Trabalho 2012 teo2010 regenerative therapy

Biochem. J. (2010) 428, 11–23 (Printed in Great Britain) doi:10.1042/BJ20100102 11

REVIEW ARTICLEEmerging use of stem cells in regenerative medicineAdrian K. K. TEO*† and Ludovic VALLIER*1

*Laboratory for Regenerative Medicine, University of Cambridge, West Forvie Building, Forvie Site, Robinson Way, Cambridge CB2 0SZ, U.K., and †Institute of Medical Biology,A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648

Stem cells represent a unique opportunity for regenerativemedicine to cure a broad number of diseases for which currenttreatment only alleviates symptoms or retards further diseaseprogression. However, the number of stem cells available hasspeedily increased these past 10 years and their diversity presentsnew challenges to clinicians and basic scientists who intend to usethem in clinics or to study their unique properties. In addition, therecent possibility to derive pluripotent stem cells from somaticcells using epigenetic reprogramming has further increased the

clinical interest of stem cells since induced pluripotent stem cellscould render personalized cell-based therapy possible. The presentreview will attempt to summarize the advantages and challengesof each type of stem cell for current and future clinical applicationsusing specific examples.

Key words: cell replacement therapy, human, regenerativemedicine, stem cell.

INTRODUCTION

During the past decade, stem cells have become a major focus fortranslational medicine. These cell types combine two propertiesthat make them uniquely attractive for regenerative medicine. Astem cell is defined as a cell type which has the property to self-renew while maintaining the capacity to differentiate into diversecell types. Stem cells are therefore different from progenitor cellswhich can differentiate into mature cell types but are incapable ofself-renewing, or somatic cells which are capable of proliferatingbut unable to differentiate. By combining these properties of self-renewal and differentiation, stem cells could allow the productionof an infinite quantity of cell types with a clinical interest. Stemcells are also becoming increasingly attractive for basic researchsince they can be expanded in vitro while maintaining their nativeproperties, allowing basic studies which are impossible withprimary tissue cultures or biopsy material. Consequently, specificstem cells such as ESCs (embryonic stem cells) have becomea reference model for understanding key molecular mechanismswhich control cell fate choice and organ differentiation.

Importantly, stem cells are already present in clinics especiallyHSCs (haematopoietic stem cells), which have been successfullyused for more than 40 years in bone marrow transplantations totreat diverse blood disorders such as leukaemia [1]. Future clinicalapplications of stem cells concern a broad number of degenerativediseases (i.e. disease in which one cell type or part of an organfails) which could potentially be treated using stem-cell-basedtherapy. This includes major metabolic diseases such as T1D(Type 1 diabetes), caused by the destruction of insulin-secretingβ-cells [2], diverse brain and myelin disorders in which specificneural 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 specificsubtype of cells are not functional. The number of diseases thatcould be cured using stem cells is extremely broad. Thus thepresent review will only focus on specific examples of currentapplications which illustrate the recent progress towards the useof stem cells in clinics.

Human stem cells can be classified into three broad categories inaccordance with their origin and their capacity of differentiation.Somatic stem cells are multipotent stem cells which originate fromdifferentiated organs (adult or foetal). They can reside specificallyin in vivo niches (endogenous somatic stem cells) and sometimescan be isolated and expanded in vitro (exogenous somatic stemcells). The third category (pluripotent stem cells) includes ESCswhich are derived from embryos at the blastocyst stage andhiPSCs [human iPSCs (induced pluripotent stem cells)] whichare generated from reprogrammed somatic cells. In the presentreview, we will examine the properties of each stem cell categoryand discuss their advantages for clinical application by describingrelevant examples. In addition, we will define their drawbacks andthe necessary progress for their future clinical applications.

TOTIPOTENCE, PLURIPOTENCE AND MULTIPOTENCE

The clinical use of a specific type of stem cell is ultimately definedby its capacity to generate a broad number of cell types. Thiscapacity of differentiation can be divided into three categories.Totipotent stem cells are capable of differentiating into all adultand embryonic tissues, including extra-embryonic tissues suchas trophectoderm. Embryos at the one and two cell stages andGCs (germ cells) are likely to be the only cells which display thiscapacity naturally in an embryonic environment [3]. So far, there isno known stem cell in vitro which has no restriction in its capacityof differentiation. The major issue here is to define the assays and

