Despite significant improvements, it appears that these surface markers are not exclusively specific for CMs as these antibodies also mark other cell types including smooth muscle cells and endothelial cells. Furthermore, they are also known to be expressed in the brain and the lung, which raises concerns whether these surface proteins can be used as sole markers for the purification of hPSC-CMs compatible for clinical applications.

More recently, Protze et al. reported successful differentiation and enrichment of sinoatrial node-like pacemaker cells (SANLPCs) from differentiating hPSCs by using cell surface markers and an NKX2-5-reporter hPSC line [68]. They found that BMP signaling specified cardiac mesoderm toward the SANLPC fate and retinoic acid signaling enhanced the pacemaker phenotype. Furthermore, they showed that later inhibition of the FGF pathway, the TFG pathway, and the WNT pathway shifted cell fate into SANLPCs, and final cell sorting for SIRPA-positive and CD90-negative cells resulted in enrichment of SANLPCs up to ~83%. These SIRPA+CD90- cells showed the molecular, cellular and electrophysiological characteristics of SANLPCs [68]. While this study makes important progress in enriching SANLPCs by modulating signaling pathways, no specific surface markers for SANLPCs were identified and the yield was still short of what is usually expected for cells purified via FACS.

Hattori et al. developed a highly efficient non-genetic method for purifying hPSC-derived CMs, in which they employed a red fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), that can label active mitochondria. Since CMs contain a large number of mitochondria, CMs from mice and marmosets (monkey) could be strongly stained with TMRM [69]. They further found that primary CMs from several different types of animals and CMs derived from both mESCs and hESCs were successfully purified by FACS up to 99% based on the TMRM signals. In addition to its efficiency for CM enrichment, TMRM did not affect cell viability and disappeared completely from the cells within 24 hrs. Importantly, injected hPSC-CMs purified in this way did not form teratoma in the heart tissues. However, since TMRM only functions in CMs with high mitochondrial density, this method cannot purify entire populations of hPSC-CMs [64]. While originally TMRM was claimed to be able to isolate mature hPSC-CMs, mounting evidence indicates that hPSC-CMs are similar to immature human CMs at embryonic or fetal stages. Therefore, both the exact phenotype of the cells isolated by TMRM and its utility are rather questionable [33, 34]. Two subsequent studies demonstrated that TMRM failed to accurately distinguish hPSC-CMs due to the insufficient amounts of mitochondria [64].

Employing the unique metabolic properties of CMs, Tohyama et al. developed an elegant purification method to enrich PSC-CMs [70]. This approach is based on the remarkable biochemical differences in lactate and glucose metabolism between CMs and non-CMs, including undifferentiated cells. Mammalian cells use glucose as their main energy source [71]. However, CMs are capable of energy production from different sources such as lactate or fatty acids [71]. A comparative transcriptome analysis was performed to detect metabolism-related genes which have different expression patterns between newborn mouse CMs and undifferentiated mouse ESCs. These results showed that CMs expressed genes encoding tricarboxylic acid (TCA) cycle enzymes more than genes related to lipid and amino acid synthesis and the pentose phosphate cycle compared to undifferentiated ESCs. To further prove this observation, they compared the metabolites of these pathways using fluxome analysis between CMs and other cell types such as ESCs, hepatocytes and skeletal muscle cells, and found that CMs have lower levels of metabolites related to lipid and amino acid synthesis and pentose phosphate. Subsequently, authors cultured newborn rat CMs and mouse ESCs in media with lactate, forcing the cells to use the TCA cycle instead of glucose, and they observed that CMs were the only cells to survive this condition for even 96 hrs. They further found that when PSC derivatives were cultured in lactate-supplemented and glucose-depleted culture medium, only CMs survived. Their yield of CM population was up to 99% and no tumors were formed when these CMs were transplanted into hearts. This lactate-based method has many advantages: its simple procedures, ease of application, no use of FACS for cell sorting, and relatively low cost. More recently, this method was applied to large-scale CM aggregates to ensure scalability. As a follow-up study, the same group recently reported a more refined lactate-based enrichment method which further depletes glutamine in addition to glucose [72]. The authors found that glutamine is essential for the survival of hPSCs since hPSCs are highly dependent on glycolysis for energy production rather than oxidative phosphorylation. The use of glutamine- and glucose-depleted lactate-containing media resulted in more highly purified hPSC-CMs with less than 0.001% of residual PSCs [72]. One concern of this lactate-based enrichment method is the health of the purified hPSC-CMs, because physiological and functional characteristics of hPSC-CMs cultured in glucose- and glutamine-depleted media for a long time may have functional impairment since CMs with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate [72]. In addition, this lactate-based strategy can only be applied to hPSC- CMs, but not other hPSC derived cells such as neuron or -cells.

Our group also recently reported a new method to isolate hPSC-CMs by directly labelling cardiac specific mRNAs using nano-sized probes called molecular beacons (MBs) [29, 73, 74]. Designed to detect intracellular mRNA targets, MBs are dual-labeled antisense oligonucleotide (ODN) nano-scale probes with a DNA or RNA backbone, a Cy3 fluorophore at the 5' end, and a Black Hole quencher 2 (BHQ2) at the 3' end [75, 76]. They form a stem-loop (hairpin) structure in the absence of a complementary target, quenching the fluorescence of the reporter. Hybridization with the target mRNA opens the hairpin and physically separates the reporter from the quencher, allowing a fluorescence signal to be emitted upon excitation. The MB-based method can be applied to the purification of any cell type that has known specific gene(s) [77].

In one study [29], we designed five MBs targeting unique sites in TNNT2 or MYH6/7 mRNA in both mouse and human. To determine the most efficient transfection method to deliver MBs into living cells, various methods were tested and nucleofection was found to have the highest efficiency. Next, we tested the sensitivity and specificity of MBs using an immortalized mouse CM cell line, HL-1, and other cell types. Finally, we narrowed it down to one MB, MHC-MB, which showed >98% sensitivity and > 95% specificity. This MHC-MB was applied to cardiomyogenically differentiated mouse and human PSCs and FACS sorting was performed. The resultant MHC-MB-positive cells expressed cardiac proteins at ~97% when measured by flow cytometry. These sorted cells also demonstrated spontaneous contraction and all the molecular and electrophysiological signatures of human CMs. Importantly, when these purified CMs were injected into the mouse infarcted myocardium, they were well integrated into the myocardium without forming any tumors, and they improved cardiac function.

In a subsequent study [74], we refined a method to enrich ventricular CMs from differentiating PSCs (vCMs) by targeting a transcription factor which is not robustly expressed in cells. Since vCMs are the main source for generating cardiac contractile forces and the most frequently damaged in the heart, there has been great demand to develop a method that can obtain a pure population of vCMs for cardiac repair. Despite this critical unmet need, no studies have demonstrated the feasibility of isolating ventricular CMs without permanently altering their genome. Accordingly, we first designed MBs targeting the Iroquois homeobox protein 4 (Irx4) mRNA, a vCM specific transcription factor [78, 79]. After testing sensitivity and specificity, one IRX4-MB was selected and applied to myogenically differentiated mPSCs. The FACS-sorted IRX4-MB-positive cells exhibited vCM-like action potentials in more than 98% of cells when measured by several electrophysiological analyses including patch clamp and Ca2+ transient analyses. Furthermore, these cells maintained spontaneous contraction and expression of vCM-specific proteins.

The MB-based cell purification method is theoretically the most broadly applicable technology among the purification methods because it can isolate any target cells expressing any specific gene. Thus, the MB-based sorting technique can be applied to the isolation of other cell types such as neural-lineage cells or islet cells, which are critical elements in regenerative medicine but do not have specific surface proteins identified to date. In addition, theoretically, this technology may have the highest efficiency when MBs are designed to have the maximum sensitivity and specificity for the cells of interest, but not others. These characteristics are particularly important for cell therapy. Despite these advantages, the delivery method of MB into the cells needs to be improved. So far, nucleofection is the best delivery method, but caused some cell damage with < 70% cell viability. Thus, development of a safer delivery method will enable wider application of MB-based cell enrichment.

Recently, Miki and colleagues reported a novel method for purifying cells of interest based on endogenous miRNA activity [80]. Miki et al. employed several synthetic mRNA switches (= miRNA switch), which consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame that codes a desired gene, such as a regulatory protein that emits fluorescence or promotes cell death. If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, thus distinguishing the cell type from others that do not contain the miRNA and express the protein.

Briefly, the authors first identified 109 miRNA candidates differentially expressed in distinct stages of hPSC-CMs (differentiation day 8 and 20). Next, they found that 14 miRNAs were co-expressed in hPSC-CMs at day 8 and day 20 and generated synthetic mRNAs that recognize these 14 miRNA, called miRNA switches. Among those miRNA switches, miR-1-, miR-208a-, and miR-499a-5p-switches successfully enriched hPSC-CMs with purity of sorted cells up to 96% determined by TNNT2 intracellular flow cytometry. Particularly, hPSC-CMs enriched by the miR-1-switch showed substantially higher expression of several cardiac specific genes/proteins and lower expression of non-CM genes/proteins compared with control cells. Patch clamp confirmed that these purified hPSC-CMs possessed both ventricular-like and atrial-like action potentials.

One of the major advantages of this technology is its wider applicability to other cell types. miRNA switches have the flexibility to design the open reading frame in the mRNA sequence such that any desired transgene can be incorporated into the miRNA switches to regulate the cell phenotype based on miRNA activity. The authors tested this possibility by incorporating BIM sequence, an apoptosis inducer, into the cardiac specific miR-1- and miR-208a switches and tested whether they could selectively induce apoptosis in non-CMs. They found that miR-1- and miR-208a-Bim-switches successfully enriched cTNT-positive hPSC-CMs without cell sorting. Enriched hPSC-CMs by 208a-Bim-switch were injected into the hearts of mice with acute MI and they engrafted, survived, expressed both cTNT and CX43, and formed gap junctions with the host myocardium. No teratoma was detected. In addition, other miRNA switches such as miR-126-, miR-122-5p-, and miR-375-switches targeting endothelial cells, hepatocytes, and -cells, respectively, successfully enriched these cell types differentiated from hPSCs. However, identification of specific miRNAs expressed only in the specific cell type of interest and verification of their specificity in target cells will be key issues for continuing to use this miRNA-based cell enrichment method.

Recent advances in biomedical engineering have contributed to developing systems that can isolate target cells using physicochemical properties of the cells. Microfluidic systems have been intensively applied for cell separation due to recent improvements in miniaturizing a cell culture system [81-83]. These advances made possible the design of automated microfluidic devices with cellular microenvironments and controlled fluid flows that save time and cost in experiments. Thus, there have been an increasing number of studies seeking to apply the microfluidic system for cell separation. Among the first, Singh et al. tested the possibility of using a microfluidic system for the separation of hPSC [84] by preparative detachment of hPSCs from differentiating cultures based on differences in the adhesion properties of different cell types. Distinct streams of buffer that generated varying levels of shear stress further allowed selective enrichment of hPSC colonies from mixed populations of adherent non-hPSCs, achieving up to 95% purity. Of note, this strategy produced hPSC survival rates almost two times higher than FACS, reaching 80%.

Subsequently, for hPSC-CMs purification, Xin et al. developed a microfluidic system with integrated ridge-like flow derivations and fishnet-like microcolumns for the enrichment of hiPSC-CMs [85]. This device is composed of a 250 mm-long microfluidic channel, which has two integrated parallel microcolumns with surfaces functionalized with anti-human TRA-1 antibody for undifferentiated hiPSC trapping. Aided by the ridge-like surface patterns on the upper wall of the channel, micro-streams are generated so that the cell suspension of mixed undifferentiated hiPSCs and hiPSC-CMs are forced to cross the functionalized fishnet-like microcolumns, resulting in trapping of undifferentiated hiPSCs due to the interaction between the hiPSCs and the columns, and the untrapped hiPSC-CMs are eventually separated. By modulating flow and coating with anti-human TRA-1 antibody, they were able to enrich CMs to more than 80% purity with 70% viability. While this study demonstrated that a microfluidic device could be used for purifying hPSC-CMs, it was not realistic because the authors used a mixture of only undifferentiated hiPSCs and hiPSC-CMs. In real cardiomyogenically differentiated hiPSCs, undifferentiated hiPSCs are rare and many intermediate stage cells or other cell types are present, so the idea that this simple device can select only hiPSC-CMs from a complex mixture is uncertain.

Overall, the advantages of microfluidic system based cell isolation include fast speed, improved cell viability and low cost owing to the automated microfluidic devices that can control cellular microenvironments and fluid flows [86-88]. However, microfluidic-based cell purification methods have limitations in terms of low purity and scalability [89-92]. In fact, there have been only a few studies demonstrating the feasibility that microfluidic device-based cell separation could achieve higher than 80% purity of target cells. Furthermore, currently available microfluidic devices allow only separation of a small number of cells (< 1011). To employ microfluidic devices for large-scale cell production, we need to develop a next generation of microfluidic devices that can achieve a throughput greater than 1011 sorted cells per hour with > 95% purity.

Having available a large quantity of a homogeneous population of cells of interest is an important factor in advancing biomedical research and clinical medicine, and is especially true for hPSC-CMs. While remarkable progress has been made in the methods for differentiating hPSCs into CMs, technologies to enrich hPSC-CMs, particularly those which are clinically applicable, have been emerging only over the last few years. Contamination with other cell types and even the heterogeneous nature of hPSC-CMs significantly hinder their use for several future applications such as cardiac drug toxicology screening, human cardiac disease modeling, and cell-based cardiac repair. For instance, cardiac drug-screening assays require pure populations of hPSC-CMs, so that the observed signals can be attributed to effects on human CMs. Studies of human cardiac diseases can also be more adequately interpreted with purified populations of patient derived hiPSC-CMs. Clinical applications with hPSC-CMs will need to be free of other PSC derivatives to minimize the risk of teratoma formation and other adverse outcomes.

