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Reprogramming Cellular Identity for Regenerative Medicine

2012; Cell Press; Volume: 148; Issue: 6 Linguagem: Inglês

10.1016/j.cell.2012.02.031

1097-4172

Anne Cherry, George Q. Daley,

Biomedical Ethics and Regulation

Although development leads unidirectionally toward more restricted cell fates, recent work in cellular reprogramming has proven that one cellular identity can strikingly convert into another, promising countless applications in biomedical research and paving the way for modeling diseases with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. Here, we review evidence demonstrating that, because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. We also discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. Although development leads unidirectionally toward more restricted cell fates, recent work in cellular reprogramming has proven that one cellular identity can strikingly convert into another, promising countless applications in biomedical research and paving the way for modeling diseases with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. Here, we review evidence demonstrating that, because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. We also discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. As a zygote cleaves and then develops into a complex organism, cells transition inexorably from one identity to another. Gene expression from a single genome naturally evolves and adapts via a carefully choreographed and directed set of inductive and selective events until lineages become segregated and tissue fates are fixed. This ability of a multicellular organism to create diverse cell types from a single stable genome provides versatility of function, permitting an organism to adapt and thrive in more varied environments than their single-cell predecessors. Although a few complex organisms, such as salamanders, regenerate large portions of their bodies by dedifferentiating their tissues, most multicellular organisms demonstrate very little reversibility of cellular identity after completing embryogenesis. Adult mammals are unable to regenerate anatomically correct organ systems after significant damage or loss, demonstrating that cellular identities in the unaffected tissues are largely stable. Even in the few mammalian organs with high rates of cell turnover, such as the skin, blood system, and gut, the range of possible cell fates is rigidly restricted to those cellular identities comprising the specific tissue. Evolution has invested heavily in maintaining and restricting cellular identities in mammals. Once a mammalian cell has progressed through its natural developmental and regenerative transitions, its final, specialized state is sustained by a loss of self-renewal and inevitable senescence. Mutations in the genetic mechanisms of cellular identity, stability, and senescence predispose cells to the development of malignancy. For example, when granulocyte macrophage precursors acquire self-renewal, these otherwise normal progenitors are transformed into leukemic stem cells (Krivtsov et al., 2006Krivtsov A.V. Twomey D. Feng Z.H. Stubbs M.C. Wang Y.Z. Faber J. Levine J.E. Wang J. Hahn W.C. Gilliland D.G. et al.Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (1163) Google Scholar). Pathologic conditions that encourage fluidity of cellular identity can similarly predispose individuals to cancer. Patients with gastresophageal reflux are a classic example of this phenomenon, in which exposure to stomach acid causes affected regions of the esophagus to transform into stomach-like tissue. This tissue metaplasia, while protecting the integrity of the esophagus, also predisposes patients to adenocarcinoma (Lagergren et al., 1999Lagergren J. Bergström R. Lindgren A. Nyrén O. Symptomatic gastroesophageal reflux as a risk factor for esophageal adenocarcinoma.N. Engl. J. Med. 1999; 340: 825-831Crossref PubMed Scopus (2586) Google Scholar). The in vivo mechanisms by which a differentiated cell transitions to another cell type (metaplasia) or to a more undifferentiated phenotype (dysplasia) are under investigation. Current research suggests that these in vivo alterations of cellular identities are brought about by changes in the epigenome and gene expression of the affected cells, which in turn provide fertile ground for the appearance of mutations that promote malignant transformation (Kang et al., 2003Kang G.H. Lee H.J. Hwang K.S. Lee S. Kim J.H. Kim J.S. Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation.Am. J. Pathol. 