Abbreviations used: AFP, α foetal protein; ALB, albumin; AT, adipose tissue; BM, bone marrow; CK, cytokeratin; DE, definitive endoderm; EGF, epidermalgrowth 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, oligodendrocyteprogenitor 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.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2010 Biochemical Society

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12 A. K. K. Teo and L. Vallier

Table 1 Types of pluripotent stem cells derived from embryos and thegermline

mEpiSC, mouse epiblast stem cell; m(h)EGC, mouse (human) embryonic GC; mGSC, multipotentgermline 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 [182–184]

mEpiSC Post-implantation embryo [185,186]mEGC PGC [187,188]mGSC Neonatal and adult mouse testis [189,190]hEGC Cultured human PGCs [191]

standards that would allow the definition of totipotency of humanstem cells in vitro. Obvious ethical considerations preclude theuse of chimaera formation and germline transmission in humans(see below) and the development of in vitro assays to demonstratethat one cell type can differentiate into all the cell types existingin the human body will be extremely challenging.

Pluripotent stem cells are capable of differentiating into thederivatives of the three germ layers (ectoderm, mesoderm andendoderm) and GCs. However, they are not totipotent as theycannot differentiate into certain cell types especially extra-embryonic tissues such as trophectoderm. Pluripotent stem cellshave been derived from embryos at different developmentalstages and from the adult germline [4] (Table 1). Pluripotencyis currently defined by specific assays. The most commonly usedassay in non-human species is germline transmission. In this case,pluripotent stem cells are injected into embryos at the blastocyststage and the resulting chimaeric embryo is re-transplanted intoa pseudo-pregnant female to continue its development. Theoffspring is chimaeric in all the tissues, including the germline,which is validated by the transmission of the genetic backgroundoriginating from the injected stem cells to the next generation.The best example of pluripotent stem cells fulfilling this criteriaremains to be mESCs (mouse ESCs) [5,6], which can differentiateinto various tissues in vitro and can contribute to the germlinein vivo after blastocyst injection. However, mESCs are unableto differentiate into trophectoderm in the absence of geneticmodifications and thus are defined as pluripotent stem cells.Concerning hESCs (human ESCs), their pluripotent state hasbeen demonstrated by a broad number of studies showing thatthey can differentiate into a large number of cell types includingthe trophectoderm and germ cells [7,8]. However, germlinetransmission assays are unfeasible for obvious ethical reasonsand pluripotency in human cells is usually demonstrated by theircapacity to form teratomas. Indeed, pluripotent stem cells canform teratomas containing derivatives of the three germ layersonce injected into an immunodeficient host. However, this assayis not quantitative and relies on histological analyses. Thereforethe lack of proper standards to define pluripotency in human stemcells remains an issue which could explain the current confusionregarding the pluripotent status of somatic stem cells such asMAPCs (multipotent adult progenitor cells) [9,10].

Multipotent stem cells are able to differentiate into all the celltypes constituting an organ. The most prominent example remainsHSCs, which are capable of differentiating into all the cell types ofthe haematopoietic system. Two types of assays are currentlyused to demonstrate the multipotent state of stem cells. The firstis to demonstrate that one stem cell can reconstitute an

entire organ. For instance, HSCs can reconstitute an entirehaematopoietic system after transplantation into sub-lethallyirradiated animals. However, this type of assay is difficult toperform in humans since they require the destruction of thetargeted organ and also require clonal analysis which can betechnically challenging. Indeed, adult stem cells often requirea complex environment to survive and to self-renew in, and thisniche can be difficult to mimic in vitro. The second type of assayis based on lineage tracing experiments in which the stem cellof interest is genetically tagged and hence marks its progeny.Thus, using this approach, one can follow the origin of a celltype in a specific organ. The existence of gut stem cells [11] andskin stem cells [12] has been characterized using this approach.Multipotent stem cells have been identified in a broad numberof organs in rodents. However, the existence and functionalityof their human counterpart remains in some cases controversialsince the lack of appropriate technology renders their isolationand thus characterization extremely challenging.

SOMATIC STEM CELLS

Endogenous somatic stem cells

Somatic stem cells are multipotent and can usually differentiateinto all the cell types constituting their organ of origin. Somesomatic stem cells such as hepatic progenitor cells proliferatevery slowly and can be activated by specific conditions suchas stress response or injury [13]. Other somatic stem cellssuch as vascular progenitor cells are proliferating constantlyand are in high numbers [14]. They are often localized in aspecific niche that is essential to maintain their property ofself-renewal and differentiation [15]. Ultimately, their normalfunction could be to maintain a constant number of cells inan organ. Somatic stem cells have been reported to be foundin 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 forregenerative medicine since their mobilization would not requireinvasive methods or immunosuppressive treatments.

