overcoming clinical hurdles for autologous pluripotent ... · infertility treatments. however,...

Tran

slat

iona

l Res

earc

h of 8

For citation purposes: Byrne JA. Resolving clinical hurdles for autologous pluripotent stem cell-based therapies. OA Stem Cells 2013 Sep 01;1(1):3.

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.

Critical Review

AbstractIntroductionHuman induced pluripotent stem cells can now be derived by using factor-mediated reprogramming to revert an individual’s somatic cells, such as skin cells, back into a pluripo-tent epigenetic state. These autolo-gous cells hold immense potential for cell-based therapeutics. Not only are they capable of forming any cell type in the human body, but they are also immunologically matched to the patient. Such cells thus have a variety of possible applications in the treat-ment of a wide range of diseases and injuries, including those associated with age-related tissue degeneration. Autologous cells also hold great promise for use in other areas, including in vitro research, develop-mental biology, drug screening and potential future reproductive appli-cations, such as the development of infertility treatments. However, there are five obstacles which thus far have prevented the safe clinical applica-tion of personalised human induced pluripotent stem cell therapeutic derivatives. These are (1) the high levels of genetic instability observed in human induced pluripotent stem cells;(2) reports of aberrant epige-netic reprogramming or epigenetic memory or both;(3) post-transplan-tation issues surrounding efficacy of the transplanted cells, including low levels of survival, functionality,

Overcoming clinical hurdles for autologous pluripotent stem cell-based therapies

JA Byrne*

reports that human induced pluripo-tent stem cell derivatives are foetal in nature, and reports of immuno-genicity gene expression or immune rejection (or both) in perfectly matched hosts;(4) issues surrounding post-transplantation safety, including an increased risk of neoplasms from transplanted cells; and (5) issues surrounding the feasibility of human induced pluripotent stem cell-based therapeutics, including the time, cost and regulatory hurdles and the diffi-culties associated with consistently generating a therapeutic cellular product on an individualised basis. This review conducts a detailed examination of these hurdles and evaluates the feasibility of various possible solutions.ConclusionInduced pluripotent stem cells possess numerous possible applica-tions, such as cell-based therapeutics for the treatment of a wide range of diseases, injuries and age-related tissue degeneration, in addition to in vitro research, developmental biology, drug screening and potential future reproductive applications, such as possible treatments for infer-tility. In this review, we have exam-ined the five hurdles to be overcome in safely moving towards personal-ised human induced pluripotent stem cell-based the apeutics.

IntroductionJohn Gurdon first discovered that a differentiated somatic cell could be reprogrammed back into a pluripo-tent state capable of generating an entirely new organism, in this case the African clawed frog Xenopus laevis1,2. However, this somatic cell nuclear transfer (SCNT) procedure

proved extremely inefficient; over 700 nuclear transfers were performed and only approximately 1% directly resulted in swimming tadpoles1. Despite these difficulties, this repre-sented the first demonstration of a differentiated cell’s remarkable ability to revert backwards in devel-opment when directed by exogenous factors, such as (in this case) the enucleated frog egg. The scientific concept that vertebrate oocytes contain factors capable of reprogram-ming somatic cell nuclei into a pluri-potent epigenetic state was then extended from amphibians to mammals in 1997, when the first mammal cloned from an adult somatic cell was born3.

This was followed by the first derivation of pluripotent stem cells (PSCs) from oocyte-reprogrammed murine somatic cells in 20004, non-human primate somatic cells in 20075 and human somatic cells in 20136. Notwithstanding this success, the primary disadvantage of the SCNT reprogramming approach is the significantly limited supply of human oocytes for SCNT-based research because of practical, legal and ethical constraints. In 2006, Shinya Yamanaka and colleagues discovered that mouse and, subse-quently, human somatic cells could be directly induced into a pluripo-tent epigenetic state with the use of exogenously delivered reprogram-ming factors7,8, which generated ‘induced pluripotent stem cells’ (iPSCs). This discovery represented a fortunate development for the advancement of autologous PSC-based therapies generated by using exogenously delivered factors. It provided a feasible alternative to

*Corresponding authorEmail: [email protected]

Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, 615 Charles E. Young Drive South, UCLA, Los Angeles, CA 90095, USA

mailto:[email protected]

Figure 1: Scientific concept behind human induced pluripotent stem cell(hiPSC)-based therapeutics. The long-term goal of the hiPSC-based therapeutic field is to use hiPSCs and their derivatives to develop personalised cellular therapies for treating diseases, injuries, age-related tissue degeneration and infertility. The underlying concept is that a patient’s own skin cells could be epigenetically reprogrammed back into pluripotent stem cells. Typically, a minimally invasive skin punch biopsy is obtained from the patient and provides a reliable source of autologous cells with extensive proliferation potential (left image). These primary dermal fibroblasts then are reprogrammed into hiPSCs (bottom image), which can be differentiated into any therapeutic cell type (right image) and then autologously transplanted back into the original individual, without the risk of immune rejection.

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

oocyte-based reprogramming, thus circumventing the practical, ethical and legal constraints associated with it.

Perhaps the most impressive demonstration of the pluripotent capacity of iPSCs was observed through a specific embryological process, referred to as tetraploid complementation. In this process, entire fertile ‘cloned’ mice could be derived from iPSCs9. Although only a

small number of iPSC lines demon-strated this capacity10, it was suffi-cient to establish that repro-grammed cell lines possessed the capacity to generate all of the func-tional tissues of an adult organism, thus highlighting their potential for multiple applications in the future (Figure 1). The aim of this review is to discuss overcoming clinical hurdles for personalized hiPSC-based therapeutics.

DiscussionThe author has referenced some of his own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.

Potential uses of human induced pluripotent stem cells(hiPSCs)hiPSCs can be used for both in vitro and in vivo applications. In vitro appli-cations include a wide range of possible in vitro research areas, such as ageing research, developmental biology, drug screening, toxicology and disease modelling; for reviews, please read11,12. In vivo applications include the promising use of hiPSC derivatives for the treatment of diseases and injuries. However, in vivo applications also include poten-tially more controversial applica-tions. One such example is the possible use of hiPSCs and their derivatives to rejuvenate human tissues and organs after age-related tissue degeneration, thereby increasing the quality and length of human life13. Moreover, hiPSCs and their derivatives may be used for reproductive applications, such as treating infertility. Such infertility treatments could be achieved through the generation of germ cells14 with or without gene correction (genetic manipulations that would pass to all subsequent generations) or possibly through the generation of ‘cloned’ individuals through embryological manipulations like tetraploid comple-mentation9, the profound bioethical considerations of which are beyond the scope of this article.

Advantages of hiPSC derivatives for autologous cellular therapiesThe advantages of hiPSC derivatives for autologous cellular therapies are the following: (1) iPSCs are pluripo-tent and thus are theoretically capable of forming any therapeuti-cally useful cell type;(2) iPSCs possess unlimited proliferation capacity (i.e.,

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

they are immortal), facilitating genetic manipulation, such as is necessitated in the case of patients with genetic disorders;(3) iPSC deri-vation does not involve the destruc-tion of human embryos and therefore is not subject to the same ethical considerations as embryonic stem cell (ESC)-based research; and (4) iPSCs can be derived from a patient’s own somatic cells (Figure 1), placing iPSC derivatives at a reduced risk of immune rejection and eliminating the cost, inconvenience and negative health consequences associated with long-term immuno suppression15. The capacity for murine iPSC deriva-tives to restore physiological function has already been established in mouse models of heart disease16, sickle cell anaemia17, Parkinson’s disease18 and haemophilia A19, providing pre-clinical data in support of the underlying promise of these cells. However, despite the obvious advantages and successes of previous animal studies, a number of remaining hurdles must be overcome before the promise of hiPSCs for regenerative medicine can be safely realised.

Current hurdles in advancing personalised pluripotent stem cells (PSCs)There are five hurdles to be overcome in safely advancing PSCs derivatives into personalised human therapeu-tics. These hurdles are (1) high levels of genetic instability, (2) aberrant epigenetic reprogramming, (3) issues surrounding post-transplantation efficacy, (4) issues surrounding post-transplantation safety, and (5) issues surrounding feasibility.

