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Footprint-free human fetal foreskin derived iPSCs: a tool for modeling hepatogenesis associated gene regulatory networks

ABSTRACT: Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). E-iPSCs were fully characterized and their transcriptomes are more similar to that of hESCs (R2 = 0.9363) in comparison to viral-derived HFF1-iPSCs (R2 = 0.8176). We used an E-iPSC-line to model hepatogenesis in vitro. The differentiation of iPSCs into hepatocyte-like cells (HLCs) is a three-step process, from the undifferentiated E-iPSC to definitive endoderm (DE), hepatic endoderm (HE) and ultimately HLCs. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4a and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. The lack of viral DNA integrations in these E-iPSCs endow them superior to viral-derived iPSCs for (i) modeling gene regulatory networks associated with hepatogenesis and gastrulation in general, (ii) toxicology studies and (iii) drug screening platforms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE66282 | GEO | 2017/09/06

SECONDARY ACCESSION(S): PRJNA276409

REPOSITORIES: GEO

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Project description:Background: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and genetic liver diseases. However the derivation of iPSCs is restricted by ethical and procedural constraints. Furthermore, incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. If liver derived iPSCs (Li-iPSCs) maintain a liver specific gene expression profile is not known. It is also not known, if Li-iPSCs would allow the generation of more fully differentiated HLCs.Methods: Primary liver cells (PLCs) were expanded from liver needle biopsies and reprogrammed into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles.Results: PLCs were obtained from small pieces of liver biopsies and could be reprogrammed into Li-iPSCs. Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues.Conclusions: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.

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Footprint-free human fetal foreskin derived iPSCs: a tool for modeling hepatogenesis associated gene regulatory networks

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