Stem cells represent a unique opportunity for regenerative medicine to cure a broad number of diseases for which current treatment only alleviates symptoms or retards further disease progression. However, the number of stem cells available has speedily increased these past 10 years and their diversity presents new challenges to clinicians and basic scientists who intend to use them in clinics or to study their unique properties. In addition, the recent possibility to derive pluripotent stem cells from somatic cells using epigenetic reprogramming has further increased the clinical interest of stem cells since induced pluripotent stem cells could render personalized cell-based therapy possible. The present review will attempt to summarize the advantages and challenges of each type of stem cell for current and future clinical applications using specific examples.

INTRODUCTION

During the past decade, stem cells have become a major focus for translational medicine. These cell types combine two properties that make them uniquely attractive for regenerative medicine. A stem cell is defined as a cell type which has the property to self-renew while maintaining the capacity to differentiate into diverse cell types. Stem cells are therefore different from progenitor cells which can differentiate into mature cell types but are incapable of self-renewing, or somatic cells which are capable of proliferating but unable to differentiate. By combining these properties of self-renewal and differentiation, stem cells could allow the production of an infinite quantity of cell types with a clinical interest. Stem cells are also becoming increasingly attractive for basic research since they can be expanded in vitro while maintaining their native properties, allowing basic studies which are impossible with primary tissue cultures or biopsy material. Consequently, specific stem cells such as ESCs (embryonic stem cells) have become a reference model for understanding key molecular mechanisms which control cell fate choice and organ differentiation.

Importantly, stem cells are already present in clinics especially HSCs (haematopoietic stem cells), which have been successfully used for more than 40 years in bone marrow transplantations to treat diverse blood disorders such as leukaemia [1]. Future clinical applications of stem cells concern a broad number of degenerative diseases (i.e. disease in which one cell type or part of an organ fails) which could potentially be treated using stem-cell-based therapy. This includes major metabolic diseases such as T1D (Type 1 diabetes), caused by the destruction of insulin-secreting β-cells [2], diverse brain and myelin disorders in which specific neural cells are targeted such as MS (multiple sclerosis), PD (Parkinson's disease) or HD (Huntington's disease), heart disease, where some cardiac cells need to be replaced upon myocardial infarction, and genetic diseases like myopathy, where a specific subtype of cells are not functional. The number of diseases that could be cured using stem cells is extremely broad. Thus the present review will only focus on specific examples of current applications which illustrate the recent progress towards the use of stem cells in clinics.

Human stem cells can be classified into three broad categories in accordance with their origin and their capacity of differentiation. Somatic stem cells are multipotent stem cells which originate from differentiated organs (adult or foetal). They can reside specifically in in vivo niches (endogenous somatic stem cells) and sometimes can be isolated and expanded in vitro (exogenous somatic stem cells). The third category (pluripotent stem cells) includes ESCs which are derived from embryos at the blastocyst stage and hiPSCs [human iPSCs (induced pluripotent stem cells)] which are generated from reprogrammed somatic cells. In the present review, we will examine the properties of each stem cell category and discuss their advantages for clinical application by describing relevant examples. In addition, we will define their drawbacks and the necessary progress for their future clinical applications.

TOTIPOTENCE, PLURIPOTENCE AND MULTIPOTENCE

The clinical use of a specific type of stem cell is ultimately defined by its capacity to generate a broad number of cell types. This capacity of differentiation can be divided into three categories. Totipotent stem cells are capable of differentiating into all adult and embryonic tissues, including extra-embryonic tissues such as trophectoderm. Embryos at the one and two cell stages and GCs (germ cells) are likely to be the only cells which display this capacity naturally in an embryonic environment [3]. So far, there is no known stem cell in vitro which has no restriction in its capacity of differentiation. The major issue here is to define the assays and standards that would allow the definition of totipotency of human stem cells in vitro. Obvious ethical considerations preclude the use of chimaera formation and germline transmission in humans (see below) and the development of in vitro assays to demonstrate that one cell type can differentiate into all the cell types existing in the human body will be extremely challenging.

Pluripotent stem cells are capable of differentiating into the derivatives of the three germ layers (ectoderm, mesoderm and endoderm) and GCs. However, they are not totipotent as they cannot differentiate into certain cell types especially extra-embryonic tissues such as trophectoderm. Pluripotent stem cells have been derived from embryos at different developmental stages and from the adult germline [4] (Table 1). Pluripotency is currently defined by specific assays. The most commonly used assay in non-human species is germline transmission. In this case, pluripotent stem cells are injected into embryos at the blastocyst stage and the resulting chimaeric embryo is re-transplanted into a pseudo-pregnant female to continue its development. The offspring is chimaeric in all the tissues, including the germline, which is validated by the transmission of the genetic background originating from the injected stem cells to the next generation. The best example of pluripotent stem cells fulfilling this criteria remains to be mESCs (mouse ESCs) [5,6], which can differentiate into various tissues in vitro and can contribute to the germline in vivo after blastocyst injection. However, mESCs are unable to differentiate into trophectoderm in the absence of genetic modifications and thus are defined as pluripotent stem cells. Concerning hESCs (human ESCs), their pluripotent state has been demonstrated by a broad number of studies showing that they can differentiate into a large number of cell types including the trophectoderm and germ cells [7,8]. However, germline transmission assays are unfeasible for obvious ethical reasons and pluripotency in human cells is usually demonstrated by their capacity to form teratomas. Indeed, pluripotent stem cells can form teratomas containing derivatives of the three germ layers once injected into an immunodeficient host. However, this assay is not quantitative and relies on histological analyses. Therefore the lack of proper standards to define pluripotency in human stem cells remains an issue which could explain the current confusion regarding the pluripotent status of somatic stem cells such as MAPCs (multipotent adult progenitor cells) [9,10].