Summary of representative methods for hPSC-CM purification

Schematic pictures of microfluidic device for enriching hiPSC-CMs. (A) The part of the device designed for trapping undifferentiated hiPSCs. (B) (Left) Illustration of the overall microfluidic device assembled with peristaltic pump, cell suspension reservoirs, and a serpentine channel. (Right) Magnified image showing a channel combining microcolumns and ridge-like flow derivation structures. Modified from Li et al. On chip purification of hiPSC-derived cardiomyocytes using a fishnet-like microstructure. Biofabrication. 2016 Sep 8;8(3): 035017

Therefore, development of reproducible, effective, non-mutagenic, scalable, and economical technologies for purifying hPSC-CMs, independent of hPSC lines or differentiation protocols, is a fundamental requirement for the success of hPSC-CM applications. Fortunately, new technologies based on the biological specificity of CMs such as MITO-tracker, molecular beacons, lactate-enriched-glucose depleted-media, and microRNA switches have been developed. In addition, technologies based on engineering principles have recently yielded a promising platform using microfluidic technology. While due to the short history of this field, more studies are needed to verify the utility of these technologies, the growing attention toward this research is a welcome move.

Another important question raised recently is how to non-genetically purify chamber-specific subtypes of CMs such as ventricular-like, atrial-like and nodal-like hPSC-CMs. So far, only a few studies have addressed this potential with human PSCs. We also showed that a molecular beacon-based strategy could enrich ventricular CMs differentiated from PSCs [74]. Another study demonstrated generation of SA-node like pacemaker cells by using a stepwise treatment of various morphogens and small molecules followed by cell sorting with several sub-specific surface markers. However, the yield of both studies was relatively low (<85%). Given the growing clinical importance of chamber-specific CMs, the strategies for purifying specific subtypes of CM that are independent of hPSC lines or differentiation protocols should be continuously developed. A recently reported cell surface capture-technology [93, 94] may facilitate identification of chamber specific CM proteins that will be useful for target CM isolation.

In summary, technological advances in the purification of hPSC-CMs have opened an avenue for realistic application of hPSC-CMs. Although initial success was achieved for purification of CMs from differentiating hPSC cultures, questions such as scalability, clinical compatibility, and cellular damage remain to be answered and isolation of human subtype CMs has yet to be demonstrated. While there are other challenges such as maturity, in vivo integration, and arrhythmogenecity, this development of purification technology represents major progress in the field and will provide unprecedented opportunities for cell-based therapy, disease modeling, drug discovery, and precision medicine. Furthermore, the availability of chamber-specific CMs with single cell analyses will facilitate more sophisticated investigation of human cardiac development and cardiac pathophysiology.

This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (No 2015M3A9C6031514), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2782, HI16C2211) and grants from NHLBI (R01HL127759, R01HL129511), NIDDK (DP3-DK108245). This work was also supported by a CityU Start-up Grant (No 7200492), a CityU Research Project (No 9610355), and a Georgia Immuno Engineering Consortium through funding from Georgia Institute of Technology, Emory University, and the Georgia Research Alliance.

The authors have declared that no competing interest exists.

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56. MLLER M, FLEISCHMANN BK, SELBERT S, JI GJ, ENDL E, MIDDELER G. et al. Selection of ventricular-like cardiomyocytes from ES cells in vitro. FASEB J. 2000;14:2540-8

57. Zhang Q, Jiang J, Han P, Yuan Q, Zhang J, Zhang X. et al. Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals. Cell Res. 2011;21:579-87

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Current Strategies and Challenges for Purification of ...

Cardiac Stem Cells Comments Off on Current Strategies and Challenges for Purification of … | January 25th, 2019

VetStem Technology: Summary

VetStem Regenerative Cell Therapy is based on a clinical technology licensed from Artecel Inc. Original patents are from the University of Pittsburgh and Duke University.

Adipose-derived regenerative cells are:

VetStem Regenerative Cell (VSRC) therapy delivers a functionally diverse cell population able to communicate with other cells in their local environment. Until recently, differentiation was thought to be the primary function of regenerative cells. However, the functions of regenerative cells are now known to be much more diverse and are implicated in a highly integrated and complex network. VSRC therapy should be viewed as a complex, yet balanced, approach to a therapeutic goal. Unlike traditional medicine, in which one drug targets one receptor, Regenerative Medicine, including VSRC therapy, can be applied in a wide variety of traumatic and developmental diseases. Regenerative cell functions include:

In general, in vitro studies demonstrate that MSCs limit inflammatory responses and promote anti-inflammatory pathways.

Multiple studies demonstrate that MSCs secrete bioactive levels of cytokines and growth factors that support angiogenesis, tissue remodeling, differentiation, and antiapoptotic events.25,28 MSCs secrete a number of angiogenesis-related cytokines such as:28

Adipose-derived MSC studies demonstrate a diverse plasticity, including differentiation into adipo-, osteo-, chondro-, myo-, cardiomyo-, endothelial, hepato-, neuro-, epithelial, and hematopoietic lineages, similar to that described for bone marrow derived MSCs.22 These data are supported by in vivo experiments and functional studies that demonstrated the regenerative capacity of adipose-derived MSCs to repair damaged or diseased tissue via transplant engraftment and differentiation.6,9,30

Homing (chemotaxis) is an event by which a cell migrates from one area of the body to a distant site where it may be needed for a given physiological event. Homing is an important function of MSCs and other progenitor cells and one mechanism by which intravenous or parenteral administration of MSCs permits an auto-transplanted therapeutic cell to effectively target a specific area of pathology.

Adipose-derived regenerative cells contain endothelial progenitor cells and MSCs that assist in angiogenesis and neovascularization by the secretion of cytokines, such as hepatic growth factor (HGF), vascular endothelial growth factor (VEGF), placental growth factor (PGF), transforming growth factor (TGF), fibroblast growth factor (FGF-2), and angiopoietin.25

Apoptosis is defined as a programmed cell death or cell suicide, an event that is genetically controlled.35 Under normal conditions, apoptosis determines the lifespan and coordinated removal of cells. Unlike during necrosis, apoptotic cells are typically intact during their removal (phagocytosis).

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What is VetStem Regenerative Medicine? | Why Use Adipose ...

Cardiac Stem Cells Comments Off on What is VetStem Regenerative Medicine? | Why Use Adipose … | January 19th, 2019

Cardiovascular disease, also called heart disease, is a broad medical term used to describe a group of conditions that affect the blood vessels or the heart. It is the most common cause of death worldwide.1

Conditions of cardiovascular disease include:

The Stem Cells Transplant Institutein Costa Rica, uses adult autologous stem cells for the treatment of cardiovascular disease (heart disease). The symptoms of cardiovascular disease will depend on the specific type of heart disease.

Treatment at the Stem Cells Transplant Institute could help improve the symptoms of cardiovascular disease such as:

Heart disease and cardiovascular disease are often used interchangeably. These terms refer to a group of conditions that affect the blood vessels and heart. Valvular heart disease affects how the valves pump blood flow in and out of the heart. Cardiomyopathy affects the contractions of the heart muscle. Heart arrhythmias are disturbances in the electrical conduction making the heart beat irregular. Coronary artery disease is the most common cause of cardiovascular disease and stem cell therapy may be an effective treatment.

Coronary artery disease is caused by atherosclerosis, the buildup of plaque, causing a narrowing or blocking the blood vessels in the coronary arteries. Coronary artery disease is the leading cause of cardiovascular disease. Atherosclerosis can lead to chest pain, heart attack or stroke.

Coronary arteries carry oxygen rich blood to the heart. Plaque is caused by the presence of cholesterol, calcium, fat, and other substances in the blood. When plaque builds up in the blood vessels it narrows the arteries causing them to harden and weaken, reducing the amount of oxygen rich blood to the heart. As a result, the heart cannot pump blood effectively to the rest of the body potentially leading to heart failure and ultimately death.

If the plaque building up in the coronary arteries breaks, a blood clot forms around the plaque. If the clot cuts off the blood flow to the heart muscle completely, the heart muscle is unable to get the necessary oxygen and nutrients causing a part of the heart muscle to die. The result is a heart attack or myocardial infarction,

Coronary artery disease, high blood pressure or a previous heart attack can lead to the onset of heart failure. Heart failure is a chronic, progressive disease typically caused by another heart condition resulting in the heart muscle losing its ability to supply the rest of body with enough blood and oxygen.

Atherosclerosis can also cause peripheral artery disease. Peripheral arterial disease occurs when the narrowed peripheral arteries cannot send enough blood flow to the extremities, usually the legs. The most common symptoms of peripheral artery disease are; cramping, pain, and/or tiredness in the leg or hip muscles during exertion. The most severe symptom of peripheral artery disease is critical limb ischemia, pain at rest due to reduced blood flow to the limb.

Approximately 85% of strokes are ischemic strokes. Atherosclerosis is the most common cause of ischemic stroke. If the arteries become too narrow due to plaque buildup, the blood cells may collect and form a clot. A larger clot can block the artery where it is formed (thrombotic stroke) while a smaller clot may travel until it reaches an artery closer to the brain (embolic stroke). When the arteries to your brain become narrow or blocked, the required blood flow is reduced resulting in stroke. Other causes of ischemic stroke are clots due to an irregular heartbeat or heart attack.

Stem cell therapy at the Stem Cells Transplant Institute may be a good alternative for patients seeking a safe, non-surgical treatment for cardiovascular disease.

Notably, adult stem and progenitor cells including.mesenchymal stem cells have progressed into clinical trials and have shown positive benefits.5

Stem cell transplantation uses healthy cells to promote the repair of damaged cells and regeneration of healthy and functional cells to repair injured tissue.1 The therapeutic effect of stem cell transplantation in patients with cardiovascular disease may be due to the paracrine effect. The theory is transplanted stem cells repair damaged tissue by releasing factors that promote regeneration of healthy stem cells, reduce inflammation, promote the growth of new blood vessels, inhibit cell death, and reduce hypertrophy.1

The results of initial research using mesenchymal stem cell transplantation:

Heart Failure

Adipose derived stem cells improve left ventricular function, promote angiogenesis, lower fibrosis, and decrease inflammation. Several months following treatment, stem cells continue to migrate to the heart muscle regenerating and renewing healthy heart function. Stem cell therapy cannot help all patients with cardiovascular disease but for many patients stem cell therapy combined with lifestyle modification may be a safe, effective, non-surgical alternative treatment.

Lifestyle changes that can help improve cardiovascular disease include:

The Stem Cells Transplant Institute uses autologous mesenchymal stem cells for the treatment of cardiovascular disease. Autologous means the stem cells are collected from the recipient so the risk of rejection is virtually eliminated. Mesenchymal stem cells are one type of adult stem cells that are found in a variety of tissues including; adipose tissue, lung, bone marrow, and blood. Mesenchymal stem cells have several advantages over other types of stem cells; ability to migrate to sites of tissue injury, strong immunosuppressive effect, and better safety after infusion.2,3 Mesenchymal stem cells are a promising treatment for cardiovascular disease. Treatment at the Stem Cells Transplant Institute may improve the symptoms and long-term complications of cardiovascular disease.

A team of stem cell experts developed an FDA approved method and protocol for harvesting and isolating adipose derived stem cells for autologous reimplantation. The collection and use of adult stem cells does not require the destruction of embryos and for this reason, more U.S. federal funding is being spent on stem cell research.

The stem cells are administered intravenously.

Costa Rica has one of the best healthcare systems in world and is ranked among the highest for medical tourism. Using the most advanced technologies, the team of experts at The Stem Cells Transplant Institute believes in the potential of stem cell therapy for the treatment of cardiovascular disease. We are committed to providing personalized service and the highest quality of care to every patient. Contact us to see if stem cell therapy may be a treatment option for you.

1.Sun R.Advances in stem cell therapy for cardiovascular disease (Review). National Journal of Mol. Med. 38: 23-29, 2016. 2 Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, Tracy M, Ghersin E, Johnston PV, Brinker JA, et al: Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA 308: 2369-2379, 2012.3 Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, Ishino K, Ishida H, Shimizu T, Kangawa K, et al: Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 12: 459-465, 2006. 4 Mazo M, Planat-Bnard V, Abizanda G, Pelacho B, Lobon B, Gavira JJ, Peuelas I, Cemborain A, Pnicaud L, Laharrague P, et al: Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction. Eur J Heart Fail 10: 454-462, 2008. 5 Stem cell-based therapies to promote angiogenesis in ischemic cardiovascular disease Luqia Hou,1,2 Am J Physiol Heart Circ Physiol 310: H455H465, 2016. 6 Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 94: 678685, 2004. 7 Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S, Epstein SE. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 109: 15431549, 2004.

8 Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, Tracy M, Ghersin E, Johnston PV, Brinker JA, Breton E, Davis-Sproul J, Schulman IH, Byrnes J, Mendizabal AM, Lowery MH, Rouy D, Altman P, Wong Po Foo C, Ruiz P, Amador A, Da Silva J, McNiece IK, Heldman AW, George R, Lardo A. Comparison of allogeneic vs autologous bone marrowderived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA 308: 23692379, 2012.

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Stem Cell Treatment Cardiovascular Disease, Heart Disease ...

Cardiac Stem Cells Comments Off on Stem Cell Treatment Cardiovascular Disease, Heart Disease … | January 13th, 2019

A cell-by-cell search for cardiac stem cells has come up empty, suggesting that previous studies hinting at the existence of cardiac stem cells were mistaken. More significantly, the absence of cardiac stem cells indicates that heart muscle that is lost due to a heart attack cannot be replaced.

The sobering finding was reported by scientists based at the Hubrecht Institute, which is located in the Netherlands. The scientists, led by Hans Clevers, group leader at the Hubrecht Institute and professor of molecular genetics at the University Medical Center Utrecht, published their work this week in the Proceedings of the National Academy of Sciences.

