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Abstract

Stem cells are self-renewable cells with the differentiation capacity to develop into somatic cells with biological functions. This ability to sustain a renewable source of multi- and/or pluripotential differentiation has brought new hope to the field of regenerative medicine in terms of cell therapy and tissue engineering. Moreover, stem cells are invaluable tools as in vitro models for studying diverse fields, from basic scientific questions such as developmental processes and lineage commitment, to practical application including drug screening and testing. The stem cells with widest differentiation potential are pluripotent stem cells (PSCs), which are rare cells with the ability to generate somatic cells from all three germ layers. PSCs are considered the most optimal choice for therapeutic potential of stem cells, bringing new impetus to the field of regenerative medicine. In this article, we discuss the therapeutic potential of human PSCs (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), reviewing the current preclinical and clinical data using these stem cells. We describe the classification of different sources of hPSCs, ongoing research, and currently encountered clinical obstacles of these novel and versatile human stem cells.

Keywords

Pluripotent stem cells (PSCs)Human embryonic stem cells (hESCs)Induced pluripotent stem cells (iPSCs)Differentiation Cell therapy Clinical trial Drug development

Introduction

Stem cells are the originating cells of a living multi-cellular organism, with the capability to self-renew and differentiate. Increasing research in recent years has demonstrated the wide range of applications possible for these versatile cells. Therapeutically and practically, stem cells are often categorized by their differentiation capacity (101) (i.e., their “potency”). Totipotent stem cells are able to differentiate to all cell types possible, including embryonic and extra-embryonic cell types (such as trophoblast and placenta). While such stem cells exist, it is quite difficult to demonstrate totipotency definitively, thus the term “pluripotency” has come to indicate experimentally proven, near-totipotent differentiation capacity to differentiate into embryonic germ layer somatic cells, including germ cells. By definition, therefore, pluripotent stem cells (PSCs) differentiate toward nearly all lineages of the three embryonic germ layers (i.e., ectoderm, mesoderm, and endoderm) as well as germ cells, but lack the ability to develop into extra-embryonic lineages. A third group of more restricted stem cells, termed multipotent stem cells, can usually differentiate into at least two cell types, often of the same germ layer but sometimes across germ layers. Most adult stem cells (ASCs) and somatic stem cells fall into this category. The most restricted stem cells are the oligopotent and monopotent stem cells, which are sometimes considered as progenitor cells for certain cell types (i.e., hematopoietic lymphoid progenitors) and these cells can only differentiate to only a few lineage-specific cell types and may be rapidly depleted.

The clinical use of stem cells is not new, as seen in the success of bone marrow-derived hematopoietic stem cell (HSC) transplantation for leukemia and other blood-related diseases (35). In addition, multipotent mesenchymal stem cells (MSCs) are also studied for their applications on the treatment of diseases of the central nervous system (43,130). However, indications for HSC transplantation are restricted to treatment for a few types of diseases, and nearly all are lineage specific for the hematopoietic system. Furthermore, the need for invasive procedures to obtain HSCs, and difficulty in isolation and maintenance of these cells and somatic/ASCs in general, also restrict prevalent use in regenerative medicine. These factors and the increasing information on PSCs, especially in the past few years, have allowed researchers to focus on PSCs and their applications. Differences between pluripotent and multipotent stem cells can be seen in terms of surface marker expression profile and, most importantly, by functional differences. PSCs will express a panel of pluripotency-related genes, usually transcription factors, and surface markers, including octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), Nanog, TRA-1-60 and TRA-1-81, the stage-specific embryonic antigens (SSEAs), and functional telomerase. Multipotent stem cells may express pluripotency-related markers or genes with unclear functional significance (149), but will always express lineage-specific progenitor markers. Both the proliferation and differentiation capability of multipotent stem cells are much more limited than PSCs. Most importantly, PSCs should demonstrate in vivo pluripotency, which ranges from the most minimal criteria of teratoma formation (the growth of a benign tumor with contributions of cell types from all three germ layers) to chimera formation, germline contribution, all the way to the most stringent test of tetraploid complementation (47). With the isolation of human embryonic stem cells (hESCs) and the new discovery of induced PSCs (iPSCs), the hope of using PSCs in the clinic has moved to the forefront of biomedical research.

Type of Endogenously Derived hPSCs

Studies on PSCs first began in the 1960s, initially using cells isolated from the mouse such as embryonal carcinoma cells (ECCs) (56) and ESCs (51). PSCs isolated from humans in terms of ECC sources were not reported until the 1980s, and this ushered in a new era in studying the biology of PSCs.