2003; 163: 1551-1556Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, Nardone et al., 2007Nardone G. Compare D. De Colibus P. de Nucci G. Rocco A. Helicobacter pylori and epigenetic mechanisms underlying gastric carcinogenesis.Dig. Dis. 2007; 25: 225-229Crossref PubMed Scopus (42) Google Scholar, Herfs et al., 2009Herfs M. Hubert P. Delvenne P. Epithelial metaplasia: adult stem cell reprogramming and (pre)neoplastic transformation mediated by inflammation?.Trends Mol. Med. 2009; 15: 245-253Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The orderly progression of cell differentiation during development has been well described by in vivo studies, but some questions can be addressed more directly in the highly controlled environment of in vitro tissue culture. Human embryonic stem (ES) cells, derived from the inner cell masses of human blastocysts, were first successfully derived less than 15 years ago by the Thomson group from the University of Wisconsin (Thomson et al., 1998Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12274) Google Scholar). Pluripotent cells are unique in that they can be grown indefinitely while retaining the ability to differentiate into all three embryonic tissue lineages. Human ES cell derivation has inspired biomedical scientists to use stem cells to address questions of human developmental biology, to study disease processes in vitro, and even to attempt to replace ailing tissues in human patients. All of these hopes have been pinned on the ability of scientists to engineer specific cellular identities. This is an ambitious goal. Murine ES cells have been in widespread use for three decades, and yet attempts to generate functional mouse blood cells, pancreatic cells, and highly specialized neurons have so far proven only partly successful. Nonetheless, biologists remain confident that ES cells can be differentiated to specific cell types if culture conditions can be identified that precisely mimic the organizational and signaling events of the developing embryo. This approach necessitates an in-depth understanding of the cellular identity changes that take place in normal development and requires direct translation of basic developmental biology into painstakingly developed protocols for directed differentiation. During in vitro differentiation, stem cells are induced to form aggregates with predictable structures (i.e., embryoid bodies) that echo embryonic organization, and growth factors have been identified that coax pluripotent cells toward one lineage or another. These approaches attempt to recapitulate the epigenetic changes that occur during embryogenesis, with the aim of creating tissue types analogous to those generated during embryonic development. In the years since the first human ES cell derivation, scientists have crafted differentiation protocols to generate many cell types, including motor neurons, retinal pigment epithelium, and hematopoietic precursors from human ES cells (Wichterle et al., 2002Wichterle H. Lieberam I. Porter J.A. Jessell T.M. Directed differentiation of embryonic stem cells into motor neurons.Cell. 2002; 110: 385-397Abstract Full Text Full Text PDF PubMed Scopus (1410) Google Scholar, Klimanskaya et al., 2004Klimanskaya I. Hipp J. Rezai K.A. West M. Atala A. Lanza R. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.Cloning Stem Cells. 2004; 6: 217-245Crossref PubMed Scopus (344) Google Scholar, Ng et al., 2005Ng E.S. Davis R.P. Azzola L. Stanley E.G. Elefanty A.G. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation.Blood. 2005; 106: 1601-1603Crossref PubMed Scopus (326) Google Scholar). These protocols exemplify the reigning paradigm that in vitro manipulations of cellular identity should follow the course of the natural, unidirectional changes that occur during development. This paradigm was overthrown in 2006, when Takahashi and Yamanaka published the distinctly unnatural conversion of murine fibroblasts into induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar). Their approach was blatantly not based on mimicking developmental events, and the cellular fate change that they engineered went backward—the implausible reversion of a differentiated, specialized somatic cell to a pluripotent embryonic progenitor. Although conversion of differentiated cells to an embryonic state had previously been accomplished by somatic cell nuclear transfer, that process was and remains to this day inefficient, cumbersome, and poorly understood (Rideout et al., 2001Rideout III, W.M. Eggan K. Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome.Science. 2001; 293: 1093-1098Crossref PubMed Scopus (630) Google Scholar). The Yamanaka reprogramming approach, on the other hand, used a few defined factors to convert a cell to a radically different identity. This landmark study compelled a bold paradigm shift and introduced the engineering of cell identity as a powerful new strategy for biomedical research and regenerative medicine. The pluripotency of murine iPS cells has been established in many ways, including gene expression and epigenome profiling, chimera formation, and tetraploid embryo complementation (Okita et al., 2007Okita K. Ichisaka T. Yamanaka S. Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3532) Google Scholar, Zhao et al., 2009Zhao X.Y. Li W. Lv Z. Liu L. Tong M. Hai T. Hao J. Guo C.L. Ma Q.W. Wang L. et al.iPS cells produce viable mice through tetraploid complementation.Nature. 2009; 461: 86-90Crossref PubMed Scopus (642) Google Scholar). Soon after the publication of reprogramming in murine cells, multiple labs confirmed that ectopic expression of defined factors could also generate iPS cells from human tissues (Yu et al., 2007Yu J.Y. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Induced pluripotent stem cell lines derived from human somatic cells.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8146) Google Scholar, Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14964) Google Scholar, Park et al., 2008bPark I.H. Zhao R. West J.A. Yabuuchi A. Huo H.G. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Reprogramming of human somatic cells to pluripotency with defined factors.Nature. 2008; 451: 141-146Crossref PubMed Scopus (2374) Google Scholar). Both gene expression and epigenetic studies revealed that iPS cells are strikingly more similar to ES cells than they are to their starting cell type (Hawkins et al., 2010Hawkins R.D. Hon G.C. Lee L.K. Ngo Q. Lister R. Pelizzola M. Edsall L.E. Kuan S. Luu Y. Klugman S. et al.Distinct epigenomic landscapes of pluripotent and lineage-committed human cells.Cell Stem Cell. 2010; 6: 479-491Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, Doi et al., 2009Doi A. Park I.H. Wen B. Murakami P. Aryee M.J. Irizarry R. Herb B. Ladd-Acosta C. Rho J.S. Loewer S. et al.Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.Nat. Genet. 2009; 41: 1350-1353Crossref PubMed Scopus (943) Google Scholar, Kim et al., 2010Kim K. Doi A. Wen B. Ng K. Zhao R. Cahan P. Kim J. Aryee M.J. Ji H. Ehrlich L.I.R. et al.Epigenetic memory in induced pluripotent stem cells.Nature. 2010; 467: 285-290Crossref PubMed Scopus (1714) Google Scholar, Polo et al., 2010Polo J.M. Liu S. Figueroa M.E. Kulalert W. Eminli S. Tan K.Y. Apostolou E. Stadtfeld M. Li Y.S. Shioda T. et al.Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells.Nat. Biotechnol. 2010; 28: 848-855Crossref PubMed Scopus (943) Google Scholar), showing that transduction of four transcription factors (i.e., Oct4, Sox2, Klf4, and c-Myc) indeed alters mammalian cellular identities in the direction opposite to that of development. Although multiple groups had induced modest cell fate changes between mesodermal or hematopoietic lineages by manipulating transcription factors (Davis et al., 1987Davis R.L. Weintraub H. Lassar A.B. Expression of a single transfected cDNA converts fibroblasts to myoblasts.Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2470) Google Scholar, McNagny and Graf, 2002McNagny K. Graf T. Making eosinophils through subtle shifts in transcription factor expression.J. Exp. Med. 2002; 195: F43-F47Crossref PubMed Scopus (95) Google Scholar, Cobaleda et al., 2007Cobaleda C. Jochum W. Busslinger M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors.Nature. 2007; 449: 473-477Crossref PubMed Scopus (380) Google Scholar), engineering cell fate changes as dramatic as reversion of differentiated cells to pluripotency was not envisioned as plausible before Yamanaka's work. The original publication of iPS cell reprogramming has inspired researchers to attempt manipulations of cellular identity in new and unexpected directions. Ectopic transcription factor expression is now being investigated as a tool to perform direct conversion, or “transdifferentiation,” of one differentiated cell type to another, including neurons and cardiomyocytes from fibroblasts and β cells from exocrine cells (Zhou et al., 2008Zhou Q. Brown J. Kanarek A. Rajagopal J. Melton D.A. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.Nature. 2008; 455: 627-632Crossref PubMed Scopus (1634) Google Scholar, Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar, Ieda et al., 2010Ieda M. Fu J.D. Delgado-Olguin P. Vedantham V. Hayashi Y. Bruneau B.G. Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.Cell. 2010; 142: 375-386Abstract Full Text Full Text PDF PubMed Scopus (1884) Google Scholar, Efe et al., 2011Efe J.A. Hilcove S. Kim J. Zhou H. Ouyang K. Wang G. Chen J. Ding S. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy.Nat. Cell Biol. 2011; 13: 215-222Crossref PubMed Scopus (520) Google Scholar). Protocols continue to be published for the generation of differentiated cell types from pluripotent stem cells, for more effective generation of stem cells from somatic cells, and for the conversion of one differentiated cell type to another. This diversity of goals represents a radical change in thinking about the categories of identity changes that can be effected in vitro (Figure 1): adult mammalian cellular identity can be manipulated not only in the direction of stem cell differentiation (a correlate of normal development), but also dedifferentiation (a correlate of dysplasia) and transdifferentiation (a correlate of metaplasia). The recent appreciation for the plasticity of cellular identity has made this area one of the most exciting topics in modern stem cell biology. Takahashi and Yamanaka's seminal publication has compelled a new and creative open-mindedness about cellular identity and has paved the way for the development of iPS cell-based disease models, drug screens, and cellular therapies. The prospects for cellular therapies are still on the horizon, but use of reprogramming technology for disease modeling and drug testing has already