Along with colleagues from cole Normale Suprieure de Lyon and the Francis Crick Institute London, the Hubrecht Institute scientists described how they applied the broadest and most direct definition of stem cell function in the mouse heart: the ability of a cell to replace lost tissue by cell division. In the heart, this means that any cell that can produce new heart muscle cells after a heart attack would be termed a cardiac stem cell.

In an attempt to find cardiac stem cells, the scientists generated a cell-by-cell map of all dividing cardiac cells before and after a myocardial infarction using advanced molecular and genetic technologies. Details of this work appeared in the PNAS article, which is titled, Profiling proliferative cells and their progeny in damaged murine hearts.

Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types, the articles authors indicated in a prepublication version of their paper. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of FSTL1 in cardiac fibroblasts results in postdamage cardiac rupture.

Ultimately, the researchers found no evidence for the existence of a quiescent circulating stem cell population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.

Most tissues of animals and humans contain stem cells that come to the rescue upon tissue damage: they rapidly produce large numbers of daughter cells to replace lost tissue cells. Cardiac tissues, however, appear to behave differently. According to the new study, the damaged heart incorporates many types of dividing cells, but none that are capable of generating new heart muscle. In fact, many of the false leads of past studies can now be explained: cells that were previously named cardiac stem cells now turn out to produce blood vessels or immune cells, but never heart muscle. Thus, the sobering conclusion is drawn that heart stem cells do not exist.

The authors make a second important observation. Connective tissue cells (also known as fibroblasts) that are intermingled with heart muscle cells respond vigorously to a myocardial infarction by undergoing multiple cell divisions. In doing so, they produce scar tissue that replaces the lost cardiac muscle.

While this scar tissue contains no muscle and thus does not contribute to the pump function of the heart, the fibrotic scar holds together the infarcted area. Indeed, when the formation of the scar tissue is blocked, the mice succumb to acute cardiac rupture. Thus, while scar formation is generally seen as a negative outcome of myocardial infarction, the authors stress the importance of the formation of scar tissue for maintaining the integrity of the heart.

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Adult Hearts Lack Cardiac Stem Cells - genengnews.com

Cardiac Stem Cells Comments Off on Adult Hearts Lack Cardiac Stem Cells – genengnews.com | January 13th, 2019

Theme Leaders

James Martin, M.D. Ph.D.Professor, Molecular PhysiologyResearch Interest - Hippo, Wnt, Bmp signaling in development, regeneration, heart disease

Todd Rosengart, M.D., F.A.C.S.Chair/Professor, SurgeryResearch Interest - Cardiac regeneration, cardiac gene therapy, angiogenesis

Members of Theme Six are developing stem cell and cellular reprogramming strategies to treat cardiovascular diseases such as infarction in situ. The goal is to use viral vectors to induce transdifferentiation of cardiac fibroblasts and myofibroblasts into functionalcardiomyocytes in situ in a patients heart. We are modeling and developing the processes in rats, pigs, and in human cardiac fibroblasts. The hope is to have options available for clinical trials within 3-5 years.

Members of Theme Six are involved in research aimed at improving heart function after different types of injury and in particular the devastating loss of heart muscle after myocardial infarction. One current approach is to investigate gene pathways, many of which are important in heart development, that enhance the ability of cardiac muscle to respond to injury. Recent exciting findings have shown that manipulations of specific genetic pathways, such as the Hippo pathway, enhance heart repair. Current investigations in this area include uncovering the molecular mechanisms underlying improved heart repair in order to develop novel therapies.

Another exciting approach involves in vivo reprogramming of cardiac fibroblasts into cardiac muscle as a way to enhance heart function after ischemic injury. This novel method was inspired by the observation by Yamanaka that fibroblasts can be reprogrammed to pluripotent cells in cultured cells. Important recent work has shown that providing a cocktail of factors to cardiac fibroblasts results in conversion of those fibroblasts into cardiac muscle. Current efforts are directed at improving the efficiency of in vivo reprogramming with the goal of using this approach in therapy, recognizing that use of this strategy in human cells will likely be more challenging than in rodent and other non-human strains. The combination of angiogenic pretreatment of scar with this strategy appears to be critical to its success.

Sadek HA, Martin JF, Takeuchi JK, Leor J, Nei Y, Giacca M, Lee RT. Multi-investigator letter on reproducibility of neonatal heart regeneration following apical resection. Stem Cell Reports. 2014; 3(1): 1.

Yen ST, Zhang M, Deng JM, Usman SJ, Smith CN, Parker-Thornburg J, Swinton PG, Martin JF, Behringer RR. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol. 2014; 393(1): 3-9.

C. Thomas Caskey, M.D. - FACP, FRSC Schizophrenia disease genes

Katarzyna Cieslik, Ph.D. - Cardiac mesenchymal progenitors

Austin Cooney, Ph.D. - Nuclear receptor regulation of embryonic stem cell function

Thomas Cooper, M.D. - Alternative splicing in cardiac development and disease

Mary Dickinson, Ph.D. - Role of fluid-derived mechanical forces in vascular remodeling and heart morphogenesis

Mark Entman, M.D. - Molecular mechanisms of cardiac injury and repair, inflammatory signaling

Charles Fraser, M.D. - Congenital heart surgery outcomes, bioengineering and assist devices

Peggy Goodell, M.D. - Hematopoietic stem cells, epigenetic modifications

Jeffrey Jacot, Ph.D. - Regenerative therapies for congenital heart disease

Sandra Haudek, Ph.D. - Circulating monocytic fibroblast precursors, cardiac hypertrophy

George Noon, M.D. - Transplant and assist devices

JoAnn Trial, Ph.D. - Origins of fibroblasts in cardiac injury healing

Peter Tsai, M.D., FACS - Custom-fenestrated endovascular stents to repair aortic transections or aneurysms

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Cardiac Regeneration, Stem Cells | Research | Baylor ...

Cardiac Stem Cells Comments Off on Cardiac Regeneration, Stem Cells | Research | Baylor … | December 23rd, 2018

Preclinical Research

Scientists are developing novel therapeutics for the treatment of cardiovascular diseases using cardiac-derived stem cells in mice and large-animal models. Three current projects are studying:

ExosomesOur researchers are isolating exosomes from specialized human cardiac-derived stem cells and finding that they have the same beneficial effects as other types of stem cells. In mice models, our research shows that exosomes produce the same post-surgery benefits, such as decreasing scar size, increasing healthy heart tissue and reducing levels of chemicals that lead to inflammation. This research suggests that exosomes convey messages that reduce cell death, promote growth of new heart muscle cells and encourage the development of healthy blood vessels.

Mechanisms of Heart Regeneration by Cardiosphere-Derived CellsInvestigators seek to understand the basic mechanisms of coronary artery disease in preclinical disease models. We hope to gather novel mechanistic insights, enabling us to boost the efficacy of stem cell-based treatments by bolstering the regeneration of injured heart muscle.

Biological PacemakersUsing an engineered virus carrying T-box (TBx18), Cedars-Sinai researchers are reprogramming heart muscle cells (cardiomyoctes) into induced sinoatrial node cells in pigs. Cedars-Sinai research shows that these new cells generate electrical impulses spontaneously and are indistinguishable from sinoatrial node or native pacemaker cells. Investigators believe this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications.

Researchers hope to someday incorporate therapeutic regeneration as a regular treatment option for a broad range of cardiovascular disorders, such as myocardial infarctions, heart failure, refractory angina and peripheral vascular disease. Through the Regenerative Medicine Clinic at the Cedars-Sinai Heart Institute, several cardiac stem cell trials are underway. They include:

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Cardiac Stem Cells - Cedars-Sinai

Cardiac Stem Cells Comments Off on Cardiac Stem Cells – Cedars-Sinai | December 20th, 2018

Human life is dependent upon the hearts ability to pump forcefully and frequently enough, but heart failure signs can disturb its normal function. Most humans cannot live more than four minutes without a heartbeat or continuous blood-flow. At that time, brain cells begin to die because they lack adequately oxygenated blood-flow.

The human adult body requires, on average, 5.0 liters of re-circulated blood per minute. In the cardiology field, this metric is called the Cardiac Output, which is calculated as Stroke Volume (SV) x Heart Rate (HR). Another key metric is a patients Ejection-Fraction (EF %). A patients EF tells a cardiologist and other physicians if his or her heart is functioning normally or low normally. It is a measurement of ones heart contraction, with a normal EF range being 55-70%.

This number can also be combined with a patients heart rate to provide physicians with a baseline of a patients cardiac status. A normal range for an adult is 60-100 beats per minute, and this can be significantly higher during a normal pregnancy.

In this article:

For a cardiologist, cardiac metrics indicate if their services are required and allowthem to sign-off on pre-operative cardiac clearances. For other physicians, it tells them if the organ which they specialize in is being perfused adequately (for example, a nephrologist would be interested to know kidney perfusion). It can also indicate the degree to which decreased heart function may affect the severity or spread of disease.

When the heart fails to contract forcefully enough and its performance decreases to the point where its ability to circulate blood adequately is compromised (the EF% falls below 40%), this is considered heart failure. The clinical parameters of heart failure are clearly defined by the New York Heart Association (NYHA), which places patients in NYHA Class III & IV into the heart failure category.

An echocardiogram (often called an Echo), as opposed to an Electrocardiogram (EKG or ECG), allows technicians and physicians to visualize the beating heart. Video clips of the heart contracting are digitally recorded, and a patients EF and Cardiac Output (CO) can be measured with several diagnostic tools (Fractional Shortening via 2D or M-Mode measurements and Simpsons Method via 2D and 3D Quantification) on a cardiovascular ultrasound system.

When an experienced echo tech or cardiologist views a failing heart, it is immediately apparent. Based on my experience reading echocardiograms, I can see that the heart walls or heart muscles (myocardium) are not contracting as vigorously as they should.

For patients with a 5% EF range, any physical movement is extremely strenuous, and they can go into cardiac arrest at any moment, which is why they are usually on cardiac telemetry in a hospital setting. Most likely, a patient with 5% EF range would be awaiting a heart transplant, unless there is a medical condition preventing them from being eligible.

Once a patient falls into the heart failure range, they will be lethargic and have severe limits on activities. Other clinical manifestations of heart failure can include peripheral edema (i.e. swelling in the feet, legs, ankles, or stomach), pulmonary edema, and shortness of breath. In many cases, this can lead to depression.

In evaluating the frequency of heart failure in the U.S, statistics from the U.S. Centers for Disease Control (CDC) find that approximately 5.7 million adults are afflicted with this condition. Additionally, care for congestive heart failure costs an estimated $30.7B per year. Furthermore, the mortality rates of patients suffering from heart failure indicate its clinical severity, with 1 in 5 patients with this condition dying within a year of receiving the diagnosis.

A patient experiencing severe heart failure has limited treatment options, which are expensive, complicated, and have major lifestyle implications.

These limited options include:

Consequently, physicians need more effective weapons for treating heart failure in order to improve patients lives and reduce healthcare-related costs. CHF patients have disproportionate hospital readmission rates when compared to other major diseases.

Enter in the growing field of cardiac stem cell treatments, which introduce fundamentally new treatment options for heart failure patients. In cardiac stem cell treatments, stem cells are taken from a patients bone marrow or fat tissue in a sterile surgical procedure and injected via a catheter-wire into infarcted or poorly contracting muscular segments of the hearts main pumping chamber, the left ventricle (LV).

Over the course of a few months, the stem cells impact myocardial cells and begin to improve the contractility of the affected segments, most likely through paracrine signaling mechanisms and impacting the local microenvironment. This can bring a patients EF to low-normal or even normal levels. As a result, a patient can live a more normal life and return to many activities.

A very early clinical trial aimed at evaluating the potential and effectiveness of cardiac stem cell therapy in humans was conducted in 2006 utilizing a commercial product, VesCellTM. The parameters and results of this trial were documented in the American Heart Associations Circulation, Abstract 3682: Treatment of Patients with Severe Angina Pectoris Using Intracoronarily Injected Autologous Blood-Borne Angiogenic Cell Precursors.The subjects of this trial received an intracoronary injection of VesCellTM, an Autologous Angiogenic Cell Precursor (ACP)-based product.

The authors drew their conclusion regarding this study. VesCell therapy for chronic stable angina seems to be safe and improves anginal symptoms at 3 and 6 months. Larger studies are being initiated to evaluate the benefit of VesCell for the treatment of this and additional severe heart diseases. (Source: Tresukosol et al. Abstract 3682: Treatment of Patients with Severe Angina Pectoris Using Intracoronarily Injected Autologous Blood-Borne Angiogenic Cell Precursors. Circulation. October 31, 2006. Vol. 114, Issue Suppl 18. Link: http://circ.ahajournals.org/content/114/Suppl_18/II_786.4 )

Another early cardiac stem cell clinical trial was performed in 2009 by a Cedars-Sinai team based on technologies and discoveries made by Eduardo Marban, MD, PhD, and led by Raj Makkar, MD. In this study, they explored the safety of harvesting, expanding, and administering a patients cardiac stem cells to repair heart tissue injured by myocardial infarction.

Recently, the American College of Cardiology (ACC) also announced results of a ground-breaking clinical study to evaluate the efficacy and effectiveness of cardiac stem cell treatment for heart failure patients. As stated by Timothy Henry, M.D., Director of Cardiology at Cedars-Sinai Heart Institute and one of the studys lead authors, This is the largest double-blind, placebo-controlled stem cell trial for treatment of heart failure to be presentedBased on these positive results, we are encouraged that this is an attractive potential therapy for patients with class III and class IV heart failure.