High levels of genetic instabilityRecent studies have demonstrated a predisposition to genetic instability within in vitro cultured PSCs20,21. Specifically, both human ESCs and hiPSCs demonstrate inherent genetic instability during in vitro culture22–26. This is a concern for the therapeutic use of these cells, as the frequently

observed genetic (karyotypic) abnor-malities in human PSCs are charac-teristic of malignant tumours27,28. Moreover, even differentiated astro-cytes, derived from karyotypically abnormal human PSCs, have demon-strated characteristics analogous to malignant tumours29. One extremely common genetic aberration is the duplication of chromosome 12. This chromosomal duplication is often associated with a proliferative growth advantage and potential tumourigen-esis30, rendering these cells unaccep-table for personalised cellular thera-peutics. One potential solution to this genomic instability obstacle is the optimisation of culture conditions to promote genomic stability. The candi-date supplements required for addi-tion to the culture media will be dependent upon the underlying causes of the genomic stability. It is thus necessary to determine whether the instability is caused by oxidative or replicative stress or both, with chemicals such as antioxidants and culture under lower oxygen levels, offering potential solutions for the ‘oxidative stress/genomic instability hypothesis’, and exogenous nucleo-side supplementation, offering solu-tions for the ‘replicative stress/genomic instability hypothesis’ (Caius Radu, personal communica-tion). Regardless of the success of these and other culture condition optimisation approaches for the promotion of genomic stability in hiPSCs and their derivatives, it would be prudent to screen each and every iPSC clone for genetic aberrations31p-rior to any therapeutic applications.

Aberrant epigenetic reprogrammingStudies comparing the DNA methyl-omes of mouse and human iPSCs with their respective species-specific ESCs revealed that many iPSC lines retain aberrant iPSC-specific differential methylation patterns. This phenom-enon is referred to as ‘epigenetic memory’32,33. The epigenetic memory of iPSCs was observed to impair the

differentiation capacity of the iPSCs, with iPSCs demonstrating a reduced capacity to differentiate into cells from lineages different from that of the donor cell type32,33. There is signif-icant evidence to suggest that most, if not all, iPSC lines can gradually resolve some, if not most, of their transcriptional and epigenetic differ-ences with ESCs over time via increased passaging34. However, it has also been observed that a subset of iPSC lines continue to retain epige-netic memory, even after extended passaging33. A recent study discov-ered that all of the examined iPSC lines (9 out of 9) demonstrated incomplete hydroxy methylation levels in multiple subtelomeric regions when compared with control human ESC lines (4 out of 4)35. It has yet to be established how significant a hurdle this residual epigenetic memory and aberrant epigenetic reprogramming will prove to be for future autologous cellular therapeu-tics. However, it may be prudent to err on the side of caution and continue to investigate novel approaches to augment the epigenetic reprogram-ming process, including use of chro-matin-modifying chemicals, such as sodium butyrate36, 5-azacyti-dine37and valproic acid38. These represent chromatin-modifying chemicals which have been previ-ously used to (1) augment epigenetic reprogramming to pluripotency;(2) increase the inclusion of factors that promote developmental competence, such as Glis139, L-Myc40 and Tbx341;and (3) test the ‘CORF-augmented reprogramming hypoth-esis’. This hypothesis proposes that the addition of putative candidate oocyte reprogramming factors (CORFs), such as ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO, KDM6B42 and/or others, may be capable of opening up the somatic heterochromatin (including subtelo-meric regions) and increase access for epigenetic reprogramming factors, such as the demethylation

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

enzyme TET3 (which is present in the oocyte)43. This, in turn, may augment the nuclear reprogramming process.