begun. This Perspective provides a discussion on which disease categories are likely to benefit in the near future from reprogramming-based models and how these models can be used to study gene-environment interactions. Research into human disease can be performed on platforms as diverse as epidemiology, human genetics, animal modeling, and in vitro cell culture. Each of these approaches provides different kinds of information about the disease under investigation, and each has its own limitations. It is not often that a new platform for studying disease arises, but in the last few years, the advent of patient-specific pluripotent stem cells has inspired researchers to contemplate modeling diseases in a powerful new way. By differentiating patient-specific iPS lines into the cell type responsible for a specific disorder, scientists hope to gain many new research tools. For disorders in which etiology is unclear, such as type I diabetes, it is theorized that patient-specific iPS cell models may confirm current theories or inspire new hypotheses about the origins and progression of the disease (Maehr et al., 2009Maehr R. Chen S.B. Snitow M. Ludwig T. Yagasaki L. Goland R. Leibel R.L. Melton D.A. Generation of pluripotent stem cells from patients with type 1 diabetes.Proc. Natl. Acad. Sci. USA. 2009; 106: 15768-15773Crossref PubMed Scopus (453) Google Scholar). For diseases in which human-specific cardiac or renal toxicity is a limiting factor in treatment, stem cell-derived models of heart or renal tissues may be used to experimentally measure and reduce drug associated toxicity. Finally, for any disorder whose iPS cell-derived target cells show a measurable disease-specific phenotype, reprogramming-based models can be used as screening tools for development of new drugs that reverse the cellular pathology in vitro and might therefore carry a greater probability of reversing disease pathology when given to patients. Because of the inherent pluripotency of the starting cells, their potential applications for cell-autonomous disorders touch virtually every organ system. Researchers studying disorders of hematological, neurological, cardiovascular, metabolic, endocrine, and muscular cell types have already begun the process of creating disease models by reprogramming disease-specific primary cell samples from patients with cystic fibrosis, Huntington's disease, Parkinson's disease, sickle cell anemia, dyskeratosis congenita, familial amyotrophic lateral sclerosis, and a growing compendium of other conditions recently reviewed by Grskovic et al., 2011Grskovic M. 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Huo H. Okuka M. Dos Reis R.M. Loewer S. et al.Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.Nature. 2010; 464: 292-296Crossref PubMed Scopus (258) Google Scholar). These inexhaustible sources of patient-specific cells are then differentiated toward the lineage affected by the disorder, be it neural, hematopoietic, cardiac, or hepatic. Once the cell type that is responsible for the disease has been generated, researchers attempt to identify a disease-associated phenotype that manifests characteristics relevant to the disorder. Such cells represent a new research platform for studying both mechanisms and genotype-phenotype interactions of the disease. For example, congenital long-QT syndrome has been difficult to study in vitro because of the inaccessibility of human cardiomyocytes carrying the causal genetic mutations (Behr et al., 2008Behr E.R. Dalageorgou C. Christiansen M. Syrris P. Hughes S. Tome Esteban M.T. Rowland E. Jeffery S. McKenna W.J. Sudden arrhythmic death syndrome: familial evaluation identifies inheritable heart disease in the majority of families.Eur. Heart J. 2008; 29: 1670-1680Crossref PubMed Scopus (333) Google Scholar). In a recent study by Moretti et al., iPS cells were derived from individuals with monogenic congenital long-QT syndrome type I, differentiated to cardiac lineages, and assayed for characteristic electrophysiological traits (Moretti et al., 2010Moretti A. Bellin M. Welling A. Jung C.B. Lam J.T. Bott-Flügel L. Dorn T. Goedel A. Höhnke C. Hofmann F. et al.Patient-specific induced pluripotent stem-cell models for long-QT syndrome.N. Engl. J. Med. 2010; 363: 1397-1409Crossref PubMed Scopus (961) Google Scholar). The patient-derived cardiomyocytes showed longer-lasting action potentials than the healthy controls, as well as an altered protein localization pattern. These findings allowed the authors to identify a dominant-negative mechanism of disease. A paper released shortly afterward described similar studies on another variant of congenital long-QT syndrome, caused by a mutation in a different gene (Itzhaki et al., 2011Itzhaki I. Maizels L. Huber I. Zwi-Dantsis L. Caspi O. Winterstern A. Feldman O. Gepstein A. Arbel G. Hammerman H. et al.Modelling the long QT syndrome with induced pluripotent stem cells.Nature. 