Additionally, Dr. Charles Goldthwaite, Jr, published a whitepaper titled, Mending a Broken Heart: Stem Cells and Cardiac Repair, in which he draws the conclusion, Given the worldwide prevalence of cardiac dysfunction and the limited availability of tissue for cardiac transplantation, stem cells could ultimately fulfill a large-scale unmet clinical need and improve the quality of life for millions of people with CVD. However, the use of these cells in this setting is currently in its infancymuch remains to be learned about the mechanisms by which stem cells repair and regenerate myocardium, the optimal cell types, and modes of their delivery, and the safety issues that will accompany their use.

Clearly, there is a trend toward acceptance of cardiac stem cell therapies as an emerging treatment option. Several world-renowned institutes are now conducting clinical studies involving cardiac stem cell treatment, as well as applying for intellectual property protection (patents) pertaining to the techniques required in administrating the therapies.

The key questions at this point in time appear to be:

An important whitepaper pertaining to cardiac stem cells is Ischemic Cardiomyopathy Patients Treated with Autologous Angiogenic and Cardio-Regenerative Progenitor Cells, written by Dr. Athina Kyritsis, et al. In it, the physicians describe their objective as investigating the feasibility, safety, and clinical outcome of patients with Ischemic Cardiomyopathy treated with Autologous Angiogenic and Cardio-Regenerative Progenitor cells (ACPs).

The researchers state: In numerous human trials there is evidence of improvement in the ejection fractions of Cardiomyopathy patients treated with ACPs. Animal experiments not only show improvement in cardiac function, but also engraftment and differentiation of ACPs into cardiomyocytes, as well as neo-vascularization in infarcted myocardium. In our clinical experience, the process has shown to be safe as well as effective.

The authors also found that patients treated with this approach gained increases in cardiac ejection fraction from their starting measurements, with improvements in their cardiac ejection fraction of 21 points (75% increase) at rest and 28.5 points (80% increase) at stress. As a result of these finding, the authors conclude, ACPs can improve the ejection fraction in patients with severely reduced cardiac function with benefits sustained to six months.

In the practice of medicine, the focus should be on delivering excellent care to patients. If there are cardiac stem cell treatments available, then regulatory obstacles should be removed when sufficient clinical trial evidence has been provided to indicate safety and efficacy.

Cardiologist Zannos Grekos, MD, a pioneer in cardiac stem cell therapy since 2006, points to the vastly untapped promise of related therapies, commenting Those of us that have been involved with cardiac stem cell treatment for the last 10-plus years can see the incredible potential this approach has.

As of 2017, the U.S. healthcare system is under enormous pressure to deliver affordable healthcareto a growing population of patients, especially those who are fully or partially covered under Medicare or Medicaid (many have secondary coverage). Although we are in the infancy of its development, cardiac stem cell treatments represent a potentially powerful treatment alternative to patients with heart failure symptoms.

To learn more, view the resources below.

1) Regenocyte http://www.regenocyte.com

2) Cleveland Clinic Stem Cell Therapy for Heart Disease my.clevelandclinic.org/health/articles/stem-cell-therapy-heart-disease

3) Harvard Stem Cell Institute (HSCI) hsci.harvard.edu/heart-disease-0

4) Cedars Sinai Cardiac Stem Cell Treatment http://www.cedars-sinai.edu/Patients/Programs-and-Services/Heart-Institute/Clinical-Trials/Cardiac-Stem-Cell-Research.aspx

5) Johns Hopkins Medicine Cardiac Stem Cell Treatments http://www.hopkinsmedicine.org/stem_cell_research/cell_therapy/a_new_path_for_cardiac_stem_cells.html

What do you think about heart failure signs and cardiac stem cell therapies? Share your thoughts in the comments section below.

Up Next:European Society of Cardiology (ESC) Congress Presentation Reveals Results From Pre-Clinical Study Using CardioCells Stem Cells for Acute Myocardial Infarction

Guest Post: This is a guest article by Clifford M. Thornton, a Certified Cardiovascular Technologist, experienced Echocardiographer Technician, and journalist in the cardiac and medical device fields. His articles have been published in Inventors Digest, Global Innovation Magazine, and Modern Health Talk. He is enthusiastic about progress with cardiac stem cell therapies and their role in heart failure treatment.He can be reached byphone at 267-524-7144 or by email at[emailprotected].

Editors Note This post was originally published on March 14, 2017, and has been updated for quality and relevancy.

Heart Failure Signs | Cardiac Stem Cell Therapies for Heart Failure Treatment

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Heart Failure Signs | Cardiac Stem Cell Therapies: Heart ...

Cardiac Stem Cells Comments Off on Heart Failure Signs | Cardiac Stem Cell Therapies: Heart … | November 15th, 2018

Embryonic stem cells (ES cells or ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo.[1][2] Human embryos reach the blastocyst stage 45 days post fertilization, at which time they consist of 50150 cells. Isolating the embryoblast, or inner cell mass (ICM) results in destruction of the blastocyst, a process which raises ethical issues, including whether or not embryos at the pre-implantation stage should have the same moral considerations as embryos in the post-implantation stage of development.[3][4] Researchers are currently focusing heavily on the therapeutic potential of embryonic stem cells, with clinical use being the goal for many labs. These cells are being studied to be used as clinical therapies, models of genetic disorders, and cellular/DNA repair. However, adverse effects in the research and clinical processes have also been reported.

Embryonic stem cells (ESCs), derived from the blastocyst stage of early mammalian embryos, are distinguished by their ability to differentiate into any cell type and by their ability to propagate. It is these traits that makes them valuable in the scientific/medical fields. ESC are also described as having a normal karyotype, maintaining high telomerase activity, and exhibiting remarkable long-term proliferative potential.[5]

Embryonic stem cells of the inner cell mass are pluripotent, meaning they are able to differentiate to generate primitive ectoderm, which ultimately differentiates during gastrulation into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell types in the adult human body. Pluripotency distinguishes embryonic stem cells from adult stem cells, which are multipotent and can only produce a limited number of cell types.

Under defined conditions, embryonic stem cells are capable of propagating indefinitely in an undifferentiated state. Conditions must either prevent the cells from clumping, or maintain an environment that supports an unspecialized state.[2] While being able to remain undifferentiated, ESCs also have the capacity, when provided with the appropriate signals, to differentiate (presumably via the initial formation of precursor cells) into nearly all mature cell phenotypes.[6]

Due to their plasticity and potentially unlimited capacity for self-renewal, embryonic stem cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease. Pluripotent stem cells have shown potential in treating a number of varying conditions, including but not limited to: spinal cord injuries, age related macular degeneration, diabetes, neurodegenerative disorders (such as Parkinson's disease), AIDS, etc.[7] In addition to their potential in regenerative medicine, embryonic stem cells provide an alternative source of tissue/organs which serves as a possible solution to the donor shortage dilemma. Not only that, but tissue/organs derived from ESCs can be made immunocompatible with the recipient. Aside from these uses, embryonic stem cells can also serve as tools for the investigation of early human development, study of genetic disease and as in vitro systems for toxicology testing.[5]

According to a 2002 article in PNAS, "Human embryonic stem cells have the potential to differentiate into various cell types, and, thus, may be useful as a source of cells for transplantation or tissue engineering."[8]

However, embryonic stem cells are not limited to cell/tissue engineering.

Current research focuses on differentiating ESCs into a variety of cell types for eventual use as cell replacement therapies (CRTs). Some of the cell types that have or are currently being developed include cardiomyocytes (CM), neurons, hepatocytes, bone marrow cells, islet cells and endothelial cells.[9] However, the derivation of such cell types from ESCs is not without obstacles, therefore current research is focused on overcoming these barriers. For example, studies are underway to differentiate ESCs in to tissue specific CMs and to eradicate their immature properties that distinguish them from adult CMs.[10]

Besides becoming an important alternative to organ transplants, ESCs are also being used in field of toxicology and as cellular screens to uncover new chemical entities (NCEs) that can be developed as small molecule drugs. Studies have shown that cardiomyocytes derived from ESCs are validated in vitro models to test drug responses and predict toxicity profiles.[9] ES derived cardiomyocytes have been shown to respond to pharmacological stimuli and hence can be used to assess cardiotoxicity like Torsades de Pointes.[17]

ESC-derived hepatocytes are also useful models that could be used in the preclinical stages of drug discovery. However, the development of hepatocytes from ESCs has proven to be challenging and this hinders the ability to test drug metabolism. Therefore, current research is focusing on establishing fully functional ESC-derived hepatocytes with stable phase I and II enzyme activity.[18]

Several new studies have started to address the concept of modeling genetic disorders with embryonic stem cells. Either by genetically manipulating the cells, or more recently, by deriving diseased cell lines identified by prenatal genetic diagnosis (PGD), modeling genetic disorders is something that has been accomplished with stem cells. This approach may very well prove invaluable at studying disorders such as Fragile-X syndrome, Cystic fibrosis, and other genetic maladies that have no reliable model system.

Yury Verlinsky, a Russian-American medical researcher who specialized in embryo and cellular genetics (genetic cytology), developed prenatal diagnosis testing methods to determine genetic and chromosomal disorders a month and a half earlier than standard amniocentesis. The techniques are now used by many pregnant women and prospective parents, especially couples who have a history of genetic abnormalities or where the woman is over the age of 35 (when the risk of genetically related disorders is higher). In addition, by allowing parents to select an embryo without genetic disorders, they have the potential of saving the lives of siblings that already had similar disorders and diseases using cells from the disease free offspring.[19]

Differentiated somatic cells and ES cells use different strategies for dealing with DNA damage. For instance, human foreskin fibroblasts, one type of somatic cell, use non-homologous end joining (NHEJ), an error prone DNA repair process, as the primary pathway for repairing double-strand breaks (DSBs) during all cell cycle stages.[20] Because of its error-prone nature, NHEJ tends to produce mutations in a cells clonal descendants.

ES cells use a different strategy to deal with DSBs.[21] Because ES cells give rise to all of the cell types of an organism including the cells of the germ line, mutations arising in ES cells due to faulty DNA repair are a more serious problem than in differentiated somatic cells. Consequently, robust mechanisms are needed in ES cells to repair DNA damages accurately, and if repair fails, to remove those cells with un-repaired DNA damages. Thus, mouse ES cells predominantly use high fidelity homologous recombinational repair (HRR) to repair DSBs.[21] This type of repair depends on the interaction of the two sister chromosomes formed during S phase and present together during the G2 phase of the cell cycle. HRR can accurately repair DSBs in one sister chromosome by using intact information from the other sister chromosome. Cells in the G1 phase of the cell cycle (i.e. after metaphase/cell division but prior the next round of replication) have only one copy of each chromosome (i.e. sister chromosomes arent present). Mouse ES cells lack a G1 checkpoint and do not undergo cell cycle arrest upon acquiring DNA damage.[22] Rather they undergo programmed cell death (apoptosis) in response to DNA damage.[23] Apoptosis can be used as a fail-safe strategy to remove cells with un-repaired DNA damages in order to avoid mutation and progression to cancer.[24] Consistent with this strategy, mouse ES stem cells have a mutation frequency about 100-fold lower than that of isogenic mouse somatic cells.[25]

On January 23, 2009, Phase I clinical trials for transplantation of oligodendrocytes (a cell type of the brain and spinal cord) derived from human ES cells into spinal cord-injured individuals received approval from the U.S. Food and Drug Administration (FDA), marking it the world's first human ES cell human trial.[26] The study leading to this scientific advancement was conducted by Hans Keirstead and colleagues at the University of California, Irvine and supported by Geron Corporation of Menlo Park, CA, founded by Michael D. West, PhD. A previous experiment had shown an improvement in locomotor recovery in spinal cord-injured rats after a 7-day delayed transplantation of human ES cells that had been pushed into an oligodendrocytic lineage.[27] The phase I clinical study was designed to enroll about eight to ten paraplegics who have had their injuries no longer than two weeks before the trial begins, since the cells must be injected before scar tissue is able to form. The researchers emphasized that the injections were not expected to fully cure the patients and restore all mobility. Based on the results of the rodent trials, researchers speculated that restoration of myelin sheathes and an increase in mobility might occur. This first trial was primarily designed to test the safety of these procedures and if everything went well, it was hoped that it would lead to future studies that involve people with more severe disabilities.[28] The trial was put on hold in August 2009 due to FDA concerns regarding a small number of microscopic cysts found in several treated rat models but the hold was lifted on July 30, 2010.[29]

In October 2010 researchers enrolled and administered ESTs to the first patient at Shepherd Center in Atlanta.[30] The makers of the stem cell therapy, Geron Corporation, estimated that it would take several months for the stem cells to replicate and for the GRNOPC1 therapy to be evaluated for success or failure.