Embryonic Carcinoma Cells

ECCs were the first PSCs to be studied in the human system. In 1984, the first ECC line was isolated from a human teratocarcinoma, known as tera-2 (4), and since then more clonal lines of ECCs have been derived (92,93). These cells display pluripotency and are used for studies in directed differentiation to various somatic cell types but mainly to neuron-like cells (3,126). While ECCs are pluripotent and can sometimes be cultivated without the aid of feeder layers, the proliferation of these cells is poor. As demonstration of the pluripotency of these cells, ECCs are able to form teratomas when transplanted subcutaneously in immunocompromised mice as well as contribute to chimera formation (shown in mouse ECCs). This ability of these abnormal cells to contribute to normal organogenesis in the right microenvironment—that of a blastocyst as opposed to subcutaneous tissue—has made ECCs attractive in vitro models to study early development (5). ECCs, however, are not suitable sources of PSCs for clinical use, since they originate from tumors and are further able to induce tumors. But as the first in vitro model of human PSCs (hPSCs), ECCs have made great contribution to understanding the biology of hPSCs and paved the way for isolation of normal hPSCs from the embryo.

Embryonic Germ Cells

In 1998, Gearhart and colleagues derived a pluripotent cell type from the gonadal ridge and mesenchyme of fetal tissue after abortion (109). These cells were termed embryonic germ cells (EGCs). EGCs proliferate better than in vitro-cultured ECCs, but required feeders for cultivation. Differentiation of EGCs was studied first by the formation of embryoid bodies and subsequently to multiple lineages of differentiated cells (108). However, teratoma formation by EGCs in animal studies has not been reported. Moreover, EGCs are isolated from aborted fetuses, which are ethically and technically difficult to obtain; thus, these PSCs are not an often used source.

Embryonic Stem Cells

The most representative type of hPSCs is the embryonic stem cell (ESC), which so far represents the “gold standard” of ex vivo-cultured human stem cells. First isolated in 1998 by James Thomson and colleagues (127), studies on human ESCs (hESCs) have revealed many useful concepts and demonstrated that hPSCs can be applied towards clinical use. hESCs are isolated from the inner cell mass of preimplantation blastocysts, and there are now approximately over 80 cell lines that have been registered at the Stem Cell Registry of the National Institute of Health in the United States with the number of hESC cell lines submitted for approval still growing (84). Biologically, hESCs renew rapidly under cultivation and can proliferate without differentiation for a long period of time (2), which are advantages over ECCs and EGCs. Moreover, hESCs can be directed to differentiate toward cells of all three germ layers under suitable in vitro conditions (46,102,127). ESCs derived from mouse are known to be germline transmissible (63), and functional germ cell development by ESCs has been proven in the mouse system (42,132) but not in hESCs due to ethical considerations. However, in vitro germ cell differentiation of hESCs has been reported (17), and germ cell-like cells can be derived from hESCs by additional treatment of bone morphogenic proteins (BMPs) (52). In addition, primate ESCs can successfully engraft and contribute to somatic cells of fetal tissues when injected to allogeneic fetus (6). hESCs were also reported to integrate and undergo differentiation when transplanted to chicken embryos (31), showing in vivo differentiation capacity. These findings have fueled the hope that hESCs can provide the basis for cell replacement at the clinical level. However, similar to ECCs and other primate ESCs that form teratoma after transplantation (6), undifferentiated hESCs form teratomas when injected into immunodeficient mice (102). While teratoma formation is a marker for pluripotency (48), it is highly undesirable and a major obstacle for the clinical use of hESCs. Since there is no method presently to maintain pluripotency without risk of teratoma formation when hESCs are transplanted for the purpose of tissue regeneration, it is currently not possible to use undifferentiated hESCs directly for therapy. Thus, clinical trials of hESCs are based on using completely differentiated hESCs to the lineage of interest to avoid teratoma formation. This requires PSCs such as hESCs to be first differentiated into homogeneous, lineage-specific cell populations prior to therapeutic application, since just a few undifferentiated hESCs are sufficient to form teratomas (28). Thus, in this review, we will discuss the therapeutic applications of PSCs in terms of their differentiated progeny.

