engleski

Development of pancreatic precursor cells from mouse embryonic stem cells in vitro

Stem cells are self-renewing elements that can generate the many cell types in the body. These cells are derived from an early stage of the embryo and are named embryonic stem (ES) cells. ES cells isolated from 129/sv mice can be differentiate into insulin-producing cells (Lumelsky et al, 2001). When injected into diabetic mice, these cells undergo rapid vascularization and maintain a clustered, islet-like organization. There is no data about such work on NOD (non-obesity) mice, which spontaneously developed Diabetes mellitus. The aim of our work was generated structure like this from blastocysts of NOD mice. The procedure involved some steps and until today we successful generated embryoid bodies, which presents the base levels for expansion of insulin-producing cells. Blastocysts are isolated from uterus of 3.5 days pregnant NOD mice. After 48 hours of culturing, on mouse embryonic fibroblasts feeder layer, the zona pellucida was dissolved in acid media. Inner cell mass of blastocysts was expanded on gelatin-coated tissue culture surface in medium for ES cells in the presence of leukemia inhibitory factor (LIF). Four days later, in medium for embryonic cells without LIF were generated embryoid bodies. Such embryoid bodies were pick up and total cellular RNA was isolated using RNAzol B. The cDNA synthesis was carried out using Moloney murine leukemia virus. The amount of cDNA was normalized based on the signal from expressed β -actin mRNA. As markers of immature pancreatic cells we examined the expression of nestin and OCT-4 genes. In the future work, these cells positive to both tested genes should be propagated in insulin- secreting cells by using specific condition. This is a good way for producing of immunocompatible tissue for transplantation in diabetic recipients. It presents powerful tool for prevention of problems associated with diabetes late complications.

embryonic stem cells; pancreas; NOD mice

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