Fig. 1: Generation of functional human hepatocytes from EPS cells.

a Scheme of the five-stage protocol for the differentiation of EPS cells into human hepatocytes. b Hierarchical clustering of the gene expression profiles of epiblast cells (EPI) at different days, iPSCs, H1 ESCs and differentiated EPS cells at the end of stage 1 (EPS-S1 cells-1 and EPS-S1 cells-2). c RT-qPCR analysis of major hepatic genes and transcription factors in EPS cells (n = 3), HepG2 cells (n = 3), EPS-Heps matured for 5 weeks (n = 3) and F-PHHs (n = 5). Gene expression was normalized to F-PHHs and housekeeping gene. d Coimmunofluorescence staining of major hepatic functional markers and transcription factors with ALB in EPS-Heps. e UPLC/MS/MS analysis in HepG2 cells, EPS-Heps and F-PHHs for the drug metabolic activities of CYP3A4 and CYP1A2 by evaluating their metabolites, 6β-hydroxytestosterone and acetaminophen, respectively. n = 3. f ALB secretion in EPS cells, HepG2 cells, EPS-Heps matured for 4 weeks and PHHs analyzed by ELISA. PHHs were cultured for 5 days in vitro using sandwich method. n = 3. g Urea synthesis in EPS cells, HepG2 cells, EPS-Heps matured for 4 weeks and PHHs. PHHs were cultured for 5 days in vitro using sandwich method. n = 3. h Coimmunofluorescence staining of human ALB with human hepatic proteins, including CYP3A4, CYP2C9, CYP2C19, CYP1A2 and CK8, in URG mice transplanted with EPS-Heps matured for 8 weeks. i Hierarchical clustering of the gene expression profiles of EPS cells, F-PHHs and EPS-Heps. j The hepatic identities of EPS-Heps, F-PHHs, EPS cells, reprogrammed hepatocytes (hiHeps)¹² and cells from other studies (GSE103078 and GSE98710) were analyzed by CellNet using RNA-seq data. Data are presented as means ± SEM. For all measurements, n represents the number of biological replicates. Scale bars, 50 μm. Original RNA-seq data of this paper are available in the NCBI Gene Expression Omnibus under accession number GSE137569.