Application of hESCs

There has been much data accumulated on the differentiation potential of hESCs for clinical use (28,66, 69,71). However, despite much progress and accrued knowledge at the basic science research level, clinical use of hESCs is still at an early stage. This lag in application of hESCs from its isolation over 10 years ago is largely due to concerns of teratoma formation from undifferentiated cell fractions as mentioned in the previous section, as well as ethical and related legal issues (79). Here we review some of the studies on hESC differentiation for therapeutic use.

Neural Regeneration

Disorders of the nervous system may be caused by accidental injury, genetically inherited chronic disorders, or age-related degeneration. Studies on PSCs in treating neural disorder are based on the concept of cell replacement to restore the normal function of damaged cells. Differentiation of hESC to neural progenitors of the ectodermal lineage has been reported by many investigators (9,113). Neurons, astrocytes, and oligodendrocytes generated from these neural progenitors have been used in animal models for efficacious repair of stroke (19) and spinal cord injury (111). Moreover, hESCs have been reported to differentiate to neuroepithelial cells at very high efficiency (90,146). However, differentiation of hESCs to mature neural cell types usually yields a mixed cell population. Differentiation of hESCs to neural lineage cells can be controlled by the culture condition, additional treatment of growth factors (112), or aided by small molecule compounds (68). In vivo models of spinal cord injury have been shown to be repaired by hESC-differentiated oligodendrocytes, which can rebuild myelin sheaths (40). Furthermore, hESC experimental therapies on neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS) are also under investigation. The applications of hESCs on the above-mentioned disease models are all based on similar strategies (i.e., to generated functional neurons for the replacement of abnormal ones). For example, Parkinson's disease is a degenerative disorder caused by the death of dopaminecontaining cells in the midbrain. hESC-differentiated dopaminergic (DA) neurons that can secrete dopamine were derived for the treatment of Parkinson's disease and found to have therapeutic effect in animal models (9,26). hESC therapy has also been considered for other prevalent neurological diseases including Alzheimer's disease, which is caused by loss of basal forebrain cholinergic neurons (BFCNs), and ALS, which is caused by a defect in the spinal cord motor neurons. Generation of functional BFCNs and motor neurons from hESCs has been successful and used in ex vivo study of Alzheimer's disease (12) and an animal model of ALS (64), respectively. Although other neurodiseases such as Huntington's disease and Alzheimer's disease have no direct targets for hESC treatment due to the lack of clear mechanism of their pathology, scientists still strive to use hESCs for generation of functional neurons to replace dysfunctional neural cell. In fact, the first clinical trial using hESCs approved by the Food and Drug Administration (FDA) of the US is for treatment of spinal cord injury patients. In this sentinel trial, oligodendrocyte progenitor cells generated from differentiated hESCs are being used to promote nerve growth and repair the myelin sheaths of the injured nerves of patients. This project was approved in 2010, conducted by the biotechnology company Geron, which is based in California (117).

Another application of hESCs on the nervous system is in the treatment of an eye disease called Stargardt's macular dystrophy (SMD). SMD is caused by the death of photoreceptor cells in the central portion of the retina called the macula, and this is an inherited disease that can lead to permanent blindness by damage of retinal pigment epithelial (RPE) cells. hESCs had been reported to differentiate to RPE cells (45), and the hESC-derived RPE cells can be used to replace the damaged cells of the patient to regain their vision. This project was approved by FDA for human trial also in 2010 (83), conducted by the biotechnology company Advanced Cell Technology (ACT) based in Connecticut.

Cardiovascular Repair

Cardiovascular diseases (CVDs) are increasingly prevalent around the world, with heart failure being the most common cause of death in many countries. CVDs include coronary heart disease (CHD), congestive heart failure (CHF), and hypertension. The major insult of CVDs is the infarction of cardiomyocytes leading to ischemic heart failure, in which death of cardiomyocytes occurs due to the depletion of oxygen. Current treatments can reduce associated risk factors but are not curative; therefore, cell therapy is highly attractive since it offers the possibility of cure. After a successful trial of autologous skeletal myoblast implantation into the postinfarction of a heart failure patient in 2001 (77), stem cells have increasingly been considered a treatment option for ischemic heart failure. hESCs are known to differentiate to myocytes morphologically similar to cardiomyocytes, which display normal cardiomyocyte function and electrophysiological properties by culturing in END2 cell condition medium (91,139) or treatment with bone morphogenetic protein-4 (BMP-4) and activin A (138). Notably, it has been reported that hESCs can spontaneously differentiate to endothelial cells and smooth muscle cells, both of which aid in the repair of the injured heart (7,37,65). While these studies demonstrate the successful differentiation of hESCs to cardiomyocytes, a homogenous population of differentiated cardiomyocytes could not be obtained, resulting in a mixed population of differentiated cells. So far, clinical trials of hESCs have not been started for cardiac diseases, but the successful application of ESCs in repairing heart failure in the mouse system has encouraged scientists to develop such therapy for human patients. While clinical work has yet to begin, hESC-derived cardiomyocytes may already have practical use as in vitro models for cardiac drug screening and testing in the pharmaceutical industry (21).

