Directed pancreatic differentiation of CyT49 in suspension culture.

<p>(A) Schematic representation of aggregation and the four-stage differentiation protocol from hESC (ES) to mesendoderm (ME), definitive endoderm (DE), primitive gut tube (PG), posterior foregut (PF), and a mixed population comprising primarily of pancreatic endoderm (PE) and endocrine precursor/endocrine cells (EP). Culture conditions, timing and rotation speeds are indicated. Markers used to identify the different stages are shown.*TBI: TGF-β RI kinase Inhibitor IV, first day of Stage-2 only. **ITS: Insulin-Transferrin-Selenium used at different concentrations in Stage-1 and -2. HrgB, heregulin 1β; D/F12, DMEM/F12; TT, TTNPB; CYC, cyclopamine; NOG, noggin. (B,C,D) Hematoxylin and eosin staining of sections of CyT49 aggregates after (B) culture in StemPro medium for 2 days (paraffin section), or (C) at d5, or (D) at d12 of differentiation (frozen sections). Scale bars, 50 µm. (E) Immunofluorescence analysis of sections of d5 aggregates stained with OCT4/DAPI, or (F) FOXA2/DAPI. A single cluster of ∼four OCT4⁺ nuclei within the field of view is indicated (arrow). Imaged with a 20× objective. Immunofluorescence analysis of d12 aggregates for (G) NKX6-1, PDX1 and CHGA expression, and (H) FOXA2, PDX1 and NKX2-2 expression. Imaged with a 40× objective. DAPI, 4′,6-diamidino-2-phenylindole.