In November 2011 Geron announced it was halting the trial and dropping out of stem cell research for financial reasons, but would continue to monitor existing patients, and was attempting to find a partner that could continue their research.[31] In 2013 BioTime (AMEX:BTX), led by CEO Dr. Michael D. West, acquired all of Geron's stem cell assets, with the stated intention of restarting Geron's embryonic stem cell-based clinical trial for spinal cord injury research.[32]

BioTime company Asterias Biotherapeutics (NYSE MKT: AST) was granted a $14.3 million Strategic Partnership Award by the California Institute for Regenerative Medicine (CIRM) to re-initiate the worlds first embryonic stem cell-based human clinical trial, for spinal cord injury. Supported by California public funds, CIRM is the largest funder of stem cell-related research and development in the world.[33]

The award provides funding for Asterias to reinitiate clinical development of AST-OPC1 in subjects with spinal cord injury and to expand clinical testing of escalating doses in the target population intended for future pivotal trials.[33]

AST-OPC1 is a population of cells derived from human embryonic stem cells (hESCs) that contains oligodendrocyte progenitor cells (OPCs). OPCs and their mature derivatives called oligodendrocytes provide critical functional support for nerve cells in the spinal cord and brain. Asterias recently presented the results from phase 1 clinical trial testing of a low dose of AST-OPC1 in patients with neurologically-complete thoracic spinal cord injury. The results showed that AST-OPC1 was successfully delivered to the injured spinal cord site. Patients followed 23 years after AST-OPC1 administration showed no evidence of serious adverse events associated with the cells in detailed follow-up assessments including frequent neurological exams and MRIs. Immune monitoring of subjects through one year post-transplantation showed no evidence of antibody-based or cellular immune responses to AST-OPC1. In four of the five subjects, serial MRI scans performed throughout the 23 year follow-up period indicate that reduced spinal cord cavitation may have occurred and that AST-OPC1 may have had some positive effects in reducing spinal cord tissue deterioration. There was no unexpected neurological degeneration or improvement in the five subjects in the trial as evaluated by the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) exam.[33]

The Strategic Partnership III grant from CIRM will provide funding to Asterias to support the next clinical trial of AST-OPC1 in subjects with spinal cord injury, and for Asterias product development efforts to refine and scale manufacturing methods to support later-stage trials and eventually commercialization. CIRM funding will be conditional on FDA approval for the trial, completion of a definitive agreement between Asterias and CIRM, and Asterias continued progress toward the achievement of certain pre-defined project milestones.[33]

The major concern with the possible transplantation of ESC into patients as therapies is their ability to form tumors including teratoma.[34] Safety issues prompted the FDA to place a hold on the first ESC clinical trial, however no tumors were observed.

The main strategy to enhance the safety of ESC for potential clinical use is to differentiate the ESC into specific cell types (e.g. neurons, muscle, liver cells) that have reduced or eliminated ability to cause tumors. Following differentiation, the cells are subjected to sorting by flow cytometry for further purification. ESC are predicted to be inherently safer than IPS cells created with genetically-integrating viral vectors because they are not genetically modified with genes such as c-Myc that are linked to cancer. Nonetheless, ESC express very high levels of the iPS inducing genes and these genes including Myc are essential for ESC self-renewal and pluripotency,[35] and potential strategies to improve safety by eliminating c-Myc expression are unlikely to preserve the cells' "stemness". However, N-myc and L-myc have been identified to induce iPS cells instead of c-myc with similar efficiency.[36]More recent protocols to induce pluripotency bypass these problems completely by using non-integrating RNA viral vectors such as sendai virus or mRNA transfection.

Due to the nature of embryonic stem cell research, there is a lot of controversial opinions on the topic. Since harvesting embryonic stem cells necessitates destroying the embryo from which those cells are obtained, the moral status of the embryo comes into question. Scientists argue that the 5-day old mass of cells is too young to achieve personhood or that the embryo, if donated from an IVF clinic (which is where labs typically acquire embryos from), would otherwise go to medical waste anyway. Opponents of ESC research counter that any embryo has the potential to become a human, therefore destroying it is murder and the embryo must be protected under the same ethical view as a developed human being.[37]

In vitro fertilization generates multiple embryos. The surplus of embryos is not clinically used or is unsuitable for implantation into the patient, and therefore may be donated by the donor with consent. Human embryonic stem cells can be derived from these donated embryos or additionally they can also be extracted from cloned embryos using a cell from a patient and a donated egg.[49] The inner cell mass (cells of interest), from the blastocyst stage of the embryo, is separated from the trophectoderm, the cells that would differentiate into extra-embryonic tissue. Immunosurgery, the process in which antibodies are bound to the trophectoderm and removed by another solution, and mechanical dissection are performed to achieve separation. The resulting inner cell mass cells are plated onto cells that will supply support. The inner cell mass cells attach and expand further to form a human embryonic cell line, which are undifferentiated. These cells are fed daily and are enzymatically or mechanically separated every four to seven days. For differentiation to occur, the human embryonic stem cell line is removed from the supporting cells to form embryoid bodies, is co-cultured with a serum containing necessary signals, or is grafted in a three-dimensional scaffold to result.[50]

Embryonic stem cells are derived from the inner cell mass of the early embryo, which are harvested from the donor mother animal. Martin Evans and Matthew Kaufman reported a technique that delays embryo implantation, allowing the inner cell mass to increase. This process includes removing the donor mother's ovaries and dosing her with progesterone, changing the hormone environment, which causes the embryos to remain free in the uterus. After 46 days of this intrauterine culture, the embryos are harvested and grown in in vitro culture until the inner cell mass forms egg cylinder-like structures, which are dissociated into single cells, and plated on fibroblasts treated with mitomycin-c (to prevent fibroblast mitosis). Clonal cell lines are created by growing up a single cell. Evans and Kaufman showed that the cells grown out from these cultures could form teratomas and embryoid bodies, and differentiate in vitro, all of which indicating that the cells are pluripotent.[41]

Gail Martin derived and cultured her ES cells differently. She removed the embryos from the donor mother at approximately 76 hours after copulation and cultured them overnight in a medium containing serum. The following day, she removed the inner cell mass from the late blastocyst using microsurgery. The extracted inner cell mass was cultured on fibroblasts treated with mitomycin-c in a medium containing serum and conditioned by ES cells. After approximately one week, colonies of cells grew out. These cells grew in culture and demonstrated pluripotent characteristics, as demonstrated by the ability to form teratomas, differentiate in vitro, and form embryoid bodies. Martin referred to these cells as ES cells.[42]

It is now known that the feeder cells provide leukemia inhibitory factor (LIF) and serum provides bone morphogenetic proteins (BMPs) that are necessary to prevent ES cells from differentiating.[51][52] These factors are extremely important for the efficiency of deriving ES cells. Furthermore, it has been demonstrated that different mouse strains have different efficiencies for isolating ES cells.[53] Current uses for mouse ES cells include the generation of transgenic mice, including knockout mice. For human treatment, there is a need for patient specific pluripotent cells. Generation of human ES cells is more difficult and faces ethical issues. So, in addition to human ES cell research, many groups are focused on the generation of induced pluripotent stem cells (iPS cells).[54]

On August 23, 2006, the online edition of Nature scientific journal published a letter by Dr. Robert Lanza (medical director of Advanced Cell Technology in Worcester, MA) stating that his team had found a way to extract embryonic stem cells without destroying the actual embryo.[55] This technical achievement would potentially enable scientists to work with new lines of embryonic stem cells derived using public funding in the USA, where federal funding was at the time limited to research using embryonic stem cell lines derived prior to August 2001. In March, 2009, the limitation was lifted.[56]

The iPSC technology was pioneered by Shinya Yamanakas lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells.[57] He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent." [58]

In 2007 it was shown that pluripotent stem cells highly similar to embryonic stem cells can be generated by the delivery of three genes (Oct4, Sox2, and Klf4) to differentiated cells.[59] The delivery of these genes "reprograms" differentiated cells into pluripotent stem cells, allowing for the generation of pluripotent stem cells without the embryo. Because ethical concerns regarding embryonic stem cells typically are about their derivation from terminated embryos, it is believed that reprogramming to these "induced pluripotent stem cells" (iPS cells) may be less controversial. Both human and mouse cells can be reprogrammed by this methodology, generating both human pluripotent stem cells and mouse pluripotent stem cells without an embryo.[60]

This may enable the generation of patient specific ES cell lines that could potentially be used for cell replacement therapies. In addition, this will allow the generation of ES cell lines from patients with a variety of genetic diseases and will provide invaluable models to study those diseases.

However, as a first indication that the induced pluripotent stem cell (iPS) cell technology can in rapid succession lead to new cures, it was used by a research team headed by Rudolf Jaenisch of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, to cure mice of sickle cell anemia, as reported by Science journal's online edition on December 6, 2007.[61][62]

On January 16, 2008, a California-based company, Stemagen, announced that they had created the first mature cloned human embryos from single skin cells taken from adults. These embryos can be harvested for patient matching embryonic stem cells.[63]

The online edition of Nature Medicine published a study on January 24, 2005, which stated that the human embryonic stem cells available for federally funded research are contaminated with non-human molecules from the culture medium used to grow the cells.[64] It is a common technique to use mouse cells and other animal cells to maintain the pluripotency of actively dividing stem cells. The problem was discovered when non-human sialic acid in the growth medium was found to compromise the potential uses of the embryonic stem cells in humans, according to scientists at the University of California, San Diego.[65]

However, a study published in the online edition of Lancet Medical Journal on March 8, 2005 detailed information about a new stem cell line that was derived from human embryos under completely cell- and serum-free conditions. After more than 6 months of undifferentiated proliferation, these cells demonstrated the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem cell lines.[66]

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Embryonic stem cell - Wikipedia

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Stem cell, an undifferentiated cell that can divide to produce some offspring cells that continue as stem cells and some cells that are destined to differentiate (become specialized). Stem cells are an ongoing source of the differentiated cells that make up the tissues and organs of animals and plants. There is great interest in stem cells because they have potential in the development of therapies for replacing defective or damaged cells resulting from a variety of disorders and injuries, such as Parkinson disease, heart disease, and diabetes. There are two major types of stem cells: embryonic stem cells and adult stem cells, which are also called tissue stem cells.

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cardiovascular disease: Cardiac stem cells

Cardiac stem cells, which have the ability to differentiate (specialize) into mature heart cells and therefore could be used to repair damaged or diseased heart tissue, have garnered significant interest in the development of treatments for heart disease and cardiac defects. Cardiac stem

Embryonic stem cells (often referred to as ES cells) are stem cells that are derived from the inner cell mass of a mammalian embryo at a very early stage of development, when it is composed of a hollow sphere of dividing cells (a blastocyst). Embryonic stem cells from human embryos and from embryos of certain other mammalian species can be grown in tissue culture.

The most-studied embryonic stem cells are mouse embryonic stem cells, which were first reported in 1981. This type of stem cell can be cultured indefinitely in the presence of leukemia inhibitory factor (LIF), a glycoprotein cytokine. If cultured mouse embryonic stem cells are injected into an early mouse embryo at the blastocyst stage, they will become integrated into the embryo and produce cells that differentiate into most or all of the tissue types that subsequently develop. This ability to repopulate mouse embryos is the key defining feature of embryonic stem cells, and because of it they are considered to be pluripotentthat is, able to give rise to any cell type of the adult organism. If the embryonic stem cells are kept in culture in the absence of LIF, they will differentiate into embryoid bodies, which somewhat resemble early mouse embryos at the egg-cylinder stage, with embryonic stem cells inside an outer layer of endoderm. If embryonic stem cells are grafted into an adult mouse, they will develop into a type of tumour called a teratoma, which contains a variety of differentiated tissue types.

Mouse embryonic stem cells are widely used to create genetically modified mice. This is done by introducing new genes into embryonic stem cells in tissue culture, selecting the particular genetic variant that is desired, and then inserting the genetically modified cells into mouse embryos. The resulting chimeric mice are composed partly of host cells and partly of the donor embryonic stem cells. As long as some of the chimeric mice have germ cells (sperm or eggs) that have been derived from the embryonic stem cells, it is possible to breed a line of mice that have the same genetic constitution as the embryonic stem cells and therefore incorporate the genetic modification that was made in vitro. This method has been used to produce thousands of new genetic lines of mice. In many such genetic lines, individual genes have been ablated in order to study their biological function; in others, genes have been introduced that have the same mutations that are found in various human genetic diseases. These mouse models for human disease are used in research to investigate both the pathology of the disease and new methods for therapy.

Extensive experience with mouse embryonic stem cells made it possible for scientists to grow human embryonic stem cells from early human embryos, and the first human stem cell line was created in 1998. Human embryonic stem cells are in many respects similar to mouse embryonic stem cells, but they do not require LIF for their maintenance. The human embryonic stem cells form a wide variety of differentiated tissues in vitro, and they form teratomas when grafted into immunosuppressed mice. It is not known whether the cells can colonize all the tissues of a human embryo, but it is presumed from their other properties that they are indeed pluripotent cells, and they therefore are regarded as a possible source of differentiated cells for cell therapythe replacement of a patients defective cell type with healthy cells. Large quantities of cells, such as dopamine-secreting neurons for the treatment of Parkinson disease and insulin-secreting pancreatic beta cells for the treatment of diabetes, could be produced from embryonic stem cells for cell transplantation. Cells for this purpose have previously been obtainable only from sources in very limited supply, such as the pancreatic beta cells obtained from the cadavers of human organ donors.

The use of human embryonic stem cells evokes ethical concerns, because the blastocyst-stage embryos are destroyed in the process of obtaining the stem cells. The embryos from which stem cells have been obtained are produced through in vitro fertilization, and people who consider preimplantation human embryos to be human beings generally believe that such work is morally wrong. Others accept it because they regard the blastocysts to be simply balls of cells, and human cells used in laboratories have not previously been accorded any special moral or legal status. Moreover, it is known that none of the cells of the inner cell mass are exclusively destined to become part of the embryo itselfall of the cells contribute some or all of their cell offspring to the placenta, which also has not been accorded any special legal status. The divergence of views on this issue is illustrated by the fact that the use of human embryonic stem cells is allowed in some countries and prohibited in others.

In 2009 the U.S. Food and Drug Administration approved the first clinical trial designed to test a human embryonic stem cell-based therapy, but the trial was halted in late 2011 because of a lack of funding and a change in lead American biotech company Gerons business directives. The therapy to be tested was known as GRNOPC1, which consisted of progenitor cells (partially differentiated cells) that, once inside the body, matured into neural cells known as oligodendrocytes. The oligodendrocyte progenitors of GRNOPC1 were derived from human embryonic stem cells. The therapy was designed for the restoration of nerve function in persons suffering from acute spinal cord injury.

Embryonic germ (EG) cells, derived from primordial germ cells found in the gonadal ridge of a late embryo, have many of the properties of embryonic stem cells. The primordial germ cells in an embryo develop into stem cells that in an adult generate the reproductive gametes (sperm or eggs). In mice and humans it is possible to grow embryonic germ cells in tissue culture with the appropriate growth factorsnamely, LIF and another cytokine called fibroblast growth factor.