2011; 471: 225-229Crossref PubMed Scopus (804) Google Scholar). This study identified additional disease-associated electrophysiological phenotypes in the patients' cells, and the authors were able to conduct a limited drug screen to investigate the potency of chemical compounds to ameliorate the disease traits. The existence of these two models will allow for direct comparison of cellular phenotypes between different genotypes of the same disease and will hopefully lead to improved, personalized therapeutic options for patients (Figure 2). Because of these many and varied benefits, reprogramming-based disease models are being rapidly adopted by translational scientists. Dozens have already been published and are the subject of recent review articles (Grskovic et al., 2011Grskovic M. Javaherian A. Strulovici B. Daley G.Q. Induced pluripotent stem cells—opportunities for disease modelling and drug discovery.Nat. Rev. Drug Discov. 2011; 10: 915-929PubMed Google Scholar, Tiscornia et al., 2011Tiscornia G. Vivas E.L. Belmonte J.C. Diseases in a dish: modeling human genetic disorders using induced pluripotent cells.Nat. Med. 2011; 17: 1570-1576Crossref PubMed Scopus (166) Google Scholar, Unternaehrer and Daley, 2011Unternaehrer J.J. Daley G.Q. Induced pluripotent stem cells for modelling human diseases.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2011; 366: 2274-2285Crossref PubMed Scopus (68) Google Scholar). However, the field of iPS cell-based disease modeling is still in its infancy, and many challenges remain. Most disease systems still face significant hurdles that need to be overcome before iPS technology can deliver on its promise. The following are specific challenges that each disease field must address. For many years, work has been underway to convert pluripotent cells into target cell types. Some highly specialized cell types like motor neurons and cardiomyocytes have been created with great fidelity using protocols that mimic pathways defined through studies of embryo development. In contrast, only close facsimiles of other much sought-after cell types, like β cells, have been created using independent protocols in different laboratories, with phenotypes that differ from actual human target cells (Kroon et al., 2008Kroon E. Martinson L.A. Kadoya K. Bang A.G. Kelly O.G. Eliazer S. Young H. Richardson M. Smart N.G. Cunningham J. et al.Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.Nat. Biotechnol. 2008; 26: 443-452Crossref PubMed Scopus (1398) Google Scholar, Zhang et al., 2009Zhang D.H. Jiang W. Liu M. Sui X. Yin X.L. Chen S. Shi Y. Deng H.K. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells.Cell Res. 2009; 19: 429-438Crossref PubMed Scopus (465) Google Scholar). Some cell types, such as definitive hematopoietic stem cells (HSCs), have never been successfully derived from pluripotent stem cells purely in vitro. In cases like HSCs, the difficulty rests in large part on the lack of suitable culture conditions to maintain and expand these evanescent cells. Where cell culture conditions remain poorly defined, directed differentiation protocols represent a critical rate-limiting step in iPS cell research. Cellular identity is a complex phenotype with many components. For research to proceed on a given iPS cell-derived cell type, the field must first agree that the in vitro product is comparable to its in vivo correlate. This measure of similarity must occur on multiple levels and include analysis of gene expression, chromatin state, and functional assays. Transcriptional activity and methylomes can be evaluated by similar methodologies in all cell types, but appropriate markers of cell identity as well as choice and validation of cell type-specific functional assays must be specifically identified for all target cell types. In some cases, target cell types derived from disease-specific iPS cells show clear and predictable phenotypes, such as the electrophysiological abnormalities in the cardiomyocytes described above. For other diseases, the appropriate assay for disease phenotype is unclear or may not show a significant difference between disease and nondisease lines. For example, recent papers have derived iPS lines from patients with idiopathic Parkinson's disease (PD) and normal controls (Soldner et al., 2009Soldner F. Hockemeyer D. Beard C. Gao Q. Bell G.W. Cook E.G. Hargus G. Blak A. Cooper O. Mitalipova M. et al.Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.Cell. 2009; 136: 964-977Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, Hargus et al., 2010Hargus G. Cooper O. Deleidi M. Levy A. Lee K. Marlow E. Yow A. Soldner F. Hockemeyer D. Hallett P.J. et al.Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.Proc. Natl. Acad. Sci. USA. 2010; 107: 15921-15926Crossref PubMed Scopus (380) Google Scholar). The researchers planned to investigate whether PD-iPS cell lines would form dopaminergic neurons at lower frequencies than WT-iPS cells during directed differentiation, as well as how dopaminergic neuron grafts would function in transplantation assays into various animal models of PD. In practice, none of these endpoints showed any difference between the disease and the control-derived iPS cells. The researchers eventually identified only a single outcome measure in the rodent model that showed a sig