Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Generation of integration-free induced hepatocytes from mouse fibroblasts

ABSTRACT: Previous studies demonstrated that hepatocyte-specific transcription factors could directly convert fibroblasts into functional hepatocytes-like cells, namely induced hepatocytes (iHeps) using viral systems. However, viral integration into host genome causes insertional mutation and risk of tumorigenecity. we showed iHeps could be generated from MEFs using the integration-free system. They were expandable in vitro and showed hepatic features, similar to primary hepatocytes. iHeps_G4H1F3 transfected (Episomal vectors) and iHeps_G4H1F3 transduced (pMXs) were duplicate, respectively. MEFs and primary hepatocytes were used as negative and positive controls, respectively.

ORGANISM(S): Mus musculus

SUBMITTER: Jonghun Kim

PROVIDER: E-GEOD-67362 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Hepatocytes that have differentiated from human embryonic stem cells have great potential for the treatment of liver disease as well as for drug testing. Moreover, in vitro hepatogenesis is a powerful model system for studying the molecular mechanisms underlying liver development. DNA methylation is an important epigenetic mechanism that influences differential gene expression during embryonic development. We profiled gene expression and DNA methylation of three cell states of in vitro hepatogenesis—human embryonic stem cells, endoderm progenitors, and mature hepatocytes—using microarray analysis. Among 525 state-specific expressed genes, 67 showed significant negative correlation between gene expression and DNA methylation. State-specific expression and methylation of target genes were validated by quantitative reverse transcription–PCR and pyrosequencing, respectively. To elucidate genome-scale methylation changes beyond the promoter, we also performed high-throughput sequencing of methylated DNA captured by MBD2 protein [see SRA link below]. We found dynamic methylation changes in intergenic regions of the human genome during differentiation. Conclusion: This study provides valuable methylation markers for the lineage commitment of in vitro hepatogenesis and should help elucidate the molecular mechanisms underlying stem cell differentiation and liver development. ES, EP, and MH expression assays were run in quadruplicate. RNA isolated from the cell line at the indicated time points was used for gene expression analysis using the Human-6 Whole-Genome Expression BeadChip (Illumina, San Diego, CA), which generates expression profiles for more than 46,000 human transcripts. Biotin-labeled cRNA was produced using a linear amplification kit (Ambion, Austin, TX) using 300 ng of quality-checked total RNA as input. Chip hybridizations, washing, Cy3-streptavidin staining, and scanning were performed on a BeadArray Reader (Illumina) platform using reagents and following protocols supplied by the manufacturer. The detection score was used to determine expression

Project description:Hepatocytes that have differentiated from human embryonic stem cells have great potential for the treatment of liver disease as well as for drug testing. Moreover, in vitro hepatogenesis is a powerful model system for studying the molecular mechanisms underlying liver development. DNA methylation is an important epigenetic mechanism that influences differential gene expression during embryonic development. We profiled gene expression and DNA methylation of three cell states of in vitro hepatogenesis—human embryonic stem cells, endoderm progenitors, and mature hepatocytes—using microarray analysis. Among 525 state-specific expressed genes, 67 showed significant negative correlation between gene expression and DNA methylation. State-specific expression and methylation of target genes were validated by quantitative reverse transcription–PCR and pyrosequencing, respectively. To elucidate genome-scale methylation changes beyond the promoter, we also performed high-throughput sequencing of methylated DNA captured by MBD2 protein [see SRA link below]. We found dynamic methylation changes in intergenic regions of the human genome during differentiation. Conclusion: This study provides valuable methylation markers for the lineage commitment of in vitro hepatogenesis and should help elucidate the molecular mechanisms underlying stem cell differentiation and liver development. ES, EP, and MH methylation assays were run in triplicate. Genomic DNA (1 µg) from each sample was bisulfite converted using the EZ DNA methylation kit (Zymo Research, Orange, CA), and 200 ng of the converted DNA was used for amplification. Amplified DNA was hybridized to the Infinium HumanMethylation27 BeadChip (Illumina), which analyzes the methylation status of 27,578 CpG sites selected from more than 14,495 well-annotated genes. The arrays were imaged using the BeadArray Reader, and image processing and extraction of intensity data were performed according to Illumina’s instructions. Each methylation signal was used to compute the β value, which is a quantitative measure of DNA methylation ranging from 0 (for completely unmethylated cytosines) to 1 (for completely methylated cytosines).