Some tissues in the adult body, such as the epidermis of the skin, the lining of the small intestine, and bone marrow, undergo continuous cellular turnover. They contain stem cells, which persist indefinitely, and a much larger number of transit amplifying cells, which arise from the stem cells and divide a finite number of times until they become differentiated. The stem cells exist in niches formed by other cells, which secrete substances that keep the stem cells alive and active. Some types of tissue, such as liver tissue, show minimal cell division or undergo cell division only when injured. In such tissues there is probably no special stem-cell population, and any cell can participate in tissue regeneration when required.

The epidermis of the skin contains layers of cells called keratinocytes. Only the basal layer, next to the dermis, contains cells that divide. A number of these cells are stem cells, but the majority are transit amplifying cells. The keratinocytes slowly move outward through the epidermis as they mature, and they eventually die and are sloughed off at the surface of the skin. The epithelium of the small intestine forms projections called villi, which are interspersed with small pits called crypts. The dividing cells are located in the crypts, with the stem cells lying near the base of each crypt. Cells are continuously produced in the crypts, migrate onto the villi, and are eventually shed into the lumen of the intestine. As they migrate, they differentiate into the cell types characteristic of the intestinal epithelium.

Bone marrow contains cells called hematopoietic stem cells, which generate all the cell types of the blood and the immune system. Hematopoietic stem cells are also found in small numbers in peripheral blood and in larger numbers in umbilical cord blood. In bone marrow, hematopoietic stem cells are anchored to osteoblasts of the trabecular bone and to blood vessels. They generate progeny that can become lymphocytes, granulocytes, red blood cells, and certain other cell types, depending on the balance of growth factors in their immediate environment.

Work with experimental animals has shown that transplants of hematopoietic stem cells can occasionally colonize other tissues, with the transplanted cells becoming neurons, muscle cells, or epithelia. The degree to which transplanted hematopoietic stem cells are able to colonize other tissues is exceedingly small. Despite this, the use of hematopoietic stem cell transplants is being explored for conditions such as heart disease or autoimmune disorders. It is an especially attractive option for those opposed to the use of embryonic stem cells.

Bone marrow transplants (also known as bone marrow grafts) represent a type of stem cell therapy that is in common use. They are used to allow cancer patients to survive otherwise lethal doses of radiation therapy or chemotherapy that destroy the stem cells in bone marrow. For this procedure, the patients own marrow is harvested before the cancer treatment and is then reinfused into the body after treatment. The hematopoietic stem cells of the transplant colonize the damaged marrow and eventually repopulate the blood and the immune system with functional cells. Bone marrow transplants are also often carried out between individuals (allograft). In this case the grafted marrow has some beneficial antitumour effect. Risks associated with bone marrow allografts include rejection of the graft by the patients immune system and reaction of immune cells of the graft against the patients tissues (graft-versus-host disease).

Bone marrow is a source for mesenchymal stem cells (sometimes called marrow stromal cells, or MSCs), which are precursors to non-hematopoietic stem cells that have the potential to differentiate into several different types of cells, including cells that form bone, muscle, and connective tissue. In cell cultures, bone-marrow-derived mesenchymal stem cells demonstrate pluripotency when exposed to substances that influence cell differentiation. Harnessing these pluripotent properties has become highly valuable in the generation of transplantable tissues and organs. In 2008 scientists used mesenchymal stem cells to bioengineer a section of trachea that was transplanted into a woman whose upper airway had been severely damaged by tuberculosis. The stem cells were derived from the womans bone marrow, cultured in a laboratory, and used for tissue engineering. In the engineering process, a donor trachea was stripped of its interior and exterior cell linings, leaving behind a trachea scaffold of connective tissue. The stem cells derived from the recipient were then used to recolonize the interior of the scaffold, and normal epithelial cells, also isolated from the recipient, were used to recolonize the exterior of the trachea. The use of the recipients own cells to populate the trachea scaffold prevented immune rejection and eliminated the need for immunosuppression therapy. The transplant, which was successful, was the first of its kind.

Research has shown that there are also stem cells in the brain. In mammals very few new neurons are formed after birth, but some neurons in the olfactory bulbs and in the hippocampus are continually being formed. These neurons arise from neural stem cells, which can be cultured in vitro in the form of neurospheressmall cell clusters that contain stem cells and some of their progeny. This type of stem cell is being studied for use in cell therapy to treat Parkinson disease and other forms of neurodegeneration or traumatic damage to the central nervous system.

Following experiments in animals, including those used to create Dolly the sheep, there has been much discussion about the use of somatic cell nuclear transfer (SCNT) to create pluripotent human cells. In SCNT the nucleus of a somatic cell (a fully differentiated cell, excluding germ cells), which contains the majority of the cells DNA (deoxyribonucleic acid), is removed and transferred into an unfertilized egg cell that has had its own nuclear DNA removed. The egg cell is grown in culture until it reaches the blastocyst stage. The inner cell mass is then removed from the egg, and the cells are grown in culture to form an embryonic stem cell line (generations of cells originating from the same group of parent cells). These cells can then be stimulated to differentiate into various types of cells needed for transplantation. Since these cells would be genetically identical to the original donor, they could be used to treat the donor with no problems of immune rejection. Scientists generated human embryonic stem cells successfully from SCNT human embryos for the first time in 2013.

While promising, the generation and use of SCNT-derived embryonic stem cells is controversial for several reasons. One is that SCNT can require more than a dozen eggs before one egg successfully produces embryonic stem cells. Human eggs are in short supply, and there are many legal and ethical problems associated with egg donation. There are also unknown risks involved with transplanting SCNT-derived stem cells into humans, because the mechanism by which the unfertilized egg is able to reprogram the nuclear DNA of a differentiated cell is not entirely understood. In addition, SCNT is commonly used to produce clones of animals (such as Dolly). Although the cloning of humans is currently illegal throughout the world, the egg cell that contains nuclear DNA from an adult cell could in theory be implanted into a womans uterus and come to term as an actual cloned human. Thus, there exists strong opposition among some groups to the use of SCNT to generate human embryonic stem cells.

Due to the ethical and moral issues surrounding the use of embryonic stem cells, scientists have searched for ways to reprogram adult somatic cells. Studies of cell fusion, in which differentiated adult somatic cells grown in culture with embryonic stem cells fuse with the stem cells and acquire embryonic stem-cell-like properties, led to the idea that specific genes could reprogram differentiated adult cells. An advantage of cell fusion is that it relies on existing embryonic stem cells instead of eggs. However, fused cells stimulate an immune response when transplanted into humans, which leads to transplant rejection. As a result, research has become increasingly focused on the genes and proteins capable of reprogramming adult cells to a pluripotent state. In order to make adult cells pluripotent without fusing them to embryonic stem cells, regulatory genes that induce pluripotency must be introduced into the nuclei of adult cells. To do this, adult cells are grown in cell culture, and specific combinations of regulatory genes are inserted into retroviruses (viruses that convert RNA [ribonucleic acid] into DNA), which are then introduced to the culture medium. The retroviruses transport the RNA of the regulatory genes into the nuclei of the adult cells, where the genes are then incorporated into the DNA of the cells. About 1 out of every 10,000 cells acquires embryonic stem cell properties. Although the mechanism is still uncertain, it is clear that some of the genes confer embryonic stem cell properties by means of the regulation of numerous other genes. Adult cells that become reprogrammed in this way are known as induced pluripotent stem cells (iPS).

Similar to embryonic stem cells, induced pluripotent stem cells can be stimulated to differentiate into select types of cells that could in principle be used for disease-specific treatments. In addition, the generation of induced pluripotent stem cells from the adult cells of patients affected by genetic diseases can be used to model the diseases in the laboratory. For example, in 2008 researchers isolated skin cells from a child with an inherited neurological disease called spinal muscular atrophy and then reprogrammed these cells into induced pluripotent stem cells. The reprogrammed cells retained the disease genotype of the adult cells and were stimulated to differentiate into motor neurons that displayed functional insufficiencies associated with spinal muscular atrophy. By recapitulating the disease in the laboratory, scientists were able to study closely the cellular changes that occurred as the disease progressed. Such models promise not only to improve scientists understanding of genetic diseases but also to facilitate the development of new therapeutic strategies tailored to each type of genetic disease.

In 2009 scientists successfully generated retinal cells of the human eye by reprogramming adult skin cells. This advance enabled detailed investigation of the embryonic development of retinal cells and opened avenues for the generation of novel therapies for eye diseases. The production of retinal cells from reprogrammed skin cells may be particularly useful in the treatment of retinitis pigmentosa, which is characterized by the progressive degeneration of the retina, eventually leading to night blindness and other complications of vision. Although retinal cells also have been produced from human embryonic stem cells, induced pluripotency represents a less controversial approach. Scientists have also explored the possibility of combining induced pluripotent stem cell technology with gene therapy, which would be of value particularly for patients with genetic disease who would benefit from autologous transplantation.

Researchers have also been able to generate cardiac stem cells for the treatment of certain forms of heart disease through the process of dedifferentiation, in which mature heart cells are stimulated to revert to stem cells. The first attempt at the transplantation of autologous cardiac stem cells was performed in 2009, when doctors isolated heart tissue from a patient, cultured the tissue in a laboratory, stimulated cell dedifferentiation, and then reinfused the cardiac stem cells directly into the patients heart. A similar study involving 14 patients who underwent cardiac bypass surgery followed by cardiac stem cell transplantation was reported in 2011. More than three months after stem cell transplantation, the patients experienced a slight but detectable improvement in heart function.

Patient-specific induced pluripotent stem cells and dedifferentiated cells are highly valuable in terms of their therapeutic applications because they are unlikely to be rejected by the immune system. However, before induced pluripotent stem cells can be used to treat human diseases, researchers must find a way to introduce the active reprogramming genes without using retroviruses, which can cause diseases such as leukemia in humans. A possible alternative to the use of retroviruses to transport regulatory genes into the nuclei of adult cells is the use of plasmids, which are less tumourigenic than viruses.

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stem cell | Definition, Types, Uses, Research, & Facts ...

Cardiac Stem Cells Comments Off on stem cell | Definition, Types, Uses, Research, & Facts … | September 16th, 2018

Cardiac stem cell research has a turbulent history. Studies revealing the presence of regenerative progenitors in adult rodents hearts formed the basis of numerous clinical trials, but several experiments have cast doubt on these cells ability to produce new tissue. Some scientists are now lauding the results of a report published in April in Circulation as undeniable evidence against the idea that resident stem cells can give rise to new cardiomyocytes.

The concept of [many] clinical trials arose from the basic science in labs of a few individuals more than 15 years ago, and that basic science is whats now being called into question, says Jeffery Molkentin, a cardiovascular biologist at Cincinnati Childrens Hospital who penned an editorial about the latest work.

The first evidence supporting the notion of cardiac stem cells in adults emerged in the early 2000s, when researchers reported that cells derived from bone marrow or adult heart expressing the protein c-kit could give rise to new muscle tissue when injected into damaged myocardium in rodents. These studies caused some controversy right from the start, Molkentin says. The main reason that this struck a raw nerve with people is because we already know that heart, in human patients, doesnt regenerate itself after an infarct.

Early skepticism arose in 2004, when two separate groups of researchers published back-to-back papers refuting the claims that bone marrowderived c-kit cells could regenerate damaged heart tissue. Still, the concept of endogenous cardiac stem cells remained a mainstream idea until Molkentin and his colleagues published a study in 2014 reporting that c-kit cells in the adult mouse heart almost never produced new cardiomyocytes, says Bin Zhou, a cell biologist at the Chinese Academy of Sciences and a coauthor of the new study.

Although Molkentins findings were replicated shortly afterwards by two independent groups (including Zhous), some researchers held fast to the idea that cardiac progenitors could regenerate injured heart tissue. Earlier this year, a team of researchersincluding Bernardo Nadal-Ginard and Daniele Torella of Magna Graecia University in Italy and several other scientists who conducted the early work on c-kit cellspublished a paper reporting the flaws in the cell lineage tracing technique employed by Molkentin, Zhou, and their colleagues. For example, they noted that the method, which involved tagging c-kitexpressing cells and their progeny with a fluorescent marker, compromised the gene required to express the c-kit protein, impairing the progenitors regenerative abilities.

In the new Circulationstudy, Zhou and his colleagues used a different approach to examine endogenous stem cell populations in mice. Instead of tagging c-kit cells, the team applied a technique that would fluorescently label nonmyocytes and newly generated muscle cells a different color from existing myocytes. This method allowed the researchers to investigate all proposed stem cell populations, rather than specifically addressing c-kit cells. We wanted to ask the broader question of whether there are any stem cells in the adult heart, Zhou says.

These experiments revealed that, while nonmyocytes generate cardiomyocytes in mouse embryos, they do not give rise to new muscle cells in adult rodents hearts. The results also address the concerns raised about c-kit lineage tracing, Zhou tells The Scientist. We think our system can conclude that nonmyocytes cannot become myocytes in adults in homeostasis and after injury.

Torella says that hes not convinced by Zhous evidence. The main issue, he explains, is that the researchers did not explicitly test whether cardiac stem cells were indeed labeled as nonmyocytes to ensure that they were not inadvertently tagging them as myocytes instead.

Molkentin disagrees with this critique, stating that the only way the system would label a myocyte progenitor as a myocyte is if it was no longer a true stem cell, but instead an immature myocyte. Zhous group uses an exhausting and very rigorous genetic approach, he adds. My opinion is that we need to go back to the bench and conduct additional research to truly understand the mechanisms at play to better inform how we design the next generation of clinical trials.