Hepatic Regeneration

Liver diseases are associated with high morbidity and mortality, especially in Asia due to the high infection rate of hepatitis viruses and the occurrence of subsequent cirrhosis and hepatocellular carcinoma. Traditionally, a number of liver diseases require organ transplantation for cure. However, the number of donor livers is not nearly sufficient for the numerous patients around the world. Thus, the use of hESCs to derive hepatocytes may be a useful solution for these patients. Differentiation of hESCs to hepatocytes-like cell has been much reported in recent years (23,61,131). In these studies, these hepatocyte-like cells derived from hESCs display metabolic functions similar to normal hepatocytes. However, the major obstacle in generating hepatocytes from hESCs is the complicated differentiation method, which requires sequential differentiation. hESCs were first differentiated to endodermal progenitor cells and then further differentiated to hepatocytes. The procedure often requires multiple culture medium supplements in different stages and long periods of cultivation time. Investigators are so far still working on improving the differentiation protocol as well as the efficiency and homogeneity of hepatocyte generation from hESCs.

Treatment of Diabetes

Diabetes is a disease in which the regulation of blood glucose concentration is not brought under control. Blood glucose concentration is regulated by insulin, a protein that is released from the β-cells of the pancreas and responds to and adjusts blood glucose fluctuation. There are two types of diabetes: Type 1 diabetes, an autoimmune disorder in which β-cells are destroyed by the body's own immune system, and type 2 diabetes, which is defined by insulin resistance of responding cells with subsequent β-cell “burn out” and dysfunction. In both types of diabetes, β-cell replacement is thought to be curative because in both types of the disease the β-cells are ultimately destroyed or lose function (151). Although the strategy of treating diabetes with β-cell transplantation is clinically possible (110), it is very much limited by the shortage of donors. Therefore, the generation of insulin-secreting cells from hESCs has gained much interest for developing a cure for diabetes (13,106). Unfortunately, similar to hepatocyte differentiation, β-cell differentiation from hESCs is quite difficult and inefficient, as is often the case for differentiation of endodermal cell types. To date, there are no reports showing direct differentiation of hESCs to β-cells without first differentiating to an endodermal progenitor stage. Recent reports show that hESCs can differentiate into insulin-secreting cells by a step-wise differentiation protocol (14,85,133), but the secretion of insulin is very inefficient and needs improvement for clinical relevance. Similar to hepatic cell derivation, the induction of hESCs into insulin-secreting cells requires multiple differentiation steps using a number of different culture conditions. Optimizing the generation of hESC-derived insulin-secreting cells will be an important strategy for the treatment of diabetes, a disease of increasing prevalence worldwide.

Differentiation of hESCs Towards Mesenchymal Lineages

Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into osteoblasts, adipocytes, chondrocytes, and myocytes (94,99). First isolated from the bone marrow as stromal cells to support hematopoiesis, MSCs can be used in a number of orthopedic applications, helping to form trabecular bone, tendon, articular cartilage, ligaments, and part of the bone marrow (10,25,29,103,105). Furthermore, MSCs has also been used in nonskeletal diseases including treating liver cirrhosis (15,53,78) and critical limb ischemia by diabetes (18). Interestingly, MSCs possess immunomodulatory properties that make these cells useful in treating immune-related diseases, including graft-versus-host disease and autoimmune diabetes (62,96, 147,148), thereby greatly expanding the therapeutic potential of these somatic stem cells. However, similar to other ASCs, MSCs are rare and in vitro expansion usually leads to senescence (134). hESCs can differentiate to mesenchymal progenitors that display MSC markers and are capable of osteogenesis, adipogenesis, and chondrogenesis (8,89). Moreover, we have found that hESC-derived MSCs also have strong immunomodulatory effects (142). Thus, hESCs can be a source of MSCs, and these hESC-derived cells could be used in similar capacities as bone marrow MSCs. Moreover, chondrocyte generation directly from hESC have recently been reported and can be used for cartilage injury repair (88,129). Adipocytes can also be differentiated from hESCs for the investigation of lipid metabolism and obesity (34).

