INTRODUCTION:

Stem cells have the ability to build every tissue in the human body, hence have great potential for future therapeutic uses in tissue regeneration and repair. In order for cells to fall under the definition of “stem cells, ” they must display two essential characteristics. First, stem cells must have the ability of unlimited self-renewal to produce progeny exactly the same as the originating cell¹. This trait is also true of cancer cells that divide in an uncontrolled manner whereas stem cell division is highly regulated. Therefore, it is important to note the additional requirement for stem cells; they must be able to give rise to a specialized cell type that becomes part of the healthy animal.

The general designation, “stem cell” encompasses many distinct cell types. Commonly, the modifiers, “embryonic,” and “adult” are used to distinguish stem cells by the developmental stage of the animal from which they come, but these terms are becoming insufficient as new research has discovered how to turn fully differentiated adult cells back into embryonic stem cells and, conversely, adult stem cells, more correctly termed “somatic” stem cells meaning “from the body”, are found in the fetus, placenta, umbilical cord blood and infants. Therefore, this review will sort stem cells into two categories based on their biologic properties – pluripotent stem cells and multipotent stem cells. Their sources, characteristics, differentiation and therapeutic applications are discussed.²

Pluripotent stem cells are so named because they have the ability to differentiate into all cell types in the body. In natural development, pluripotent stem cells are only present for a very short period of time in the embryo before differentiating into the more specialized multipotent stem cells that eventually give rise to the specialized tissues of the body. These more limited multipotent stem cells come in several subtypes: some can become only cells of a particular germ line (endoderm, mesoderm, ectoderm) and others, only cells of a particular tissue. In other words, pluripotent cells can eventually become any cell of the body by differentiating into multipotent stem cells that themselves go through a series of divisions into even more restricted specialized cells.³

Sources of Stem Cells:

Pluripotent:

Pluripotent stem cells being used in research today mainly come from embryos, hence the name, “embryonic stem cells”. Pre-implantation embryos a few days old contain only 10-15% pluripotent cells in the “inner cell mass”. Those pluripotent cells can be isolated, then cultured on a layer of “feeder” cells which provide unknown cues for many rounds of proliferation while sustaining their pluripotency.

Recently, two different groups of scientists induced adult cells back into the pluripotent state by molecular manipulation to yield “induced pluripotent stem cells” (iPS) that share some of the same characteristics as embryonic stem cells such as proliferation, morphology and gene expression (in the form of distinct surface markers and proteins being expressed).4-8 Both groups used retroviruses to carry genes for transcription factors into the adult cells. These genes are transcribed and translated into proteins that regulate the expression of other genes designed to reprogram the adult nucleus back into its embryonic state⁴. Both introduced the embryonic transcription factors known as Sox2 and Oct4. One group also added Klf4 and c-Myc4, and the other group added Lin28 and Nanog.6 Other combinations of factors would probably also work, but, unfortunately, neither the retroviral carrier method nor the use of the oncogenic transcription factor c-Myc are likely to be approved for human therapy. Consequently, a purely chemical approach to deliver genes into the cells, and safer transcription factors are being tried. Results of these experiments look promising.⁵

Multipotent:

Multipotent stem cells may be a viable option for clinical use. These cells have the plasticity to become all the progenitor cells for a particular germ layer or can be restricted to become only one or two specialized cell types of a particular tissue. The multipotent stem cells with the highest differentiating potential are found in the developing embryo during gastrulation (day 14-15 in humans, day 6.5-7 in mice). These cells give rise to all cells of their particular germ layer, thus, they still have flexibility in their differentiation capacity. They are not pluripotent stem cells because they have lost the ability to become cells of all three germ layers. On the low end of the plasticity spectrum are the unipotent cells that can become only one specialized cell type such as skin stem cells or muscle stem cells⁶. These stem cells are typically found within their organ and although their differentiation capacity is restricted, these limited progenitor cells play a vital role in maintaining tissue integrity by replenishing aging or injured cells. There are many other sub-types of multipotent stem cells occupying a range of differentiation capacities. For example, multipotent cells derived from the mesoderm of the gastrula undergo a differentiation step limiting them to muscle and connective tissue; however, further differentiation results in increased specialization towards only connective tissue and so on until the cells can give rise to only cartilage or only bone.⁷

Multipotent stem cells found in bone marrow are best known, because these have been used therapeutically since the 1960’s¹⁰ (their potential will be discussed in greater detail in a later section). Recent research has found new sources for multipotent stem cells of greater plasticity such as the placenta and umbilical cord blood.¹¹ Further, the heart, until recently considered void of stem cells, is now known to contain stem cells with the potential to become cardiac myocytes. Similarly, neuro-progenitor cells have been found within the brain.

