Fig. 2.

A Scheme of the 3-stage protocol for differentiation of SOX9⁺ MPCs into stem cell-derived beta cells. MACS was performed at d12 using the SC30 cell clone. B Analysis of CPA1 protein expression during differentiation (d0-d29) by Western Blot. C IF staining of NKX6.1/CPA1 and NEUROG3/insulin after stage 4 (d15) or 5 (d18), respectively. Scale bar = 50 µm. D IF staining of CK19/C-peptide and insulin/C-peptide at d18, d22 or d29 of differentiation. Scale bar = 50 µm (d18 CK19/C-peptide) or 100 µm. Arrowheads indicate early insulin-positive cells. E Measurement of insulin and C-peptide secretion and content in d29 sorted SC30 cells vs unsorted cells. Data are means ± SEM, n = 5–12 (content) and n = 3–7 (secretion). Two-tailed Student’s t-test. F IF staining of glucagon and insulin in d29 cells. Arrowheads mark polyhormonal cells. Scale bar = 50 µm