Table 1
Types of pluripotent stem cells derived from embryos and the germline

mEpiSC, mouse epiblast stem cell; m(h)EGC, mouse (human) embryonic GC; mGSC, multipotent germline stem cell; PGC, primordial GC.

Type of pluripotent stem cell Source of derivation Reference 
mESC Pre-implantation embryo [5,6
Primate ESC Pre-implantation embryo [176,177
hESC Pre-implantation embryo [111,178
 Morula and late blastocyst stage embryo [179,180
 Single blastomere [181
 Parthenogenetic embryo [182184
mEpiSC Post-implantation embryo [185,186
mEGC PGC [187,188
mGSC Neonatal and adult mouse testis [189,190
hEGC Cultured human PGCs [191
Type of pluripotent stem cell Source of derivation Reference 
mESC Pre-implantation embryo [5,6
Primate ESC Pre-implantation embryo [176,177
hESC Pre-implantation embryo [111,178
 Morula and late blastocyst stage embryo [179,180
 Single blastomere [181
 Parthenogenetic embryo [182184
mEpiSC Post-implantation embryo [185,186
mEGC PGC [187,188
mGSC Neonatal and adult mouse testis [189,190
hEGC Cultured human PGCs [191

Multipotent stem cells are able to differentiate into all the cell types constituting an organ. The most prominent example remains HSCs, which are capable of differentiating into all the cell types of the haematopoietic system. Two types of assays are currently used to demonstrate the multipotent state of stem cells. The first is to demonstrate that one stem cell can reconstitute an entire organ. For instance, HSCs can reconstitute an entire haematopoietic system after transplantation into sub-lethally irradiated animals. However, this type of assay is difficult to perform in humans since they require the destruction of the targeted organ and also require clonal analysis which can be technically challenging. Indeed, adult stem cells often require a complex environment to survive and to self-renew in, and this niche can be difficult to mimic in vitro. The second type of assay is based on lineage tracing experiments in which the stem cell of interest is genetically tagged and hence marks its progeny. Thus, using this approach, one can follow the origin of a cell type in a specific organ. The existence of gut stem cells [11] and skin stem cells [12] has been characterized using this approach. Multipotent stem cells have been identified in a broad number of organs in rodents. However, the existence and functionality of their human counterpart remains in some cases controversial since the lack of appropriate technology renders their isolation and thus characterization extremely challenging.

SOMATIC STEM CELLS

Endogenous somatic stem cells

Somatic stem cells are multipotent and can usually differentiate into all the cell types constituting their organ of origin. Some somatic stem cells such as hepatic progenitor cells proliferate very slowly and can be activated by specific conditions such as stress response or injury [13]. Other somatic stem cells such as vascular progenitor cells are proliferating constantly and are in high numbers [14]. They are often localized in a specific niche that is essential to maintain their property of self-renewal and differentiation [15]. Ultimately, their normal function could be to maintain a constant number of cells in an organ. Somatic stem cells have been reported to be found in the brain [16,17], haematopoietic system, skin [18,19], heart [20,21], muscle [22,23], lung [24,25], intestine [26] and liver [27,28] (Figure 1 and Table 2). They represent the ideal target for regenerative medicine since their mobilization would not require invasive methods or immunosuppressive treatments.

Endogenous and exogenous somatic stem cells for cell-based therapy

Figure 1
Endogenous and exogenous somatic stem cells for cell-based therapy

Endogenous adult stem cells (SC) can be activated with the use of drugs or growth factors to prompt regeneration in tissues or organs in vivo. Exogenous adult stem cells can be expanded in ex vivo cultures and then transplanted into the human body for cell replacement or to stimulate regeneration via paracrine actions. Stem cells which remain to be fully characterized are indicated by (?).

Figure 1
Endogenous and exogenous somatic stem cells for cell-based therapy

Endogenous adult stem cells (SC) can be activated with the use of drugs or growth factors to prompt regeneration in tissues or organs in vivo. Exogenous adult stem cells can be expanded in ex vivo cultures and then transplanted into the human body for cell replacement or to stimulate regeneration via paracrine actions. Stem cells which remain to be fully characterized are indicated by (?).

Table 2
Types of adult stem cells reportedly found
Type of adult stem cell Location found Expandable in vitroUsed in clinical trials? 
HSC BM, placenta, UCB and peripheral blood Yes Yes 
MSC BM, amniotic fluid, placenta, AT and UCB Yes Yes 
Muscle satellite stem cell Between basement membrane and cell membrane (sarcolemma) of muscle fibre No Yes 
NSC SVZ of lateral ventricles and SGL of dentate gyrus of the hippocampus in the brain Yes Going to start 
Limbal stem cell Corneoscleral limbus Yes Going to start 
Skin stem cell Basal layer of epidermis and base of hair follicles Yes No 
Gut stem cell Crypts Yes No 
Lung stem cell Between airways (bronchioles) and alveoli No No 
Cardiac stem cell Heart No No 
Liver stem cell Canals of Hering at the terminal bile ductules Yes No 
Pancreatic stem cell Pancreatic ducts No No 
Type of adult stem cell Location found Expandable in vitroUsed in clinical trials? 
HSC BM, placenta, UCB and peripheral blood Yes Yes 
MSC BM, amniotic fluid, placenta, AT and UCB Yes Yes 
Muscle satellite stem cell Between basement membrane and cell membrane (sarcolemma) of muscle fibre No Yes 
NSC SVZ of lateral ventricles and SGL of dentate gyrus of the hippocampus in the brain Yes Going to start 
Limbal stem cell Corneoscleral limbus Yes Going to start 
Skin stem cell Basal layer of epidermis and base of hair follicles Yes No 
Gut stem cell Crypts Yes No 
Lung stem cell Between airways (bronchioles) and alveoli No No 
Cardiac stem cell Heart No No 
Liver stem cell Canals of Hering at the terminal bile ductules Yes No 
Pancreatic stem cell Pancreatic ducts No No 

For instance, a broad number of studies have focused on activating endogenous NSCs (neural stem cells) to replace missing neural cells [29,30]. The use of FGF2 (fibroblast growth factor 2), EGF (epidermal growth factor) [31], TGFα (transforming growth factor α) [32], VEGF (vascular endothelial growth factor) [33,34], SCF (stem cell factor) [35], glucocorticoids and IGF1 (insulin-like growth factor 1) [36] could possibly activate neuronal replacement in neurodegenerative diseases and also in stroke injury. Another example would be the activation of in vivo muscle repair using growth factors [37]. IGF1 has been strongly linked to promote cellular recruitment and muscular repair [38]. Together with the inhibition of the NF-κB (nuclear factor κB) pathway [39] and modulation of TNFα (tumour necrosis factor α) function [40], muscular atrophy could be prevented [41].

However, the clinical use of endogenous somatic stem cells is currently limited for diverse reasons. Their existence remains controversial in several organs especially in humans where lineage tracing or reconstitution experiments are impossible. Therefore their identification and characterization is difficult and past experience has shown that results obtained in animal models might not be translated directly to humans. This limits the study of the mechanisms controlling the self-renewal and differentiation of endogenous stem cells, which is necessary for the development of future clinical treatments. Two controversial examples concern the pancreas and liver. Using lineage tracing techniques in the mouse, it has been demonstrated that pancreatic β-cells ‘regenerate’ via self-replication of existing β-cells and not from stem cells [42,43]. However, recent reports have contradicted these observations by showing that β-cells can be made de novo from islet progenitor cells expressing Ngn3 and residing in the pancreatic duct [44]. Nevertheless, most of these results were obtained in animal models of injury in which the pancreas is induced to regenerate after duct ligation, and recent studies have not been able to repeat these experiments [45]. Therefore it remains to be demonstrated that this model is physiologically relevant and that a pool of pancreatic stem cells can be identified in normal conditions, especially in humans.

Hepatic oval cells/hepatic progenitors represent another controversial adult stem cell [46,47]. These liver-specific stem cells originally identified in rodents could be used to make hepatocytes for cell-based therapy of liver failure and liver metabolic diseases [4850]. They are bipotent (i.e. they are capable to differentiate into duct cells or hepatocytes) as shown by the expression of bile duct markers [CK (cytokeratin)-7, CK-19 and OV-6], and hepatocyte markers [AFP (α foetal protein) and ALB (albumin)]. They also express HNFs (hepatocyte nuclear factors) which mark hepatic progenitors during embryonic liver development [51]. In humans, they are localized in the canals of Hering at the terminal bile ductules [52] and are reportedly activated in disease conditions [53,54]. However, they are extremely rare (if in existence) and possibly inaccessible [52]. There have been reports of the isolation of hepatic progenitor cells from the adult human liver [5557]. However, the lack of definitive markers and the dissimilarity between human and rodents renders the identification of a common hepatic progenitor cell difficult [58].

These two examples illustrate the difficulty of translating observations obtained in animal models directly to humans [5961]. Indeed, stem cells in distant species might have different functions and activity [62,63]. Another major drawback is that endogenous somatic stem cells could be targeted by the diseases, explaining why endogenous somatic stem cells appear to be incapable or insufficient in stopping the progression of degenerative diseases. Finally, the use of endogenous stem cells could be precluded in genetic diseases whereby stem cells continue to produce progeny carrying the same phenotype. Consequently, genetic correction will be necessary, implicating the use of gene therapy approaches or complex manipulations in vitro. In conclusion, mobilization of endogenous somatic stem cells represents the ultimate objective of regenerative medicine. This approach is the most ideal, ‘simple’ and natural as we are simply prompting and activating our innate body system to repair diseased tissues. There would not be complicated problems such as immunological response to exogenous foreign cells or ethical issues related to ESCs. However, their clinical use will require better identification and characterization to develop new approaches allowing their mobilization in vivo, especially in a diseased environment.

Exogenous somatic stem cells

Exogenous somatic stem cells are derived in vitro from diverse organs. They have a high capacity of proliferation and can be maintained in vitro for a prolonged period of time. Exogenous somatic stem cells are commonly recognized to exhibit lineage-restricted multipotency. However, several reports have suggested that somatic stem cells could be pluripotent since they can differentiate into a broad number of cell types unrelated to their organ of origin [64]. In addition, controversial reports have suggested that these cells could participate in germ layer formation [64]. However, these results have been difficult to replicate and the current consensus is that adult somatic stem cells have a restricted capacity of differentiation compared with those of ESCs. Furthermore, exogenous somatic stem cells often represent an in vitro state which has no equivalence in vivo. However, their use in regenerative medicine is promising since they are easy to isolate and can be expanded in vitro. In addition, immunosuppressive treatments are not required due to autologous transplantations, making them ideal candidates for clinical trials. The two best examples are NSCs [65,66] and multipotent MSCs (mesenchymal stromal cells) [67,68] which are currently being used in several clinical trials.

NSCs are progenitor cells located in the SVZ (subventricular zone) of the lateral ventricles or SGL (subgranular layer) of the dentate gyrus of the hippocampus in the brain. NSCs have been cultured in vitro as cell clumps known as ‘neurospheres’ [69,70]. The neurospheres can be passaged several times, demonstrating their capacity of self-renewal. They are also multipotent since they can differentiate into neurons and glia [71,72]. Therefore NSCs represent a unique source of cells for clinical application, and several clinical trials are currently testing their efficiency [73]. One of them is conducted by StemCells Inc. (San Francisco, CA, U.S.A.) and its objective is to use NSCs for the treatment of Batten's disease, a lysosomal storage disorder. In addition, several trials have used neuronal cells for stroke patients [74,75]. ReNeuron (Guildford, U.K.) is currently proposing to transplant myc-immortalized hNSCs (human NSCs) [76] derived from human foetal tissue into stroke patients. Therefore the first clinical applications of NSCs have started. However, several challenges have to be solved before larger trials could start, including better isolation and expansion methods for NSCs, as well as prevention of tumour formation after transplantation [77].

MSCs are fibroblast-like cells which can be isolated from diverse sources including BM (bone marrow) [78], amniotic fluid [79], placenta [80,81], AT (adipose tissue) [82,83] and UCB (umbilical cord blood) [84]. MSCs are mainly identified by their adherent property, differentiation potential, and cell-surface markers such as CD73, CD90 and CD105 [67,85,86]. However, their capacity of differentiation in vivo and in vitro is limited as they are not pluripotent. Besides MSCs, several related types of cells including MAPCs were isolated from the BM [9,10]. MSCs are multipotent [87] and have been reported to be able to differentiate into osteoblasts, adipocytes [88], chondrocytes [89,90], and epithelial [91], endothelial [92] and neural cells [93]. It should be noted that MSCs derived from different tissues may exhibit slightly different propensities and capacities to differentiate into specific cell types. For example, MSCs from synovial tissue may be more effective in repairing cartilage defects than MSCs from other tissues [94]. MSCs self-renew in vitro but not indefinitely since they do senesce after 20 population doublings and ultimately lose their potential to differentiate [95]. More importantly, they do tend to generate abnormal karyotypes in vitro as their genomes are highly unstable [96]. This could represent a major limitation for their use in cell-based therapy.

Nonetheless, the interest in MSCs for clinical applications resides less in their capacity of differentiation than in their paracrine actions on tissue repair [97]. Indeed, MSCs express low levels of MHC class II molecules and thus should be weakly immunogenic, suggesting that immunosuppressive treatment might not be required. In addition, they have been shown to be able to modulate the immune response of cells in vitro and to limit inflammation [98]. Hence they could be used for preventing graft rejections and GvHD (graft versus host disease). MSCs were first used in clinical trials as a complementary treatment for breast cancer patients [99]. Thereafter they were demonstrated further to be beneficial against GvHD [100]. On the basis of these successes, a broad number of studies are currently being performed to define their efficacy to treat Crohn's disease [101], ALS (amyotrophic lateral sclerosis) [102] and heart diseases [103]. In particular for heart therapy, autologous BM-MSCs have been used in controlled clinical trials [104]. Importantly, MSCs do not repopulate damaged tissues and cells quickly disappear after engraftment in vivo. Despite this, small but positive effects on cardiac performance have been observed, and this clinical benefit could be achieved through immune modulation and increased angiogenesis.

MSCs could also potentially be used to treat liver diseases. BM-MSCs apparently are able to differentiate into hepatocyte-like cells without cell fusion [105,106]. UCB-MSCs [107] and AT-MSCs [108] can also be differentiated into hepatocyte-like cells. hMSC (human MSC)-derived hepatocytes have recently been reported to be able to restore liver function [109]. MSCs are now being used in clinical trials for end-stage liver disease [110] and initial results appear promising. However, long-term recovery of liver function remains to be demonstrated and further studies will be required to understand the mechanisms by which MSCs can restore liver function. In summary, the mechanisms by which MSCs can achieve their immunomodulatory and detoxification functions, as well as paracrine secretions, remain to be defined. Further basic studies will be extremely helpful in validating their real function in vivo and the mechanisms by which they can alleviate so many symptoms. However, their interest for clinical application cannot be denied and the broad number of clinical trials which are currently performed with MSCs should rapidly provide some information regarding their real efficacy and long-term effect.

The examples described above illustrate the advantages of somatic stem cells for clinical applications. These cells are relatively easy to isolate and they do not require immunosuppressive treatment. In addition, they can be used to treat a broad number of diseases without major risks. All of these aspects greatly simplify their use in a clinical context and thus make them attractive for clinical trials. However, an increasing amount of data are suggesting that even a short period of time in culture in vitro can modify the genetic and epigenetic characteristics of somatic stem cells. The consequences of these modifications on cell proliferation and differentiation after transplantation and after a prolonged period of time in vivo will have to be examined carefully. Finally, the mechanisms involving somatic stem cells in tissue repair and/or in modulating immune response are not always fully understood and additional basic studies are required to fully define the clinical potential of these different cell types.