Issues surrounding post-transplanta-tion efficacyPost-transplantation efficacy is deter-mined by (1)an efficient and reliable in vitro differentiation protocol into the target therapeutic cell type;(2)sufficient post-transplantation inte-gration, maturation and survival; and (3)maintained functionality to induce a therapeutically detectable effect. Efficient and reliable differentiation protocols have been developed for motor neurons44, dopaminergic neurons45, cardiomyocytes46, hemat-opoietic precursors47 and hepato-cytes48. However, recent research by William Lowry and colleagues strongly suggests that iPSC and ESC derivatives are foetal in nature49. The ‘in vivo development recapitulation/maturation hypothesis’ proposes that the differentiation of iPSCs, into mature adult cells, can be obtained through in vitro recapitulation of in vivo developmental processes. Indeed, the derivation of every func-tional tissue in adult ‘all-iPSC’ mice serves to highlight the remarkable developmental capacity of certain, albeit rare, mammalian iPSC lines under optimal (in vivo) differentia-tion conditions9. One possible solu-tion to the low levels of survival, inte-gration and maturation of cells post-transplantation is the ‘cell delivery optimisation/pro-survival hypothesis’. This hypothesis proposes that cell delivery approaches could be optimised with pro-survival factors, such as anti-inflammatory and free radical scavengers, tissue engineering strategies, co-transplantation with carrier cells, preconditioning the cells (for example, heat-shock) genetic modification to enhance engraftment and survival or a combination of these factors50,51.

Of particular concern for future proposed iPSC-based patient-specific cellular therapeutics is that multiple

independent groups have observed what appears to be an above baseline immune response after transplanta-tion of iPSCs, or iPSC-derivatives, into perfectly matched (autologous/syngeneic) hosts52–54. It remains unclear whether this immune rejec-tion is (1) associated with expression of immunogenicity genes, such as HORMAD1 or ZG16 or both54, (2) linked to low developmental compe-tency52, or (3) linked primarily to residual transgene expression53,54. In any event, the immunogenicity issue represents an important problem that needs to be resolved, ideally in the human system, in order to ensure that transplanted hiPSC derivatives are not rejected by the recipient’s immune system.

Issues surrounding post-transplanta-tion safetyThe key post-transplantation safety concern is that hiPSC derivatives will give rise to neoplasms, particularly teratomas and teratocarcinomas55. In addition to the safety concernstradi-tionally associated with teratomas, there is the potential that other types of embryonal neoplasms, relatingto specific tissue lineages, may arise after engraftment55. These ‘mono-dermal’ teratomas have been observed after the transplantation of human PSC derivatives in rodents56, thus highlighting the post-transplan-tation safety concerns. In addition to the genomic instability issues previ-ously discussed in the context of post-transplantational safety concerns, there are three, potentially comple-mentary, putative sources of post-iPSC transplantation teratomas: (1) contaminating undifferentiated cells, (2) transgenic expression of poten-tially oncogenic reprogramming factors, and (3)potentially on cogenic insertional mutagenesis. Contami-nating PSCs (as few as two cells) can generate teratomas57, thus demon-strating the need to generate extremely pure populations of cells for therapeutic applications. This

could theoretically be achieved by using positive selection to identify and purify the target cell58 or negative selection to identify and remove undifferentiated cells59 or both. Such selections would be applied in addi-tion to the use of transgenic selection with tissue-specific promoters. This would allow fluorescent marker expression-based purification, using flow cytometry, or antibiotic resist-ance gene expression-based purifica-tion, using antibiotic selection60. However, non-genetic methods, based upon tissue-specific metabolism, might provide safer and very efficient protocols61. The concern regarding the potential for residual or reacti-vated transgene expression in the iPSC derivatives is based on the obser-vation that reprogramming factors used to generate iPSCs are also associ-ated with tumours and hyper-plasia62,63. In addition, chimeric mice generated from iPSCs containing the cMyc transgene have demonstrated a predisposition towards tumour formation64, and random integration of viruses into tumour suppressor genes may increase the risk of neoplasms. However, multiple transgene-free and integration-free approaches have now proven effec-tive in generating hiPSCs, and even if these cells are derived under research-grade conditions, they still may prove useful for therapeutic applications, after conversion to putative clinical-grade conditions65. One potential solution, which could address numerous aspects of post-transplan-tation safety, is to genetically modify iPSCs to express a suicide gene, such as her pessimplex virus 1 thymidine kinase (HSV1tk). This, remarkably, permits both positron emission tomography (PET)-based in vivo imaging (using the PET probe fluoro-3-hydroxymethyl-butyl-guanine, FHBG) of the transplanted iPSC deriv-atives66,67 and the ability to induce cell death (using the drug ganciclovir) in the event the transplanted cells result

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

in tumour formation or other negative outcomes68 amphibia to mammals.

