In vitro culture at 39° C during hepatic maturation of human ES cells facilitates hepatocyte-like cell functions
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE172227 | GEO | 2022/03/29
REPOSITORIES: GEO
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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:Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. We demonstrate the purification of a sub-population of functional HLCs differentiated from multiple hPSC lines using the hepatocyte surface marker Asialoglycoprotein Receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared to unsorted HLCs.
2016-01-22 | GSE77086 | GEO
Project description:Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) hold great promise for liver disease modeling, drug discovery, drug toxicity screens, or even regenerative therapies. Yet, several hurdles still need to be overcome, including among others decrease in the cost of goods to generate HLCs and automation of the differentiation process. We here describe that use of an automated liquid handling system results in highly reproducible HLC differentiation from hPSCs. This enabled us to screen 92 chemicals to replace expensive growth factors at each step of the differentiation protocol to reduce the cost of goods of the differentiation protocol by approximately 79%. In addition, we also evaluated several recombinant extracellular matrices (ECM) to replace Matrigel. We demonstrated that differentiation of hPSCs on Laminin-521 using an optimized small molecule combination resulted in HLCs that were transcriptionally identical to HLCs generated using current growth factor combinations. In addition, the HLCs created using the optimized small molecule combination also secreted similar concentrations of albumin and urea, and relatively low concentrations of alfa-fetoprotein (AFP), displayed similar CYP3A4 functionality and a similar drug toxicity susceptibility as HLCs generated with growth factor cocktails. The broad applicability of the new differentiation protocol was demonstrated for four different hPSC lines. This allowed the creation of a scalable, xeno-free, and cost-efficient hPSC-derived HLC culture, suitable for high throughput disease modeling and drug screenings, or even for the creation of HLCs for regenerative therapies.
2024-02-26 | GSE207569 | GEO
Project description:Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPSHLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drugmetabolizing capacity and drug responsiveness.
2014-11-12 | GSE61287 | GEO
Project description:Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver disease and to test drugs. However, despite their advantages over other hepatocyte culture systems, HLCs lack several in vivo characteristics including cell polarity, which is critical for proper hepatocyte biology. We report a stem cell-based differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. Unlike conventional HLCs, polarized HLCs secrete cargo directionally, similar to hepatocytes in vivo. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.
2020-02-25 | GSE123462 | GEO
Project description:Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). E-iPSCs were fully characterized and their transcriptomes are more similar to that of hESCs (R2 = 0.9363) in comparison to viral-derived HFF1-iPSCs (R2 = 0.8176). We used an E-iPSC-line to model hepatogenesis in vitro. The differentiation of iPSCs into hepatocyte-like cells (HLCs) is a three-step process, from the undifferentiated E-iPSC to definitive endoderm (DE), hepatic endoderm (HE) and ultimately HLCs. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4a and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. The lack of viral DNA integrations in these E-iPSCs endow them superior to viral-derived iPSCs for (i) modeling gene regulatory networks associated with hepatogenesis and gastrulation in general, (ii) toxicology studies and (iii) drug screening platforms.
2017-09-06 | GSE66282 | GEO
Project description:Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between WT HLCs and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S-mephenytoin metabolites using LC-MS. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogrel- and ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogrel- and DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict the clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome editing technologies for pharmaceutical research.
2019-05-07 | GSE123937 | GEO
Project description:The purpose of this experiment was to compared the transcriptome of hepatocyte-like cells (HLCs) generated in vitro and adult primary human hepatocytes (PHHs). HLCs were differentiated from either hESCs or hIPSCs using previously established protocols (Hannah et al 2013; Segeritz et al 2018). Undifferentiated hIPSCs were used as control to confirm differentiation status. PHHs were commercially sourced as well as freshly isolated from donors.
2022-08-11 | E-MTAB-10634 | biostudies-arrayexpress
Project description:Background & Aims: Obesity is the most important risk factor and a potential treatment target for fatty liver disease (FLD). The continuous adaptation or ‘remodelling’ of hepatic mitochondrial flexibility plays a key role in the pathogenesis of FLD. Human adipose stem cell (hASC)-derived hepatocyte-like cells (HLCs) offer attractive tools for the research and therapy of liver dysfunction. However, evidence showing the ‘remodelling’ of hepatic mitochondrial flexibility or differentiation capability of hASCs in obese patients is lacking. Methods: The differentiation capability and mitochondrial structure and function of hepatic cells were evaluated by measuring gene expression, de novo lipogenesis and mitochondrial respiration in HLCs derived from hASCs of obese patients and lean controls. The ability of the GSK3 inhibitor CHIR-99021 to modify the mitochondrial oxidative dysfunction was evaluated in the HLCs derived from the hASCs of obese patients. Results: hASCs from obese patients showed the potential to differentiate into HLCs. HLCs from the hASCs of obese patients exhibited the characteristics of hepatic steatosis, lower expression of the subunits of mitochondrial complex I and lower oxidative phosphorylation levels. CHIR-99021 promoted the expression of NDUFB8, NDUFB9, the subunits of mitochondrial complex I, and the basal oxygen consumption rate of the HLCs derived from the hASCs of obese patients by upregulating the expression of PGC-1α, the transcription factor involved in mitochondrial biogenesis and ‘remodelling’. Conclusions: The results demonstrate that obese patient ASC-derived HLCs had mitochondrial oxidative dysfunction, which may represent the consequence of hepatic steatosis.
2021-03-19 | GSE151760 | GEO
Project description:Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to primary human hepatocytes (PHH). Here, we systematically compared the activation of four different stress pathways in PHH, HepG2 liver cancer cells, hiPSCs and different stages within the differentiation towards HLCs (definitive endoderm, hepatoblast, immature hepatocytes and HLCs). We exposed all different cell types in a concentration response to four different compounds that specifically activate the oxidative stress response (diethyl-maleate), unfolded protein response (tunicamycin), DNA damage response (cisplatin) and inflammatory signalling (TNF). We used targeted RNA-sequencing to map concentration response transcriptional similarities and differences using bench-mark concentration modelling for the various stress responses in the different test systems. We observed that HLCs are more sensitive to oxidative stress than PHH showing activation at ten-fold lower concentrations and induce a strong anti-oxidant response. Although similar UPR gene sets were activated in HLCs compared to PHH, PHH were about ten times more sensitive. Furthermore, HLCs were highly sensitive to inflammation conditions similar to HepG2 cells and in sharp contrast to hiPSC, which showed hardly any response. On the contrary, hiPSC and HepG2 were highly sensitive to DNA damage signalling while HLCs resisted a strong p53 DNA damage response upon exposure. Overall, the data indicate that despite limitation in full hepatocyte maturation, HLCs did gain specific adaptive cellular stress response networks that mimic those of PHHs.
2021-02-18 | GSE155771 | GEO