Small but Significant: Inter- and Intrapatient Variations in iPS Cell–based Disease Modeling
Authors: Malte Sgodda, Tobias Cantz
Journal: Molecular Therapy (2013)

Last year the Nobel Prize in Physiology or Medicine was awarded to John B. Gurdon and Shinya Yamanaka for their groundbreaking research on reprogramming of somatic cells.1Gurdon JB The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles.J Embryol Exp Morphol. 1962; 10: 622-640PubMed Google Scholar,2Takahashi 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 (18888) Google Scholar Using a set of just four transcription factors, Yamanaka demonstrated that somatic cells could be reprogrammed into induced pluripotent stem (iPS) cells that exhibited most, if not all, of the hallmarks of bona fide pluripotent stem cells. This observation immediately raised the prospect of patient-specific pluripotent stem cells both for therapeutic applications using stem cell–based transplants and for disease modeling. However, six years after the appearance of this landmark study, the suitability of patient-specific iPS cells for disease modeling or drug screening remains challenged by the existence of clone-to-clone variability that can complicate such studies. In this issue of Molecular Therapy, Thatava et al. describe the generation of three iPS cell lines from each of three individual patients suffering from type 1 diabetes (T1D) and the subsequent differentiation of these T1D-iPS cells into pancreatic cells.3Thatava T Kudva YC Edukulla R Squillace K De Lamo JG Khan YK Intrapatient variations in type 1 diabetes–specific iPS cell differentiation into insulin producing cells.Mol Ther. 2013; 21: 228-239Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Interestingly, they observed a high degree of intrapatient variability in the capacity of the T1D-iPS cells to develop into glucose-responsive insulin-producing cells. Indeed, the iPS cell lines derived from a single donor seemed to be as different from each other as individual iPS cell lines derived from unrelated donors. These observations highlight a number of caveats to iPS cell–based disease modeling (Figure 1). These include sequence variations such as point mutations in single cells of the original nonclonal somatic cell population, the fidelity of nuclear reprogramming and the impact of residual epigenetic signatures derived from the original cells, potential genomic alterations during the initial expansion of iPS cell colonies into established iPS cell lines, potential subclone-related immunogenic properties, and, finally, the variability between differentiation protocols per se and the variability of the phenotype of the differentiated cells derived from disease-affected and control iPS cell lines. Deep-sequencing strategies had previously demonstrated that at least half the point mutations identified in established human iPS cells were present in a very small subset of the starting cell population.4Gore 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: 63-67Crossref PubMed Scopus (1000) Google Scholar,5Young MA Larson DE Sun CW George DR Ding L Miller CA et al.Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells.Cell Stem Cell. 2012; 10: 570-582Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Very recently, Abyzov et al. described "line-manifested" copy-number variations (CNVs) among dermal fibroblasts and iPS cells derived from them, which reflected a somatic mosaicism normally present in human skin.6Abyzov A Mariani J Palejev D Zhang Y Haney MS Tomasini L et al.Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.Nature. 2012; (e-pub ahead of print 18 November 2012)Google Scholar These observations underline the need for extensive genetic screening of established iPS cell lines intended for potential therapeutic applications. Moreover, such mutations might affect differentiation propensities or the cells' responses to pharmacological or genetic treatments—suggesting ramifications for disease modeling as well. Recent data have shown that a preferential stoichiometry of reprogramming factors greatly enhances the generation of iPS cells,7Papapetrou EP Tomishima MJ Chambers SM Mica Y Reed E Menon J et al.Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.Proc Natl Acad Sci USA. 2009; 106: 12759-12764Crossref PubMed Scopus (226) Google Scholar,8Tiemann U Sgodda M Warlich E Ballmaier M Schöler HR Schambach A et al.Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells.Cytometry A. 2011; 79: 426-435Crossref PubMed Scopus (54) Google Scholar and there is increasing evidence that the extent of reprogramming of the epigenetic status of the original cells toward a fully pluripotent state is also very sensitive to factor stoichiometry.9Carey BW Markoulaki S Hanna JH Faddah DA Buganim Y Kim J et al.Reprogramming factors stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.Cell Stem Cell. 2011; 9: 588-598Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar However, the latter can be ensured using elaborate polycistronic reprogramming constructs.10Warlich E Kuehle J Cantz T Brugman MH Maetzig T Galla M et al.Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming.Mol Ther. 2011; 19: 782-789Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar Bock and colleagues have developed a valuable epigenetic scorecard11Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD et al.Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.Cell. 2011; 144: 439-452Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar that assesses the transcriptional and epigenetic similarity of iPS cell lines and that might prove useful in identifying individual lines with a full capacity to differentiate in vitro. Notably, the degree of CNVs, as well as epigenetic and transcriptional differences, seems to be greater in early iPS-cell passages.12Hussein SM Batada NN Vuoristo S Ching RW Autio R Närvä E et al.Copy number variation and selection during reprogramming to pluripotency.Nature. 2011; 471: 58-62Crossref PubMed Scopus (758) Google Scholar,13Polo JM Liu S Figueroa ME Kulalert W Eminli S Tan KY 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 In these studies it was postulated that replicative stress during the initial phase of reprogramming resulted in mosaic early-passage colonies that contain cells with a high number of CNVs. Quite a few of these CNVs might give rise to a growth disadvantage that gradually removes certain clones, which could explain why later passages of iPS cell lines exhibited fewer CNVs. However, given that the number of chromosomal abnormalities increases at higher passages,14Mayshar Y Ben-David U Lavon N Biancotti JC Yakir B Clark AT et al.Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar high-passage iPS cell lines could acquire other genetic aberrations that interfere with their capacity to differentiate or with the resulting cell type–specific phenotype. The ideal differentiation protocol should lead to an iPS cell–derived, but tissue-specific, cell that closely mimics the phenotype of its in vivo counterpart, with high efficiency. So far, even the most sophisticated differentiation protocols result in a quite heterogeneous cell population and lack reproducible efficiency if different pluripotent starting cells are used. With respect to endodermal and, in particular, pancreatic cell differentiation, protocols are stepwise in nature, comprising induction of definitive endoderm through a meso-endodermal progenitor cell state followed by the specification of a more tissue-specific state (pancreatic precursor cell). These precursor cells can further mature in vivo following transplantation and can thus give rise to results that are superior to those obtained when using more mature cells, which might have failed to acquire the full metabolic capabilities of the intended target cell owing to missing or inefficient cues during in vitro differentiation.15Kroon E Martinson LA Kadoya K Bang AG Kelly OG Eliazer S 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 (1399) Google Scholar This observation casts doubt on the current concepts for in vitro disease modeling because an assumed maturation state of in vitro differentiated cells may not represent a state that provides metabolic function after engraftment in vivo. Thatava and colleagues report the reproducible generation of just such a pancreatic progenitor cell from different patient-specific iPS cells. However, the further differentiation of these cells into functional (i.e., glucose-responsive) insulin-producing pancreatic β-cells was successful in only a subset of iPS cell lines.3Thatava T Kudva YC Edukulla R Squillace K De Lamo JG Khan YK Intrapatient variations in type 1 diabetes–specific iPS cell differentiation into insulin producing cells.Mol Ther. 2013; 21: 228-239Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar This observation of high intrapatient as well as interpatient variability between functionally differentiated cell phenotypes may support a paradigm shift for differentiation strategies applied in disease modeling—rather than aim for mature cell phenotypes in a direct and rapid differentiation protocol, one might first aim to generate well-defined and expandable precursor cells as an intermediate step.16Cheng X Ying L Lu L Galvão AM Mills JA Lin HC et al.Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.Cell Stem Cell. 2012; 10: 371-384Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar,17Sakurai H Sakaguchi Y Shoji E Nishino T Maki I Sakai H et al.In vitro modeling of paraxial mesodermal progenitors derived from induced pluripotent stem cells.PLoS One. 2012; 7: e47078Crossref PubMed Scopus (52) Google Scholar After such self-renewing progenitor cells were established, one could extensively characterize their epigenetic and transcriptional profile and identify progenitor cell lines that might most efficiently respond to subsequent differentiation cues during further in vitro differentiation. Then, in a second step, these cells could be differentiated toward mature cells in a more reproducible manner and presumably with greater functionality. If the disease-related phenotype of patient-specific iPS cell derivatives was subtle compared with that of derivatives of iPS cells from healthy individuals, such intra- and intersample variation could cloud the capacity to detect disease-specific effects. Therefore, it has been postulated that syngeneic control iPS cell lines might be useful to minimize intersample differences between disease-specific and control cell lines. Such syngeneic control iPS cells could be generated by applying tailored strategies for genetic correction of disease-associated mutations, which might be achieved by homologous recombination mediated by zinc-finger nucleases or transcription activator–like effector nucleases (TALENs).18Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (834) Google Scholar,19Bedell VM Wang Y Campbell JM Poshusta TL Starker CG Krug 2nd, RG et al.In vivo genome editing using a high-efficiency TALEN system.Nature. 2012; 491: 114-118Crossref PubMed Scopus (749) Google Scholar However, off-target effects such as DNA double-strand breaks could occur using both techniques,20Carlson DF Fahrenkrug SC Hackett PB Targeting DNA with fingers and TALENs.Mol Ther Nucleic Acids. 2012; 1: e3Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar which might also affect the cells' capacity to differentiate. Considering the advantages of a syngeneic source of mutated and normal stem cell derivatives, one could argue that such tailored gene-editing approaches could be utilized to introduce disease-specific mutations in well-established human embryonic stem cell lines, which would not exhibit epigenetic or transcriptional aberrations related to iPS cell generation per se. But this approach fails to take into account that a particular genetic mutation would not necessarily cause the same severe phenotype in individual patients with different genotypic backgrounds and that the onset of the particular disorder could further depend on other individual factors. In conclusion, Thatava et al. provide good evidence that human disease–specific iPS cell lines exhibit considerable intra- and intersample variability that must be addressed if tissue-specific functional iPS cell derivatives are to be used for basic or translational research on T1D. Future research may identify additional factors that could provide improved tools that generate iPS cells with less intra- and intersample variation. Advanced profiling of the iPS cells' transcriptome and epigenome may not only assay the establishment of fully reprogrammed pluripotent stem cells but also more adequately predict the differentiation ability of a given iPS cell line such that cell lines with aberrant differentiation potential could be excluded. If more subtle disease-related phenotypes are investigated, syngeneic cell sources for the disease-specific and the control cells may provide an advantageous system, provided that relevant intersample genetic alterations could be excluded by applying array–comparative genomic hybridization or deep-sequencing techniques.