Other scientists note that stem cells may not need to become new myocytes to help repair the injured heart. According to Phillip Yang, a cardiologist at Stanford University who did not take part in the work, many scientists now agree that stem cells are not regenerating damaged cardiomyocytes. Instead, he explains, a growing body of research now supports an alternative theory, which posits that progenitor cells secrete small molecules called paracrine factors that help repair injured heart cells. (Yang is involved in several stem cell clinical trials).

When you inject these stem cells, its pretty incontrovertible that they help heart function in a mouse injury model, Yang says. But the truth is, most of these cells are dead upon arrival [to the site of injury]. So the question is: Why is heart function still improving if these cells are dying?

Y. Li et al., Genetic lineage tracing of nonmyocyte population by dual recombinases, Circulation, 138:793-805, 2018.

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Adult Cardiac Stem Cells Don't Exist: Study | The ...

Cardiac Stem Cells Comments Off on Adult Cardiac Stem Cells Don’t Exist: Study | The … | September 8th, 2018

There was a very sad example of this in the last decade.There's a wonderful drug, and a class of drugs actually,but the particular drug was Vioxx, andfor people who were suffering from severe arthritis pain,the drug was an absolute lifesaver,but unfortunately, for another subset of those people,they suffered pretty severe heart side effects,and for a subset of those people, the side effects wereso severe, the cardiac side effects, that they were fatal.But imagine a different scenario,where we could have had an array, a genetically diverse array,of cardiac cells, and we could have actually testedthat drug, Vioxx, in petri dishes, and figured out,well, okay, people with this genetic type are going to havecardiac side effects, people with these genetic subgroupsor genetic shoes sizes, about 25,000 of them,are not going to have any problems.The people for whom it was a lifesavercould have still taken their medicine.The people for whom it was a disaster, or fatal,would never have been given it, andyou can imagine a very different outcome for the company,who had to withdraw the drug.

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Susan Solomon: The promise of research with stem cells ...

Cardiac Stem Cells Comments Off on Susan Solomon: The promise of research with stem cells … | August 23rd, 2018

Stem cell therapy can be described as a means or process by which stem cells are used for the prevention, treatment or the cure of diseases. Stem cells are a special kind of cells that have features other types of cells dont have. As an illustration, stem cells are capable of proliferation. This implies that they can develop into any type of cell, and grow to start performing the functions of the tissue. In addition, they can regenerate. This means they can multiply themselves. This is most important when a new tissue has to be formed. Also, they modulate immune reactions. This has made them useful for the treatment of autoimmune diseases, especially those that affect the musculoskeletal system such as rheumatoid arthritis, systemic lupus erythematosus and so on. Stem cells can be derrived from different sources. They can be extracted from the body, and in some specific parts of the body. This includes the blood, bone marrow, umbilical cord in newborns, adipose tissue, and from embryos. There are 2 main types of stem cell transplant. These are autologous stem cell transplant, and allogeneic stem cell transplant. The autologous stem cell transplant means that stem cells are extracted from the patient, processed, and then transplanted back to the patient, for therapeutic purposes. On the other hand, allogeneic stem cell transplant means the transplant of stem cells or from another individual, known as the donor, to another person, or recipient. Some treatments must be given to the receiver to prevent any cases of rejections, and other complications. The autologous is usually the most preferred type of transplant because of its almost zero side effects. Below are some of the stem cell treatments. Our goal is to provide education, research and an opportunity to connect with Stem Cell Doctors, as well as provide stem cell reviews

Adipose Stem Cell TreatmentsAdipose stem cell treatment is one of the most commonly used. This is because large quantities of stem cells can be derrived from them. According to statistics, the number of stem cells in adipose tissue are usually hundreds of times higher than what can be obtained from other sources, such as the bone marrow stem cells. Adipose stem cells have taken the center stage in the world of stem cell therapy. Apart from the ease that comes with the harvesting of these cells from the adipose tissue, they also have some special features, that separates them from other types of cells. Adipose stem cells are capable of regulating and modulating the immune system. This includes immune suppression, which is important for the treatment of autoimmune diseases. In addition, adipose stem cells can differentiate to form other types of cells. Some of them include the bone forming cells, cardiomyocytes, and cells of the nervous system.

This process can be divided into four parts. These are

Stem cell joint injection is fast becoming the new treatment of joint diseases. Stem cells derived from bone marrow, adipose and mesenchymal stem cells are the most commonly used. The stem cells are injected into the joints, and they proceed to repair and replace the damaged tissues. The cells also modulate the inflammatory process going on. Overall, stem cell joint injections significantly reduce the recovery time of patients and also eliminates pain and risks associated with surgery. Examples of diseases where this treatment is used include osteoarthritis, rheumatoid arthritis, and so on. Researchers and physicians have rated this procedure to be the future of joint therapy.

Losing a tooth as a kid isnt news because youd eventually grow them back, but losing one as an adult isnt a pleasant experience. Youd have to go through the pains of getting a replacement from your dentist. Apart from the cost of these procedures, the pain and number of days youd have to stay at home nursing the pain is also a problem. Nevertheless, there are great teeth replacement therapies available for all kinds of dental problems. Although there are already good dental treatment methods, stem cell therapy might soon become the future of dental procedures. Currently, a lot of research is being done on how stem cells can be used to develop teeth naturally, especially in patients with dental problems. The aim of the project is to develop a method whereby peoples stem cells are used in regenerating their own teeth and within the shortest time possible. Some of the benefits of the stem cell tooth would be:

The quality of life of those that underwent serious procedures, especially those that had an allogeneic hematopoietic stem cell transplantation done was studied. It was discovered that this set of people had to cope with some psychological problems, even years after the procedure. In addition, allogeneic stem cell transplantation often comes with some side effects. However, this a small price to pay, considering that the adverse effects are not usually life-threatening. Also theses types of procedures are used for severe disorders or even terminal diseases. On the other hand, autologous stem cell transplantation bears the minimum to no side effects. Patients do have a great quality of life, both in the short term and in the long term.

This is one of the many uses of stem cells. The stem cell gun is a device that is used in treating people with wounds or burns. This is done by simply triggering it, and it sprays stem cells on the affected part. This kind of treatment is crucial for victims of a severe burn. Usually, people affected by severe burns would have to endure excruciating pain. The process of recovery is usually long, which might vary from weeks to months, depending on the severity of the burn. Even after treatment, most patients are left with scars forever. However, the stem cell gun eliminates these problems, the skin can be grown back in just a matter of days. The new skin also grows evenly and blends perfectly with the other part of the body. This process is also without the scars that are usually associated with the traditional burns therapy. The stem cell gun is without any side effects.

There is one company that focuses on the production of stem cell supplements. These stem cells are usually natural ingredients that increase the development of stem cells, and also keeps them healthy. The purpose of the stem cell supplements is to help reduce the aging process and make people look younger. These supplements work by replacing the dead or repairing the damaged tissues of the body. There have been a lot of testimonials to the efficacy of these supplements.

It is the goal of researchers to make stem cell therapy a good alternative for the millions of patients suffering from cardiac-related diseases. According to some experiments carried out in animals, stem cells were injected into the ones affected by heart diseases. A large percentage of them showed great improvement, even within just a few weeks. However, when the trial was carried out in humans, some stem cells went ahead to develop into heart muscles, but overall, the heart function was generally improved. The reason for the improvement has been attributed to the formation of new vessels in the heart. The topic that has generated a lot of arguments have been what type of cells should be used in the treatment of heart disorders. Stem cells extracted from the bone marrow, embryo have been in use, although bone marrow stem cells are the most commonly used. Stem cells extracted from bone marrow can differentiate into cardiac cells, while studies have shown that other stem cells cannot do the same. Even though the stem cell therapy has a lot of potential in the future, more research and studies have to be done to make that a reality.

The use of stem cells for the treatment of hair loss has increased significantly. This can be attributed to the discovery of stem cells in bone marrow, adipose cells, umbilical cord, and so on. Stem cells are extracted from the patient, through any of the sources listed above. Adipose tissue stem cells are usually the most convenient in this scenario, as they do not require any special extraction procedure. Adipose tissue is harvested from the abdominal area. The stem cells are then isolated from the other cells through a process known as centrifugation. The stem cells are then activated and are now ready for use. The isolated stem cells are then introduced into the scalp, under local anesthesia. The entire process takes about three hours. Patients are free to go home, after the procedure. Patients would begin to see improvements in just a few months, however, this depends largely on the patients ability to heal. Every patient has a different outcome.

Human umbilical stem cells are cells extracted from the umbilical cord of a healthy baby, shortly after birth. Umbilical cord tissue is abundant in stem cells, and the stem cells can differentiate into many types of cells such as red blood cells, white blood cells, and platelets. They are also capable of differentiating into non-blood cells such as muscle cells, cartilage cells and so on. These cells are usually preferred because its' extraction is minimally non invasive. It also is nearly painless. It also has zero risks of rejecting, as it does not require any form of matching or typing.Human umbilical stem cell injections are used for the treatment of spinal cord injuries. A trial was done on twenty-five patients that had late-stage spinal cord injuries. They were placed on human umbilical stem cell therapy, while another set of 25 patients were simultaneously placed on the usual rehabilitation therapy. The two groups were studied for the next twelve months. The results of the trial showed that those people placed on stem cell therapy by administering the human umbilical cell tissue injections had a significant recovery, as compared to the other group that underwent the traditional rehabilitation therapy. It was concluded that human umbilical tissue injections applied close to the injured part gives the best outcomes.

Stem cell therapy has been used for the treatment of many types diseases. This ranges from terminal illnesses such as cancer, joint diseases such as arthritis, and also autoimmune diseases. Stem cell therapy is often a better alternative to most traditional therapy today. This is because stem cell procedure is minimally invasive when compared to chemotherapy and so on. It harnesses the bodys own ability to heal. The stem cells are extracted from other parts of the body and then transplanted to other parts of the body, where they would repair and maintain the tissues. They also perform the function of modulating the immune system, which makes them important for the treatment of autoimmune diseases. Below are some of the diseases that stem cell therapies have been used successfully:

A stem cell bank can be described as a facility where stem cells are stored for future purposes. These are mostly amniotic stem cells, which are derived from the amnion fluid. Umbilical cord stem cells are also equally important as it is rich in stem cells and can be used for the treatment of many diseases. Examples of these diseases include cancer, blood disorders, autoimmune diseases, musculoskeletal diseases and so on. According to statistics, umbilical stem cells can be used for the treatment of over eighty diseases. Storing your stem cells should be seen as an investment in your health for future sake. Parents do have the option of either throwing away their babys umbilical cord or donating it to stem cell banks.

The adipose tissue contains a lot of stem cells, that has the ability to transform into other cells such as muscle, cartilage, neural cells. They are also important for the treatment of some cardiovascular diseases. This is what makes it important for people to want to store their stem cells. The future health benefit is huge. The only way adults can store their stem cells in sufficient amounts is to extract the stem cells from their fat tissues. This process is usually painless and fast. Although, the extraction might have to be done between 3 to 5 times before the needed quantity is gotten. People that missed the opportunity to store their stem cells, using their cord cells, can now store it using their own adipose tissues. This can be used at any point in time.

Side effects often accompany every kind of treatment. However, this depends largely on the individual. While patients might present with side effects, some other people wouldnt. Whether a patient will present with adverse effects, depends on the following factors;

Some of the common side effects of stem cell transplant are;

Stem cell treatment has been largely successful so far, however, more studies and research needs to be done. Stem cell therapy could be the future.

Stem cells are unique cells that have some special features such as self-regeneration, tissue repair, and modulation of the immune system. These are the features that are employed in the treatment of diseases.

Our doctors are certified by iSTEMCELL but operate as part of a medical group or as independent business owners and as such are free to charge what the feel to be the right fit for their practice and clients. We have seen Stem Cell Treatment costs range from $3500 upwards of $30,000 depending on the condition and protocol required for intended results. Find the Best Stem Cell Doctor Near me If you are interested in saving money, try our STEM CELL COUPON!

Travel Medcations are becoming very popular around the globe for several reasons but not for what one might think. It is not about traveling to Mexico to save money, but to get procedures or protocols that are not yet available in your home country. Many procedures are started in your home country, then the tissue is set to the tissue lab where it is then grown in a process to maximize live cells, then sent to a hospital in Mexico designed to treat or provide different therapies for different conditions. If you're ready to take a medical vacation call 972-800-6670 for our"WHITE GLOVE" service.

Chen, C. and Hou, J. (2016). Mesenchymal stem cell-based therapy in kidney transplantation. Stem Cell Research & Therapy, 7(1).

Donnelly, A., Johar, S., OBrien, T. and Tuan, R. (2010). Welcome to Stem Cell Research & Therapy. Stem Cell Research & Therapy, 1(1), p.1.

Groothuis, S. (2015). Changes in Stem Cell Research. Stem Cell Research, 14(1), p.130.

Rao, M. (2012). Stem cells and regenerative medicine. Stem Cell Research & Therapy, 3(4), p.27.

Vunjak-Novakovic, G. (2013). Physical influences on stem cells. Stem Cell Research & Therapy, 4(6), p.153.

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Stem Cell Therapy and Stem Cell Injection Provider Finder ...

Cardiac Stem Cells Comments Off on Stem Cell Therapy and Stem Cell Injection Provider Finder … | August 19th, 2018

"Muscle fiber" and "Myofiber" redirect here. For protein structures inside cells, see Myofibril.

A myocyte (also known as a muscle cell)[1] is the type of cell found in muscle tissue. Myocytes are long, tubular cells that develop from myoblasts to form muscles in a process known as myogenesis.[2] There are various specialized forms of myocytes: cardiac, skeletal, and smooth muscle cells, with various properties. The striated cells of cardiac and skeletal muscles are referred to as muscle fibers.[3] Cardiomyocytes are the muscle fibres that form the chambers of the heart, and have a single central nucleus.[4] Skeletal muscle fibers help support and move the body and tend to have peripheral nuclei.[5][6] Smooth muscle cells control involuntary movements such as the peristalsis contractions in the oesophagus and stomach.