The cardiac stem cells are present in such small numbers, that they are difficult to study and their function has not been fully determined. The second review in this series will discuss their potential in greater detail.⁸

Characteristics that Identify Stem Cells:

Pluripotent:

Since Federal funding for human embryonic stem cells is restricted in the United States, many scientists use the mouse model instead. Besides their ability to self-renew indefinitely and differentiate into cell types of all three germ layers, murine and human pluripotent stem cells have much in common. It should not be surprising that so many pluripotency traits are conserved between species given the shared genomic sequences and intra-cellular structure in mammals.⁹ Both mouse and human cells proliferate indefinitely in culture, have a high nucleus to cytoplasm ratio, need the support of growth factors derived from other live cells, and display similar surface antigens, transcription factors and enzymatic activity (i.e. high alkaline phosphatase activity). However, differences between mouse and human pluripotent cells, while subtle, are very important. Although the transcription factors mentioned above to induce pluripotency from adult cells (Oct3/4 and Sox2) are shared, the extracellular signals needed to regulate them differ. Mouse embryonic stem cells need the leukemia inhibitory factor and bone morphogenic proteins while human require the signaling proteins Noggin and Wnt for sustained pluripotency. Surface markers used to identify pluripotent cells also differ slightly between the two species as seen in the variants of the adhesion molecule SSEA (SSEA-1 in mouse, SSEA-3 and 4 in humans). Thus, while pluripotency research in mouse cells is valuable, a direct correlation to the human therapy is not likely.¹⁰

Last, but certainly not least, a big difference between mouse and human stem cells are the moral and ethical dilemmas that accompany the research. Some people consider working with human embryonic stem cells to be ethically problematic while very few people have reservations on working with the mouse models. However, given the biological differences between human and mouse cells, most scientists believe that data relevant for human therapy will be missed by working only on rodents.¹¹

Multipotent:

Cell surface markers are typically also used to identify multipotent stem cells. For example, mesenchymal stem cells can be purified from the whole bone marrow aspirate by eliminating cells that express markers of committed cell types, a step referred to as lineage negative enrichment, and then further separating the cells that express the sca-1 and c-Kit surface markers signifying mesenchymal stem cells. Both the lineage negative enrichment step and the sca-1/c-Kit isolation can be achieved by using flow cytometry and is discussed in further detail in the following review. The c-Kit surface marker also is used to distinguish the recently discovered cardiac stem cells from the rest of the myocardium¹². A great deal of recent work in cardiovascular research has centered on trying to find which markers indicate early multipotent cells that will give rise to pre-cardiac myocytes. Cells with the specific mesodermal marker, Kdr, give rise to the progenitor cells of the cardiovascular system including contracting cardiac myocytes, endothelial cells and vascular smooth muscle cells and are therefore considered to be the earliest cells with specification towards the cardiovascular lineage. Cells at this early stage still proliferate readily and yet are destined to become cells of the cardiovascular system and so may be of great value therapeutically.¹³

Stem Cell Therapy:

Pluripotent stem cells:

Pluripotent stem cells have not yet been used therapeutically in humans because many of the early animal studies resulted in the undesirable formation of unusual solid tumors, called teratomas. Teratomas are made of a mix of cell types from all the early germ layers. Later successful animal studies used pluripotent cells modified to a more mature phenotype which limits this proliferative capacity. Cells derived from pluripotent cells have been used to successfully treat animals. For example, animals with diabetes have been treated by the creation of insulin-producing cells responsive to glucose levels. Also, animals with acute spinal cord injury or visual impairment have been treated by creation of new myelinated neurons or retinal epithelial cells, respectively. Commercial companies are currently in negotiations with the FDA regarding the possibility of advancing to human trials. Other animal studies have been conducted to treat several maladies such as Parkinson’s disease, muscular dystrophy and heart failure.¹⁴

