1. Volume 10, Issue 4, Pages 371-384 (April 2012)
Self-Renewing Endodermal Progenitor Lines Generated from Human Pluripotent Stem Cells 
Xin Cheng, Lei Ying, Lin Lu, Aline M. Galvão, Jason A. Mills, Henry C. Lin, Darrell N. Kotton, Steven S. Shen, M. Cristina Nostro, John Kim Choi, Mitchell J. Weiss, Deborah L. French, Paul Gadue 
Cell Stem Cell 
Volume 10, Issue 4, Pages (April 2012)
DOI: /j.stem
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
A Putative Definitive Endoderm Progenitor Population Is Identified in Differentiation Cultures from Human ESCs
The human ESC line H9 was differentiated toward the hepatic lineage.
(A) Phase contrast image of a culture maintained for 4 weeks shows two distinct types of populations (Differentiated versus Progenitor). The scale bar represents 100 μm. Image was captured with a 20× objective.
(B) Colony types indicated in (A) were manually picked and pooled into groups, and gene expression was analyzed by quantitative real-time RT-PCR, comparing to undifferentiated ESCs. ES, embryonic stem cells; Diff, differentiated cells; Prog, progenitor colonies. The expression of indicated genes is shown normalized to the housekeeping gene PPIG. Values represent the mean of three individual pools of each population. Error bars represent the standard errors.
(C) Cultures were assayed for CD117 (KIT) versus CXCR4, SOX17 versus FOXA1, and SOX17 versus CXCR4 by flow cytometry in three separate stains.
See also Table S4.
Cell Stem Cell  , DOI: ( /j.stem )
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3. Figure 2 Purification and Maintenance of Endodermal Progenitor Cells
(A) Cultures containing endoderm progenitors as in Figure 1 were sorted based upon CXCR4 expression. Gene expression was analyzed by QRT-PCR. CXCR4+ cells were cultured as in Figure 1 and after 1 or 3 passages, cultures were assayed for SOX17 versus FOXA1 expression by intracellular flow cytometry.
(B) EP cell lines are established by sorting definitive endoderm (CXCR4+CD117high) cells from day 5 of human ESC differentiation cultures induced with high levels of Activin A, and culturing the cells in optimized conditions (see text). Images show typical morphology of EP cell colonies (H9-EP), captured using 10× and 20× objectives.
(C) EP cell lines derived from H9 ESCs or iPSCs were cultured and cell growth was analyzed by cell count at each passage. Left panel: graph shows relative cell growth over time (Square: iPSC2-derived EP cell line; Circle: H9-derived EP cell line). Right panel: SOX17 versus FOXA1 expression by intracellular flow cytometry after either 24 (H9-EP) or 20 (iPSC2-EP) passages.
See also Figures S1 and S2 and Tables S1–S4.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 3
Gene Expression Microarray Analyses of EP Cells Compared to ESCs and Transient Definitive Endoderm
(A–C) Purified ESCs (sorted SSEA3high SSEA4high), transient endoderm (day 5 high Activin-treated, sorted CXCR4high CD117high), and EP cells (sorted CXCR4high CD117high) were analyzed using gene expression microarrays. T0, ESCs; T5, transient endoderm; EPC, EP cells. (A) ESC, transient endodermal cell, and EP cell global expression profile clustered by dendrogram. (B) Principal component analysis of populations in (A). (C) Heat map expression levels of genes related to pluripotency, primitive streak/mesendoderm, definitive endoderm, and cell surface markers.
(D) Quantitative RT-PCR confirmation of a subset of genes examined in (C). Expression levels were normalized to 1 in ESCs for pluripotency genes, transient endoderm for primitive streak genes, and EP cells for definitive endoderm genes, respectively. Error bars represent the standard error of three individual pools of each population.
(E) Flow cytometry analyses distinguish EP cells from ESCs and transient endoderm cells by cell surface markers ALCAM, LGR5, and CXCR4. (ESCs: gray filled histograms; transient endoderm: red histogram; EP cells: yellow histograms.)
