Fig. 4

Islet-like clusters express specific markers of beta cells and secrete insulin in response to glucose challenge. a The mRNA expression profile of components implicated in the commitment, differentiation, and maturation of beta cells was examined in differentiated islet-like cell clusters by RT-PCR. Shown is the expression in 6-week differentiated (Diff) MDSC and in Min6 insulinoma cells as a control. b Protein expression profiles were also probed by Western blot analysis at three different time points,: undifferentiated PP8 MDSC (MDSC), early differentiated MDSC (Diff-Ea: PP8 + 2 weeks differentiation), and late differentiated MDSC (Diff-La: PP8 + 6 weeks differentiation). Shown are typical profiles blotted for Nestin, Glut2, Pdx1, Nkx2-2, and MafA. Alpha-Tubulin is shown last as a loading control. Molecular weight markers are indicated in kDa. c Glucose-responsive insulin secretion: for each assay, 4 to 5 islet-like cell clusters differentiated for 8–10 weeks were collected in 24-well dishes and starved in KREBS buffer for 2 h before incubating for 1 h in KREBS supplemented with 2.8 mM glucose and subsequently incubated for 1 h in 20 mM glucose. Secreted insulin was measured by FRET. Whereas little or no insulin was released in 2.8 mM glucose, when further challenged with 20 mM glucose, clusters secreted significant levels of insulin with regard to the small number of cells in the wells (1500 to 2000 cells/well, n = 3); *P < 0.01