Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation

Background. Porcine fetal pancreas is a potential source of beta cells for transplantation, The immaturity of the cells is a problem. We have defined the optimal conditions for in vitro propagation of this tissue before trans plantation. Methods. Porcine fetal pancreas tissue was obtained for tissue culture at v arious stages of development. Serum-containing and serum-free media and a v ariety of potential differentiation factors were tested. In vitro, the numb ers of endocrine islet cells and their proliferation were quantified and fu nctional maturity of the beta cells was assessed by perifusion. Growth and maturation of the cells was assessed 3 months after transplantation into nu de mice. Results. Highest beta cell mass was obtained from end-gestational, as compa red with early fetal or neonatal, pancreas. Nicotinamide and sodium butyrat e effectively increased the insulin content and the number of endocrine cel ls in culture. In combination, these factors led up to a 90-fold increase i n the insulin content of islet-like cell clusters (ICC) as compared with un treated controls. However, a high level of cell death through apoptosis was observed in these maximally stimulated endocrine cells, and they did not s urvive as grafts when transplanted into nude mice. Instead, a serum-free cu lture medium containing 10 mM nicotinamide and 0.1 mM isobutylmethylxanthin e was found to support both differentiation and proliferation of endocrine cells as loose ICCs. Insulin release from these ICCs was sensitive to gluco se. When transplanted under the kidney capsule of normoglycemic nude mice, a high level of beta cell differentiation and function was evident only in the ICCs cultured in the serum-free medium, and in freshly isolated ICCs, W hen transplanted to hyperglycemic nude recipients, the cells cultured in se rum-free medium for 3 weeks reversed hyperglycemia more consistently and ra pidly than freshly isolated ICCs. Conclusions. Optimal maturation of porcine fetal pancreatic cells is obtain ed in serum-free medium supplemented with nicotinamide. Butyrate is a poten t stimulus for beta cell differentiation but leads to increased apoptotic c ell death.