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

However, the clinical use of endogenous somatic stem cellsis currently limited for diverse reasons. Their existence remainscontroversial in several organs especially in humans where lineagetracing or reconstitution experiments are impossible. Thereforetheir identification and characterization is difficult and pastexperience has shown that results obtained in animal models mightnot be translated directly to humans. This limits the study of themechanisms controlling the self-renewal and differentiation ofendogenous stem cells, which is necessary for the developmentof future clinical treatments. Two controversial examples concernthe pancreas and liver. Using lineage tracing techniques inthe mouse, it has been demonstrated that pancreatic β-cells


Emerging use of stem cells in regenerative medicine 13

Table 2 Types of adult stem cells reportedly found

Type of adult stem cell Location found Expandable in vitro? Used in clinical trials?

HSC BM, placenta, UCB and peripheral blood Yes YesMSC BM, amniotic fluid, placenta, AT and UCB Yes YesMuscle satellite stem cell Between basement membrane and cell membrane (sarcolemma) of muscle fibre No YesNSC SVZ of lateral ventricles and SGL of dentate gyrus of the hippocampus in the brain Yes Going to startLimbal stem cell Corneoscleral limbus Yes Going to startSkin stem cell Basal layer of epidermis and base of hair follicles Yes NoGut stem cell Crypts Yes NoLung stem cell Between airways (bronchioles) and alveoli No NoCardiac stem cell Heart No NoLiver stem cell Canals of Hering at the terminal bile ductules Yes NoPancreatic stem cell Pancreatic ducts No No

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 exvivo 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 (?).

‘regenerate’ via self-replication of existing β-cells and not fromstem cells [42,43]. However, recent reports have contradictedthese observations by showing that β-cells can be made denovo from islet progenitor cells expressing Ngn3 and residingin the pancreatic duct [44]. Nevertheless, most of these resultswere obtained in animal models of injury in which the pancreasis induced to regenerate after duct ligation, and recent studieshave not been able to repeat these experiments [45]. Thereforeit remains to be demonstrated that this model is physiologicallyrelevant and that a pool of pancreatic stem cells can be identifiedin normal conditions, especially in humans.

Hepatic oval cells/hepatic progenitors represent anothercontroversial adult stem cell [46,47]. These liver-specific stemcells originally identified in rodents could be used to makehepatocytes for cell-based therapy of liver failure and livermetabolic diseases [48–50]. They are bipotent (i.e. they arecapable to differentiate into duct cells or hepatocytes) as shownby 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 nuclearfactors) which mark hepatic progenitors during embryonic liverdevelopment [51]. In humans, they are localized in the canals ofHering at the terminal bile ductules [52] and are reportedly activ-ated in disease conditions [53,54]. However, they are extremelyrare (if in existence) and possibly inaccessible [52]. There havebeen reports of the isolation of hepatic progenitor cells from theadult human liver [55–57]. However, the lack of definitive markersand the dissimilarity between human and rodents renders theidentification of a common hepatic progenitor cell difficult [58].

These two examples illustrate the difficulty of translatingobservations obtained in animal models directly to humans [59–61]. Indeed, stem cells in distant species might have differentfunctions and activity [62,63]. Another major drawback isthat endogenous somatic stem cells could be targeted by thediseases, explaining why endogenous somatic stem cells appearto be incapable or insufficient in stopping the progression of



degenerative diseases. Finally, the use of endogenous stemcells could be precluded in genetic diseases whereby stem cellscontinue to produce progeny carrying the same phenotype.Consequently, genetic correction will be necessary, implicatingthe use of gene therapy approaches or complex manipulationsin vitro. In conclusion, mobilization of endogenous somatic stemcells represents the ultimate objective of regenerative medicine.This approach is the most ideal, ‘simple’ and natural as weare simply prompting and activating our innate body system torepair diseased tissues. There would not be complicated problemssuch as immunological response to exogenous foreign cells orethical issues related to ESCs. However, their clinical use willrequire better identification and characterization to develop newapproaches allowing their mobilization in vivo, especially in adiseased environment.

