Project description:iHep cells maintained in two-dimensional (2D) culture can become functionally close to hepatocytes by forming cell aggregates under three-dimensional (3D) culture condition.
Project description:In order to characterize iHep cells more precisely, we conducted global gene expression analyses using microarrays to compare among the gene expression profiles of MEFs, iHep cells and adult mouse hepatocytes. Three MEF samples that were individually prepared from 13.5 days post coitum (dpc) embryos, three types of iHep cells and hepatocytes obtained from the adult mouse liver were used.
Project description:Plasticity of differentiated cells has been proved by nuclear transfer, induced pluripotent cells and transdifferentiation. Here we show that by transduction of 3 factors (Hnf1alpha, Gata4, and Foxa3) and p19Arf inactivation, tail-tip fibroblasts can be converted to hepatocyte-like (iHep) cells, expressing hepatocyte marker genes, and acquiring many mature hepatocyte functions in vitro and in vivo. p19Arf-null TTFs were tranfected with 3 liver enriched transcription factors, then changed to modified Block's medium. To enrich iHep cells, epithelial cells were enriched by partial trypsin digestion.
Project description:We have found three specific combinations of two transcription factors, comprising Hnf4alpha plus Foxa1, Foxa2 or Foxa3, could convert mouse embryonic fibroblasts (MEFs) into cells that closely resemble hepatocytes in vitro. Then we used RNA-seq to explore the conversion event to the iHep cells.
Project description:In order to characterize iHep cells more precisely, we conducted global gene expression analyses using microarrays to compare among the gene expression profiles of MEFs, iHep cells and adult mouse hepatocytes.
Project description:In the first group of samples, we are looking into the different metabolites and their concentrations within our human embryonic stem cells over the course of differentiation. Our goal is to study how the metabolism of stem cells are changing as they undergo this conversion to become hepatocyte-like cells. In the second group of samples labeled HyCell, we would like to understand the concentrations of metabolites within our CHO cells in order to update the parameters for our metabolic model. Included are two samples from different days of a suspension batch culture which may have different lactate production qualities.
Project description:Plasticity of differentiated cells has been proved by nuclear transfer, induced pluripotent cells and transdifferentiation. Here we show that by transduction of 3 factors (Hnf1alpha, Gata4, and Foxa3) and p19Arf inactivation, tail-tip fibroblasts can be converted to hepatocyte-like (iHep) cells, expressing hepatocyte marker genes, and acquiring many mature hepatocyte functions in vitro and in vivo.
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.
Project description:We have found three specific combinations of two transcription factors, comprising Hnf4alpha plus Foxa1, Foxa2 or Foxa3, could convert mouse embryonic fibroblasts (MEFs) into cells that closely resemble hepatocytes in vitro. Then we used ChIP-seq to explore the targets of Hnf4alpha during conversion event from MEFs to iHep cells.
Project description:We examined how each chemical contributed to hepatocyte revitalization by removing each component of the 5C induction cocktail individually. We then compared deach chemical contributed to hepatocyte revitalization by removing each component of the 5C induction cocktail individually