Background and aims: New sources of insulin-secreting cells are strongly required for the cure of type 1 diabetes. Recent successes in differentiating human embryonic stem cells (ESC), in combination with the discovery that it’s possible to derive human induced pluripotent stem cells (hiPSC) from somatic cells, have raised the possibility that a sufficient amount of patient-specific insulin-secreting islet-like cells might be derived from patients cells through cell reprogramming and differentiation.

Methods: We performed the differentiation of hiPSC, derived from the reprogramming of both fetal and adult fibroblasts, in insulin-producing cells, optimizing some protocols already established for ESC. The expression of marker genes of pancreas differentiation was measured through real-time PCR analysis (Taqman) and expressed as fold changes (FC) compared to undifferentiated hiPSC. Proteic expression was confirmed by cytofluorimetric analysis. HiPSC differentiated in vitro at two different stages (posterior foregut and endocrine cells) were transplanted under the kidney capsule of NOD/SCID mice. One, four, eight and twelve weeks after transplantation the secretion of human C-peptide after oral glucose administration was measured and an immunohistochemical analysis of grafts was performed.

Results: With this protocol we were able to obtain the down-regulation of the pluripotency genes Oct4 and Nanog and the up-regulation of the definitive endoderm genes Sox17 and Foxa2 (28,3±11,8 and 6,5±3,6 FC) and of the pancreatic endoderm genes Pdx1, Ngn3 and Nkx2.2 (7883±153,1, 90±6,8 and 17,6±10,5 FC). At the end of the differentiation process the production of insulin mRNA was highly increased (65484±977 FC) and 5±2,9% cells resulted insulin-positive; terminally differentiated cells also produce C-peptide in vitro (1,7±0,1 ng/mL). In mice transplanted with differentiated hiPSC secretion of human C-peptide after glucose stimulus was observed at 1 and 2 months after transplantation (C-peptide mean release at 30 minutes: 0,52±0,1ng/mL). Hystochemical analysis of the grafts showed the presence of pancreatic (Pdx1, ChgB, Syp positive) but also pluripotent cells (Sox2, Oct4, Ki67 positive).

Conclusions: In vitro results show that hiPSC, following the stages of pancreatic organogenesis, differentiate in insulin producing cells. Furthermore, in vivo study suggests that some differentiated cells engraft and survive in the recipient mice, but highlight the necessity to look for new markers in order to select and transplant only the differentiated pancreatic cells, avoiding the contamination of pluripotent cells with tumorigenic potential.