Hematopoietic Development

Differentiation of hESCs to the other branch of mesenchymal lineage cells, the hematopoietic cells, is a valuable application. The hematopoietic system contains mobile suspension cells with diverse biological functions, which is much different from solid organs. HSCs are actually the first stem cell system that was studied intensively, and they have been used clinically in the form of HSC transplantation for decades (39). However, HSCs are rare cells and cell numbers further decrease with age (100). Thus, generation of hematopoietic progenitors from hESCs is still an important endeavor. Studies have shown that both lymphoid and myeloid cells can be generated from hESC-derived hematopoietic progenitors (11,135). Functional erythrocytes derived from hESCs that can carry oxygen have been reported (75). Furthermore, hESCs can also generate macrophages (86) or megakaryocytes (57), forming functional platelets that work as well as human platelets isolated from an adult human (27). In the lymphoid lineage, hESC-derived progenitors can differentiate into B lymphocytes, T lymphocytes, as well as nature killer lymphocytes (NKs) (128,136). Interestingly, generation of NKs from hESC-derived progenitors is more efficient than the other two lymphocytes (58). Furthermore, differentiation of NK cells from hESCs can be used to treat cancer (addressed below). These hESC-derived hematopoietic cells can be used for the treatment of hematopoietic diseases such as anemia and immunodeficiency syndromes. Indeed, the efficient generation of mature nonnucleated erythrocytes from hESCs would be most exciting, since this can pave the way to obviate the need for donation of blood, the most commonly used cell therapy product.

Cancer Therapy

Cancer is one of the major causes of death in the world. Cancer cells are aggressively growing cells that proliferate unchecked. Uncontrolled growth of cancer cells results in invasion and destruction of adjacent tissues, ultimately causing death of the patient. Typical treatments of cancer include surgery, radiation, and chemotherapy, all of which cause high morbidity and are often still not curative. It has been reported recently that cell-based therapies can be used to treat cancer cells. hESC-derived HSCs can differentiate to NK cells (58,136), which are able to produce cytokines and perform antibody-mediated or direct cell-mediated cytotoxicity on target cells. The concept of hESC-derived NK cells as cancer therapy would utilize an exogenously expressed chimeric antigen receptor or T-cell receptors for the recognition of cancer cell antigens, directing the cytotoxic cells to kill cancer cells (58). In addition, dendritic cells, the professional antigen-presenting cell, have also been derived from hESCs and used for targeting cancer cells (107,119). However, these fascinating ideas for treating cancer require genetic manipulation of hESCs; therefore, therapeutic use remains to be further investigated. Recently, the cultured “conditioned” medium of hESCs was found to inhibit tumor growth (30). Postovit et al. reported that hESC microenvironment neutralized the expression of Nodal, an embryonic morphogen, in aggressive tumor cells and inhibited tumor growth (97). These findings provide very novel methods for possible use of hESCs in cancer therapy.

Other Applications of hESCs: Drug Discovery and Toxicity Testing

As mentioned above, hESC-derived cells may be used for replacing diseased or injured cells. However, therapeutic application of hESCs is still far from large-scale clinical use. One area in which hESCs can have a more immediate application is in the area of drug screening and testing. In the field of drug discovery, molecules must first be tested for their effectiveness during the screening, and furthermore for toxicity. Currently, in vitro drug studies have relied on nonhuman normal cells, which are easily obtained compared to human normal cells but nevertheless requiring constant new supplies due to replicative senescence, or human cancer cell lines, which are easily obtained and cultured, but, being abnormal cells, may be irrelevant to the physiologic process being studied. Thus, the availability of differentiated human somatic cells “on demand” from hESCs would dramatically enhance the validity of in vitro studies, as well as cut down the time required to have human-relevant physiologic data. The toxicity of a leading compound is usually first tested in animal studies before human clinical trials can be started. However, some side effects are not apparent until human clinical trials are completed, a very costly and time-consuming mistake for pharmaceutical companies. Thus, the use of relevant hESC-differentiated cells for in vitro drug studies is an important area to develop, since the toxicity of a novel drug candidate can be more specifically selected to suit human physiology. This increases the accuracy and cuts down the time and cost required for drug development (48,118). Moreover, unlike for therapeutic application, the use of hESCs for drug development does not involve any clinical issues as is the case for therapeutic use, and this will allow for more rapid application of the exciting technology of PSCs.

