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A two-step de-differentiation and maturation of primary human hepatocytes in vitro with efficient metabolic and regenerative capacities

ABSTRACT: Hepatocytes hold significant promise for applications in drug screening, disease modeling, and clinical cell transplantation. Generating large amouts of hepatocyte in vitro for those application is still challenging. We have previously established a 5C medium for long-term culture and functional maintanace human primary hepatocytes (PHHs) in vitro. However, 5C condtion could not support hepatocyte expansion, which limited its application. In this study, we established a new hepatocyte expansion medium (NHEM) for efficient hepatocyte expansion, which turned the PHHs into hepatic progenitor-like cells (HPLCs). We also generated an optimized 6C condtion for HPLCs maturation. Overall, this study provide a new strategy to generate large amounts of hepatocytes in vitro.

ORGANISM(S): Homo sapiens

PROVIDER: GSE261962 | GEO | 2024/09/23

REPOSITORIES: GEO

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Project description:Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e. personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECM) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned co-culture (iMPCC) platform in a multi-well format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel™). We assessed iHep maturity via global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal CYP450 activities, phase-II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. Conclusion: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multi-organ responses to compounds. We used Affymetrix microarrays to profile the global gene expression of co-culture stabilized iHeps (iMPCCs) relative to freshly isolated and co-culture stabilized primary human hepatocytes (2 donors). To assess the transcriptomic stability of iHeps in iMPCCs, RNA was extracted following 9 and 21 days of culture for hybridization to Affymetrix microarrays. The hepatic maturation state of iHeps was assessed by comparing gene expression against microarrays containing data from two primary human hepatocyte donors, both following hepatocyte isolation (day 0) and after stabilization in the micropatterened co-culture platform (day 6 and day 42 MPCCs), as previously described.

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A two-step de-differentiation and maturation of primary human hepatocytes in vitro with efficient metabolic and regenerative capacities

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