1. Volume 19, Issue 4, Pages 449-461 (October 2016)
Conversion of Human Gastric Epithelial Cells to Multipotent Endodermal Progenitors using Defined Small Molecules 
Yunfang Wang, Jinhua Qin, Shuyong Wang, Wencheng Zhang, Jialei Duan, Jing Zhang, Xin Wang, Fang Yan, Mingyang Chang, Xiaofang Liu, Bo Feng, Jiang Liu, Xuetao Pei 
Cell Stem Cell 
Volume 19, Issue 4, Pages (October 2016)
DOI: /j.stem
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2. Cell Stem Cell 2016 19, 449-461DOI: (10.1016/j.stem.2016.06.006)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Conversion of hGECs toward hiEndoPCs by Small Molecules in the Presence of GSEMF Feeders
(A) Schematic figure indicates the derivation of hiEndoPCs.
(B) Representative images show the generation of hiEndoPCs treated with 8M-RM and with aGSEMFs.
(C) Numbers of various-sized colonies induced using different ECM substrata or feeder cells or conditional medium (CM). Results are means and SEM for biological replicates (n = 3). fISEMFs, fetal intestinal subepithelial myofibroblasts; fGSEMFs, fetal gastric subepithelial myofibroblasts; fDCs, fetal diaphragm cells; aGSEMFs, adult gastric subepithelial myofibroblasts; MEFs, mouse embryonic fibroblasts; HFFs, human foreskin fibroblasts; MSCs, mesenchymal stem cells; fISEMFs-CM, conditioned medium of fetal intestinal subepithelial myofibroblasts; aGSEMFs-CM, conditioned medium of adult gastric subepithelial myofibroblasts.
(D) Numbers of various-sized colonies induced with combinations of small molecules as indicated. Results are means and SEM for biological replicates (n = 3).
(E) Representative morphologies of hGECs and GSEMFs cultured in BBRS for 9 days and 15 days, respectively, are shown.
(F) Fluorescence in situ hybridization (FISH) analysis of hiEndoPCs derived from male hGECs fed with female GSEMFs is shown. Red, X chromosome; green, Y chromosome.
All results were replicated in at least three independent experiments. See also Figures S1 and S2.
Cell Stem Cell  , DOI: ( /j.stem )
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4. Figure 2 hiEndoPCs Are Endowed with Properties of EndoPCs
(A) Immunostaining of hGECs and hiEndoPCs is shown.
(B) qRT-PCR of endodermal stem/progenitor-specific and gastric-specific markers. Expression levels were normalized to 1 in hGECs (left panel) or hiEndoPCs (right panel). Results are means and SEM for technical replicates (n = 3). hESCs, human embryonic stem cells; DE, definitive endoderm; PGT, primitive gut-tube; PFG, posterior foregut.
(C–F) hGECs and hiEndoPCs were analyzed using RNA-seq and genome DNA methylation analysis. Single colonies of hiEndoPCs were analyzed. (C) Cluster of RNA-seq dataset based on normalized read counts is shown. (D) Selected differentially expressed genes between hiEndoPCs and hGECs are shown. (E) Volcano plots of genes with differentially methylated promoters between hiEndoPCs and hGECs are shown. (F) DNA methylation at the promoter regions of FOXA2 and GATA4 gene locus is shown.
All results (except RNA-seq and DNA methylation analysis) were replicated in at least three independent experiments. See also Figures S3 and S4.
Cell Stem Cell  , DOI: ( /j.stem )
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5. Figure 3
Cell Cycle and Wnt Pathway Are Essential to hiEndoPCs Generation
(A and B) Secretome analysis in the supernatant of cultured GSEMFs versus MSCs. A list of secreted proteins >1.5-fold or <0.66-fold changes (A) and the functional summary of differentially secreted proteins (B) are shown.
(C) qRT-PCR analysis of cell-cycle-associated genes in hiEndoPCs versus hGECs. Results are means and SEMs for technical replicates (n = 3).
(D) Representative fluorescence-activated cell sorting (FACS) plots of CFSE-labeled GECs cultured under indicated conditions and co-cultured with or without GSEMFs in Transwell plates for 48 hr. SM, 4M medium; KM, Kubota’s medium.
(E and F) qRT-PCR analysis of Wnt-related genes in hiEndoPCs and hGECs. Results are means and SEM for technical replicates (n = 3).
(G) Quantitative analyses of colony number and area of hiEndoPCs formation treated with the indicated inhibitors are shown. Results are means and SEM for biological replicates (n = 3).