Alternative Source of PSCs

The Induced Pluripotent Stem Cells (iPSCs)

In addition to the isolation of endogenous PSCs for investigation, which may involve invasion methods or ethical issues, reprogramming of somatic cells back to a pluripotent stage is another important area of research for regenerative medicine. Several methods have been used to create reprogrammed somatic cells, including nuclear transfer (22) or direct fusion of cells with pluripotent ESCs (98). However, both methods are not without concerns such as ultra-low efficiency and tetraploid problems for nuclear transfer and cell—cell fusion methods, respectively (47). In recent years, the formation of induced pluripotent stem cells (iPSCs) by introducing four factors [c-MYC, OCT4, SOX2, and Krüppel-like factor 4 (KLF4) (121) (or LIN28 and NANOG replacing c-MYC and KLF4) (143)] into somatic cells allowed scientists to obtain reprogrammed cells much more easily and robustly than previous methods. This revolutionary method of reprogramming was first demonstrated in mouse somatic cells (122), and human iPSC (hiPSC) were reprogrammed successfully in the very next year. Characterization of hiPSCs showed that these cells share many properties with pluripotent hESCs, including similar stemness profile, self-renewal ability, and differentiation potential into cells of all the three germ layer and they can form teratomas in vivo (121,143). Unlike the isolation of hESCs, creation of hiPSCs does not require human embryos, and this new source of PSCs can be autologous, obviating the concerns of immune rejection when transplanted. In the past few years, the number of studies on hiPSCs has grown exponentially. To date, the generation of hiPSC from various cell types has been reported, from the initially used fibroblasts to keratinocytes, hematopoietic cells, endothelial cells, to even stem cells (1,60,74,140,141).

iPSCs are ES-like cells that possess pluripotency very similar to ESCs. Due to these similarities, applications of hESCs can logically be extended to include hiPSCs. Researchers are therefore now replacing hiPSCs for hESCs to generate cells used therapeutically, which include cardiomyocytes (123,145), insulin-secreting cells (124), hematopoietic cells (141), and hepatocytes (33, 49). These cells are human derived and can also have the advantage of being patient specific. Moreover, hiPSCs can immediately be used in nonclinical applications of hESCs such as in vitro drug testing studies. In addition, since hiPSC can be derived from patients, these cells can not only be used as a model of the disease but also to screen for drug candidates directly for such patients. Thus, while hiPSC-based cell therapy may be a future goal, use of these PSCs on disease modeling can be an immediate practical application.

Major Challenges for Therapeutic Use in iPSC Biology

As promising as the iPSC technology appears to be, there are still important challenges that have to be overcome before therapeutic use can be considered (73). One of the most critical is the selection and delivery of reprogramming factors. Because oncogenes were used in the initial reprogramming protocol, much research has been focused on exclusion of these genes since clinical use is not possible if oncogenes are needed. Moreover, the smaller the number of genes needed for reprogramming, the less manipulation required, decreasing the risk of introducing side effects and allowing for more rapid approval in the road for the therapeutic applications.

The most undesired factor, the oncogene c-MYC, had been shown to be dispensable in the reprogramming of mouse and human fibroblasts (81). However, it was also found that reprogramming without c-MYC in somatic cells may result in much decreased efficiency. Therefore, genes with similar effect but not producing oncogenic results have been tried, such as using l-MYC instead of c-MYC (81). Reprogramming with the withdrawal of one or more of the factors can also create less genetically modified and less oncogenic iPS cells. Cells that endogenously express one or more of the reprogramming factors can be reprogrammed with lesser factors; for example, neuron stem cells expressing SOX2 (54), and human umbilical vein endothelial cells (HUVECs) expressing KLF4 (38). To date, the only factor that appears to be indispensible for reprogramming of hiPSCs is the pluripotent transcription factor OCT4. Other factors can be removed or replaced for successful hiPSC reprogramming, depending on the type of donor cells chosen.