EMBRYONIC STEM CELLS

hESCs are pluripotent stem cells derived from the inner cell mass of embryos at the blastocyst stage [111]. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into derivatives of the three germ layers from which all adult organs are derived. Therefore hESCs represent a unique opportunity for regenerative medicine since they could be used to generate an infinite quantity of cells with a clinical interest. However, the clinical application of these unique cell types is currently limited by two challenges: the difficulty to generate fully functional cell types and also the safety issue associated with teratoma formation.

Difficulty to generate fully functional cell types from hESCs

A major focus of the past 10 years has been the development of robust protocols to differentiate hESCs into homogenous populations of cell types with a clinical interest. Consequently, a broad number of protocols have been developed to generate diverse cell types, including retina cells [112,113], dopaminergic neurons [114], motor neurons [115,116], OPCs (oligodendrocyte progenitor cells) [117], cardiomyocytes [118,119], pancreatic β-cells [120] and hepatocytes [121124] (Figure 2 and Figure 3). These methods mark a clear progress towards the generation of fully functional cell types. They have benefited from the lessons learnt from mESCs as well as from the knowledge developed by developmental biology studies. Indeed, past experiences have shown that following a natural path of development in vitro remains the best way to generate functional cell types from pluripotent stem cells of embryonic origin. Therefore most of the protocols developed to drive differentiation of hESCs respect key stages of embryonic development. However, this methodology also implies that generation of fully mature and functional cells from hESCs will require an extended period of time of in vitro culture, mimicking a complex environment constantly changing over time. Therefore such an approach is presented with unprecendented challenges which would require a strong collaboration between stem cell biologists and developmental biologists. Generation of pancreatic β-cells is a good example to illustrate the progress and challenges regarding the generation of clinically relevant cells from hESCs.

Human pluripotent stem cells for cell-based therapy

Figure 2
Human pluripotent stem cells for cell-based therapy

hESCs can be derived from the inner cell mass of the human blastocyst, whereas hiPSCs can be derived via reprogramming of human somatic cells. These human pluripotent stem cells can be differentiated into clinically useful cell types, such as neural cells, retinal cells, cardiomyocytes, hepatocytes and pancreatic β-cells, and be used for cell replacement therapy.

Figure 2
Human pluripotent stem cells for cell-based therapy

hESCs can be derived from the inner cell mass of the human blastocyst, whereas hiPSCs can be derived via reprogramming of human somatic cells. These human pluripotent stem cells can be differentiated into clinically useful cell types, such as neural cells, retinal cells, cardiomyocytes, hepatocytes and pancreatic β-cells, and be used for cell replacement therapy.

An example of an hESC differentiation protocol

Figure 3
An example of an hESC differentiation protocol

Directed differentiation of hESCs to hepatic progenitors using fully defined culture conditions [175]. hESCs were differentiated into endoderm using activin, BMP4 (bone morphogenetic protein 4), FGF2 and the PI3K (phosphoinositide 3-kinase) inhibitor LY294002. Hepatic specification was then initiated with FGF10, RA (retinoic acid) and an inhibitor of the activin/nodal receptor SB431542. Addition of FGF4, HGF and EGF resulted in maturation of the hepatic progenitors expressing some markers of mature hepatocytes, such as ALB, AFP, AAT (α1-antitrypsin) and CK18.

Figure 3
An example of an hESC differentiation protocol

Directed differentiation of hESCs to hepatic progenitors using fully defined culture conditions [175]. hESCs were differentiated into endoderm using activin, BMP4 (bone morphogenetic protein 4), FGF2 and the PI3K (phosphoinositide 3-kinase) inhibitor LY294002. Hepatic specification was then initiated with FGF10, RA (retinoic acid) and an inhibitor of the activin/nodal receptor SB431542. Addition of FGF4, HGF and EGF resulted in maturation of the hepatic progenitors expressing some markers of mature hepatocytes, such as ALB, AFP, AAT (α1-antitrypsin) and CK18.

The Edmonton Protocol has proven that islet transplantation via the hepatic portal vein can correct insulin deficiency in diabetic patients [125]. However, the lack of cadaveric donors severely limits the number of patients that could benefit from this therapy. Generating pancreatic cells from hESCs represent a unique opportunity to bypass this limitation. However, the resulting β-cells would have to synthesize and store insulin, and also maintain blood glucose in a physiological range when challenged with sporadic food intake. Past experiences [126,127] have demonstrated that strict adherence to the pancreas developmental process is essential for the production of pancreatic cells from ESCs [2,128,129]. Accordingly, a robust method of differentiation should respect four major stages: (i) formation of DE (definitive endoderm) [130,131], (ii) formation of PE (pancreatic endoderm), (iii) formation of endocrine progenitors, and (iv) maturation of endocrine progenitors into fully functional hormone-secreting cells. Several methods are now available to efficiently generate DE cells from hESCs and to further differentiate the resulting cells into pancreatic progenitors. However, the specification of these progenitors toward the endocrine pathway as well as the production of fully mature β-cells remains a challenge.

Indeed, the most advanced protocol for the differentiation of hESCs into pancreatic β-cells allows the production of multi-hormonal immature pancreatic endocrine cells which lack Nkx6.1 and MafA co-expression. Despite this major limitation, the pancreatic progenitors generated using this approach can differentiate into fully functional pancreatic cells in vivo after transplantation into immuno-deficient mice and the resulting β-cells can re-establish euglycaemia in a mouse model of diabetes [120]. These results are extremely encouraging since they provide the first demonstration that stem-cell-based therapy could be used to treat diabetes. However, partially differentiated pancreatic progenitors can easily be contaminated by pluripotent cells which further increase the risk of teratoma formation associated with hESCs (see below). In addition, non-chemically defined culture conditions containing FBS (foetal bovine serum) are still used, increasing the difficulty for such approaches to be approved for clinical trials. Finally, any cell-based therapy approach will have to address specific problems associated with the auto-immune disease involved in T1D. Therefore the results obtained with hESCs are very promising and they demonstrate the potential of pluripotent stem cells to develop new treatment for diabetes. However, more efforts are still required to generate fully functional β-cells from human pluripotent stem cells and to demonstrate that pancreatic progenitors can be used safely in a clinical context.

Importantly, the challenges described above for pancreatic cells are shared with many other cell types. For example, HSCs generated from hESCs resemble those of the yolk sac [132,133] and thus have a limited capacity of repopulation in immuno-deficient animals [134]. In addition, hESC-derived hepatocytes appear to have an embryonic identity since they have limited capacity to colonize the adult liver and they usually display limited functionality when compared with their in vivo counterparts [121124]. Therefore developing robust protocols of differentiation remains a major challenge to deliver the clinical promises of hESCs. However, this issue can be solved by directly using the progenitors already available as shown with pancreatic cells (see above) and/or by increasing our understanding of the mechanisms controlling organ maturation. This last approach is currently used successfully for a large number of cell types and especially with neural cells.

Indeed, protocols currently available to generate functional neurons, retina cells or OPCs have strongly benefited from knowledge generated by developmental biologists concerning neural tube patterning and neuronal specification [135,136]. Therefore these protocols generally respect key stages of development and require extended periods of time of in vitro culture (between 45 days and up to 3 months) to generate functional cell types. In addition, the availability of animal models for specific neurodegenerative diseases and the existence of robust in vitro functional tests allowed validation of the cells generated. For example, transplantation of ESC-derived dopaminergic neurons appeared to restore motor function in animal models of PD [114], and studies have shown that hESCs can be differentiated into retinal pigment cells which can restore retinal structure and function in animal models of retinal degeneration [137]. Finally, OPCs generated from hESCs have been transplanted into animal models of spinal cord injury and the transplanted cells appear not only to recolonize the scarred area, but also to promote the recruitment of endogenous stem cells, which ultimately results in restoration of mobility [117,138]. These exciting preliminary findings led to the first hESC-based clinical trial for treatment of spinal cord injuries by Geron Corporation (Menlo Park, CA, U.S.A.). This clinical trial, despite being put on hold at the moment, represents an important step towards clinical applications of human pluripotent stem cells and the outcome will bring essential information concerning the feasibility of stem-cell-based therapy for a large number of diseases.

Safety issue with hESCs

Safety issues continue to be the main limitation for the use of hESCs in clinics, and teratoma formation represents one of the most difficult challenges (Figure 4). Indeed, hESCs are naturally tumourous by definition. hESC lines are considered to be pluripotent based on the capacity to form teratomas once injected into an immunodeficient host. Therefore there is a risk of tumour formation if the transplanted cells are contaminated by residual pluripotent stem cells. Importantly, several reports have shown that this risk is increased when early progenitors are used [120,139] and that methods producing heterogeneous populations of cells favour contamination by undifferentiated cells. Hence teratoma formation can easily be avoided using robust methods of differentiation allowing the generation of a homogenous population of fully differentiated cells. A second cause of teratoma formation could be more problematic. Differentiated cells generated from ESCs could have the capacity to dedifferentiate into pluripotent stem cells when grown in a specific environment in vivo. However, there is no evidence that fully differentiated cells generated from hESCs can be reprogrammed in vivo or in vitro without overexpression of pluripotency factors. Systematic studies regarding the occurrence of teratoma formation after transplantation of hESC-derived somatic cells could be necessary to completely rule out such a hypothesis.

Roadmap from human stem cells to cell replacement therapy

Figure 4
Roadmap from human stem cells to cell replacement therapy

Some selection criteria for the type of human stem cells (SCs) and the methodology of differentiation prior to approval for clinical use. Thereafter the end-products can be used for cell replacement therapy or for drug testing. FDA, Federal Drug Administration.

Figure 4
Roadmap from human stem cells to cell replacement therapy

Some selection criteria for the type of human stem cells (SCs) and the methodology of differentiation prior to approval for clinical use. Thereafter the end-products can be used for cell replacement therapy or for drug testing. FDA, Federal Drug Administration.

Graft overgrowth represents another issue associated with progenitors generated from hESCs. Indeed, the injection of early progenitors in the adult environment can cause uncontrolled proliferation of these progenitors, resulting in a tumour-like structure incompatible with the function of the transplanted organ. For example, a recent report has described neuronal progenitors generated from hESCs to continue proliferating after transplantation in the brain, leading to abnormal growth [140]. This issue illustrates once again the importance of generating fully differentiated cell types from hESCs. The issue of tumour formation associated with hESCs is also heightened by their relative karyotypic instability [141]. Indeed, hESCs are prone to acquire chromosomal translocations when grown over a prolonged period of time in culture. This is a common feature with any primary cell culture [142], which can be solved by karyotyping hESCs regularly and by maintaining robust culture conditions [143]. However, the most robust method currently available to expand hESCs involves co-culture with feeder cells and serum, which are not always fully compatible with clinical applications.

To conclude, the risk of tumour formation by hESCs remains an issue, but it can be easily controlled by using methods of differentiation allowing the generation of homogenous population of fully differentiated cells. Importantly, technical solutions can be used to guarantee the absence of pluripotent stem cells in transplanted cells. Methods of purification allowing transplantation of homogenous populations of differentiated cells are currently available and are developed by several laboratories [144147]. This can be achieved with FACS using specific cell-surface markers for pluripotent stem cells and/or for differentiated cells. hESCs can also be engineered to contain a selection gene which can be used to avoid the presence of any residual undifferentiated cells [148]. Finally, encapsulation of differentiated cells when compatible with their functionality can also be an advantageous solution. Therefore there are multiple solutions to increase the safety of cells generated from hESCs, which should accelerate their use in clinic.

HUMAN-INDUCED PLURIPOTENT STEM CELLS

Pluripotent stem cells can also be generated by reprogramming somatic cells using either SCNT (somatic cell nuclear transfer) or overexpression of pluripotency factors. However, SCNT has never been performed successfully in humans and the difficulty in obtaining human oocytes remains a major limitation. The recent possibility to generate hiPSCs from somatic cells by overexpressing pluripotency factors such as Oct4, Sox2, Klf4 and c-Myc represents a more immediate opportunity for regenerative medicine. Indeed, this approach can be used to generate hiPSCs from a large number of patients of different age and gender [149,150]. In addition, a broad number of somatic cells can be reprogrammed by the four factors approach, including skin fibroblasts [151], keratinocytes [152], NSCs [153], B-lymphocytes [154], pancreatic β-cells [155], hepatocytes and intestinal cells [156]. Therefore hiPSCs could enable the production of patient-specific cell types which are fully immune-compatible with the original donor, thereby avoiding the use of immunosuppressive treatment during cell-based therapy (Figure 2). In addition, hiPSCs can be generated from somatic cells isolated from patients suffering from diverse diseases. Then, the resulting ‘diseased’ hiPSCs can be differentiated into the cell type targeted by the disease and thus provide in vitro models to perform large-scale studies impossible with primary cell culture or with biopsy material. Two recent studies have shown that hiPSCs can be used to study SMA (spinal muscular atrophy) and FD (familial dysautonomia) [156a,156b]. In both studies, ‘diseased’ hiPSCs were differentiated into neuronal cells which then display phenotype characteristic of cells from patients with SMA or FD. Therefore these culture systems provide, for the first time, an in vitro model to study these complex diseases and also to screen pharmaceutical compounds in vitro using relevant tissues. Hence hiPSCs represent a unique opportunity to develop new treatments for a large number of diseases which are lacking relevant in vitro models (Figure 4).

Limitations to in vivo use

The use of hiPSCs in vivo is currently limited by several issues. hiPSCs are commonly generated using viruses overexpressing the factors necessary to achieve epigenetic reprogramming. Therefore the resulting hiPSCs contain multiple copies of each transgene in their genome and these insertions can provoke mutations in key genes, such as oncogenes or cell-cycle regulators. This phenomenon has been well described in gene therapy clinical trials for SCID (severe combined immunodeficiency) [157]. Indeed, several patients developed leukaemia after insertion of the lentiviruses in proto-oncogene LMO2 (LIM domain only 2) [158,159]. Therefore the use of viruses to generate hiPSCs represents a major limitation for their in vivo use. A solution could be to sequence the entire genome of each hiPSC to select lines that only integrate viruses in regions of the genome devoid of genes. Alternatively, a large number of groups are now developing non-integrative methods to generate hiPSCs, including the use of transitory transfection of episomal viruses [160162]. The development of small molecules represents the ultimate solution to the problems associated with genetic modifications and several reports have shown that c-Myc and Sox2 could be replaced respectively, by molecules such as the histone modifier inhibitor BIX-01294 [163] and the TGFβ receptor inhibitor SB431542 [164]. However, all of these methods have been used on a limited number of somatic cells of embryonic origin and more work is required to demonstrate that they can be used with a broad number of adult patients.

Generation of hiPSCs also require the use of c-Myc and/or the inhibition of the p53 pathway, both of which present safety issues. Indeed, studies in the mouse have shown that reactivation of the exogenous c-Myc transgene can induce tumour formation [165]. Recent reports have also shown that inhibition of the p53 pathway strongly increases the efficiency of epigenetic reprogramming and thus suggests that the main pathway protecting cells against DNA damage needs to be turned off to allow the generation of hiPSCs [166171]. The consequences on the genomic integrity of hiPSCs remain to be demonstrated, but inhibition of p53 could increase the karyotypic instability already observed with hESCs. Finally, the pluripotent status of hiPSCs confers upon them the capacity to form teratomas in vivo and this safety issue, which is shared with hESCs, could be aggravated in hiPSCs. Indeed, a recent report has shown that the capacity of iPSCs to form teratomas can vary in function of their origin [172]. This phenomenon could be explained by the persistence of undifferentiated cells after differentiation, suggesting that some iPSC lines are more difficult to differentiate than others. Alternatively, differentiated cells generated from iPSCs could re-acquire a pluripotent status after transplantation in vivo. Therefore further studies are necessary to fully demonstrate that differentiated cells generated from iPSCs are epigenetically stable over a prolonged period of time in vitro and in vivo.

Generation of fully differentiated cells

hiPSCs and hESCs share many characteristics, including their capacity of differentiation, the expression of pluripotency markers and their dependence on activin/nodal signalling to maintain their pluripotent status [173]. These functional similarities also imply that hiPSCs and hESCs suffer from the same limitations, which include the risk of teratoma formation (see above), variability in their capacity to differentiate and difficulty in generating fully functional cells. Moreover, incomplete epigenetic reprogramming, epigenetic memory, transgene insertion and genetic instability could worsen the variability between hiPSCs and thus render its use almost impossible for personalized therapy. Indeed, each hiPSC line could require the development of a specific method of differentiation. Consequently, further studies are needed to definitively demonstrate that hiPSCs generated from a broad number of patients can all be efficiently differentiated into one particular cell type using a unique protocol. In addition, the absence of robust protocols to generate fully differentiated cells from hiPSCs is currently limiting the development of in vitro models of disease. Indeed, the cells generated from diseased hiPSCs display features of embryonic progenitors which cannot be used to mimic disease only occurring in the adult [174]. Therefore much more effort will be required to deliver the clinical promises of hiPSCs. However, this major objective could be achieved very rapidly since hiPSCs have become the main focus of many laboratories and they will plainly benefit from the experience accumulated over the past 10 years with hESCs.

CONCLUSIONS AND PERSPECTIVES

The increasing types of stem cells available represent a unique opportunity for regenerative medicine as they will offer new treatment for a broad number of diseases which cannot be fully cured by drug-based therapy. Importantly, stem cells are already reaching the clinic as shown by the large number of clinical trials using diverse types of stem cells. However, stem cells present new challenges that will need to be solved to fully realise their clinical promises. The broad diversity of stem cells available is now offering a large choice for clinicians and a universal stem cell for ‘all the diseases’ is unlikely to exist. Therefore, the ‘right’ stem cell will have to be used for the ‘right’ disease. In addition, safety could block or delay a large number of clinical applications. Indeed, the necessity to demonstrate that a stem cell is not harmful represents a necessary hurdle, especially with pluripotent stem cells capable of forming teratomas. Therefore regulatory agencies will have to define new criteria to evaluate the risk associated with specific stem cells and their differentiated progeny. This process is often delayed by the difficulty to define what is an acceptable risk/benefit with stem-cell-based therapy. Is teratoma an acceptable risk compared with a terminal disease? In addition, novel animal models are urgently needed to reinforce the work currently performed in rodents. Indeed, the mouse presents clear advantages but also important limitations. Their size is not relevant for human treatment and mouse models rarely reproduce, in full, the human disease. Finally, monitoring functionality of human cells in a fully immunosuppressed xeno-environment present obvious issues. Therefore the use of large animals or non-human primates will be necessary to validate the functionality and safety of the cells generated from hESCs/hiPSCs and also other stem cells. To conclude, clinical applications of stem cells are becoming a reality and future applications will continue to require a strong collaboration between basic scientists, clinicians, regulatory agencies and, more importantly, patients, whose support is essential.

Abbreviations

     
  • AFP

    α foetal protein

  •  
  • ALB

    albumin

  •  
  • AT

    adipose tissue

  •  
  • BM

    bone marrow

  •  
  • CK

    cytokeratin

  •  
  • DE

    definitive endoderm

  •  
  • EGF

    epidermal growth factor

  •  
  • ESC

    embryonic stem cell

  •  
  • FD

    familial dysautonomia

  •  
  • FGF

    fibroblast growth factor

  •  
  • GC

    germ cell

  •  
  • GvHD

    graft versus host disease

  •  
  • hESC

    human ESC

  •  
  • HNF

    hepatocyte nuclear factor

  •  
  • HSC

    haematopoietic stem cell

  •  
  • IGF1

    insulin-like growth factor 1

  •  
  • iPSC

    induced pluripotent stem cell

  •  
  • hiPSC

    human iPSC

  •  
  • MAPC

    multipotent adult progenitor cell

  •  
  • mESC

    mouse ESC

  •  
  • MSC

    mesenchymal stromal cell

  •  
  • NSC

    neural stem cell

  •  
  • OPC

    oligodendrocyte progenitor cell

  •  
  • PD

    Parkinson's disease

  •  
  • SCF

    stem cell factor

  •  
  • SCNT

    somatic cell nuclear transfer

  •  
  • SGL

    subgranular layer

  •  
  • SMA

    spinal muscular atrophy

  •  
  • SVZ

    subventricular zone

  •  
  • T1D

    Type 1 diabetes

  •  
  • TGF

    transforming growth factor

  •  
  • UCB

    umbilical cord blood

We thank Dr Tamir Rashid for helpful discussions, Ms Stephanie Brown for proof-reading prior to submission, and Dr Nicholas Hannan for providing the immunostaining figures.

FUNDING

A. K. K. T. is supported by A*STAR Graduate Academy of A*STAR (Agency for Science, Technology and Research), Singapore, and L.V. is supported by the Medical Research Council (U.K.).

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