Exogenous somatic stem cells

Exogenous somatic stem cells are derived in vitro from diverseorgans. They have a high capacity of proliferation and can bemaintained in vitro for a prolonged period of time. Exogenoussomatic stem cells are commonly recognized to exhibit lineage-restricted multipotency. However, several reports have suggestedthat somatic stem cells could be pluripotent since they candifferentiate into a broad number of cell types unrelated totheir organ of origin [64]. In addition, controversial reportshave suggested that these cells could participate in germ layerformation [64]. However, these results have been difficult toreplicate and the current consensus is that adult somatic stemcells have a restricted capacity of differentiation compared withthose of ESCs. Furthermore, exogenous somatic stem cells oftenrepresent an in vitro state which has no equivalence in vivo.However, their use in regenerative medicine is promising sincethey are easy to isolate and can be expanded in vitro. In addition,immunosuppressive treatments are not required due to autologoustransplantations, 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 beingused in several clinical trials.

NSCs are progenitor cells located in the SVZ (subventricularzone) of the lateral ventricles or SGL (subgranular layer) of thedentate gyrus of the hippocampus in the brain. NSCs have beencultured in vitro as cell clumps known as ‘neurospheres’ [69,70].The neurospheres can be passaged several times, demonstratingtheir capacity of self-renewal. They are also multipotent sincethey can differentiate into neurons and glia [71,72]. ThereforeNSCs 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 treatmentof 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 transplantmyc-immortalized hNSCs (human NSCs) [76] derived fromhuman foetal tissue into stroke patients. Therefore the first clinicalapplications of NSCs have started. However, several challengeshave to be solved before larger trials could start, including betterisolation and expansion methods for NSCs, as well as preventionof tumour formation after transplantation [77].

MSCs are fibroblast-like cells which can be isolated fromdiverse sources including BM (bone marrow) [78], amnioticfluid [79], placenta [80,81], AT (adipose tissue) [82,83] andUCB (umbilical cord blood) [84]. MSCs are mainly identifiedby 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 vitrois limited as they are not pluripotent. Besides MSCs, severalrelated types of cells including MAPCs were isolated from theBM [9,10]. MSCs are multipotent [87] and have been reportedto be able to differentiate into osteoblasts, adipocytes [88],chondrocytes [89,90], and epithelial [91], endothelial [92] andneural cells [93]. It should be noted that MSCs derived fromdifferent tissues may exhibit slightly different propensities andcapacities to differentiate into specific cell types. For example,MSCs from synovial tissue may be more effective in repairingcartilage defects than MSCs from other tissues [94]. MSCs self-renew in vitro but not indefinitely since they do senesce after20 population doublings and ultimately lose their potential todifferentiate [95]. More importantly, they do tend to generateabnormal 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 applicationsresides less in their capacity of differentiation than in theirparacrine actions on tissue repair [97]. Indeed, MSCs expresslow levels of MHC class II molecules and thus should be weaklyimmunogenic, suggesting that immunosuppressive treatmentmight not be required. In addition, they have been shown to beable to modulate the immune response of cells in vitro and tolimit inflammation [98]. Hence they could be used for preventinggraft rejections and GvHD (graft versus host disease). MSCswere first used in clinical trials as a complementary treatment forbreast cancer patients [99]. Thereafter they were demonstratedfurther to be beneficial against GvHD [100]. On the basis ofthese successes, a broad number of studies are currently beingperformed 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 havebeen used in controlled clinical trials [104]. Importantly, MSCs donot repopulate damaged tissues and cells quickly disappear afterengraftment in vivo. Despite this, small but positive effects oncardiac performance have been observed, and this clinical benefitcould be achieved through immune modulation and increasedangiogenesis.

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] andAT-MSCs [108] can also be differentiated into hepatocyte-likecells. hMSC (human MSC)-derived hepatocytes have recentlybeen reported to be able to restore liver function [109]. MSCs arenow being used in clinical trials for end-stage liver disease [110]and initial results appear promising. However, long-term recoveryof liver function remains to be demonstrated and further studieswill be required to understand the mechanisms by which MSCscan restore liver function. In summary, the mechanisms by whichMSCs can achieve their immunomodulatory and detoxificationfunctions, as well as paracrine secretions, remain to be defined.Further basic studies will be extremely helpful in validating theirreal function in vivo and the mechanisms by which they canalleviate so many symptoms. However, their interest for clinicalapplication cannot be denied and the broad number of clinicaltrials which are currently performed with MSCs should rapidlyprovide some information regarding their real efficacy and long-term effect.

