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Conversion of terminally committed hepatocytes to culturable bipotent progenitor cells with regenerative capacity

ABSTRACT: A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and if such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01 and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus our study advances the goals of liver regenerative medicine. Transcriptomic analyses for freshly isolated MHs and cells cultured for the designated periods with or without YAC stimulation (Matrix 1). Transcriptomic analysis for CLiPs which underwent hepatic induction (Matrix 2). Transcriptomic comparison of hepatic inducibility between CLiPs at early passage and those at late passages (Matrix 3). Transcriptomic comparison between chimera-derived rat cells (designated as “2nd”) and primary rat MH-derived cells (designated as “1st”) (Matrix 4).

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE87611 | GEO | 2016/11/15

SECONDARY ACCESSION(S): PRJNA345379

REPOSITORIES: GEO

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Project description:Background: HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. Treatment with HGF has been shown to accelerate resolution of fibrotic liver lesions in experimental animal models. To formally address the importance of c-Met signaling in hepatocytes in the context of chronic liver injury, we have used hepatocyte-specific Metfl/fl;Alb-Cre+/- conditional knockout mice (KO) and a model of liver fibrosis. Methods: CCl4 was administrated biweekly over a period of 4 weeks (injury phase), and the animals were followed over the next 4 weeks (healing phase). Macroscopic and microscopic changes during the injury and healing phases were monitored by IHC. Deposition of ECM was assessed by Sirius red staining and hydroxiproline content. Activation of hepatic stellate cells (HSC) was estimated by a-SMA using WB and IHC. Expression levels of the selected key fibrotic molecules were evaluated by RT-qPCR and WB. Time-dependent global transcriptomic changes from whole livers and isolated hepatocytes were examined using gene expression microarrays. Results: Loss of HGF/c-Met signaling in hepatocytes altered the hepatic microenvironment and dramatically aggravated hepatic fibrogenesis. Increased liver damage was associated with decreased hepatocyte proliferation, progressive accumulation of HSCs, and delayed fibrinolysis causing increased collagen deposit. Dystrophic calcification of necrotic areas impaired phagocytosis, resulting in sustained inflammatory and fibrogenic signaling further augmenting severity of fibrogenesis. Global gene-expression analysis demonstrated upregulation of key fibrogenic molecules, such as Tgf-Ã¢ and Pdgf-Ã¢, paralleled by a decreased expression of genes important for cell cycle, stress response and regeneration, which could be attributed to the c-Met deficiency in hepatocytes. Additionally, key chemotactic and inflammatory cytokines, including Ccl2, SDF1/Cxcr4 and Spp1, were upregulated in Metfl/fl;Alb-Cre+/- hepatocytes. However, the major pro-fibrotic signaling originated from the non-parenchymal cell compartments, as revealed by cell type-specific gene expression signatures. Conclusion: These results indicate that lack of c-Met signaling in hepatocytes disrupts the balance between extracellular matrix production and degradation and establish a protective role for c-Met against adverse microenvironment leading to the development of fibrotic liver disease. In the present study, we reported a detailed and comprehensive dynamic characterization of the cellular and molecular alterations involved in fibrosis in the liver of c-Met transgenic mice. Liver samples from female animals were collected at various time-points after fibrosis induction using CCL4 ranging from 0 weeks to 3 weeks. Tissue samples were divided into two parts; one was fixed in 10% formalin for histological evaluation and the other was used for RNA analysis.

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Conversion of terminally committed hepatocytes to culturable bipotent progenitor cells with regenerative capacity

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