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Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types. ispartof: PLOS ONE vol:13 issue:5 ispartof: location:United States status: published

Summary Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes., Highlights • RMCE using positive/negative selection allows generation of transgenic hPSC lines in 15 days • The AAVS1 locus exerts transgene inhibition by inducing de novo DNA methylation • AAVS1-mediated silencing appears sequence and lineage-specific in vitro, yet is not present in vivo • AAVS1 does not meet the requirements to be considered a universal safe harbor locus in vitro, Verfaillie, Ordovás, and colleagues report an efficient and fast method to genetically engineer hPSC in the AAVS1 safe harbor locus free of random integration events. Their results demonstrate the suitability of the locus for several applications to study the hepatic lineage, but they uncover a so-far-unknown variable AAVS1-mediated transgene inhibition in undifferentiated and hepatocyte differentiating hESC. Therefore, AAVS1 cannot be considered a safe harbor locus.

Summary Although pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro., Graphical Abstract, Highlights • Epigenetic studies to understand hepatocyte differentiation from human PSC • Dynamics in histone profile correlate with alterations in gene transcription • hESC-derived HLCs have higher H3K27me3 mark at regulatory regions compared with PHHs • Reducing H3K27me3 by EZH2 inhibition did not improve hepatocyte differentiation, Vanhove, Pistoni, and colleagues. report that inhibitory H3K27me3 marks at regulatory regions of hepatocyte marker genes were higher in hESC-derived HLCs in vitro compared with uncultured PHHs. Reducing H3K27me3 by EZH2 inhibition failed to induce hepatocyte gene expression and further differentiation, demonstrating that H3K27me3 does not repress transcription and appears dispensable for maturation of hESCs to hepatocytes in vitro.