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Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor.

ABSTRACT: A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.

SUBMITTER: Rodriguez-Aguilera JR

PROVIDER: S-EPMC7210258 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor.

Rodríguez-Aguilera Jesús Rafael JR Ecsedi Szilvia S Goldsmith Chloe C Cros Marie-Pierre MP Domínguez-López Mariana M Guerrero-Celis Nuria N Pérez-Cabeza de Vaca Rebeca R Chemin Isabelle I Recillas-Targa Félix F Chagoya de Sánchez Victoria V Hernández-Vargas Héctor H

Scientific reports 20200508 1

A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region ...[more]

PMID: 32385352

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Project description:BackgroundResearch directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study.MethodsE12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression.ResultsE12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described.ConclusionThe availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.

Dataset Information

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor.

Publications

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor.

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