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Endothelial and cardiac progenitor cells for …

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JavaScript is disabled on your browser. Please enable JavaScript to use all the features on this page.Abstract

Stem cells have the potential to differentiate into cardiovascular cell lineages and to stimulate tissue regeneration in a paracrine/autocrine manner; thus, they have been extensively studied as candidate cell sources for cardiovascular regeneration. Several preclinical and clinical studies addressing the therapeutic potential of endothelial progenitor cells (EPCs) and cardiac progenitor cells (CPCs) in cardiovascular diseases have been performed. For instance, autologous EPC transplantation and EPC mobilization through pharmacological agents contributed to vascular repair and neovascularization in different animal models of limb ischemia and myocardial infarction. Also, CPC administration and in situ stimulation of resident CPCs have been shown to improve myocardial survival and function in experimental models of ischemic heart disease. However, clinical studies using EPC- and CPC-based therapeutic approaches have produced mixed results. In this regard, intracoronary, intra-myocardial or intramuscular injection of either bone marrow-derived or peripheral blood progenitor cells has improved pathological features of tissue ischemia in humans, despite modest or no clinical benefit has been observed in most cases. Also, the intriguing scientific background surrounding the potential clinical applications of EPC capture stenting is still waiting for a confirmatory proof. Moreover, clinical findings on the efficacy of CPC-based cell therapy in heart diseases are still very preliminary and based on small-size studies.

Despite promising evidence, widespread clinical application of both EPCs and CPCs remains delayed due to several unresolved issues. The present review provides a summary of the different applications of EPCs and CPCs for cardiovascular cell therapy and underlies their advantages and limitations.

bone marrow-derived cells

cardiac progenitor cells

C-X-C chemokine receptor type 4

1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-acetylated low density lipoprotein

endothelial colony forming cells

endothelial progenitor cells

granulocyte colony-stimulating factor

individual patient data

induced pluripotent stem cells

platelet-derived growth factor receptor

stromal cell derived factor-1

stage specific embryonic antigen-1

ST-segment elevation myocardial infarction

vascular endothelial growth factor

Ulex europaeus agglutinin-1

Endothelial progenitor cells

Stem cells



Myocardial infarction

Regenerative medicine

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Heart Disease A Closer Look at Stem Cells

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Overview of current stem cell-based approaches to treat heart disease

Since heart failure after heart attacks results from death of heart muscle cells, researchers have been developing strategies to remuscularize the damaged heart wall in efforts to improve its function. Researchers are transplanting different types of stem cell and progenitor cells (see above) into patients to repair the damaged heart muscle. These strategies have mainly used either adult stem cells (found in bone marrow, fat, or the heart itself) or pluripotent (ES or iPS) cells.

Preliminary results from experiments with adult stem cells showed that they appeared to improve cardiac function even though they died shortly after transplantation. This led to the idea that these cells can release signals that can improve function without replacing the lost muscle. Clinical trials began in the early 2000s transplanting adult stem cells from the bone marrow and then from the heart. These trials demonstrated that transplanting cells into damaged hearts is feasible and generally safe for patients. However, larger trials that were randomized, blinded, and placebo-controlled, showed fewer indications of improved function. The consensus now is that adult stem cells have modest, if any, benefit to cardiac function.

Research shows that pluripotent stem cell-derived cardiomyocytes can form beating human heart muscle cells that both release the necessary signals and replace muscle lost to heart attack. Transplantation of pluripotent stem cell-derived cardiac cells have demonstrated substantial benefits to cardiac function in animal models of heart disease, from mice to monkeys. Recently, pluripotent stem cell-derived interventions were used in clinical trials for the first time. Patches of human heart muscle cells derived from the stem cells were transplanted onto the surface of failing hearts. Early results suggest that this approach is feasible and safe, but it is too early to know whether there are functional benefits.Research is ongoing to test cellular therapies to treat heart attacks by combining different types of stem cells, repeating transplantations, or improving stem cell patches. Clinical trials using these improved methods are currently targeted to begin around 2020.Unfortunately, many unscrupulous clinics are making unsubstantiated claims about the efficacy of stem cell therapies for heart failure, creating confusion about the current state of cellular approaches for heart failure. To learn more about warning signs of these unproven interventions, please visit Nine Things to Know About Stem Cell Treatments.

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Vancouver Stem Cell Treatment Centre | Stem Cells

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How do Stem Cellsfunction?Stem cells have the capacity to migrate to injured tissues, a phenomenon calledhoming. This occurs by injury or disease signals that are released from the distressed cells and tissue. Once stem cells arrive,they dock on adjacent cells to commence performing their job to repair the problem.

