Cell Journal (Jan 2009)
Effect of Extracellular Matrix on Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells
Abstract
Objective: Evaluation of extracellular matrics (ECMs) effect on differentiation ofembryonic stem cells (ESCs) to pancreatic β-cell.Materials and Methods: Mouse ESC line, Royan B1, was subjected to differentiationinto β-like cells in a three-step method: generation of embryoid bodies(EBs), spontaneous differentiation and induction by Nicotinamide onto differentmatrices including poly L-ornithine/laminin, gelatin, and two different dilution ofmatrigel (1:30, 1:100) and control group (no ECM). At the final step, differentiatedcells were analyzed for expression of some pancreas-specific genes using "semiquantitativeRT-PCR ", for detection of insulin and C-peptide presence in cells using"immunocytochemistry" and for the evaluation of the amount of secretd insulinin response to glucose Using "insulin secretion assay".Results: The semi-quantitative RT-PCR analysis of differentiated cells on 1:30matrigel coated-plates showed consistent higher expression of β-cell specificmarkers including Insulin I, Insulin II, Slc2a2 in comparison to the other ECMs.The results of immunostainig for C-peptide showed no significant differences betweenthe experimental groups and finally insulin secretion assay revealed thatdifferentiated cells on 1:30 matrigel coated-plates secreted more insulin in responseto glucose in comparison to the other ECMs.Conclusion: Our results suggest that type of ECM may influence ESC differentiationinto insulin-secreting cells and 1:30 matrigel was more effective. However, thesuccess rate of differentiation needs further investigations using other appropriateECMs.
