Figure 4.

In vitro β-cell differentiation of human pancreatic duct-derived cells (HDDCs) . (A): HDDCs individually transfected with PDX1, NGN3, and MAFA show upregulation of INS and SYN gene expression with highest detection levels observed in MAFA transfection conditions (n = 3–6). ∗∗, p < .01 compared with PDX1 and NGN3 conditions. (B): Real-time polymerase chain reaction analysis also shows upregulation of several β-cell markers (NeuroD, NKX2.2, GATA4, PAX6, GCK, GLUT-2, FOXA2, NGN3, MAFA, PDX1, NKX6.1) and MAFA biological target genes (Pc, Prlr) after transfections with MAFA synthetic modified mRNA (n = 3–11). Logarithmic scale is presented as powers of 10. (C): INS expression on HDDCs after 5 transfections with NGN3 smRNA. (D, E): Immunostaining for INS (green) in HDDCs after 5 transfections with MAFA smRNA. (F–I): After MAFA differentiation, HDDCs coexpressed insulin and MAFA localized into the nucleus. (J–L): Positive staining for PDX1 (J) and coexpression of INS (green) and GLUT-2 (red) (K and L) were observed on HDDCs after 5 transfections with MAFA smRNA. Staining images are representative of 3–5 different experiments. Scale bars = 200 µm (D, F, J–L), 100 µm (C), 50 µm (G, H), and 20 µm (E, I). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; INS, insulin; MAFA, V-Maf musculoaponeurotic fibrosarcoma oncogene homolog A; PDX1, pancreatic duodenal homeobox 1.