Issues surrounding feasibilityAlthough hundreds of clinical trials are currently being performed using hematopoietic and mesenchymal stem cells, no clinical trial has yet been performed using hiPSCs. Two clinical trials have been performed using human ESCs. These trials, in particular, thus serve to highlight the difficulties facing the safe transition of human PSC-based therapeutics into clinical trials. Specifically, the phase 1 clinical study by Geron (Menlo Park, CA, USA)to use human ESC-derived oligodendrocytes to treat patients with spinal cord injury69 was discontinued for busi-ness reasons, and the long-term clin-ical benefits of the phase 1 clinical trial of Advanced Cell Technology (Marlborough, MA, USA)to treat macular degeneration70 are unknown. Both companies faced rigorous implementation of regulatory proto-cols by the Food and Drug Adminis-tration (FDA) before the trials were eventually approved, thus high-lighting the FDA’s safety concerns that contaminated PSCs could possibly generate teratomas in the recipients. The safety measures proposed herein may prove helpful in reducing some of the regulatory burden for future PSC-based clinical trials. However, other feasibility considerations, including the amount of time taken to reprogram, charac-terise and differentiate the iPSC derivatives prior to clinical applica-tion, must also be taken into account. Most reprogramming and characteri-sation studies require several months, and certain differentiation protocols can take many additional months (for example, differentiation into retinal pigmented epithelium). The extended time required to reprogram, charac-terise and differentiate a therapeutic population of cells may prove a limi-tation to the suitability of this person-alised iPSC derivative approach in

certain cell-based treatments, particularly those in which trans-plantation to the patient is required within a short time frame. The time taken to reprogram a therapeutically useful iPSC colony is prolonged by the traditionally low iPSC reprogram-ming efficiency(<0.1%). However, technological advances, such as combinations of synthetic mRNAs71, non-integrated micro RNAs72 and various small-molecule reprogram-ming enhancers73, may sufficiently augment the reprogramming process to a level at which reprogramming efficiency is no longer a concern. In addition to time considerations, high financial costs are involved in performing this multi-step repro-gramming, characterisation and differentiation approach, particularly when performed with the use of expensive, defined, good manufac-turing practice-grade media under clinical-grade conditions. These factors thus may reduce the commer-cial feasibility of the approach. These time and financial considerations are further exacerbated when genetic modification of the iPSCs is also performed, such as when performing gene correction in the case of inher-ited genetic diseases, transgenic insertion to augment cellular func-tion and/or the genetic modification of the iPSCs for in vivo tracking and suicide cell responses, as previously discussed. The overall feasibility of the iPSC-based therapeutic approach will be determined essentially by whether the technical, financial and temporal issues can be adequately resolved. With the extensive amount of research currently being conducted in the iPSC field, in addition to the ongoing scientific investigation of the mechanisms of nuclear reprogram-ming, it is plausible to consider that these feasibility issues will be adequately addressed in due course. However, when this will occur remains to be seen.

ConclusionDefined factors can reprogram differ-entiated cells back into pluripotency. The discovery of iPSCs has provided a broad range of possible applications. Such potential in vivo applications include cell-based therapeutics to treat a wide range of diseases, injuries and age-related tissue degeneration. More-over, iPSCs hold significant promise for in vitro research, developmental biology, drug screening and potential future reproductive applications, such as a possible treatment for infertility. In this review, we have examined the five hurdles to be overcome in safely moving personalised hiPSC thera-peutic derivatives into the clinic and discussed various possible solutions.

AbbreviationsCORF, candidate oocyte reprogram-ming factor; ESC, embryonic stem cell; FDA, Food and Drug Administra-tion; FHBG, fluoro-3-hydroxyme-thyl-butyl-guanine; hiPSC, humanin duced pluripotent stem cell; iPSC, induced pluripotent stem cell; PCS, pluripotent stem cell; PET, positron emission tomography; SCNT, somatic cell nuclear transfer.

AcknowledgementsThe study was performed in associa-tion with the Clinical Investigations for Dermal Mesenchymally-Obtained Derivatives (CIDMOD) Initiative, and supported by funding from the Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, the Phelps Family Foundation, Fibro cell Science, Inc. (Exton, PA, USA) and the UCLA Clinical and Translational Science Institute.

References1. Gurdon JB. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol.1962 Dec;10:622–40.2. Gurdon JB, Uehlinger V. ‘Fertile’ intes-tine nuclei. Nature. 1966 Jun;210(5042):1240–1.

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

3. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997;385(6619):810–3.4. Munsie MJ, Michalska AE, O’Brien CM, Trounson AO, Pera MF, Mountford PS. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Curr Biol.2000 Aug;10(16):989–92.5. Byrne JA, Pedersen DA, Clepper LL, Nelson M, Sanger WG, Gokhale S et al. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature. 2007 Nov;450(7169):497–502.6. Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-Hedges R, Ma H, et al. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell. 2013 Jun;153(6):1228–38.7. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov;131(5):861–72.8. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embry-onic and adult fibroblast cultures by defined factors. Cell. 2006 Aug;126(4):663–76.9. Zhao XY, Li W, Lv Z, Liu L, Tong M, Hai T, et al. iPS cells produce viable mice through tetraploid complementation. Nature. 2009 Jul;461(7260):86–90.10. Stadtfeld M, Hochedlinger K. Induced pluripotency: history, mechanisms, and applications. Genes Dev. 2010;24(20):2239–63.11. Colman A, Dreesen O. Pluripotent stem cells and disease modeling. Cell Stem Cell. 2009 Sep;5(3):244–7.12. Grskovic M, Javaherian A, Strulovici B, Daley GQ, et al. Induced pluripotent stem cells—opportunities for disease modelling and drug discovery. Nat Rev Drug Discov. 2011 Dec;10(12):915–29.13. Svendsen CN. Back to the future: how human induced pluripotent stem cells will transform regenerative medicine. Hum Mol Genet. 2013 Aug;22(R1):R32–8.14. Ishii T, Pera RA, Greely HT. Ethical and legal issues arising in research on inducing human germ cells from pluripo-tent stem cells. Cell Stem Cell. 2013 Aug;13(2):145–8.15. Filler G. Challenges in pediatric trans-plantation: the impact of chronic kidney disease and cardiovascular risk factors on long-term outcomes and recommended

management strategies. Pediatr Trans-plant.2011 Feb;15(1):25–31.16. Nelson TJ, Martinez-Fernandez A, Yamada S, Perez-Terzic C, Ikeda Y, Terzic A. Repair of acute myocardial infarction by human stemness factors induced pluri-potent stem cells. Circulation. 2009 Jul;120(5):408–16.17. Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007 Dec;318(5858):1920–3.18. Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F, et al. Neurons derived from reprogrammed fibroblasts function-ally integrate into the fetal brain and improve symptoms of rats with Parkin-son’s disease. Proc Natl Acad Sci USA. 2008 Jan;105(15):5856–61.19. Xu D, Alipio Z, Fink LM, Adcock DM, Yang J, Ward DC, et al. Phenotypic correc-tion of murine hemophilia A using an iPS cell-based therapy. Proc Natl Acad Sci USA. 2009 Dec;106(3):808–13.20. Ben-David U, Benvenisty N, Mayshar Y. Genetic instability in human induced pluripotent stem cells: classification of causes and possible safeguards. Cell Cycle. 2010 Dec;9(23):4603–4.21. Ben-David U, Mayshar Y, Benvenisty N. Large-scale analysis reveals acquisition of lineage-specific chromosomal aberra-tions in human adult stem cells. Cell Stem Cell. 2011 Aug;9(2):97–102.22. Ben-David U, Benvenisty N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer. 2011 Apr;11(4):268–77.23. Chen LW, Kuang F, Wei LC, Ding YX, Yung KK, Chan YS. Potential application of induced pluripotent stem cells in cell replacement therapy for Parkinson’s disease. CNS Neurol Disord Drug Targets. 2011;10(4):449–58.24. Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011;471(7336):63–7.25. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Närvä E, et al. Copy number variation and selection during reprogramming to pluripotency. Nature. 2011 Mar;471(7336):58–62.26. Mayshar Y, Ben-David U, , Lavon N, Biancotti JC, Yakir B, Clark AT, et al. Identi-fication and classification of chromo-somal aberrations in human induced