The examples described above illustrate the advantagesof somatic stem cells for clinical applications. These cellsare relatively easy to isolate and they do not requireimmunosuppressive treatment. In addition, they can be used totreat a broad number of diseases without major risks. All ofthese aspects greatly simplify their use in a clinical context



and thus make them attractive for clinical trials. However, anincreasing amount of data are suggesting that even a shortperiod of time in culture in vitro can modify the genetic andepigenetic characteristics of somatic stem cells. The consequencesof these modifications on cell proliferation and differentiationafter transplantation and after a prolonged period of timein vivo will have to be examined carefully. Finally, the mechanismsinvolving somatic stem cells in tissue repair and/or in modulatingimmune response are not always fully understood and additionalbasic studies are required to fully define the clinical potential ofthese different cell types.

EMBRYONIC STEM CELLS

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

Difficulty to generate fully functional cell types from hESCs

A major focus of the past 10 years has been the developmentof robust protocols to differentiate hESCs into homogenouspopulations of cell types with a clinical interest. Consequently,a broad number of protocols have been developed to generatediverse cell types, including retina cells [112,113], dopaminergicneurons [114], motor neurons [115,116], OPCs (oligodendrocyteprogenitor cells) [117], cardiomyocytes [118,119], pancreatic β-cells [120] and hepatocytes [121–124] (Figure 2 and Figure 3).These methods mark a clear progress towards the generation offully functional cell types. They have benefited from the lessonslearnt from mESCs as well as from the knowledge developedby developmental biology studies. Indeed, past experiences haveshown that following a natural path of development in vitroremains the best way to generate functional cell types frompluripotent stem cells of embryonic origin. Therefore most of theprotocols developed to drive differentiation of hESCs respect keystages of embryonic development. However, this methodologyalso implies that generation of fully mature and functionalcells from hESCs will require an extended period of time ofin vitro culture, mimicking a complex environment constantlychanging over time. Therefore such an approach is presentedwith unprecendented challenges which would require a strongcollaboration between stem cell biologists and developmentalbiologists. Generation of pancreatic β-cells is a good exampleto illustrate the progress and challenges regarding the generationof clinically relevant cells from hESCs.

The Edmonton Protocol has proven that islet transplantation viathe hepatic portal vein can correct insulin deficiency in diabeticpatients [125]. However, the lack of cadaveric donors severelylimits the number of patients that could benefit from this therapy.Generating pancreatic cells from hESCs represent a uniqueopportunity to bypass this limitation. However, the resultingβ-cells would have to synthesize and store insulin, and alsomaintain blood glucose in a physiological range when challengedwith sporadic food intake. Past experiences [126,127] have

demonstrated that strict adherence to the pancreas developmentalprocess is essential for the production of pancreatic cells fromESCs [2,128,129]. Accordingly, a robust method of differentiationshould respect four major stages: (i) formation of DE (definitiveendoderm) [130,131], (ii) formation of PE (pancreatic endoderm),(iii) formation of endocrine progenitors, and (iv) maturation ofendocrine progenitors into fully functional hormone-secretingcells. Several methods are now available to efficiently generateDE cells from hESCs and to further differentiate the resultingcells into pancreatic progenitors. However, the specification ofthese progenitors toward the endocrine pathway as well as theproduction of fully mature β-cells remains a challenge.

Indeed, the most advanced protocol for the differentiationof hESCs into pancreatic β-cells allows the production ofmulti-hormonal immature pancreatic endocrine cells which lackNkx6.1 and MafA co-expression. Despite this major limitation,the pancreatic progenitors generated using this approach candifferentiate into fully functional pancreatic cells in vivo aftertransplantation into immuno-deficient mice and the resultingβ-cells can re-establish euglycaemia in a mouse model ofdiabetes [120]. These results are extremely encouraging sincethey provide the first demonstration that stem-cell-based therapycould be used to treat diabetes. However, partially differentiatedpancreatic progenitors can easily be contaminated by pluripotentcells which further increase the risk of teratoma formationassociated with hESCs (see below). In addition, non-chemicallydefined culture conditions containing FBS (foetal bovine serum)are still used, increasing the difficulty for such approaches tobe approved for clinical trials. Finally, any cell-based therapyapproach will have to address specific problems associated withthe auto-immune disease involved in T1D. Therefore the resultsobtained with hESCs are very promising and they demonstratethe potential of pluripotent stem cells to develop new treatmentfor diabetes. However, more efforts are still required to generatefully functional β-cells from human pluripotent stem cells and todemonstrate that pancreatic progenitors can be used safely in aclinical context.