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:Studies in mice have shown that PPARalpha is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARalpha in human liver. Here we set out to compare the function of PPARalpha in mouse and human hepatocytes via analysis of target gene regulation. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARalpha agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARalpha expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARalpha in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARalpha targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARalpha in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARalpha targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARalpha activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARalpha as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARalpha regulates a mostly divergent set of genes in mouse and human hepatocytes. Experiment Overall Design: Primary hepatocytes from 6 human subjects were treated with the PPARalpha agonist Wy14643 for 6 and 24 hours, and gene expression profiling was performed using Affymetrix GeneChips. Human hepatocytes and Hepatocyte Culture Medium Bulletkit were purchased from Lonza Bioscience (Verviers, Belgium). Primary hepatocytes were isolated from surgical liver biopsies obtained from six individual donors who underwent surgery after informed consent was obtained for surgery with subsequent use of samples in experiments. Hepatocytes were isolated with two-step collagenase perfusion method and the viability of the cells was over 80%. Cells were plated on collagen-coated six-well plates and filled with maintenance medium. Upon arrival of the cells, the medium was discarded and was replaced by Hepatocyte Culture Medium (HCM) with additives. The additives included Gentamicin sulphate/Amphotercin-B, Bovine serum albumin (Fatty acid free), Transferrin, Ascorbic acid, Insulin, Epidermal growth factor, Hydrocortisone hemisuccinate. The next day, cells were incubated in fresh medium in the presence or absence of Wy14643 (50 microM) dissolved in DMSO for 6 and 24 hours, followed by RNA isolation.

Project description:Studies in mice have shown that PPARÎ± is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARÎ± in human liver. Here we set out to compare the function of PPARÎ± in mouse and human hepatocytes via analysis of target gene regulation. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARÎ± agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARÎ± expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARÎ± in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARÎ± targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARÎ± in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARÎ± targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARÎ± activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARÎ± as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARÎ± regulates a mostly divergent set of genes in mouse and human hepatocytes. GSE17250: Comparative analysis of gene regulation by the transcription factor PPARÎ±_mouse; GSE17251: Comparative analysis of gene regulation by the transcription factor PPARÎ±_human Experiment Overall Design: Refer to individual Series

Project description:The liver is one of most important organs in our bodies. It performs many essential functions including metabolism, synthesis, secretion, detoxification, and storage. Hepatocytes are the principal cell type in the liver and are involved in multiple liver-specific functions. There have been several efforts to develop in vitro culture systems capable of maintaining hepatocyte-specific phenotype over long time periods. In hepatic tissue engineering, two commonly used culture systems are the collagen sandwich and monolayers of cells. ‚àö√á¬¨‚Ä†In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and analyzed via Gene Set Enrichment Analysis (GSEA), which is a powerful method to elicit biologically meaningful information from microarray data at the level of gene sets. Results indicate that the gene expression in hepatocytes in collagen sandwich cultures gradually diverges from that in monolayer cultures. Gene sets up-regulated in collagen sandwich cultures include those associated with liver metabolic and synthetic functions. ‚àö√á¬¨‚Ä†These functions are associated with lipid, amino acid, carbohydrate, and alcohol metabolism and bile acid synthesis. Nuclear receptors are up-regulated in collagen sandwiches 24 hours after seeding. Signals transmitted from these receptors may cause the up-regulation of other processes in subsequent days. Cytochrome-P450 monooxygenase expression was initially down-regulated but exhibited up-regulation after 72 hours. Our results provide a baseline for further explorations into the systems biology of engineered liver mimics as well as 2D and 3D co-cultures of primary hepatocytes and non-parenchymal cells. To better understand differences in quality of in vitro growth of rat hepatocytes between culture on a monolayer of collagen gel and sandwiched between two layers of gel, we measured gene expression in hepatocytes in these two culture conditions in triplicate for four time points: 1 day, 2 days, 3 days, and 8 days of culturing. Overall, we obtained Affymetrix microarray data for 24 samples, divided to 12 samples from monolayer and double layer cultures each, each of which are divided into four time points with 3 sample replicates.

Dataset Information

Generation of integration-free induced hepatocytes from mouse fibroblasts

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure