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Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells.

ABSTRACT: Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating in vitro has been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated morphology and exhibit gene expression profiles similar to adult PHHs, but importantly, they retain their phenotype over several passages. Further, unlike previous reports, where extensive manipulations of culture conditions are required to convert SHPCs to metabolically functional hepatocytes, SHPCs in our co-culture system maintain expression of xenobiotic metabolism-associated genes. We show that SHPCs in co-culture are able to perform xenobiotic metabolism at rates equal to their parent PHHs as evidenced by the metabolism of acetaminophen to all of its major metabolites. In summary, we present an improved co-culture system that allows generation of SHPCs from adult PHHs that maintain their differentiated phenotype over multiple passages. Our findings would be useful for expansion of limited PHHs for use in studies of drug metabolism and toxicity testing.

SUBMITTER: Sengupta S

PROVIDER: S-EPMC8320630 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells.

Sengupta Srikumar S Johnson Brian B Seirup Morten M Ardalani Hamisha H Duffin Bret B Barrett-Wilt Gregory A GA Stewart Ron R Thomson James A JA

Current research in toxicology 20200601

Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating <i>in vitro</i> has been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated ...[more]

PMID: 34345838

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Project description:Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

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Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells.

Publications

Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells.

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