Importantly, the challenges described above for pancreaticcells are shared with many other cell types. For example, HSCsgenerated 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 hepatocytesappear to have an embryonic identity since they have limitedcapacity to colonize the adult liver and they usually display limitedfunctionality when compared with their in vivo counterparts [121–124]. Therefore developing robust protocols of differentiationremains a major challenge to deliver the clinical promises ofhESCs. However, this issue can be solved by directly using theprogenitors already available as shown with pancreatic cells (seeabove) and/or by increasing our understanding of the mechanismscontrolling organ maturation. This last approach is currently usedsuccessfully for a large number of cell types and especially withneural cells.

Indeed, protocols currently available to generate functionalneurons, retina cells or OPCs have strongly benefited fromknowledge generated by developmental biologists concerningneural tube patterning and neuronal specification [135,136].Therefore these protocols generally respect key stages ofdevelopment and require extended periods of time of in vitroculture (between 45 days and up to 3 months) to generatefunctional cell types. In addition, the availability of animalmodels for specific neurodegenerative diseases and the existenceof robust in vitro functional tests allowed validation of thecells generated. For example, transplantation of ESC-deriveddopaminergic neurons appeared to restore motor function in



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 bedifferentiated into clinically useful cell types, such as neural cells, retinal cells, cardiomyocytes, hepatocytes and pancreatic β-cells, and be used for cell replacement therapy.

animal models of PD [114], and studies have shown thathESCs can be differentiated into retinal pigment cells whichcan restore retinal structure and function in animal models ofretinal degeneration [137]. Finally, OPCs generated from hESCshave been transplanted into animal models of spinal cord injuryand the transplanted cells appear not only to recolonize thescarred area, but also to promote the recruitment of endogenousstem cells, which ultimately results in restoration of mobility[117,138]. These exciting preliminary findings led to the firsthESC-based clinical trial for treatment of spinal cord injuries byGeron Corporation (Menlo Park, CA, U.S.A.). This clinical trial,despite being put on hold at the moment, represents an importantstep towards clinical applications of human pluripotent stem cellsand the outcome will bring essential information concerningthe feasibility of stem-cell-based therapy for a large number ofdiseases.

Safety issue with hESCs

Safety issues continue to be the main limitation for the useof hESCs in clinics, and teratoma formation represents oneof the most difficult challenges (Figure 4). Indeed, hESCs arenaturally tumourous by definition. hESC lines are consideredto be pluripotent based on the capacity to form teratomasonce injected into an immunodeficient host. Therefore thereis a risk of tumour formation if the transplanted cells arecontaminated by residual pluripotent stem cells. Importantly,several reports have shown that this risk is increased whenearly progenitors are used [120,139] and that methods producingheterogeneous populations of cells favour contamination byundifferentiated cells. Hence teratoma formation can easily beavoided using robust methods of differentiation allowing thegeneration of a homogenous population of fully differentiatedcells. A second cause of teratoma formation could be more

problematic. Differentiated cells generated from ESCs could havethe capacity to dedifferentiate into pluripotent stem cells whengrown in a specific environment in vivo. However, there is noevidence that fully differentiated cells generated from hESCs canbe reprogrammed in vivo or in vitro without overexpression ofpluripotency factors. Systematic studies regarding the occurrenceof teratoma formation after transplantation of hESC-derivedsomatic cells could be necessary to completely rule out such ahypothesis.

Graft overgrowth represents another issue associated withprogenitors generated from hESCs. Indeed, the injection of earlyprogenitors in the adult environment can cause uncontrolledproliferation of these progenitors, resulting in a tumour-likestructure incompatible with the function of the transplantedorgan. For example, a recent report has described neuronalprogenitors generated from hESCs to continue proliferating aftertransplantation in the brain, leading to abnormal growth [140].This issue illustrates once again the importance of generatingfully differentiated cell types from hESCs. The issue of tumourformation associated with hESCs is also heightened by theirrelative karyotypic instability [141]. Indeed, hESCs are proneto acquire chromosomal translocations when grown over aprolonged period of time in culture. This is a common featurewith any primary cell culture [142], which can be solved bykaryotyping hESCs regularly and by maintaining robust cultureconditions [143]. However, the most robust method currentlyavailable to expand hESCs involves co-culture with feeder cellsand serum, which are not always fully compatible with clinicalapplications.

