Embryonic stem (ES) cells represent a potential source of transplantable cells for the treatment of diabetes because of their almost unlimited proliferation capacity and the potential to differentiate into insulin-producing cells. The differentiation conditions significantly affect the development of pancreatic cells from ES cells in vitro. Here, we describe an efficient strategy for the in vitro generation of insulin-producing cells. The protocol includes the spontaneous generation of multilineage progenitor cells and their differentiation induction by growth and extracellular matrix factors into C-peptide/insulin-positive islet-like clusters. The differentiated cells release insulin in response to high glucose concentrations. No specific selection for nestin-positive cells is performed at any time of ES cell differentiation.
