Fig. 3

Immunofluorescence analysis of beta cell markers in islet-like cell clusters differentiated from MDSC. a Islet-like clusters were differentiated in vitro for 6 weeks from MDSC derived from nestin-eGFP transgenic mice. Photomicrographs show staining for eGFP and insulin (red) and DNA (blue). The bottom panels show a merged view of GFP, insulin, and DNA (left panel) and the phase-contrast image of the cluster with staining for insulin and DNA (right panel). Scale bar = 50 μm. b–d MDSC differentiated into islet-like clusters for 6 weeks were dissociated by short enzymatic treatment and collected by cytospin centrifugation, before fixation and staining for the expression of transcription factors implicated in beta-cell differentiation and insulin. Shown are fluorescence micrographs of dissociated cells stained for DNA and Nkx2.2 or DNA and Pax6 (b), co-stained for PDX1 and insulin (c), and for Nkx6.1 and MafA (d). Scale bars = (b) 20 μm, (c,d) 10 μm