ABSTRACT: Genome-wide analysis of mRNA expression alterations in human primary hepatocytes compared with cAMP treated and non-treated human embryonic stem cell (hESC) derived hepatic populations
Project description:Genome-wide analysis of mRNA expression alterations in human primary hepatocytes compared with cAMP treated and non-treated human embryonic stem cell (hESC) derived hepatic populations
Project description:Microarrays were used to examine gene expression differences between primary human hepatocytes and hESC derived hepatoblast cultures treated or non-treated with cAMP. hESC's were differentiated with a hepatic cell lineage specific protocol that included the use of a 3-dimensional cell aggregation culturing technique. Followed by cAMP signaling, this promoted the maturation of hepatoblasts into a more hepatocyte-like population. Importantly, key enzymes related to liver function show that cAMP induced populations have a gene expression profile that is similar to primary human hepatocytes. Total RNA obtained from cultured primary hepatocytes and hESC derived hepatic populations.
Project description:Microarrays were used to examine gene expression differences between primary human hepatocytes and hESC derived hepatoblast cultures treated or non-treated with cAMP. hESC's were differentiated with a hepatic cell lineage specific protocol that included the use of a 3-dimensional cell aggregation culturing technique. Followed by cAMP signaling, this promoted the maturation of hepatoblasts into a more hepatocyte-like population. Importantly, key enzymes related to liver function show that cAMP induced populations have a gene expression profile that is similar to primary human hepatocytes.
Project description:Expression data from undifferentiated human embryonic stem cells (hESC) and Day 3.5 mesodermal progenitor (CD326neg CD56+) population
Project description:The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.
Project description:Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival, proliferation, differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2, previously shown to promote Cx32 expression in mature hepatocytes, up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation, resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast, negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast, the p38 MAPK activator, anisomycin, blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.