See also Figure S3 and Tables S1–S4.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 4 Hepatic Differentiation of EP Cells In Vitro
(A–C) Cells from the H9-EP line and from one single-cell-derived subclone (clone 2) were differentiated into hepatocytes as indicated, and were harvested for analyses at days 14, 20, and 27 (T14, T20, and T27). (A) Gene expression was analyzed by QRT-PCR on samples harvested at three time points from H9-EP and clone 2 lines, comparing to undifferentiated EP cells (T0). The expression of indicated genes is normalized to the housekeeping gene PPIG. Values represent the mean of three independent differentiation experiments. Error bars represent the standard error. (B) Flow cytometry analyses of AFP versus FOXA1, AAT versus FOXA1, or ASGPR1 versus CXCR4 at day 20 of differentiation, comparing to undifferentiated EP cells (T0 EP cell). Percentage of cells within each quadrant is indicated. Shown are the representative data from H9-EP (passage 10) and clone 2 EP (passage 6) cells. (C) To assess CYP3A4 activity of EP cell-derived hepatocytes, H9-EP cells (passage 15) were differentiated into hepatocytes for 24 days and then cultured in the presence (Rifampicin) or absence (DMSO) of Rifampicin for 3 days, and compared to HepG2 cells. Values represent means of measured luminescence units normalized to 6 × 104 cells, and error bars represent standard error (n = 3). See also Figure S4 and Tables S1–S4.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 5 Pancreatic Differentiation of EP Cells In Vitro
(A–D) Cells from the H9-EP line (passages 6–12) and from one single-cell-derived subclone (clone 2) (passages 5–11) were differentiated into pancreatic cells as indicated in the text. Cells were harvested for analyses at day 12 (T12) to day 18. (A) Immunofluorescence staining of human islets (Islet) and day 14 EP cell-derived pancreatic cultures (EP) for c-peptide (red) and GCG (green) or PDX1 (green). Scale bars represent 100 μm. (B) Intracellular flow cytometric analyses of c-peptide versus GCG and c-peptide versus SST at day 12 of differentiation. (C) Gene expression was analyzed by QRT-PCR on samples harvested at day 18 of differentiation and compared to adult islets. The expression of indicated genes is normalized to 1 in islets. Values represent the mean of three independent differentiation experiments or two batches of adult islets. Error bars represent the standard error. (D) The percentage of c-peptide+ cells that coexpress either SST or GCG was quantified. Data are shown for endocrine cells generated from EP cells (H9-EP, n = 12; and iPS2-EP, n = 5) and directly from H9 ESCs (ESC, n = 4).
(E) Intracellular flow cytometric analyses of c-peptide expression, gated on c-peptide+ cells from day 12 EP cell-derived pancreatic cultures (blue histogram) and from human islets (red filled histogram). The values shown are mean fluorescence intensity of c-peptide in H9-EP-derived cells (blue, n = 6) and human islets (red, n = 2).
(F) The H9 EP cells were differentiated into β-cells and stimulated with D-glucose at 2 mM (Basal) or 20 mM (glucose) at day 14, and were compared to adult islets. C-peptide release was measured at various time points (5, 10, 15, 20, and 40 min) (H9-EP, n = 3; islets, n = 2; error bars represent the standard error). Asterisk indicates statistical significance as determined by t test. p =
See also Figures S5 and Tables S1–S4.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 6
Tumor Potential and Spontaneous Differentiation of EP cells In Vivo
(A) To determine if EP cells retain teratoma forming potential, 0.5 million H9 ESCs or H9 EP cells were transplanted intramuscularly into immune compromised mice. Images show teratoma formation in a mouse injected with ESCs (ES, left panel) and lack of tumor formation in a mouse injected with EP cells (EP, right panel).
(B) A summary of teratoma-forming ability of pluripotent stem cells (ES/iPSC), day 5 (T5) transient endoderm, ESC-derived day 20 hepatocyte cultures (ES-derived hepatocytes), and EP cells (EPC) and EP cell-derived day 9 hepatocyte cultures (EP cell-derived hepatocytes). For transient endoderm, both bulk cultures and CXCR4+CD117high sorted cells were used for transplantation.
(C–F) Analyses of EP cell transplants. Eight to ten million EP cells mixed with matrigel were injected subcutaneously into immunodeficient mice, and the resultant matrigel plugs were isolated 3–12 weeks posttransplantation. Scale bars represent 100 μm. (C) Sections of matrigel plugs were stained with hematoxylin and eosin. Shown are endoderm-like structures at lower (10×, left panel) and higher magnifications (20×, right panel). (D) Immunohistochemistry using an anti-human antibody reveals human-cell-derived endodermal structures surrounded by nonhuman mesenchyme and adipocytes. (E) Immunohistochemistry for intestinal markers reveals gut/intestinal structures in the matrigel plug. (F) Immunohistochemistry for hepatocyte markers in the matrigel plug.
See also Figure S6 and Tables S1–S3.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2012 Elsevier Inc. Terms and Conditions