Scientists hope that stem cell therapy can improve cardiac function by integration of newly formed beating cardiac myocytes into the myocardium to produce greater force. Patches of cardiac myocytes derived from human embryonic stem cells can form viable human myocardium after transplantation into animals with some showing evidence of electrical integration. Damaged rodent hearts showed slightly improved cardiac function after injection of cardiac myocytes derived from human embryonic stem cells. The mechanisms for the gain in function are not fully understood but it may be only partially due to direct integration of new beating heart cells. It is more likely due to paracrine effects that benefit other existing heart cells (see next review).¹⁵

Multipotent stem cells:

Multipotent stem cells harvested from bone marrow have been used since the 1960’s to treat leukemia, myeloma and lymphoma. Since cells there give rise to lymphocytes, megakaryocytes and erythrocytes, the value of these cells is easily understood in treating blood cancers. Recently, some progress has been reported in the use of cells derived from bone marrow to treat other diseases. For example, the ability to form whole joints in mouse models has been achieved starting with mesenchymal stem cells that give rise to bone and cartilage. In the near future multipotent stem cells are likely to benefit many other diseases and clinical conditions. Bone marrow-derived stem cells are in clinical trials to remedy heart ailments. This is discussed in detail in the next review of this series.¹⁶

Pluripotent vs. Multipotent:

Pluripotent and multipotent stem cells have their respective advantages and disadvantages. The capacity of pluripotent cells to become any cell type is an obvious therapeutic advantage over their multipotent kin. Theoretically, they could be used to treat diseased or aging tissues in which multipotent stem cells are insufficient. Also, pluripotent stem cells proliferate more rapidly so can yield higher numbers of useful cells. However, use of donor pluripotent stem cells would require immune suppressive drugs for the duration of the graft while use of autologous multipotent stem cells (stem cells from ones’ self) would not. This ability to use one’s own cells is a great advantage of multipotent stem cells. The immune system recognizes specific surface proteins on cells/objects that tell them whether the cell is from the host and is healthy. Autologous, multipotent stem cells have the patient’s specific surface proteins that allow it to be accepted by the host’s immune system and avoid an immunological reaction. Pluripotent stem cells, on the other hand, are not from the host and therefore, lack the proper signals required to stave off rejection from the immune system. Research is ongoing trying to limit the immune response caused by pluripotent cells and is one possible advantage that iPS cells may have.¹⁷

Stem-cell therapy for cardiac disease:

Heart failure is the leading cause of death worldwide, and current therapies only delay progression of the Disease. Laboratory experiments and recent clinical trials suggest that cell-based therapies can improve Cardiac function, and the implications of this for cardiac regeneration are causing great excitement. Bone-Marrow-derived progenitor cells and other progenitor cells can differentiate into vascular cell types, restoring Blood flow. More recently, resident cardiac stem cells have been shown to differentiate into multiple cell types Present in the heart, including cardiac muscle cells, indicating that the heart is not terminally differentiated. These new findings have stimulated optimism that the progression of heart failure can be prevented or even Reversed with cell-based therapy.¹⁸

Cardiac regeneration:

Few questions in cardiac regeneration are definitively resolved. But it is widely agreed that the regenerative capacity of human myocardium is grossly inadequate to compensate for the severe loss of heart muscle Presented by catastrophic myocardial infarction or other myocardial Diseases. By contrast, skeletal muscle in mammals can regenerate efficiently, even after widespread injury. Satellite cells and other types of Myoblast reside in skeletal muscle and form large numbers of new myo-Tubes within days of muscle injury. However, a regenerative response Does occur in the hearts of some vertebrates, such as zebrafish and Newts, after injury. In the normal state, newt cardiomyocytes, like those of mice and humans, rarely divide. But after a substantial injury, Remaining cardiomyocytes initiate DNA synthesis and re-enter the cell Cycle. Division of existing cardiomyocytes seems to be the most important factor for cardiac regeneration in this animal¹⁹. Dedifferentiation of Cardiomyocytes near the injured zone occurs before their proliferation and is characterized by loss of expression of cardiac contractile proteins Such as α-myosin heavy chain and troponin T. Cardiac regeneration in zebrafish might be initiated predominantly by undifferentiated Stem or progenitor cells from the outer (epicardial) layer of the heart8. Further study of newts and zebrafish will define more clearly whether Cardiac regeneration in these organisms requires dedifferentiation, proliferation and subsequent differentiation of existing cardiomyocytes, or Whether regeneration is driven by the recruitment of stem cells to the Injured site. By contrast, in mammalian hearts cardiomyocytes bordering a myocardial infarction rarely divide after injury, although transgenic overexpression of specific genes in mice can increase cardio-Myocyte division cardiomyocytes²⁰ but This probably occurs at a very low rate in the absence of injury1. CSCs Have a high proliferation and differentiation potential in vitro and the possibilities of expanding autologous CSCs ex vivo or stimulating The regeneration capacity of these cells in vivo are exciting options for Therapeutic regeneration.²¹

