Characterization of iTS-P as pancreatic progenitors.

<p>(A) Teratoma/tumorigenic Assay. One Balb/c-nude mouse approximately 1.5 months after grafting is shown as an example. The site into which ES cells (indicated by “ES cell”) were inoculated exhibited the presence of a solid tumor, whereas the site into which iTS-P (indicated by “iTS-P 4–2”) were inoculated did not show any sign of solid tumor generation. (B) RT-PCR analysis for mRNA expression of endodermal cell markers (Hnf1, Hnf4, Hnf6, Foxa2, Sox17, and HB9) in iTS-P lines (4–2, 4–3, and 4–7) and differentiated ES cells at Stages 1 to 3 (Stage 1–3 ES; used as a positive control). Abbreviations: Hnf1, hepatocyte nuclear factor-1-; Hnf4, hepatocyte nuclear factor-4-; Hnf6, hepatocyte nuclear factor-6; Foxa2, forkhead box protein A2; Sox17, SRY (sex determining region Y)-box 17; HB9, homeobox protein HB9. (C) Immunostaining of iTS-P 4–2 Stages 4–5 using anti-Pdx1 antibody. Scale bar = 100 μm. (D) Electron microscopy of dense-core vesicles (arrows) in iTS-P 4–2 Stages 4–5. Scale bar = 5 μm. (E) Schematic representation of stepwise differentiation of ES cells towards insulin-producing cells. Cells of the definitive endoderm (DE) express Foxa2 and Sox17; cells of gut tube endoderm (GTE) express Hnf1β and Hnf4α; cells of pancreatic progenitors (PP) express Pdx1 and Hnf6; and insulin-producing cells (IPC) express insulin (Ins), glucose transporter 2 (Glut2), and glucokinase (GK). Abbreviations: KAAD-cyclopamine, 3-keto-N-aminoethyl-N'-aminocaproyldihydrocinnamoyl cyclopamine; FGF10, fibroblast growth factor 10; DAPT, N-(S)-phenyl-glycine-t-butyl ester; IFG-1, insulin-like growth factor 1.