Stem cells serve as a cell replacementwhere they change into the required cell type such as a muscle cell, bone orcartilage. This is ideal for traumaticinjuries and many orthopedic indications.

They do not express specific human leukocyte antigens (HLAs) which helpthem avoid the immune system. Stemcells dock on adjacent cells and release proteins called growth factors, cytokines and chemokines. These factors help control many aspects of the healing and repairprocess systemically.

Stem cells control the immune system and regulate inflammation which is a keymediator of disease, aging, and is ahallmark of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

They help to increase new blood vesselformation so that tissues receive proper blood flow and the correct nutrients needed to heal as in stroke, peripheral arterydisease and heart disease.

Stem cells provide trophic support forsurrounding tissues and help hostendogenous repair. This works wellwhen used for orthopedics. In case ofdiabetes, it may help the remaining beta cells in the pancreas to reproduce orfunction optimally.

As CSN research evolves, the field ofregenerative medicine and stem cells offers the greatest hope for those suffering from degenerative diseases, conditions for which there is currently no effective treatment or conditions that have failed conventional medical therapy.

Stem cell treatment is a complex process allowing us to harvest the bodys own repair mechanism to fight against degeneration, inflammation and general tissue damage. Stem cells are cells that can differentiate into other types of tissue to restore function and reduce pain.

Adult stem cells are found in abundance in adipose (fat) tissue, where more than 5million stem cells reside in every gram. These stem cells are called adult mesenchymal stem cells.

Our medical doctors extract stromal vascular fraction (SVF) from your own body to provide treatment using your very own cells. This process is calledautologous mesenchymal stem celltherapy. Our multi-specialty team deploys SVF under an institutional review board (IRB). This is an approved protocol that governs investigational work and the focus is to maintain safety of autologous use of SVF for various degenerative conditions.

How do we perform the stem cell treatment?Our procedure is very safe and completed in a single visit to our clinic. On the day of treatment, our physicians inject a localanaesthetic and harvest approximately 60 cc (2 oz.) of stromalvascular fraction (SVF) from under the skin of your flanks or abdomen. The extracted SVF is then refined in a closed system using strictCSN protocols to produce pure stromalvascular fraction (SVF). SVF containsregenerative cells including mesenchymal and hematopoietic stem cells, macrophages, endothelial cells, immune regulatory cells, and important growth factors that facilitate your stem cell activity. CSN technology allows us to isolate high numbers of viable stem cells that we can immediately deploy directly into a joint, trigger point, and/or byintravascular infusion. Specific deployment methods have been developed that are unique for each condition being treated.

During the refinement process, thesubcutaneous harvested cells andtheir connecting collagen matrix willbe separated, leaving purified free stem cells. About half of the SVF will be pure stem cells, while the remainder will be acombination of other regenerative cellsand growth factors. Before the SVF isre-injected into your body during the final part of the process we perform a qualityand quantity test which will examine the cell count and viability.

Perfecting the stem cell treatmentOur team records cell numbers and viability so that we can gain a better understanding of what constitutes a successful treatment. Although it is not yet possible to predict what number of cells that will be recovered in a harvest, it is very important that we know the total cell count and cell viability. It is only with this data that we will beginto understand why treatments are verysuccessful, only slightly successful orunsuccessful.

While vigilant about patient safety, we are also learning and sharing with the CSN data bank about which diseases respond best and which deployment methods are most effective with over 80 other clinics.

This data collection from all over the world makes the Cell Surgical Network the worlds largest regenerative medicineclinical research organization.

Network physicians have the opportunity to share their data, as well as their clinical experiences, thus helping one anotherto achieve higher levels of scientificunderstanding and optimizing medical protocols.

Injecting into thevascular system and/ora jointWe will administer the stem cell treatment with two methods:

The belief is that for many degenerative joint conditions IV and intra-articulardeployment is superior because each of these conditions have a local pathology and a central pathology. The local resident stem cell population has been working very hard to repair the damage and over the course of time these stem cells have become worn out, depleted and slowly die. This essentially causes a state of stem cell depletion. When we inject our mix of stem cells, cytokines and growth factors (known as SVF)inflammation is decreased and theregenerative process improved.

The stem cells that we have injected will then bring the level of stem cells closer to the normal level, thus restoring the natural balance and allow the body to heal itself.

Caplan et al, The MSC: An Injury Drugstore, DOI 10.1016/j .stem.2011.06.008

How long does it last?Many studies have shown the healing and regenerative ability of stem cells. Forexample, a study in World Journal of Plastic Surgery (Volume 5[2]; May 2016) followed a woman with knee arthritis. Before and after analysis of MRI images confirmed new growth of cartilage tissue. Unlike steroids, lubricants, and other injectable treatments, stem cells actually repair damaged tissue.

As published in Experimental andTherapeutic Medicine (Volume 12[2]; August 2016), numerous studies with hundreds of patients showed continuous improvement of arthritis for two years. Patients showed improvement three months after a single treatment and they continued to show improvement for two full years. This is why stem cells are often referred to as regenerative medicine.

No one can guarantee results for this or any other treatment. Outcomes will vary from patient to patient. Each potential patient must be assessed individually to determine the potential for optimum results from this regenerative therapy. To learn more about stem cell therapy, please contact us by clicking here or calling our clinic at 604-708-CELL (604-708-2355).

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Vancouver Stem Cell Treatment Centre | Stem Cells

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Creating Embryonic Stem Cells Without Embryo Destruction

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By: Ian Murnaghan BSc (hons), MSc - Updated: 12 Sep 2015| *Discuss

One of the biggest hurdles in stem cell research involves the use of embryonic stem cells. While these stem cells have the greatest potential in terms of their ability to differentiate into many different kinds of cells in the human body, they also bring with them enormous ethical controversies. The extraction of embryonic stem cells involves the destruction of an embryo, which upsets and outrages some policy makers and researchers as well as a number of public members. Not only that, but actually obtaining them is a challenge in itself and one that has become more difficult in places such as the United States, where policies have limited the availability of embryonic stem cells for use.

Although researchers have focused on harnessing the power of adult stem cells, there have still been many difficulties in the practical aspects of these potential therapies. In an ideal world, we would be able to use embryonic stem cells without destroying an embyro. Now, however, this ideal hope may actually have some realistic basis. In recent medical news, there has been important progress in the use of embryonic stem cells.

There are still many more tests and research that must be conducted to verify the safety and reliability of the procedure but it is indeed hopeful that funding can now increase for stem cell research. If you are an avid reader of health articles, you will probably be able to stay up-to-date on the latest developments related to this medical news. This newest research into embryonic stem cells holds promise and hope for appeasing the controversy around embryonic stem cell use and allowing for research to finally move forward with fewer challenges and controversies. For those who suffer from one of the many debilitating diseases and conditions that stem cell treatments may help or perhaps cure one day, this is welcome news.

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Stem Cell Basics VII. |

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There are many ways in which human stem cells can be used in research and the clinic. Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. Predictably controlling cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. While recent developments with iPS cells suggest some of the specific factors that may be involved, techniques must be devised to introduce these factors safely into the cells and control the processes that are induced by these factors.

Human stem cells are currently being used to test new drugs. New medications are tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines have a long history of being used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists must be able to precisely control the differentiation of stem cells into the specific cell type on which drugs will be tested. For some cell types and tissues, current knowledge of the signals controlling differentiation falls short of being able to mimic these conditions precisely to generate pure populations of differentiated cells for each drug being tested.

Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including maculardegeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

Figure 3. Strategies to repair heart muscle with adult stem cells. Click here for larger image.

2008 Terese Winslow

For example, it may become possible to generate healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic heart disease. Preliminary research in mice and other animals indicates that bone marrow stromal cells, transplanted into a damaged heart, can have beneficial effects. Whether these cells can generate heart muscle cells or stimulate the growth of new blood vessels that repopulate the heart tissue, or help via some other mechanism is actively under investigation. For example, injected cells may accomplish repair by secreting growth factors, rather than actually incorporating into the heart. Promising results from animal studies have served as the basis for a small number of exploratory studies in humans (for discussion, see call-out box, "Can Stem Cells Mend a Broken Heart?"). Other recent studies in cell culture systems indicate that it may be possible to direct the differentiation of embryonic stem cells or adult bone marrow cells into heart muscle cells (Figure 3).

Cardiovascular disease (CVD), which includes hypertension, coronary heart disease, stroke, and congestive heart failure, has ranked as the number one cause of death in the United States every year since 1900 except 1918, when the nation struggled with an influenza epidemic. Nearly 2,600 Americans die of CVD each day, roughly one person every 34 seconds. Given the aging of the population and the relatively dramatic recent increases in the prevalence of cardiovascular risk factors such as obesity and type 2 diabetes, CVD will be a significant health concern well into the 21st century.

Cardiovascular disease can deprive heart tissue of oxygen, thereby killing cardiac muscle cells (cardiomyocytes). This loss triggers a cascade of detrimental events, including formation of scar tissue, an overload of blood flow and pressure capacity, the overstretching of viable cardiac cells attempting to sustain cardiac output, leading to heart failure, and eventual death. Restoring damaged heart muscle tissue, through repair or regeneration, is therefore a potentially new strategy to treat heart failure.

The use of embryonic and adult-derived stem cells for cardiac repair is an active area of research. A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived cells including mesenchymal cells (bone marrow-derived cells that give rise to tissues such as muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells, have been investigated as possible sources for regenerating damaged heart tissue. All have been explored in mouse or rat models, and some have been tested in larger animal models, such as pigs.

A few small studies have also been carried out in humans, usually in patients who are undergoing open-heart surgery. Several of these have demonstrated that stem cells that are injected into the circulation or directly into the injured heart tissue appear to improve cardiac function and/or induce the formation of new capillaries. The mechanism for this repair remains controversial, and the stem cells likely regenerate heart tissue through several pathways. However, the stem cell populations that have been tested in these experiments vary widely, as do the conditions of their purification and application. Although much more research is needed to assess the safety and improve the efficacy of this approach, these preliminary clinical experiments show how stem cells may one day be used to repair damaged heart tissue, thereby reducing the burden of cardiovascular disease.

In people who suffer from type1 diabetes, the cells of the pancreas that normally produce insulin are destroyed by the patient's own immune system. New studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually could be used in transplantation therapy for persons with diabetes.

To realize the promise of novel cell-based therapies for such pervasive and debilitating diseases, scientists must be able to manipulate stem cells so that they possess the necessary characteristics for successful differentiation, transplantation, and engraftment. The following is a list of steps in successful cell-based treatments that scientists will have to learn to control to bring such treatments to the clinic. To be useful for transplant purposes, stem cells must be reproducibly made to:

Also, to avoid the problem of immune rejection, scientists are experimenting with different research strategies to generate tissues that will not be rejected.

To summarize, stem cells offer exciting promise for future therapies, but significant technical hurdles remain that will only be overcome through years of intensive research.

Previous|VII. What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized?|Next

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Why are Adult Stem Cells Important? Boston Children’s …

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Adult stem cells are the bodys toolbox, called into action by normal wear and tear on the body, and when serious damage or disease attack. Researchers believe that adult stem cells also have the potential, as yet untapped, to be tools in medicine. Scientists and physicians are working towards being able to treat patients with their own stem cells, or with banked donor stem cells that match them genetically.

Grown in large enough numbers in the lab, then transplanted into the patient, these cells could repair an injury or counter a diseaseproviding more insulin-producing cells for people with type 1 diabetes, for example, or cardiac muscle cells to help people recover from a heart attack. This approach is called regenerative medicine.

A number of challenges must be overcome before the full therapeutic potential of adult stem cells can be realized. Scientists are exploring practical ways of harvesting and maintaining most types of adult stem cells. Right now, scientists do not have the ability to grow the cells in the amounts needed for treatment. More work is also needed to find practical ways to direct the different kinds of cells to where theyre needed in the body, preferably without the need for surgery or other invasive methods.

Research in all aspects of adult stem cells and their potential is underway at Childrens Hospital Boston. Realizing that potential will require years of research, but promising strides are being made.

Why are Adult Stem Cells Important? Boston Children's ...

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Banking Menstrual Stem Cells | What are Menstrual Stem …

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Stem cells in menstrual blood have similar regenerative capabilities as thestem cells in umbilical cord blood and bone marrow. Cryo-Cell's patent-pendingmenstrual stem cell service offers women in their reproductive years the ability to store and preserve these cells for potential use by herself or a family memberfree from ethical or political controversy.

Cryo-Cell is the only stem cell bank in the world that can offer womenthe reassurance and peace of mind that comes with this opportunity.

What are menstrual stem cells?Stem cells in menstrual blood are highly proliferativeandpossess the unique ability to develop into various other types of healthy cells. During a womans menstrual cycle, these valuable stem cells are discarded.

Cryo-Cell'smenstrual stem cell bankingservice captures those self-renewing stem cells, processes and cryopreserves them for emerging cellular therapies that hold the promise of potentially treatinglife-threatening diseases.

How are menstrual stem cells collected, processed and stored?The menstrual blood is collected in a physicians officeusing a medical-grade silicone cup in place of a tampon orsanitary napkin. The sample is shipped to Cryo-Cell via a medical courier and processed in our state-of-the-art ISO Class 7 clean room.

The menstrual stem cells are stored in two cryovials that are overwrapped to safeguard them during storage. The overwrapped vials are cryogenically preserved in a facility that isclosely monitored at all times to ensure that your menstrual stem cells are safe and ready for future use.

What are the benefits of banking menstrual stem cells?Cryo-Cell's innovative menstrual stem cell banking service provides women with the exclusive opportunity to build their own personal healthcare portfolio with stem cells that will be a 100% match for the donor. Menstrual stem cells have demonstrated the capability of differentiating into many other types of stem cells such as cardiac, neural, bone, fat and cartilage.

Bankingmenstrual stem cells now is an investment in your future medical needs. Currently, they are being studied to treat stroke, heart disease, diabetes, neurodegenerative disease, and ischemic wounds in pre-clinical and clinical models.

Cryo-Cells activities for New York State residents are limited to collection, processing, and long-term storage ofmenstrual stem cells. Cryo-Cells possession of a New York State license for such collection, processing, and long-term storage does not indicate approval or endorsement of possible future uses or future suitability of these cells.

Banking Menstrual Stem Cells | What are Menstrual Stem ...

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Advanced maturation of human cardiac tissue grown from …

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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).

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

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Advanced maturation of human cardiac tissue grown from ...

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

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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 ( 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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New insights into cardiac stem cells could lead to heart …

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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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Adult Stem Cells in Vascular Remodeling – Theranostics

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


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 ( See 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 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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2. Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R. et al. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation. 2017;135:e146-e603

3. Ross R. Atherosclerosis an inflammatory disease. N Engl J Med. 1999;340:115-26

4. Libby P, Hansson GK. Inflammation and immunity in diseases of the arterial tree: players and layers. Circ Res. 2015;116:307-11

5. Li S, Sengupta D, Chien S. Vascular tissue engineering: from in vitro to in situ. Wiley Interdiscip Rev Syst Biol Med. 2014;6:61-76

6. Seifu DG, Purnama A, Mequanint K, Mantovani D. Small-diameter vascular tissue engineering. Nat Rev Cardiol. 2013;10:410-21

7. Chiu J-J, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91:327-87

8. Yoder MC. Is endothelium the origin of endothelial progenitor cells?. Arterioscler Thromb Vasc Biol. 2010;30:1094-103

9. Bautch VL. Stem cells and the vasculature. Nat Med. 2011;17:1437-43

10. Hu Y, Xu Q. Adventitial biology: differentiation and function. Arterioscler Thromb Vasc Biol. 2011;31:1523-9

11. Stenmark KR, Yeager ME, El Kasmi KC, Nozik-Grayck E, Gerasimovskaya EV, Li M. et al. The adventitia: essential regulator of vascular wall structure and function. Annu Rev Physiol. 2013;75:23-47

12. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K. Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med. 2006;203:1273-82

13. Houtkamp MA, de Boer OJ, van der Loos CM, van der Wal AC, Becker AE. Adventitial infiltrates associated with advanced atherosclerotic plaques: structural organization suggests generation of local humoral immune responses. J Pathol. 2001;193:263-9

14. Pasquinelli G, Pacilli A, Alviano F, Foroni L, Ricci F, Valente S. et al. Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics. Cytotherapy. 2010;12:275-87

15. Psaltis PJ, Harbuzariu A, Delacroix S, Holroyd EW, Simari RD. Resident vascular progenitor cells - diverse origins, phenotype and function. J Cardiovasc Transl Res. 2011;4:161-76

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Adult Stem Cells in Vascular Remodeling - Theranostics

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Regenerative Medicine – AABB

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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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Regenerative Medicine - AABB

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Adult stem cell – Wikipedia

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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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StemCell Maxum Longevity Support | Make America Well

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A Natural formula designed to prevent premature aging. Feel better and look younger! Your cells lose function as you age. Adult stem cells rejuvenate old damaged tissues, but adult stem cells are also aging. Now you can do something about it. StemCell Maxum supports adult stem cells and their functions.

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StemCell Maxum is designed to prevent premature aging. This can help you feel better and look younger. Your cells lose function as you age. Adult stem cells rejuvenate damaged and old tissues, but adult stem cells are also aging. Now you can do something about it. StemCell Maxum supports adult stem cells and their functions.

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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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Current Strategies and Challenges for Purification of …

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


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 ( See 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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What is VetStem Regenerative Medicine? | Why Use Adipose …

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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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Stem Cell Treatment Cardiovascular Disease, Heart Disease …

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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 ...

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Adult Hearts Lack Cardiac Stem Cells –

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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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Cardiac Regeneration, Stem Cells | Research | Baylor …

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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 ...

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Cardiac Stem Cells – Cedars-Sinai

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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

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