RESUMEN
Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or ß-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.
Asunto(s)
Diferenciación Celular , Endodermo/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Secretoras de Insulina/metabolismo , Técnicas de Reprogramación Celular , Endodermo/citología , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Secretoras de Insulina/citologíaRESUMEN
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.
Asunto(s)
Biomarcadores/metabolismo , Linaje de la Célula , Hepatocitos/citología , Hepatocitos/metabolismo , Histonas/metabolismo , Células Madre Embrionarias Humanas/citología , Lisina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Dimetilsulfóxido/farmacología , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Metilación , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Células Madre Embrionarias/metabolismo , Marcación de Gen/métodos , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/metabolismo , Recombinasas/metabolismo , Transgenes , Células Cultivadas , Metilación de ADN , Dependovirus/genética , Células Madre Embrionarias/citología , Silenciador del Gen , Sitios Genéticos , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Recombinasas/genéticaRESUMEN
Expression of NKX2-1 is required to specify definitive endoderm to respiratory endoderm. However, the transcriptional regulation of NKX2-1 is not fully understood. Here we demonstrate that aside from specifying undifferentiated human embryonic stem cell (hESC) to definitive endoderm, high concentrations of Activin-A are also necessary and sufficient to induce hESC-derived definitive endodermal progeny to a FOXA2/NKX2-1/GATA6/PAX9 positive respiratory epithelial fate. Activin-A directly mediates the induction of NKX2-1 by interacting with ALK4, leading to phosphorylation of SMAD2, which binds directly to the NKX2-1 promoter and activates its expression. Activin-A can be replaced by GDF11 but not transforming growth factor ß1. Addition of Wnt3a, SHH, FGF2, or BMP4 failed to induce NKX2-1. These results suggest that direct binding of Activin-A-responsive SMAD2 to the NKX2-1 promoter plays essential role during respiratory endoderm specification.