Robust, long-term culture of endoderm derived hepatic organoids (eHEPOs) for disease modeling

Many liver pathologies ranging from cancer to genetic metabolic diseases are amenable to cellular replacement therapies. One of the main difficulties associated with cellular therapy approaches is the generation and expansion of mature autologous hepatocytes. The ability to derive patient-specific induced pluripotent stem cells (iPSCs) have solved the problem of generating autologous cells but differentiation and expansion of mature hepatocytes remain a challenge. Here, we report the generation of human iPSC-derived hepatic organoid (eHEPO) culture system for producing functional hepatocytes using EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within two weeks and expanded long-term without any lose of differentiation capacity to mature hepatocyte (>12 months with passage number 25) and are resilient to freeze-thaw cycles. The hepatic identity of eHEPOs was confirmed by morphological analyses, expression of mature hepatocyte markers and in vitro functional assays. Extensive global transcriptome analyses of all intermediate stages demonstrated the stepwise differentiation iPSCs to mature hepatocytes in endoderm-derived organoids. Finally, starting form patient-specific iPSCs, we created an organoid-based model of Citrullinemia type 1, an inherited urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild type ASS1 gene which also indicated that this model is amenable to genetic manipulation. Our results demonstrate that iPSC-derived EpCAM-positive endodermal cells are excellent cell sources to generate patient-specific hepatic organoids in a fast and efficient manner. Overall design: We analysed the cell cultures at different stages of eHEPO differentiation from control and ASS1 mutant patients using RNA-sequencing. RNA from 26 samples including iPS, endoderm progenitors, organoids under expansion (EM) and differentiation (DM) conditions were isolated. Libraries were prepared using the Celseq2 method, which is originally designed for single cell sequencing, however these samples are bulk RNA. We aimed for low coverage sequencing (~2 million mapped to the coding regions) per sample to investigate the gross changes in the transcriptome during lineage specification and differentiation. Libraries (rpi small primer) were sequenced on an Illumina HiSeq 4000 using 151-bp paired-end sequencing at the Genomescan. All the samples were sequenced in a single run to an average depth of ~10 million reads per sample. Reads were mapped to the latest human coding transcriptome, normalized and analyzed using the standard DESEQ2 package DK1 and DK3 are different citrullimenia patients with ASS1 mutation