pluripotent stem cells. Cell Stem Cell. 2010 Oct;7(4):521–31.27. Oosterhuis JW, Looijenga LH. Testicular germ-cell tumours in a broader perspective. Nat Rev Cancer. 2005 Mar;5(3):210–22.28. Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell. 2006 Mar;124(6):1169–81.29. Gopalakrishna-Pillai S, Iverson LE. Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors. BMC Med Genomics. 2010;3:12.30. Baker DE, Harrison NJ, Maltby E, Smith K, Moore HD, Shaw PJ, et al. Adaptation to culture of human embry-onic stem cells and oncogenesis in vivo. Nat Biotechnol. 2007 Feb;25(2): 207–15.31. Baker M. Stem cells: fast and furious. Nature. 2009;458(7241):962–5.32. Kim K, Doi A, Wen B, Ng K, Zhao R, et al. Epigenetic memory in induced pluri-potent stem cells. Nature. 2010 Jul;467(7313):285–90.33. Kim K, Zhao R, Doi A, Ng K, Unter-naehrer J, Cahan P, et al. Donor cell type can influence the epigenome and differ-entiation potential of human induced pluripotent stem cells. Nat Biotechnol. 2011 Nov;29(12):117–9.34. Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, et al. Cell type of origin influences the molecular and func-tional properties of mouse induced pluri-potent stem cells. Nat Biotechnol. 2010 Jul;28(8):848–55.35. Wang T, Wu H, Li Y, Szulwach KE, Lin L, Li X, et al. Subtelomeric hotspots of aberrant 5-hydroxymethyl cytosine-mediated epigenetic modifications during reprogramming to pluripotency. Nat Cell Biol. 2013 May;15(6):700–11.36. Ware CB, Wang L, Mecham BH, Shen L, Nelson AM, Bar M, et al. Histone deacet-ylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells. Cell Stem Cell. 2009 Apr;4(4):359–69.37. Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, et al. Dissecting direct reprogramming through integra-tive genomic analysis. Nature. 2008 May;454(7200):49–55.

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

38. Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, et al. Induc-tion of pluripotent stem cells from primary human fibroblasts with only Oct 4 and Sox 2. Nat Biotechnol. 2008 Oct;26(7):1269–75.39. Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T, et al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature. 2011 Jun;474(7350):225–9.40. Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S. Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci USA. 2010;107(32):14152–7.41. Han J, Yuan P, Yang H, Zhang J, Soh BS, Li P, et al. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature. 2010;463(7284):1096–100.42. Awe JP, Byrne JA. Identifying candi-date oocyte reprogramming factors using cross-species global transcriptional anal-ysis. Cell Reprogram. 2013;15(2):126–33.43. Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, et al. The role of Tet 3 DNA dioxyge-nase in epigenetic reprogramming by oocytes. Nature. 2011;477(7366):606–10.44. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, et al. Induced pluripotent stem cells gener-ated from patients with ALS can be differ-entiated into motor neurons. Science. 2008;321(5893):1218–21.45. Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, et al. Parkinson’s disease patient-derived induced pluripo-tent stem cells free of viral reprogram-ming factors. Cell. 2009;136(5):964–77.46. Carvajal-Vergara X, Sevilla A, D’Souza SL, Ang YS, Schaniel C, Lee DF, et al. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature. 2010;465(7299):808–12.47. Grigoriadis AE, Kennedy M, Bozec A, Brunton F, Stenbeck G, Park IH, et al. Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells. Blood. 2010;115(14):2769–76.48. Sullivan GJ, Hay DC, Park IH, Fletcher J, Hannoun Z, Payne CM, et al. Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology. 2010;51(1):329–35.