To conclude, the risk of tumour formation by hESCs remainsan issue, but it can be easily controlled by using methods ofdifferentiation allowing the generation of homogenous populationof fully differentiated cells. Importantly, technical solutionscan be used to guarantee the absence of pluripotent stem



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 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 replacementtherapy or for drug testing. FDA, Federal Drug Administration.



cells in transplanted cells. Methods of purification allowingtransplantation of homogenous populations of differentiatedcells are currently available and are developed by severallaboratories [144–147]. This can be achieved with FACS usingspecific cell-surface markers for pluripotent stem cells and/or fordifferentiated cells. hESCs can also be engineered to contain aselection gene which can be used to avoid the presence of anyresidual undifferentiated cells [148]. Finally, encapsulation ofdifferentiated cells when compatible with their functionality canalso be an advantageous solution. Therefore there are multiplesolutions 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 reprogrammingsomatic cells using either SCNT (somatic cell nuclear transfer)or overexpression of pluripotency factors. However, SCNThas never been performed successfully in humans and thedifficulty in obtaining human oocytes remains a major limitation.The recent possibility to generate hiPSCs from somatic cellsby overexpressing pluripotency factors such as Oct4, Sox2,Klf4 and c-Myc represents a more immediate opportunity forregenerative medicine. Indeed, this approach can be used togenerate hiPSCs from a large number of patients of differentage and gender [149,150]. In addition, a broad number ofsomatic cells can be reprogrammed by the four factors approach,including skin fibroblasts [151], keratinocytes [152], NSCs[153], B-lymphocytes [154], pancreatic β-cells [155], hepatocytesand intestinal cells [156]. Therefore hiPSCs could enablethe production of patient-specific cell types which are fullyimmune-compatible with the original donor, thereby avoiding theuse of immunosuppressive treatment during cell-based therapy(Figure 2). In addition, hiPSCs can be generated from somaticcells isolated from patients suffering from diverse diseases. Then,the resulting ‘diseased’ hiPSCs can be differentiated into the celltype targeted by the disease and thus provide in vitro models toperform large-scale studies impossible with primary cell culture orwith biopsy material. Two recent studies have shown that hiPSCscan 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 displayphenotype characteristic of cells from patients with SMA or FD.Therefore these culture systems provide, for the first time, anin vitro model to study these complex diseases and also to screenpharmaceutical compounds in vitro using relevant tissues. HencehiPSCs represent a unique opportunity to develop new treatmentsfor a large number of diseases which are lacking relevant in vitromodels (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 thefactors necessary to achieve epigenetic reprogramming. Thereforethe resulting hiPSCs contain multiple copies of each transgenein their genome and these insertions can provoke mutationsin key genes, such as oncogenes or cell-cycle regulators. Thisphenomenon has been well described in gene therapy clinicaltrials for SCID (severe combined immunodeficiency) [157].Indeed, several patients developed leukaemia after insertion ofthe lentiviruses in proto-oncogene LMO2 (LIM domain only2) [158,159]. Therefore the use of viruses to generate hiPSCsrepresents a major limitation for their in vivo use. A solutioncould be to sequence the entire genome of each hiPSC to select

lines that only integrate viruses in regions of the genome devoid ofgenes. Alternatively, a large number of groups are now developingnon-integrative methods to generate hiPSCs, including the useof transitory transfection of episomal viruses [160–162]. Thedevelopment of small molecules represents the ultimate solutionto the problems associated with genetic modifications and severalreports have shown that c-Myc and Sox2 could be replacedrespectively, by molecules such as the histone modifier inhibitorBIX-01294 [163] and the TGFβ receptor inhibitor SB431542[164]. However, all of these methods have been used on a limitednumber of somatic cells of embryonic origin and more work isrequired to demonstrate that they can be used with a broad numberof adult patients.

Generation of hiPSCs also require the use of c-Myc and/or theinhibition of the p53 pathway, both of which present safety issues.Indeed, studies in the mouse have shown that reactivation of theexogenous c-Myc transgene can induce tumour formation [165].Recent reports have also shown that inhibition of the p53 pathwaystrongly increases the efficiency of epigenetic reprogramming andthus suggests that the main pathway protecting cells against DNAdamage needs to be turned off to allow the generation of hiPSCs[166–171]. The consequences on the genomic integrity of hiPSCsremain to be demonstrated, but inhibition of p53 could increasethe karyotypic instability already observed with hESCs. Finally,the pluripotent status of hiPSCs confers upon them the capacityto form teratomas in vivo and this safety issue, which is sharedwith hESCs, could be aggravated in hiPSCs. Indeed, a recentreport has shown that the capacity of iPSCs to form teratomascan vary in function of their origin [172]. This phenomenon couldbe explained by the persistence of undifferentiated cells afterdifferentiation, suggesting that some iPSC lines are more difficultto differentiate than others. Alternatively, differentiated cellsgenerated from iPSCs could re-acquire a pluripotent status aftertransplantation in vivo. Therefore further studies are necessary tofully demonstrate that differentiated cells generated from iPSCsare epigenetically stable over a prolonged period of time in vitroand in vivo.

Generation of fully differentiated cells

hiPSCs and hESCs share many characteristics, including theircapacity of differentiation, the expression of pluripotency markersand their dependence on activin/nodal signalling to maintaintheir pluripotent status [173]. These functional similarities alsoimply 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 ingenerating fully functional cells. Moreover, incomplete epigeneticreprogramming, epigenetic memory, transgene insertion andgenetic instability could worsen the variability between hiPSCsand thus render its use almost impossible for personalized therapy.Indeed, each hiPSC line could require the development of aspecific method of differentiation. Consequently, further studiesare needed to definitively demonstrate that hiPSCs generated froma broad number of patients can all be efficiently differentiatedinto one particular cell type using a unique protocol. In addition,the absence of robust protocols to generate fully differentiatedcells from hiPSCs is currently limiting the development ofin vitro models of disease. Indeed, the cells generated fromdiseased hiPSCs display features of embryonic progenitors whichcannot be used to mimic disease only occurring in the adult[174]. Therefore much more effort will be required to deliverthe clinical promises of hiPSCs. However, this major objectivecould be achieved very rapidly since hiPSCs have become themain focus of many laboratories and they will plainly benefit



from the experience accumulated over the past 10 years withhESCs.

CONCLUSIONS AND PERSPECTIVES

The increasing types of stem cells available represent a uniqueopportunity for regenerative medicine as they will offer newtreatment for a broad number of diseases which cannot be fullycured by drug-based therapy. Importantly, stem cells are alreadyreaching the clinic as shown by the large number of clinical trialsusing diverse types of stem cells. However, stem cells presentnew challenges that will need to be solved to fully realise theirclinical promises. The broad diversity of stem cells available isnow offering a large choice for clinicians and a universal stemcell for ‘all the diseases’ is unlikely to exist. Therefore, the‘right’ stem cell will have to be used for the ‘right’ disease. Inaddition, safety could block or delay a large number of clinicalapplications. Indeed, the necessity to demonstrate that a stemcell is not harmful represents a necessary hurdle, especially withpluripotent stem cells capable of forming teratomas. Thereforeregulatory agencies will have to define new criteria to evaluatethe risk associated with specific stem cells and their differentiatedprogeny. This process is often delayed by the difficulty to definewhat is an acceptable risk/benefit with stem-cell-based therapy. Isteratoma an acceptable risk compared with a terminal disease? Inaddition, novel animal models are urgently needed to reinforcethe work currently performed in rodents. Indeed, the mousepresents clear advantages but also important limitations. Theirsize is not relevant for human treatment and mouse modelsrarely reproduce, in full, the human disease. Finally, monitoringfunctionality of human cells in a fully immunosuppressed xeno-environment present obvious issues. Therefore the use of largeanimals or non-human primates will be necessary to validate thefunctionality and safety of the cells generated from hESCs/hiPSCsand also other stem cells. To conclude, clinical applications ofstem cells are becoming a reality and future applications willcontinue to require a strong collaboration between basic scientists,clinicians, regulatory agencies and, more importantly, patients,whose support is essential.

ACKNOWLEDGEMENTS

We thank Dr Tamir Rashid for helpful discussions, Ms Stephanie Brown for proof-readingprior 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 ResearchCouncil (U.K.).

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Received 15 January 2010/4 March 2010; accepted 9 March 2010Published on the Internet 28 April 2010, doi:10.1042/BJ20100102


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