Improved maturity and yield of pluripotent stem cell-derived hepatocytes for 2D and 3D modeling of human liver pathophysiology
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE152390 | GEO | 2022/02/03
REPOSITORIES: GEO
Similar Datasets
Project description:Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2×106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6×106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.
2024-10-23 | GSE267210 | GEO
Project description:Improved maturity and yield of pluripotent stem cell-derived hepatocytes for 2D and 3D modeling of human liver pathophysiology
| PRJNA639504 | ENA
Project description:Human pluripotent stem cells (hPSCs) can differentiate into all cell types in the body that may replace current cell sources applied in regenerative medicine, cell therapy, drug discovery and development and general research. Human PSC-derived hepatocyte-like cells (HLCs) have the potential to replace primary hepatocytes and other cell models applied in liver disease treatment and drug discovery and development. These cells share many features with their in vivo counterparts however, the generation of fully functional hPSC-derive HLCs is still lacking, which prevent their application in the previously mentioned fields. This study followed the transcriptome dynamics during the differentiation of hPSC-derived HLCs at definitive endoderm, hepatoblast, early HLC and late HLC developmental stages and the controls hPSCs and human liver tissues which consists of at least 70% hepatocytes. The aim is to reveal expression deviations between hPSC-derived hepatocytes and their in vivo counterparts that may contribute to the modification of differentiation protocols to generate fully functional hepatocytes.
2017-08-09 | E-MTAB-5367 | biostudies-arrayexpress
Project description:The differentiation of pluripotent stem cells to hepatocyte-like cells offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of hepatic-like cells (HLC) remains controversial. To obtain an unbiased characterization, we performed a transcriptomics study using the Affymetrix Gene Chip® HG-U133 plus 2.0, with HLC derived from embryonic and induced stem cells (ESC, hiPSC) from two different laboratories. Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14 days cultivated primary human hepatocytes (see submission E-MTAB-3994) obtained under patient informed consent from three male donors. Three biological replicas were used for each ESC and HLC models. (associated data set: E-MTAB-3994 - Gene expression profile of human embryonic stem cell-derived hepatocyte-like cells in matrigel and recombinant laminins)
2016-02-24 | E-MTAB-4442 | biostudies-arrayexpress
Project description:Hepatocytes isolated from DILI patient's liver (#2064) were cultured for a long term using irrMEF and EMUKK-05, and comprehensive gene expression was compared between Puromycin-treated and non-treated groups. In addition, comprehensive gene expression analysis of human mature hepatocytes, primary cultured cells, and ips cell-derived hepatocyte-like cells were performed as controls. Two-condition experiment, Proliferating hepatocytes (ProilHH) vs. puromycin-treated ProliHH. Primary human hepatocytes (PHH) and isolated humen mature hepatocytes (MH) and human iPSC-derived hepatocyte-like cells (HLC) as controls.
2021-12-30 | GSE192653 | GEO
Project description:In both the regulatory and commercial arenas, the goal is to move away from time consuming, costly in-life rodent studies and towards safety assessment strategies that rely on testing species-relevant cells in vitro. The liver has been a major focus of these efforts, yet there are currently no in vitro alternatives for hepatotoxicity testing accepted by regulators, and the assays that do exist typically utilize hepatocyte monolayer culture. While hepatocytes have been the primary component of in vitro hepatotoxicity assay development, the non-parenchymal cells (NPCs) (i.e., hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells) also play a critical role in the progression of liver pathologies. The goal of this study was to develop an organotypic co-culture system that could read out the various mechanisms of action observed in vivo, as well as maintain metabolic capability and extended viability to allow for repeat dosing. We developed a two-dimensional 96-well plate-based co-culture system that includes primary rat hepatocytes, stellate, Kupffer, and endothelial cells that supports hepatocyte viability and phenotype stability for up to eight days. Importantly, markers of hepatocyte differentiation and polarity are maintained in co-culture, but hepatocyte monocultures with no other cell types lose phenotypic markers after prolonged culture. This co-culture model was leveraged to assess the effects of known hepatotoxic stimuli, and the co-culture model resulted in responses that were more representative of the in vivo phenotypes.
2021-07-01 | GSE133717 | GEO
Project description:Recent advances towards the efficient and reproducible differentiation of stem cells into hepatocyte-like cells (HLC) have created potential opportunities in clinical transplantation. For this approach to be successfully translated, however, it is necessary to understand the immune response to stem cell-derived cellular products. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. Here we determine the expression of immune recognition molecules on HLC generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluate the impact on recognition by NK cells. We report that HLC lack MHC class I expression, and that embryonic stem cell derived-HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I render embryonic stem cell derived-HLC, and induced pluripotent stem cell derived-HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell derived-HLC, and induced pluripotent stem cell derived-HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell killing of embryonic stem cell derived-HLC, but not induced pluripotent stem cell-derived HLC. Hepatocytes were isolated from FVB/NJ mice and C57Bl/6 mice using a modification of the Seglen’s perfusion technique.
2017-11-14 | GSE106829 | GEO
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.
2015-02-01 | E-GEOD-62962 | biostudies-arrayexpress
Project description:The zebrafish is as a powerful vertebrate model system for modeling human disease including liver pathology. In ZFE, hepatic responses can be expected after exposure to hepatotoxicants, because hepatocytes are present from 36-hpf and at 72-hpf the liver is fully functioning. These characteristics make the whole ZFE an attractive alternative model for compound-induced hepatotoxicity screening. Therefore, the main objective of this study is to further strengthen the applicability of whole ZFE as an alternative model for hepatotoxicity testing, with a special focus on the ability to identify gene expression responses in whole ZFE that are suggestive of hepatotoxicity. Deep sequence technology is applied to assess whether hepatotoxicity-specific transcripts that are identified in livers of hepatotoxicant treated adult zebrafish can be detected in whole ZFE as well.
2015-05-15 | GSE38042 | GEO
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).
2015-02-01 | GSE62962 | GEO