Additional file 2 of High-efficiency c-Myc-mediated induction of functional hepatoblasts from the human umbilical cord mesenchymal stem cells
Creators: name: Deng, Jie
name: Luo, Kai
name: Xu, Pengchao
name: Jiang, Qingyuan
name: Wang, Yuan
name: Yao, Yunqi
name: Chen, Xiaolei
name: Cheng, Fuyi
name: Xie, Dan
name: Deng, Hongxin
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 2: Figure S1. Characterization of HUMSCs. (A) Morphology of Primary HUMSCs (P0), HUMSCs in passage 5 (P5) and HUMSCs in passage 10 (P10). The cells showed homogeneous fibroblastic morphology. (B) Expression of cell surface markers on HUMSCs. HUMSCs were positive for mesenchymal stem markers (CD166, CD29,CD90, CD105 and CD44) and negative for hematopoietic marker (CD45, CD34 and HLA-DR) or endothelial marker (CD31). (D) Multiple differentiation potential of HUMSCs. HUMSCs could differentiate into adipocytes, osteocytes and chondrocytes. Scale bar: 50 μm (A) 100 μm (D). Figure S2. HSFs-iHeps harbored hepatocyte characteristics.(A) cell morphology of HSF, HSF-iheps and PHHs. (B) Hepatic gene expression in HSF-iHeps detected by western blot.(C) Expression of hepatic genes in HSF-iHeps measured by qPCR. (D) Co-expression of ALB and HNF4A, TF, AAT, MRP2, CK18 and ASS1 in HSF-iHeps revealed by immunofluorescence staining. Figure S3. HSF-iHeps possessed hallmark functions of mature hepatocytes. (A) Basic liver function analysis of HSF-iHep, including oil red O staining (i) PAS staining (ii) uptake of ICG (iii) LDL uptake. (B) ELISA was used to detect the secretion of ALB (left) and (C) AAT (right) during hepatic induction of HSF-iHeps. (D) The mRNA levels of CYP genes were determined by qPCR in PHHs and HSF-iHeps cultured for 2 days before inducer treatment. Data are normalized to PHHs. (E) The mRNA levels of the induced CYP enzymes were measured by qPCR. Data are represented as the mean ± SD. (F) Expression of drug transporter genes in HSF-iHeps determined by qPCR. Data are normalized to PHHs. Figure S4. Transcriptome pattern of HUMSC-iHep cells. (A) Whole-genome expression analysis shows the gene expression of HUMSC, PHH, HUMSC-iHep, HSF-iHep, Hepatoblast and Fetal liver cells. HUMSC-iHep and HSF-iHep are grouped with hepatoblast, but closer to Fetal liver more than PHH, as shown in the cluster tree.Color representation represents the level of expression.(B) Heat maps of the expression of fibroblast genes, hepatic transcription factors, functional hepatocyte genes involved in glucose metabolism, lipid cholesterol metabolism, fatty acid metabolism, coagulation and drug metabolism in various cells. (C) qPCR results showed that the functional genes of the mature bile duct, such as cystic fibrosis transmembrane conduction regulator (CFTR), secretin receptor (SCTR), somatostatin receptor 2 (SSTR2), and aquaprin-1 (AQP1), are not expressed in HUMSC-iHeps, suggesting that the cells are heterogeneous but do not differentiate into bile duct epithelial cells. Data are normalized to PHHs. Figure S5. Gene Set Enrichment Analysis (GSEA) of HUMSC-iHeps and PHHs. GSEA analysis revealed remarkably enriched hepatic gene expression in both PHHs and HUMSC-iHeps. Gene sets were compiled from BIOCARTA, the Kyoto Encyclopedia of Genes and Genomes (KEGG). A peak shift to the left side indicates the enrichment of the indicated set of hepatic genes in PHHs or HUMSC-iHeps. Figure S6. In vivo safety assessment. (A) HUMSC-iHep (2×106) and HepG2 (2×106) cells were subcutaneously transplanted into the flank areas of nude mice. HUMSC-iHeps did not form tumors 3 months after transplantation. But HepG2 cells form tumors 2 weeks after transplantation. (B) The karyotypes of HUMSC-iHep cells at passage 10 were analyzed by chromosome analysis during mitosis. Figure S7. c-Myc promoted reprogramming mainly at the early stage (0-6 days). (A) c-Myc levels in HUMSCs cells before and after induction with doxycycline (Dox) as measured by western blot. (B) Six combinations of doxycycline-induced c-Myc expression during the reprogramming of HUMSCs into HLCs. The whole induction 18 days were divided into 3 phases. Red bars represent the DOX addition and gray bars represent Dox withdrawn. (C) The proportion of red fluorescent cells in six groups was detected by flow cytometry after induction for 18 days. Figure S8. Regulatory network altered by c-Myc. (A) Schematic diagram depicts the experiment strategy. (B) Transcriptome correlation analysis of HUMSCs inducted by 3TFs(KT1-3) and 3TFs+c-Myc(CKT1-3). (C) Expression of genes from two gene sets: E2F targets and G2M checkpoint in two groups of HUMSCs inducted by 3TFs(KT1-3) and 3TFs+c-Myc(CKT1-3). (D) GSEA enrichment plot of two gene sets. (E) Genomic distribution of c-Myc binding sites.