Most studies on iPSC generation use viral methods to introduce the genetic factors. For in vitro studies, this method of gene transduction is acceptable and allows for the highest efficiency of reprogramming. However, the initial studies with integrating viral vectors have been associated with introduction of chromosomal aberrations, and thus cannot be used in clinical applications. This is especially problematic for human cells since the low efficiency of gene transduction intrinsic to such cells often required the use of lentiviral vectors, which have been the vector with the most reports of inducing chromosomal aberrations (76,115). Some researchers have used nonintegrating viruses, such as adenovirus for reprogramming to prevent the genomic insults caused by gene insertion (116). However, the reprogramming is still viral based and should be handled very carefully for clinical safety concern. Other methods for reprogramming without virus infection have been reported in recent years. Transfection of transient expression vectors by traditional gene delivery methods (87) or a transposome system (137), with the combination of polycistronic gene expression vectors (32) can also generate hiPSCs successfully. Furthermore, direct transduction of the reprogramming factor proteins instead of genes is also another method for hiPSC generation (144,150). In addition, small molecular compounds have been studied to facilitate the reprogramming process with reduced number of factors (24,44,67,72), and these compounds include DNA methylation inhibitors, histone methylation inhibitors, and histone deacetylase inhibitors. Modifications of gene delivery methods all aim for the same goal, which is to replace the use of viruses. hiPSCs generated by nonviral, minimal artificial gene manipulations that are safe to use clinically is an urgent goal for the field.

While the transcription factor-mediated reprogramming is quite remarkable, emerging data are showing that hiPSCs are similar but not identical to hESCs, as initially believed (50). Research has found that the epigenetic background of the reprogrammed hiPSCs does not correspond exactly to hESCs. The “completeness” of the reprogramming process is an important issue since not all of the hiPSCs studies define exactly the criteria of “complete” reprogramming of somatic cells to pluripotency. In fact, the characterization of hiPSCs as PSCs has not yet been unified. Recent studies show that epigenetic memory of the cell-of-origin in reprogrammed hiPSCs is retained, in which the DNA methylation “signature” of the somatic cell used for reprogramming still exists in the reprogrammed cells (41,55,95,120). In other words, these hiPSCs still have active expression of specific genes originating from the donor cells. This phenomenon is apparently not seen in other methods of reprogramming such as nuclear transfer. However, the epigenetic memory of hiPSCs can be corrected by drug-induced chromatin modification or by serial reprogramming (55). Such epigenetic retention of a cell-of-origin gene expression pattern presumably indicates an incomplete reprogramming of the hiPSCs, which may be a major concern for preparation of PSCs.

However, this phenomenon raises an interesting question: If the main purpose for generating the hiPSCs is to differentiate these cells toward a particular somatic cell type, is it necessary for reprogramming back to full pluripotency? The existence of epigenetic memory might conversely be manipulated advantageously for hiPSCs instead of being a disadvantage, because the hiPSC would be biased to differentiate toward the cell type of the donor cell. This brings out a new concept in reprogramming, of choosing the cell of origin for hiPSCs reprogramming based on the type of differentiated cell desired, such as to reprogram hiPSCs from cells of the heart to achieve differentiation toward cardiomyocytes. While this may sound counterintuitive, because differentiated cell types cannot be easily cultured or expanded in vitro, reprogramming to pluripotency would solve this issue, since hiPSCs are able to be expanded ex vivo and manipulated using standard cell culture methods. This would allow easy storage and allow for on-demand availability of end-differentiated cell types, which previously has not been possible for most somatic cell types. The detailed definition and manipulation of the epigenetic memory of hiPSCs for differentiation to specific cell type is an unexplored area of research and may have unexpected therapeutic application. Despite the issues of defining full reprogramming of hiPSCs and the completeness of their pluripotency, these cells still appear to have therapeutic applicability and warrant further elucidation.

Roadblocks to Therapeutic use of PSCs in General

Despite the powerful differentiation capacity and relatively easily ex vivo culture characteristics, several key hurdles must be overcome before hESCs and hiPSCs can be used for clinical applications. Ethical issues related to hESC isolation have limited research on this cell type. Although hiPSCs do not have the ethical concerns of hESCs, the generation methodology and degree of pluripotency of these cells still need to be well established. Both hESCs and iPSCs were originally derived and cultured on inactivated mouse embryonic fibroblast (MEF) feeders, which obstructs clinical use of these hPSCs due to xenogeneic component contamination. Recent studies had improved the culture system by generating feeder-free conditions suitable for the maintenance of hPSCs (70). In addition, efficient differentiation is also an issue, since PSCs in general will induce teratoma formation. Moreover, the formation of teratomas remains a major obstacle to therapeutic application of hPSCs. Within a population of differentiated hPSCs, there needs only to be a few remaining undifferentiated hPSCs for teratoma formation (104). Kooreman and Wu demonstrated a system to monitor hPSC teratoma formation (59); however, the incidence and mechanism of this process is still unclear, which gives us few tools to solve this problem. Unless the pluripotency of these PSCs can be completely shut down or controlled, contamination of undifferentiated hESCs or hiPSCs will be a dangerous problem in human subjects. Intense research is ongoing to develop better and more efficient methods for generating homogeneous populations of fully end-differentiated cells from hPSCs (16). Furthermore, the use of hESC- or hiPSC-derived cells as therapeutic treatment may still encounter immune rejection. While hiPSCs that are generated from and used in the same donor should theoretically evade this problem, it has not been established whether during the differentiation process, immunogenic molecules might be induced and expressed along the way. While immune-suppressing drugs can be used, or genetic modification of the PSCs can be made to be immune tolerant, these methods all have undesirable side effects. One other solution is to create a cell bank of hPSCs based on human leukocyte antigen (HLA) types for matching of PSCs to patients (20,80,82). Although wide and large-scale processing of information would be necessary for creating such a useful database, it would increase the safety of using PSCs therapeutically. The banking of hiPSC would also open up use of third-party cells for reprogramming, which may actually be a benefit since it is known that not all somatic cells can be reprogrammed with the same efficiency. Thus, cell sources that are abundant and more efficiently reprogrammed can be used for hiPSC generation, such as fetal somatic cells (20,38). In this way, cell sources for hiPSC generation can be further expanded, increasing the applicability of this revolutionary technology.

Conclusions

Since the first discovery and study of stem cells, researchers have been fascinated by these versatile cells. Stem cell-based therapies have created new directions for treating human disease. Much effort has been made towards the therapeutic applications of both somatic stem cells and PSCs [reviewed in (36,114,125)]. Clearly, the clinical use of somatic stem cells is at a more mature stage (e.g., HSCs for blood diseases), but the applicable range of diseases is much narrow than that for PSCs. hPSCs provide powerful tools for regenerative medicine (Fig. 1), as these cells can potentially differentiate into all types of functional somatic cells. Thus, in our review, we have focused on the therapeutic potential of hPSCs including hESCs and hiPSCs, highlighting current preclinical and clinical data of these stem cells, and the robust differentiation of these stem cells into MSCs, which are a prevalent stem cell source for clinical use (53,62,105). While the problems with using xenogeneic feeder cells has been largely eliminated by the development of feeder-free culture systems, the risk of teratoma formation still exists if incompletely differentiated hPSCs are transplanted into patients. The ethical concerns regarding hESCs have been obviated by the creation of iPSCs, and while the efficiency of reprogramming is low, this may be overcome by a number of methods including using a more “reprogramming-efficient” cell source from fetal tissue. However, tumorigenicity issues still preclude clinical use of these novel stem cells for now, and the new findings of epigenetic memory add another level of complexity in the road to clinical use of iPSCs. Nevertheless, despite these hurdles to overcome, PSCs likely represent the best source of stem cells for regenerative medicine based on their pluripotency, and intense investigation is ongoing for the use of hPSCs in many different diseases. While many issues remain to be resolved, the differentiation potential and ex vivo expansion capacity of hPSCs make these stem cells invaluable tools for biomedical science.

Figure 1.

Applications of human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs).

Footnotes

Acknowledgments

This work was supported in part by grants (NSC99-3111-B-002-009, NSC99-3111-B-400-004, and NSC100-2321-B-400-022) from the National Science Council of Taiwan. The authors declare no conflict of interest.

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Current Applications of Human Pluripotent Stem Cells: Possibilities and Challenges

Abstract

Keywords

Introduction

Type of Endogenously Derived hPSCs

Embryonic Carcinoma Cells

Embryonic Germ Cells

Embryonic Stem Cells

Application of hESCs

Neural Regeneration

Cardiovascular Repair

Hepatic Regeneration

Treatment of Diabetes

Differentiation of hESCs Towards Mesenchymal Lineages

Hematopoietic Development

Cancer Therapy

Other Applications of hESCs: Drug Discovery and Toxicity Testing

Alternative Source of PSCs

The Induced Pluripotent Stem Cells (iPSCs)

Major Challenges for Therapeutic Use in iPSC Biology

Roadblocks to Therapeutic use of PSCs in General

Conclusions

Footnotes

Acknowledgments

References