Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

Broad Institute, Cambridge, MA 02142, USA.
Cell (Impact Factor: 32.24). 02/2011; 144(3):439-52. DOI: 10.1016/j.cell.2010.12.032
Source: PubMed


The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

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Available from: Mackenzie W Amoroso, Oct 11, 2015
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    • "We demonstrated that certain hiPSC-derived cells such as RPE cells are much less immunogenic than other hiPSCderived cell types such as SMCs. The mechanisms underlying the differential immunogenicity can be very complex, depending on the intrinsic immune characteristics of the cell types and the impact of epigenetic abnormality of hiPSCs on differentiated cell types as previously noted (Bar-Nur et al., 2011; Bock et al., 2011; Kim et al., 2010, 2011; Polo et al., 2010; Ruiz et al., 2012). In support of this notion, we demonstrate that the abnormal expression of immunogenic antigens in some hiPSCderived cells, but not in others, contributes to the differential immunogenicity. "
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    ABSTRACT: The breakthrough of induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific iPSCs may become a renewable source of autologous cells for cell therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood. Using a humanized mouse model (denoted Hu-mice) reconstituted with a functional human immune system, we demonstrate that most teratomas formed by autologous integration-free hiPSCs exhibit local infiltration of antigen-specific T cells and associated tissue necrosis, indicating immune rejection of certain hiPSC-derived cells. In this context, autologous hiPSC-derived smooth muscle cells (SMCs) appear to be highly immunogenic, while autologous hiPSC-derived retinal pigment epithelial (RPE) cells are immune tolerated even in non-ocular locations. This differential immunogenicity is due in part to abnormal expression of immunogenic antigens in hiPSC-derived SMCs, but not in hiPSC-derived RPEs. These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell stem cell 08/2015; 17(3). DOI:10.1016/j.stem.2015.07.021 · 22.27 Impact Factor
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    • "Furthermore, by sequencing and combining dozens of low-input methylomes from the same sample (e.g., one-cell, four-cell, or 20-cell pools), it will be possible to create composite methylomes that provide excellent genomewide coverage based on relatively few cells and include an inherent assessment of variation. Essentially, composite methylomes redefine the concept of reference epigenome corridors (Bock et al., 2011) in the context of individual samples, thus providing a new type of reference methylome map that can account for tissue heterogeneity and cell-to-cell variation. "
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    ABSTRACT: Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 03/2015; 10(8):1386-97. DOI:10.1016/j.celrep.2015.02.001 · 8.36 Impact Factor
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    • "Directed differentiation of human pluripotent stem cells into hepatocytes, typically called hepatocyte-like cells (HLCs), with a mature functional phenotype, could in theory provide a readily available source of metabolically competent cells for use in drug screening (Greenhough et al., 2010; Yildirimman et al., 2011). However, the differentiation efficiency of HLCs from human pluripotent stem cells can be variable, which is believed to be mainly due to differences of the differentiation protocols being employed and the propensity of the selected pluripotent stem cell line to differentiate toward a hepatic lineage (Baxter et al., 2010; Bock et al., 2011). The differentiation efficiency of HLCs from a starting culture of undifferentiated pluripotent stem cells can range from 9% to 90%, as determined by the percentage of cells in the culture that express the hepatocyte protein marker albumin (Hay et al., 2008; Rashid et al., 2010; Shiraki et al., 2008). "
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    ABSTRACT: Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular co-culture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology.
    Toxicological Sciences 12/2014; 144(1). DOI:10.1093/toxsci/kfu269 · 3.85 Impact Factor
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