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Necroptosis Is a Mechanism of Death in Mouse Induced Hepatocyte-Like Cells Reprogrammed from Mouse Embryonic Fibroblasts.

ABSTRACT: Liver transplantation is recommended for patients with liver failure, but liver donors are limited. This necessitates the development of artificial livers, and hepatocytes are necessary to develop such artificial livers. Although induced hepatocyte-like cells are used in artificial livers, the characteristics of mouse induced hepatocyte-like cells (miHeps) reprogrammed with embryonic fibroblasts have not yet been clarified. Therefore, this study investigated the mechanisms underlying the survival, function, and death of miHeps. miHeps showed decreased cell viability, increased cytotoxicity, decreased hepatic function, and albumin and urea secretion at passage 14. Addition of necrostatin-1 (NEC-1) to miHeps inhibited necrosome formation and reactive oxygen species generation and increased cell survival. However, NEC-1 did not affect the hepatic function of miHeps. These results provide a basis for development of artificial livers using hepatocytes.

SUBMITTER: Lee YS

PROVIDER: S-EPMC6078850 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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Necroptosis Is a Mechanism of Death in Mouse Induced Hepatocyte-Like Cells Reprogrammed from Mouse Embryonic Fibroblasts.

Lee Yun-Suk YS Park Kyung-Mee KM Yu Lina L Kwak Ho-Hyun HH Na Hee-Jun HJ Kang Kyung-Sun KS Woo Heung-Myong HM

Molecules and cells 20180710 7

Liver transplantation is recommended for patients with liver failure, but liver donors are limited. This necessitates the development of artificial livers, and hepatocytes are necessary to develop such artificial livers. Although induced hepatocyte-like cells are used in artificial livers, the characteristics of mouse induced hepatocyte-like cells (miHeps) reprogrammed with embryonic fibroblasts have not yet been clarified. Therefore, this study investigated the mechanisms underlying the surviva ...[more]

PMID: 29991669

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Project description:BackgroundThe liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male's liver are good in alcohol clearance and lipid metabolism, while female's liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently.MethodsMouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model.ResultsIn this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex.ConclusionIn conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Project description:BACKGROUND:The development of somatic reprogramming, especially purely chemical reprogramming, has significantly advanced biological research. And chemical-induced extraembryonic endoderm-like (ciXEN) cells have been confirmed to be an indispensable intermediate stage of chemical reprogramming. They resemble extraembryonic endoderm (XEN) cells in terms of transcriptome, reprogramming potential, and developmental ability in vivo. However, the other characteristics of ciXEN cells and the effects of chemicals and bFGF on the in vitro culture of ciXEN cells have not been systematically reported. METHODS:Chemicals and bFGF in combination with Matrigel were used to induce the generation of ciXEN cells derived from mouse embryonic fibroblasts (MEFs). RNA sequencing was utilised to examine the transcriptome of ciXEN cells, and PCR/qPCR assays were performed to evaluate the mRNA levels of the genes involved in this study. Hepatic functions were investigated by periodic acid-Schiff staining and indocyanine green assay. Lactate production, ATP detection, and extracellular metabolic flux analysis were used to analyse the energy metabolism of ciXEN cells. RESULTS:ciXEN cells expressed XEN-related genes, exhibited high proliferative capacity, had the ability to differentiate into visceral endoderm in vitro, and possessed the plasticity allowing for their differentiation into induced hepatocytes (iHeps). Additionally, the upregulated biological processes of ciXEN cells compared to those in MEFs focused on metabolism, but their energy production was independent of glycolysis. Furthermore, without the cocktail of chemicals and bFGF, which are indispensable for the generation of ciXEN cells, induced XEN (iXEN) cells remained the expression of XEN markers, the high proliferative capacity, and the plasticity to differentiate into iHeps in vitro. CONCLUSIONS:ciXEN cells had high plasticity, and energy metabolism was reconstructed during chemical reprogramming, but it did not change from aerobic oxidation to glycolysis. And the cocktail of chemicals and bFGF were non-essential for the in vitro culture of ciXEN cells.

Dataset Information

Necroptosis Is a Mechanism of Death in Mouse Induced Hepatocyte-Like Cells Reprogrammed from Mouse Embryonic Fibroblasts.

Publications

Necroptosis Is a Mechanism of Death in Mouse Induced Hepatocyte-Like Cells Reprogrammed from Mouse Embryonic Fibroblasts.

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