The unusual microstructure of muscle cells has led cell biologists to create specialized terminology. However, each term specific to muscle cells has a counterpart that is used in the terminology applied to other types of cells:

The sarcoplasm is the cytoplasm of a muscle fiber. Most of the sarcoplasm is filled with myofibrils, which are long protein cords composed of myofilaments. The sarcoplasm is also composed of glycogen, a polysaccharide of glucose monomers, which provides energy to the cell with heightened exercise, and myoglobin, the red pigment that stores oxygen until needed for muscular activity.[7]

There are three types of myofilaments:[7]

Together, these myofilaments work to produce a muscle contraction.

The sarcoplasmic reticulum, a specialized type of smooth endoplasmic reticulum, forms a network around each myofibril of the muscle fiber. This network is composed of groupings of two dilated end-sacs called terminal cisternae, and a single transverse tubule, or T tubule, which bores through the cell and emerge on the other side; together these three components form the triads that exist within the network of the sarcoplasmic reticulum, in which each T tubule has two terminal cisternae on each side of it. The sarcoplasmic reticulum serves as reservoir for calcium ions, so when an action potential spreads over the T tubule, it signals the sarcoplasmic reticulum to release calcium ions from the gated membrane channels to stimulate a muscle contraction.[7][8]

The sarcolemma is the cell membrane of a striated muscle fiber and receives and conducts stimuli. At the end of each muscle fiber, the outer layer of the sarcolemma combines with tendon fibers.[9] Within the muscle fiber pressed against the sarcolemma are multiple flattened nuclei; this multinuclear condition results from multiple myoblasts fusing to produce each muscle fiber, where each myoblast contributes one nucleus.[7]

The cell membrane of a myocyte has several specialized regions, which may include the intercalated disk and the transverse tubular system. The cell membrane is covered by a lamina coat which is approximately 50nm wide. The laminar coat is separable into two layers; the lamina densa and lamina lucida. In between these two layers can be several different types of ions, including calcium.[10]

The cell membrane is anchored to the cell's cytoskeleton by anchor fibers that are approximately 10nm wide. These are generally located at the Z lines so that they form grooves and transverse tubules emanate. In cardiac myocytes this forms a scalloped surface.[10]

The cytoskeleton is what the rest of the cell builds off of and has two primary purposes; the first is to stabilize the topography of the intracellular components and the second is to help control the size and shape of the cell. While the first function is important for biochemical processes, the latter is crucial in defining the surface to volume ratio of the cell. This heavily influences the potential electrical properties of excitable cells. Additionally deviation from the standard shape and size of the cell can have negative prognostic impact.[10]

Each muscle fiber contains myofibrils, which are very long chains of sarcomeres, the contractile units of the cell. A cell from the biceps brachii muscle may contain 100,000 sarcomeres.[11][verification needed] The myofibrils of smooth muscle cells are not arranged into sarcomeres. The sarcomeres are composed of thin and thick filaments. Thin filaments are made of actin and attach at Z lines which help them line up correctly with each other.[12] Troponins are found at intervals along the thin filaments. Thick filaments are made of the elongated protein myosin.[13] The sarcomere does not contain organelles or a nucleus. Sarcomeres are marked by Z lines which show the beginning and the end of a sarcomere. Individual myocytes are surrounded by endomysium.

Myocytes are bound together by perimysium into bundles called fascicles; the bundles are then grouped together to form muscle tissue, which is enclosed in a sheath of epimysium. The perimysium contains blood vessels and nerves which provide for the muscle fibers. Muscle spindles are distributed throughout the muscles and provide sensory feedback information to the central nervous system. Myosin is shaped like a long shaft with a rounded end pointed out towards the surface. This structure forms the cross bridge that connects with the thin filaments.[13]

A myoblast is a type of embryonic progenitor cell that differentiates to give rise to muscle cells.[14] Differentiation is regulated by myogenic regulatory factors, including MyoD, Myf5, myogenin, and MRF4.[15] GATA4 and GATA6 also play a role in myocyte differentiation.[16]

Skeletal muscle fibers are made when myoblasts fuse together; muscle fibers therefore are cells with multiple nuclei, known as myonuclei, with each cell nucleus originating from a single myoblast. The fusion of myoblasts is specific to skeletal muscle (e.g., biceps brachii) and not cardiac muscle or smooth muscle.

Myoblasts in skeletal muscle that do not form muscle fibers dedifferentiate back into myosatellite cells. These satellite cells remain adjacent to a skeletal muscle fiber, situated between the sarcolemma and the basement membrane[17] of the endomysium (the connective tissue investment that divides the muscle fascicles into individual fibers). To re-activate myogenesis, the satellite cells must be stimulated to differentiate into new fibers.

Myoblasts and their derivatives, including satellite cells, can now be generated in vitro through directed differentiation of pluripotent stem cells.[18]

Kindlin-2 plays a role in developmental elongation during myogenesis.[19]

Muscle fibers grow when exercised and shrink when not in use. This is due to the fact that exercise stimulates the increase in myofibrils which increase the overall size of muscle cells. Well exercised muscles can not only add more size but can also develop more mitochondria, myoglobin, glycogen and a higher density of capillaries. However muscle cells cannot divide to produce new cells, and as a result we have fewer muscle cells as an adult than a newborn.[20]

When contracting, thin and thick filaments slide with respect to each other by using adenosine triphosphate. This pulls the Z discs closer together in a process called sliding filament mechanism. The contraction of all the sarcomeres results in the contraction of the whole muscle fiber. This contraction of the myocyte is triggered by the action potential over the cell membrane of the myocyte. The action potential uses transverse tubules to get from the surface to the interior of the myocyte, which is continuous within the cell membrane. Sarcoplasmic reticula are membranous bags that transverse tubules touch but remain separate from. These wrap themselves around each sarcomere and are filled with Ca2+.[13]

Excitation of a myocyte causes depolarization at its synapses, the neuromuscular junctions, which triggers action potential. With a singular neuromuscular junction, each muscle fiber receives input from just one somatic efferent neuron. Action potential in a somatic efferent neuron causes the release of the neurotransmitter acetylcholine.[21]

When the acetylcholine is released it diffuses across the synapse and binds to a receptor on the sarcolemma, a term unique to muscle cells that refers to the cell membrane. This initiates an impulse that travels across the sarcolemma.[20]

When the action potential reaches the sarcoplasmic reticulum it triggers the release of Ca2+ from the Ca2+ channels. The Ca2+ flows from the sarcoplasmic reticulum into the sarcomere with both of its filaments. This causes the filaments to start sliding and the sarcomeres to become shorter. This requires a large amount of ATP, as it is used in both the attachment and release of every myosin head. Very quickly Ca2+ is actively transported back into the sarcoplasmic reticulum, which blocks the interaction between the thin and thick filament. This in turn causes the muscle cell to relax.[20]

There are four main different types of muscle contraction: twitch, treppe, tetanus and isometric/isotonic. Twitch contraction is the process previously described, in which a single stimulus signals for a single contraction. In twitch contraction the length of the contraction may vary depending on the size of the muscle cell. During treppe (or summation) contraction muscles do not start at maximum efficiency; instead they achieve increased strength of contraction due to repeated stimuli. Tetanus involves a sustained contraction of muscles due to a series of rapid stimuli, which can continue until the muscles fatigue. Isometric contractions are skeletal muscle contractions that do not cause movement of the muscle. However, isotonic contractions are skeletal muscle contractions that do cause movement.[20]

Specialized cardiomyocytes located in the sinoatrial node are responsible for generating the electrical impulses that control the heart rate. These electrical impulses coordinate contraction throughout the remaining heart muscle via the electrical conduction system of the heart. Sinoatrial node activity is modulated, in turn, by nerve fibres of both the sympathetic and parasympathetic nervous systems. These systems act to increase and decrease, respectively, the rate of production of electrical impulses by the sinoatrial node.

There are numerous methods employed for fiber-typing, and confusion between the methods is common among non-experts. Two commonly confused methods are histochemical staining for myosin ATPase activity and immunohistochemical staining for Myosin heavy chain (MHC) type. Myosin ATPase activity is commonlyand correctlyreferred to as simply "fiber type", and results from the direct assaying of ATPase activity under various conditions (e.g. pH).[22] Myosin heavy chain staining is most accurately referred to as "MHC fiber type", e.g. "MHC IIa fibers", and results from determination of different MHC isoforms.[22] These methods are closely related physiologically, as the MHC type is the primary determinant of ATPase activity. Note, however, that neither of these typing methods is directly metabolic in nature; they do not directly address oxidative or glycolytic capacity of the fiber. When "type I" or "type II" fibers are referred to generically, this most accurately refers to the sum of numerical fiber types (I vs. II) as assessed by myosin ATPase activity staining (e.g. "type II" fibers refers to type IIA + type IIAX + type IIXA... etc.).

Below is a table showing the relationship between these two methods, limited to fiber types found in humans. Note the sub-type capitalization used in fiber typing vs. MHC typing, and that some ATPase types actually contain multiple MHC types. Also, a subtype B or b is not expressed in humans by either method.[23] Early researchers believed humans to express a MHC IIb, which led to the ATPase classification of IIB. However, later research showed that the human MHC IIb was in fact IIx,[23] indicating that the IIB is better named IIX. IIb is expressed in other mammals, so is still accurately seen (along with IIB) in the literature. Non human fiber types include true IIb fibers, IIc, IId, etc.

Further fiber typing methods are less formally delineated, and exist on more of a spectrum. They tend to be focused more on metabolic and functional capacities (i.e., oxidative vs. glycolytic, fast vs. slow contraction time). As noted above, fiber typing by ATPase or MHC does not directly measure or dictate these parameters. However, many of the various methods are mechanistically linked, while others are correlated in vivo.[26][27] For instance, ATPase fiber type is related to contraction speed, because high ATPase activity allows faster crossbridge cycling.[22] While ATPase activity is only one component of contraction speed, type I fibers are "slow", in part, because they have low speeds of ATPase activity in comparison to type II fibers. However, measuring contraction speed is not the same as ATPase fiber typing.

Because of these types of relationships, Type I and Type II fibers have relatively distinct metabolic, contractile, and motor-unit properties. The table below differentiates these types of properties. These types of propertieswhile they are partly dependent on the properties of individual fiberstend to be relevant and measured at the level of the motor unit, rather than individual fiber.[22]

Traditionally, fibers were categorized depending on their varying color, which is a reflection of myoglobin content. Type I fibers appear red due to the high levels of myoglobin. Red muscle fibers tend to have more mitochondria and greater local capillary density. These fibers are more suited for endurance and are slow to fatigue because they use oxidative metabolism to generate ATP (adenosine triphosphate). Less oxidative type II fibers are white due to relatively low myoglobin and a reliance on glycolytic enzymes.

Fibers can also be classified on their twitch capabilities, into fast and slow twitch. These traits largely, but not completely, overlap the classifications based on color, ATPase, or MHC.

Some authors define a fast twitch fiber as one in which the myosin can split ATP very quickly. These mainly include the ATPase type II and MHC type II fibers. However, fast twitch fibers also demonstrate a higher capability for electrochemical transmission of action potentials and a rapid level of calcium release and uptake by the sarcoplasmic reticulum. The fast twitch fibers rely on a well-developed, short term, glycolytic system for energy transfer and can contract and develop tension at 23 times the rate of slow twitch fibers. Fast twitch muscles are much better at generating short bursts of strength or speed than slow muscles, and so fatigue more quickly.[28]

The slow twitch fibers generate energy for ATP re-synthesis by means of a long term system of aerobic energy transfer. These mainly include the ATPase type I and MHC type I fibers. They tend to have a low activity level of ATPase, a slower speed of contraction with a less well developed glycolytic capacity. They contain high mitochondrial volumes, and the high levels of myoglobin that give them a red pigmentation. They have been demonstrated to have high concentrations of mitochondrial enzymes, thus they are fatigue resistant. Slow twitch muscles fire more slowly than fast twitch fibers, but are able to contract for a longer time before fatiguing.[28]

Individual muscles tend to be a mixture of various fiber types, but their proportions vary depending on the actions of that muscle and the species. For instance, in humans, the quadriceps muscles contain ~52% type I fibers, while the soleus is ~80% type I.[29] The orbicularis oculi muscle of the eye is only ~15% type I.[29] Motor units within the muscle, however, have minimal variation between the fibers of that unit. It is this fact that makes the size principal of motor unit recruitment viable.

The total number of skeletal muscle fibers has traditionally been thought not to change.It is believed there are no sex or age differences in fiber distribution; however, proportions of fiber types vary considerably from muscle to muscle and person to person.Sedentary men and women (as well as young children) have 45% type II and 55% type I fibers.[citation needed]People at the higher end of any sport tend to demonstrate patterns of fiber distribution e.g. endurance athletes show a higher level of type I fibers.Sprint athletes, on the other hand, require large numbers of type IIX fibers.Middle distance event athletes show approximately equal distribution of the two types. This is also often the case for power athletes such as throwers and jumpers.It has been suggested that various types of exercise can induce changes in the fibers of a skeletal muscle.[30]It is thought that if you perform endurance type events for a sustained period of time, some of the type IIX fibers transform into type IIA fibers. However, there is no consensus on the subject.It may well be that the type IIX fibers show enhancements of the oxidative capacity after high intensity endurance training which brings them to a level at which they are able to perform oxidative metabolism as effectively as slow twitch fibers of untrained subjects. This would be brought about by an increase in mitochondrial size and number and the associated related changes, not a change in fiber type.

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Myocyte - Wikipedia

Cardiac Stem Cells Comments Off on Myocyte – Wikipedia | July 31st, 2018