Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of human pluripotent stem cell-derived hepatocytes with adult features and potential for modeling metabolic diseases

ABSTRACT: Liver disease and toxicity that causes impaired liver functionality have severe effects on normal body functions. There is a strong need for better and more predictable in vitro models for studying disease and improving mechanistic understanding of adverse effects of drugs in humans. This experiment aimed to characterize human induced pluripotent stem cell-derived hepatocytes (hiPS-HEP), in order to assess their potential as in vitro tools for metabolism studies and disease modeling. hiPS-HEP from three cell lines were compared to human primary hepatocytes from three donors, before and after plating.

ORGANISM(S): Homo sapiens

SUBMITTER: Benjamin Ulfenborg

PROVIDER: E-MTAB-8286 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Rifampicin and efavirenz are commonly used medicines to treat tuberculosis (rifampicin) and HIV/AIDS (efavirenz). Rifampicin and efavirenz are metabolized in the liver and exposure to these medicines can alter microRNA expression in hepatocytes. Changes in microRNA expression may affect metabolism of these medicines and potentially influence how patient's respond to therapy. It is important to understand changes in microRNA expression in a hepatic cell-based model. Differentiated HepaRG cells were used because mRNA expression and induction of drug metabolizing enzymes are comparable to that of primary human hepatocytes yet differentiated HepaRG cells have an extended lifespan. Differentiated HepaRG cells were obtained from Merck Millipore and cultured as an adherent cell line at a density of 315000 cells/well using collagen I coated 24-well plates. Clinically relevant concentrations of rifampicin and efavirenz were selected as 6.4uM efavirenz (dissolved in 100% dimethyl sulfoxide) and 24.4uM rifampicin (dissolved in 100% dimethyl sulfoxide), while 0.02% dimethyl sulfoxide was used as control to minimize toxicity. HepaRG serum-free induction medium was used to dilute efavirenz and rifampicin. On day 7 since thawing differentiated HepaRG cells, these cells were treated with 6.4uM efavirenz or 24.4uM rifampicin or 0.02% dimethyl sulfoxide for a period of 24 hours. Three biological replicates were available for each treatment condition. Total RNA was isolated from the cells, after treatment, using the Quick-RNATM MiniPrep Kit from Zymo Research Corporation. MicroRNA expression profiling was performed using the TaqMan® OpenArray® Human MicroRNA Panel and QuantStudioTM 12K Flex system. The R/Bioconductor package “Automated Analysis of High-Throughput qPCR Data” were used for data analyses. CT values were used for differential expression analysis and microRNAs with a CT value > 35 was considered undetected. MicroRNAs undetected in any of the replicate samples or microRNAs with AmpScore < 1.24 or CqConf < 0.8 were excluded. Quantile normalization was followed by limma analyses to identify differentially expressed microRNAs for efavirenz versus dimethyl sulfoxide and rifampicin versus dimethyl sulfoxide.

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Characterization of human pluripotent stem cell-derived hepatocytes with adult features and potential for modeling metabolic diseases

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