49. Patterson M, Chan DN, Ha I, Case D, Cui Y, Van Handel B, et al. Defining the nature of human pluripotent stem cell progeny. Cell Res. 2012;22(1):178–93.50. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007;25(9):1015–24.51. Robey TE, Saiget MK, Reinecke H, Murry CE. Systems approaches to preventing transplanted cell death in cardiac repair. J Mol Cell Cardiol. 2008;45(4):567–81.52. Araki R, Uda M, Sunayama M, Naka-mura M, Ando S, Sugiura M, et al. Negli-gible immunogenicity of terminally differ-entiated cells derived from induced pluripotent or embryonic stem cells. Nature. 2013;494(7435):100–4.53. Morizane A, Doi D, Kikuchi T, Okita K, Hotta A, Kawasaki T, et al. Direct compar-ison of autologous and allogeneic trans-plantation of iPSC-derived neural cells in the brain of a nonhuman primate. Stem Cell Reports 2013;1(4):283–92.54. Zhao T, Zhang Z-N, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells. Nature. 2011;474(7350):212–5.55. Cunningham JJ, Ulbright TM, Pera MF, Looijenga LH. Lessons from human tera-tomas to guide development of safe stem cell therapies. Nat Biotechnol. 2012;30(9):849–57.56. Roy NS, Cleren C, Singh SK, Yang L, Beal MF, Goldman SA. Functional engraft-ment of human ES cell-derived dopamin-ergic neurons enriched by coculture with telomerase-immortalized midbrain astro-cytes. Nat Med. 2006;12(11):1259–68.57. Lawrenz B, Schiller H, Willbold E, Ruediger M, Muhs A, Esser S. Highly sensi-tive biosafety model for stem-cell-derived grafts. Cytotherapy. 2004;6(3):212–22.58. Huber I, Itzhaki I, Caspi O, Arbel G, Tzukerman M, Gepstein A, et al. Identifi-cation and selection of cardiomyocytes during human embryonic stem cell differ-entiation. FASEB J. 2007;21(10):2551–63.59. Tang C, Lee AS, Volkmer JP, Sahoo D, Nag D, Mosley AR, et al. An antibody against SSEA-5 glycan on human pluripo-tent stem cells enables removal of tera-toma-forming cells. Nat Biotechnol. 2011;29(9):829–34.

60. Elliott DA, Braam SR, Koutsis K, Ng ES, Jenny R, Lagerqvist EL, et al. NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods. 2011;8(12):1037–40.61. Tohyama S, Hattori F, Sano M, Hishiki T, Nagahata Y, Matsuura T, et al. and Fukuda K. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell. 2013;12(1):127–37.62. Masip M, Veiga A, Izpisúa Belmonte JC, Simón C. Reprogramming with defined factors: from induced pluripotency to induced transdifferentiation. Mol Hum Reprod. 2010;16(11):856–68.63. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448(7151):313–7.64. AweJP, Lee PC, Ramathal C, Vega-Crespo A, Durruthy-Durruthy J, Cooper A, et al. and Byrne JA. Generation and char-acterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status. Stem Cell Res Ther. 2013;4(4):87.65. Herschman HR. Micro-PET imaging and small animal models of disease. Curr Opin Immunol. 2003;15(4):378–84.66. Phelps ME. Positron emission tomog-raphy provides molecular imaging of biological processes. Proc Natl Acad Sci USA. 2000;97(16):9226–33.67. Cao F, Drukker M, Lin S, Sheikh AY, Xie X, Li Z, et al. Molecular imaging of embry-onic stem cell misbehavior and suicide gene ablation. Cloning Stem Cells. 2007;9(1):107–17.68. Strauss S. Geron trial resumes, but standards for stem cell trials remain elusive. Nat Biotechnol. 2010;28(10):989–90.69. Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, Ostrick RM, et al. Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet. 2012;379(9817): 713–20.70. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010;7(5):618–30.71. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, et al. Highly

of 8

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

.A

ll au

thor

s co

ntrib

uted

to c

once

ption

and

des

ign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

All

auth

ors

abid

e by

the

Ass

ocia

tion

for M

edic

al E

thic

s (A

ME)

eth

ical

rule

s of

dis

clos

ure.



Critical Review

efficient miRNA-mediated repro-gramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 2011;8(4):376–88.72. Li W, JiangK, Ding S. Concise review: a chemical approach to control cell fate and function. Stem Cells. 2012(1);30:61–8.

overcoming clinical hurdles for autologous pluripotent ... · infertility treatments. however,...

Documents