All results were replicated in at least three independent experiments. See also Figures S5 and S6.
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4 Endodermal Lineage-Restricted Potential of hiEndoPCs
(A and B) Representative images (A) and summary (B) of teratoma formation are shown.
(C) Karyotype of hiEndoPCs at passages 3 and 5, respectively, is shown.
(D and E) HE staining shows sections of hiEndoPC outgrowths in mouse epididymal fat pads at lower (D) and higher (E) magnifications.
(F and G) Immunostaining shows hiEndoPC outgrowths for human-LGR5 (F) and isotype control (G).
(H) Relative mRNA expression of neuroectoderm and mesoderm markers in hESCs and hiEndoPCs cultured for the indicated days under corresponding conditions. Results are means and SEM for technical replicates (n = 3).
All results were replicated in at least three independent experiments.
Cell Stem Cell  , DOI: ( /j.stem )
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7. Figure 5
hiEndoPCs Can Give Rise to Pancreatic, Intestinal, and Hepatic Cells
(A) Bright-field images show hiEndoPC-Pans induced with the three-dimensional (3D) differentiation protocol.
(B) Immunostainings show pancreatic markers of hiEndoPC-Pans organoid sections.
(C) Dithizone (DTZ) staining of hiEndoPC-Pans is shown.
(D) Expressions of the pancreas-specific genes of hiEndoPC-Pans were measured by qRT-PCR. hGECs-P, hGECs were induced in the medium tailored for pancreatic islet. Results are means ± SEM for technical replicates (n = 3).
(E) Secreted C-peptide in response to low (2 mM) or high (20 mM) concentrations of D-glucose, 30 mM potassium chloride (KCl), or 100 μM tolbutamide (Tol) was measured with a C-peptide ELISA kit. Results are means and SEM for biological replicates (n = 3). hESC-Pans, hESC-derived pancreatic cells.
(F) Bright-field images show hiEndoPC-Ints.
(G) Immunostainings show intestinal markers in hiEndoPC-Ints.
(H) Expressions of the intestinal-specific genes were measured by qRT-PCR. hGECs-I, hGECs were induced in the medium tailored for intestine. Results are means and SEM for technical replicates (n = 3).
(I) Representative morphology, immunostaining for hepatic markers, and ICG uptake of hiEndoPC-Heps are shown.
(J) qRT-PCR of hepatic-lineage markers. Expression levels were normalized to 1 in hGECs. hGECs-H, hGECs were induced in the medium tailored for hepatocytes. Results are means and SEM for technical replicates (n = 3). hESC-Heps, hESC-derived hepatocytes; PHHs, primary human hepatocytes.
(K) Albumin secretion was measured for hiEndoPC-Heps (∗∗∗p < 0.001). Results are means and SEM for biological replicates (n = 3).
(L) CYP3A4 activity was measured for hiEndoPC-Heps (∗p < 0.05). Results are means and SEM for biological replicates (n = 3).
Scale bar, 50 μm. All results were replicated in at least three independent experiments. See also Figure S6.
Cell Stem Cell  , DOI: ( /j.stem )
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8. Figure 6
Transcriptome and Epigenome Analysis and In Vivo Transplantation of hiEndoPC-Heps
(A) Principal component analysis of hiEndoPC-Heps, hiEndoPCs, and hGECs based on global DNA methylation profile is shown.
(B) Volcano plots show genes with differentially methylated promoters between hiEndoPC-Heps and hiEndoPCs.
(C) DNA methylation at the promoter regions of hepatic-associated genes locus is shown.
(D) Principal component analysis of hiEndoPC-Heps, hiEndoPCs, and hGECs based on global gene expression profiles is shown.
(E) Heatmap of 1,834 genes differentially expressed between hiEndoPC-Heps and hiEndoPCs (a minimal fold change of 1 with adjusted p values below 0.01). Genes that exhibited significantly different expression levels among genes involved in liver development and maturation, fatty acid metabolism, and drug metabolism were extracted.
(F) Human serum albumin levels in recipients of hiEndoPC-Heps (n = 12) or hESC-Heps (n = 5) is shown.
(G) Fah staining shows repopulated hiEndoPC-Heps in F/R liver sections; magnifications are whole mount.
(H) Immunostaining shows that the engrafted hepatocytes in livers of F/R mice were positive for human hepatocyte antigen (Hepar) and human ALB. Scale bar, 50 μm.
See also Figure S6.
Cell Stem Cell  , DOI: ( /j.stem )
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