Which stem cells should be used for cardiac therapy?

Perhaps the most stunning aspect of current progress towards cardiac Regeneration is the wide variety of cell types that have been considered as candidates for therapeutic delivery in humans. This myriad of cell types reflects the unmet medical need for treating heart disease, and hence the large amount of experimental effort being put into Devising cell-based therapies. It also points to the lack of mechanistic Understanding at many levels. The ideal cell type has not yet emerged, and few studies have directly compared different stem-cell types.²²

Skeletal myoblasts:

One of the first cell-based cardiac regeneration strategies was injection of autologous skeletal myoblasts into ischaemic myocardium. Myoblasts are resistant to ischaemia, can differentiate into myotubes in Vivo (but not into cardiomyocytes) and improve ventricular function In laboratory animal experiments. Myotubes do not integrate electrically with surviving cardiomyocytes and thus do not beat in synchrony With the surrounding myocardium. Human trials of myoblasts in heart Failure are ongoing; however, some have been terminated because of Lack of efficacy and it is unlikely that skeletal myoblasts will be able to Truly regenerate myocardium. Mouse skeletal muscle contains a population of non-satellite cells that can differentiate into spontaneously Beating cells with cardiomyocyte features but an equivalent population of cardiac-committed cells in human skeletal muscle has not yet Been described.²³

Bone-marrow-derive:

A subset of bone-marrow-derived haematopoietic cells were the first Adult stem cells or progenitor cells reported to differentiate into cardio-Myocytes when transplanted into infarcted hearts of mice. The first Evidence that adult bone-marrow-derived progenitor cells participate in the formation of cardiomyocytes in adult human hearts was based on reports of Y-chromosome-positive cardiomyocytes in female donor Hearts transplanted in male recipients. Animal studies of bone-mar-Row transplantation with labelled haematopoietic stem cells followed by Myocardial infarction revealed cardiomyocytes derived from the trans-Planted cells, but at an exceptionally low rate²⁴. However, other studies in animals have not demonstrated differentiation of haematopoietic Progenitor cells into cardiomyocytes or improvement in cardiac Function. Currently, no consensus exists on whether bone-marrow-Derived progenitor cells differentiate into cardiomyocytes in vivo. Endothelial progenitor cells (EPCs) are a subset of haematopoietic Cells found in the bone marrow that have the potential to differentiate into endothelial cells. EPCs have not been shown to differentiate into cardiomyocytes in vivo, but they probably have a role in promoting Angiogenesis. In addition to directly contributing to the vasculature required to deliver nutrients to new cardiomyocytes, endothelial cells Can also provide paracrine survival signals to cardiomyocytes²⁵. EPCs are Readily isolated from the blood and the bone marrow, and clinical studies Suggest that cell-based therapy with EPCs can improve myocardial function. But definitions of EPCs vary such that different studies probably Use different types of cell, making comparisons difficult. So far, most clinical studies have used bone-marrow mononuclear Cells and showed either no benefit or small (but possibly clinically important) improvements in cardiac function³². The mechanisms of these functional improvements are unknown, but it is unlikely that the improvements result from differentiation of the injected cells into Cardio myocytes. Growth factor and cytokine release by injected cells is Frequently suggested as a potential mechanism of action, and improved Microvascular function has been shown in the REPAIR-AMI study.²⁶

Embryonic stem cells:

