RNA-sequencing of multicellular human liver organoids derived from 3 different iPS cells
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ABSTRACT: By taking advantage of the foregut generation method, we initially differentiated iPSCs to foregut spheroids through definitive endoderm specification as described. The foregut spheroids were embedded in Matrigel and cultured with retinoic acid (RA). Following 4-day RA treatment, we switched into hepatocyte maturation media for the induction of the hepatocyte differentiation process to establish human liver organoids, hereafter defined as HLO, as early as day 20. To characterize the lipid metabolism related genes in HLO, we conducted RNA sequencing to unbiasedly identify and visualize coordinately expressed gene signatures among groups of three different iPSC derived HLO, and various stage-derived primary-liver cells and tissues as previously described. Correlation spanning tree based on self-organizing maps analysis using 18,338 genes organized into 400 metagenes confirmed overall similarities between HLO and primary hepatocytes, yet distinct from human fetal liver samples. Highly comparable signatures to primary hepatocytes contained major hepatocyte associated genes including lipid metabolism, suggesting that the HLO continue to progress towards a hepatocyte-like fate.
Project description:By taking advantage of the foregut generation method, we initially differentiated iPSCs to foregut spheroids through definitive endoderm specification as described. The foregut spheroids were embedded in Matrigel and cultured with retinoic acid (RA). Following 4-day RA treatment, we switched into hepatocyte maturation media for the induction of the hepatocyte differentiation process to establish human liver organoids, hereafter defined as HLO, as early as day 20. To gain quantitative insights regarding the cellular composition in HLO, single-cell RNA sequencing was used to analyze their mRNA expression from 4,059 cells. t-distributed stochastic neighbor embedding analysis confirmed the five distinct major clusters among the cells in HLO, containing hepatocyte-, biliary cell-, hepatic stellate cell-, Kupffer cell-, biliary tree (or peribiliary gland) stem cell-like populations.
Project description:We described a highly efficient method to establish the immortalized HepLPC cell, these cell lines could efficiently expand and able to readily differentiate back into metabolically functional hepatocytes. To further characterize them, we compare the global expression profiles among immortalized human liver progenitor-like cells (iHepLPCs), hepatocyte-like cells differentiated from iHepLPCs (iHepLPCs-Hep) and 3D cell spheroids of the iHepLPCs-Hep (iHepLPCs-Hep-3D).
Project description:Hepatoblasts emerging at E8.5 from the foregut endoderm proliferate vigorously and differentiate to hepatocytes and biliary epithelial cells. To find genes important for hepatocyte differentiation during development, we compared gene expression profiles of hepatoblasts/immature hepatocytes at E12.5 and E17.5. As Dlk, also known as Pref-1, is expressed in hepatoblasts/immature hepatocytes, we performed a microarray analysis of the Dlk+ cells isolated from livers at E12.5 and E17.5. Keywords: fetal liver cells comparing
Project description:We established a human liver organoid (HLO) based screening model for analyzing DILI pathology at organoid resolution. HLO contains polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNAseq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. By developing a 384 well based high-speed live imaging platform, we successfully developed a Liver organoid-based Toxicity screen (LoT) with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity. We functionally validated LoT with 238 marketed drugs at 4 different concentrations. LoT positively predicts genomic predisposition (CYP2C9*2) for Bosentan-induced cholestasis. Thus, LoT is a high-fidelity model for drug safety with a cost-effective platform, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.
Project description:Hepatoblasts emerging at E8.5 from the foregut endoderm proliferate vigorously and differentiate to hepatocytes and biliary epithelial cells. To find genes important for hepatocyte differentiation during development, we compared gene expression profiles of hepatoblasts/immature hepatocytes at E12.5 and E17.5. As Dlk, also known as Pref-1, is expressed in hepatoblasts/immature hepatocytes, we performed a microarray analysis of the Dlk+ cells isolated from livers at E12.5 and E17.5. Keywords: fetal liver cells comparing Mouse hepatoblasts were isolated from E12.5 and E17.5 fetal liver using anti-mouse Dlk monoclonal antibody (mAb) according to a previous report and dissolved in Trizol reagent. The cDNA samples synthesized from total RNA were used for a microarray analysis with the mouse GEM2 microarray. One array, no replicates.
Project description:The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M cyclosporin A (CsA); SFM + 1 mM Def + 1 ?M CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def.
Project description:Robust and scalable differentiation of human pluripotent stem cells into high-quality hepatocyte-like cells (HLC) is achieved by acting on Wnt/b-catenin and TGFb pathways and taking advantage of latest knowledge on human liver development. Obtained HLC are comparable to primary human hepatocytes and superior to stem cell-derived hepatocytes generated with previously described protocols. As a proof of concept, such HLC were used to assess drug hepatotoxicity and study human ductal plate and bile duct morphogenesis in a complex liver organoid model.
Project description:We generated intrahepatic cholangiocyte organoids and fetal hepatocyte organoids to compare their transcriptomic profile with liver tissue, primary human hepatocytes, and other hepatocyte model systems.
Project description:Primary human hepatocytes (PHH) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture and resistance to genetic manipulation have limited their use. Here we show that retrorsine improves human hepatocyte repopulation of chimeric mice to levels where poor donor PHH can be isolated for ex vivo cultures. Mouse-passaged (mp)PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months, as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized and then re-cultured as spheroids or re-transplanted to create highly humanized mice carrying a genetically altered hepatocyte graft. Together, these advances provide flexible tools for studying human liver disease and evaluating hepatocyte-targeted gene therapy approaches
Project description:Longitudinal monitoring of liver function in vivo is hindered by the lack of high-resolution non-invasive imaging techniques. Using the anterior chamber of the mouse eye as a transplantation site, we have established a platform for longitudinal in vivo imaging of liver spheroids at cellular resolution. Transplanted liver spheroids engraft on the iris, become vascularized and innervated, retain hepatocyte-specific and liver-like features and can be studied by in vivo confocal microscopy. Employing fluorescent probes administered intravenously or spheroids formed from reporter mice, we showcase potential use of this platform exemplified by monitoring hepatocyte cell cycle activity, bile secretion and lipoprotein uptake. Moreover, we show that hepatic lipid accumulation during diet-induced hepatosteatosis is mirrored in intraocular grafts in vivo. The here described technology will provide a crucial tool to study liver physiology and disease progression in both pre-clinical and basic research as well as a drug screening platform in the pharma industry.