Induction of differentiation of embryonic stem cells into insulin secreting cells by fetal soluble factors

¹ Institute of Bioengineering, University Miguel Hernández, E-03550, San Juan de Alicante, Spain

^* To whom correspondence should be addressed. E-mail: Franz.Martin{at}umh.es.

	Abstract

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that developing pancreas releases soluble factors responsible for "in vitro" endocrine pancreatic differentiation from embryonic stem (ES) cells. A mouse D3 ES cell line was transfected with a human insulin promoter--geo/pGK-hygro^r construct. To direct differentiation cells were cultured for 7 days to form embryoid bodies (EB) and then plated for an additional 7 days. During this 14-day period, besides eliminating LIF, cells were cultured in low serum concentration with the addition of conditioned media from e16.5 pancreatic buds (PB). Islet cell differentiation was studied by: i) X-gal staining after neomycin selection; ii) BrdU studies; iii) simple and double immunohistochemistry for insulin, C-peptide and Glut-2; iv) RT-PCR for insulin and PDx-1; v) insulin and C-peptide content and secretion assays; vi) intraperitoneal glucose tolerance test; vii) electrophysiology (patch-clamp studies in inside-out configuration) and viii) transplantation of differentiated cells under the kidney capsule of streptozotocin (STZ)-diabetic mice. The differentiated ES cells showed: changes in the mRNA levels of insulin and PDX-1; co-expression of insulin, c-peptide and glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study we establish a battery of techniques that could be used together to properly characterise islet-cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental to engineer cells from stem cells.