Embryonic stem (ES) cells are the prototypical stem cells. They unambiguously fulfil all requirements of stem cells: clonality, selfrenewal and multipotentiality. ES cells can differentiate into any cell present in the adult organism and have the potential to completely regenerate the Myocardium. Two of the obstacles that stand in the way of the therapeutic use of ES cells are immunological rejection and the propensity Of ES cells to form teratomas when injected in vivo. As knowledge of pathways for ES-cell differentiation and for heart embryonic Development increases, ES-cell differentiation might become more Controllable27. Methods to limit teratoma formation include genetic Selection of differentiated ES cells, or differentiation of ES cells in Vitro into cardiomyocytes or endothelial cells before injection for Example, tumour-necrosis factor promotes the differentiation of ES Cells into cardiomyocytes. Differentiated ES cells can survive and Improve myocardial function if delivered to the myocardium in a rich Prosurvival cocktail. An inherent difficulty in controlling the growth and differentiation of ES cells and other pluripotent stem cells is that The timing with which specific signalling pathways are activated might Be crucial. For example, recent studies on mouse and zebrafish embryos Reveal that the role of the Wnt–β-catenin pathway in cardiac develop-Ment varies depending on the developmental stage.²⁸

Endogenous cardiac stem cells:

Because allogeneic cells face immunological challenges that would probably require immunosuppression, the isolation of endogenous adult mammalian CSCs on the basis of cell-surface markers has generated great enthusiasm. However, a definitive marker for CSCs has not yet been identified. Mammalian myocardium includes a small proportion of stem cells that express the cell-surface markers Kit or Sca. Side-population cells, identified by their ability to exclude Hoechst dye, were first described in the bone marrow as being enriched in haematopoietic stem cells, but they are also found in other organs, including the heart. Some side-population cells express Kit and/or Sca1, and like Kit+ CSCs and Sca1+ CSCs, side-population cells can generate cardiomyocytes in vitro and in vivo. In addition to Kit+ CSCs, Sca1+ CSCs and side-population cells, a fourth population of CSCs expresses the transcription factor is l129. Lineage-tracing experiments have shown that Isl1-expressing cells can differentiate into endothelial, endocardial, smooth muscle, conduction system, right ventricular and atrial myogenic lineages during the development of the embryonic heart Isl1-expressing cells are also present in the adult mammalian heart, but they are limited to the right atrium, are found in smaller numbers than in embryonic hearts13 and have an unknown physiological role. Recently, epicardium-derived progenitor cells have been described that show angiogenic potential. CSCs can be isolated and expanded from human myocardial samples obtained using a minimally invasive biopsy procedure. Thus, from autologous CSCs, it might be possible to generate enough cells to transplant into patients with heart failure, a procedure that would have minimal risk of immune rejection or teratoma formation. But no clinical data using CSCs are available yet, and many important questions about CSCs remain unanswered. Can their in vitro proliferative and differentiation potential translate to long-term in vivo function? Do they retain their cardiogenic potential in disease states, or with advancedstem cells to the injured myocardium³⁰. For example, local myocardial delivery of the chemoattractant cytokine CXCL12 can improve homing of EPCs to the heart. Furthermore, mesenchymal stem cells can be found in small numbers in peripheral blood, indicating that nonhaematopoietic stem cells or progenitor cells circulate and might have the capability to home to injured tissues. Like stem cells or progenitor cells in other tissues, CSCs reside in clusters consistent with the existence of cardiac niches53. The factors that attract these cells out of their putative niches to an injury site remain to be defined. The question also remains whether CSCs stably reside in the heart or are derived from other tissues such as the bone marrow, as has been suggested for Kit+ cells.

How to develop a stem-cell therapy for Parkinson’s disease:

A clinically competitive cell therapy must provide advantages over current treatments for PD. Cell-based approaches should induce long-lasting, major improvements of mobility and suppression of dyskinesias. Alternatively, the new cells should improve symptoms that are resistant to other treatments, such as balance problems. Improvements after fatal grafts have not exceeded those found with deep brain stimulation, and there is no convincing evidence for reversal of drug-resistant symptoms. Incomplete recovery could be due to only part of the striatum having been reinnervated Even in animals with good reinnervation, however, improvements are only partial, indicating that the ectopic graft placement in the striatum may be of crucial importance³¹. Grafts implanted in the substantia nigra give some improvements in animals and have been tested clinically but they are not able to reconstruct the nigrostriatal pathwa. Even if stem cell technology can generate large numbers of dopaminergic neurons, the development of effective cell therapy for PD will require three additional advances. First, better criteria for selecting the patients suitable for cell therapy have to be defined. Dopaminergic cell therapy will most likely be successful only in those affected individuals who show marked symptomatic benefit in response to L-dopa and in whom the main pathology is a loss of dopaminergic neurons³². Debilitating symptoms in PD and related disorders are also caused by pathological changes in non-dopaminergic systems. Until we know how to repair these systems, enrollment of individuals with such symptoms in clinical trials with dopaminergic cell therapy should be carefully considered. Second, the functional efficacy of grafts must be improved. On the basis of imaging before surgery, the transplantation procedure should be customized with respect to the dose and location of grafted cells so that the repair of the dopamine system will be as complete as possible in each patient’s brain. There is so far no evidence that stem cell–derived dopaminergic neurons will induce more pronounced improvement as compared with primary neurons in fetal grafts³³. One advantage with stem cells is the possibility for controlled genetic modification, which, for example, could be used to increase survival, differentiation, migration and function of their progeny. For more complete reversal of Parkinson’s symptoms, it may be necessary to stimulate regrowth of axons from grafts in the substantia nigra to the striatum; this would probably require modulation of host growthinhibitory mechanisms. The ability of grafted NSCs to rescue dysfunctional dopaminergic neurons, through release of neurotrophic molecules, could also promote symptomatic relief³⁴. It is unknown whether immunosuppressive treatment is needed in patients with human stem cell grafts. Results with ALLOGENEIC fetal grafts suggest that effective immunosuppression, at least for 1 year after transplantation, is necessary to optimize functional outcome. If immune reactions constitute a substantial problem, alternative solutions could be to generate transgenic ESCs or NSCs. ISOGENIC stem cells seem ideal but require therapeutic cloning or the use of adult stem cells from the patient.

Third, strategies to avoid adverse effects must be developed. New animal models are needed to reveal the pathophysiological mechanisms of graft-induced dyskinesia. The risk for teratoma from ESCs as well as the consequences of introducing new genes in stem cell–derived neurons should be carefully evaluated. Implantation of mouse ESCs into rat striatum caused teratomas in 20% of the animals. However, the risk is reduced if the cells are differentiated beforehand in vitro. Importantly, ESCs seem more prone to generate tumors when implanted into the same species from which they were derived. Thus, an absence of tumors after implantation of human ESCs into rodents does not exclude their occurrence in the human brain. To improve safety it may be necessary to engineer ESCs with relatable suicide genes.³⁵

Can cell therapy work in stroke?

In stroke, occlusion of a cerebral artery leads to focal ischemia in a restricted CNS region. Many different types of neurons and glial cells degenerate in stroke. It has not yet been convincingly demonstrated that neuronal replacement induces symptomatic relief in individuals who have suffered strokes. In the only reported clinical trial, persons with stroke affecting basal ganglia received implants of neurons generated from the human NT-2 teratocarcinoma cell line into the infarcted area38. Improvements in some affected individuals correlated with increased metabolic activity at the graft site. This finding could be interpreted as graft function but might as well reflect inflammation or increased activity in host neurons. Autopsy in one individual who had suffered a stroke revealed a population of grafted cells expressing a neuronal marker 2 years after surgery.³⁶

How to develop a stem cell therapy for stroke:

To repair the stroke-damaged brain may seem unrealistic because of atrophy and loss of many cell types. However, even re-establishment of only a fraction of damaged neuronal circuitries could have important implications. In the ideal scenario, NSCs implanted in the damaged area will differentiate in situ into those cells that have died. This strategy requires that the largely unknown developmental mechanisms instructing stem cells to differentiate into specific cell types will work also in the brain of the affected individual. For maximum functional recovery, transplantation should probably be combined with stimulation of neurogenesis from endogenous NSCs. Neurogenesis occurs from NSCs in the human SVZ, and neuronal precursors are found in human subcortical white matter³⁷. Adequate blood supply will be crucial for survival and development of the new neurons. Neurogenesis is closely associated with angiogenesis from endothelial precursors. Angiogenesis occurs in the human brain after stroke but may have to be further stimulated to increase the yield of surviving new neurons. Administration of vascular endothelial growth factor (VEGF) promotes SVZ neurogenesis and angiogenesis in the penumbra region (region at risk) after stroke. VEGF can also guide directed migration of undifferentiated SVZ neural progenitors. For efficient repair it may be necessary to provide NSCs with a platform so that they can re-form appropriate brain structure. In neonatal mice, NSCs seeded on synthetic extracellular matrix and implanted into the ischemia-damaged area generated new vascularized parenchyma comprising neurons and glia. Research should now aim to identify and improve efficacy of different mechanisms, which may underlie the benefit of stem cells after stroke³⁸. For developing the neuronal replacement strategy toward clinical application, three different tasks can be distinguished: (i) Proof of principle should be obtained that neurons generated from NSCs can survive in large numbers in animals subjected to stroke, migrate to appropriate locations, show morphological and functional properties of those neurons that have died and establish afferent and efferent synaptic interactions with neurons that survived the insult. Magnetic resonance imaging seems ideal for noninvasive imaging at high spatial and temporal resolution of the survival, migration and differentiation of grafted cells. (ii) Behavioral recovery must be optimized in animal models. Strategies to improve survival, differentiation and integration of NSCs will require detailed knowledge of the regulation of these processes. The time window after the insult when the generation of new neurons will lead to maximum restitution of neuronal circuitries and functional recovery should be determined. (iii) There is a need to define which patients are suitable for stem cell therapy. The occurrence of striatal neurogenesis after stroke focuses the interest on individuals with basal ganglia infarcts. If stem cells can also generate cortical neurons and repair axonal damage, individuals with lesions in the cerebral cortex may be included. A strategy for repair of infarcted white matter was suggested recently by the observation that NEUROSPHERES derived from adult tissue injected intravenously or intraventricularly in mice66 gave rise to cells that migrated to demyelinated areas and remyelinated axons.³⁹

Risks Factor of stem cells therapy:

Risks associated with stem cell therapy depend on many risk factors. A risk is defined as a combination of the probability of occurrence of harm and the severity of that harm. A risk factor or hazard is defined as a potential source of harm. Examples of risk factors are the type of stem cells used, their procurement and culturing history, the level of manipulation and site of injection. Because of the variety of risk factors, the risks associated with different stem cell based medicinal products may differ widely as well. For an adequate benefit/risk assessment of a stem cell based medicinal product, all important identified risks (i.e. risks or adverse events identified in clinical experience) as well as potential/theoretical risks (e.g. non-clinical safety concerns that have not been observed in clinical experience) should be thoroughly evaluated. Such an evaluation at the start and during the development of a stem cell based therapy may help to determine the extent and focus of the product development and safety evaluation plans. Here we discuss several risks associated with stem cell based medicinal products, and the risk factors contributing to these risks.

Different categories of risk factors can be distinguished Firstly, risk factors associated with the intrinsic cellular properties of a particular cell type or class of stem cells); secondly extrinsic risk factors introduced by procurement, handling, culturing, or storage of the cells; and finally the risk factors associated with the clinical characteristics (e.g. surgical procedures, immunosuppression, site and mode of administration, or co-morbidities) will be discussed. It is important to realize that multiple risk factors from these different categories can contribute to the risk to the patient. In principle, knowledge on potential risks and risk factors obtained with other/existing stem cell based medicinal products may contribute to the risk evaluation of new stem cell based therapies.

Initial clinical experience with somatic stem cell therapy may appear promising. However, many questions regarding the potential risks have not yet been answered. The amount of data and the knowledge of risks associated with the use of stem cell therapy are expanding. However, due to the large variation amongst the studies (e.g. study protocol, patient population, heterogeneity of the administered cell population, timing/location of injection) it is difficult to extrapolate results from one study to another, and also from one stem cell based medicinal product to another. Currently, the most extensive clinical experience has been obtained with haematopoietic stem cells and mesenchymal stem/stromal cells. The clinical experience with endothelial progenitor cells is also growing.⁴⁰

In most cases, irrespective of the treated condition or mode of administration, MSC therapy appears relatively safe However given the limited time of follow up, the low number of patients, the variation in cell preparations and characterization and mode of delivery, further studies on the safety of MSC are still needed, especially on long term effects such as tumourigenicity. Autologous stem cell transplantation is perceived as non-harmful; however this only applies for non-substantially manipulated stem cells. The risks associated with autologous stem cells that are substantially manipulated (e.g. by tissue culture or genetic modification) or cells that are not intended to be used for the same essential function or functions in the recipient as in the donor do need further evaluation.

In contrast to SSC, there is currently no clinical experience with pluripotent stem cells. This is in particular due to the assumption that the application of these cells is associated with a higher risk in particular related to tumourigenicity. Recent developments indicate that clinical experience with embryonic stem cells become available in the near future. At this moment we are aware of 3 clinical trials using human ESC-derived cells that have been approved by the FDA. The first approved trial is using oligodendrocyte progenitor cells aimed at the treatment of spinal cord injury. This trial has been temporarily put on hold before the first patient was included due to non-clinical findings of microscopic cysts in the regenerating injury site . However the hold has been lifted and recruitment is currently ongoing. The two other trials have just been cleared by the FDA, and aim to treat two eye diseases (Stargardt's macular dystrophy and dry age-related macular degeneration) with ESC-derived cells.⁴¹

As discussed earlier, the perceived risk on tumor formation is higher for iPSC than for ESC. Clinical application of iPSC is still relatively far away as the technique to generate these cells is still quite new and the methods to generate these cells more safely are rapidly developing. For iPSC, even the non-clinical information on the tumour formation in a context relevant to regenerative medicine (focal injection or iv administration) is still very limited (mouse iPSC) or essentially lacking (human iPSC) apart from their teratoma inducing capabilities.

Overall stem cell therapy may represent great hope for multiple diseases and degenerative conditions, but a thorough evaluation of the risk factors and potential risk of a stem cell based medicinal product must be a prerequisite step before wider clinical application and/or registration can be accepted⁴². For each stem cell based medicinal product the potential risks to the patient needs to be adequately evaluated and should take into account not only the specific intrinsic characteristics of a specific stem cell but also the safety data already obtained with similar type of products. In addition extrinsic risk factors like manufacturing, handling, storage- and clinical or treatment related risk factors can contribute to the overall risk to the patient. During the risk evaluation, knowledge of the safety of (similar) stem cell based medicinal products may be of great value. Documented/identified risks, and known risk factors as well as potential/theoretical risks should be considered in the risk evaluation. Presents a (non-exhaustive) overview of risk factors and risks. It should be clear that while tumour formation is an important risk associated with stem cell therapy, other risks (e.g. adverse immune modulation) as well as strategies to minimize the risks should be should be carefully evaluated.

Furthermore, for the successful development of a stem cell based medicinal product more information on the biological mechanism of stem cell therapy is needed as well as sufficient characterization of the cells and reproducible production of stem cell batches. The current knowledge on the mechanism of action of stem cell therapy is still limited and the cellular requirements necessary for a successful product are largely unknown other issues such as choice of stem cells to be used, the need/possibility for concurrent tissue regeneration in case of irreversible tissue loss, the differentiation degree and specific identity of the transplanted cells, and the long-term survival of engrafted cells in the absence of a normal supportive tissue environment should be considered as well.⁴³

CONCLUSION:

The promises of cures for human ailments by stem cells have been much touted but many obstacles must still be overcome. First, more human pluripotent and multipotent cell research is needed since stem cell biology differs in mice and men. Second, the common feature of unlimited cell division shared by cancer cells and pluripotent stem cells must be better understood in order to avoid cancer formation. Third, the ability to acquire large numbers of the right cells at the right stage of differentiation must be mastered. Fourth, specific protocols must be developed to enhance production, survival and integration of transplanted cells. Finally, clinical trials must be completed to assure safety and efficacy of the stem cell therapy. When it comes to stem cells, knowing they exist is a long way from using them therapeutically.

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Received on 02.02.2023 Modified on 28.02.2023

Res. J. Pharmacology and Pharmacodynamics.2023;15(2):77-86.

DOI: 10.52711/2321-5836.2023.00016