Here, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into insulin-producing cells. The three-step protocol comprises (i) the formation of embryoid bodies, (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage. Differentiated cells can be obtained within approximately 33 d. Differentiation induction by growth and extracellular-matrix factors, including laminin, nicotinamide and insulin, leads to the formation of ES-derived progeny that resembles cells committed to the pancreatic lineage. During differentiation, transcript levels of genes expressed in early pancreatic cells are upregulated. Continued differentiation results in the development of C-peptide/insulin-positive islet-like clusters that release insulin upon glucose stimulation. Differentiated ES cells that overexpress the pancreatic developmental control gene Pax4 develop insulin-secretory granules and reveal functional properties with respect to the pancreas-specific ATP-modulated K+ channel and the normalization of glycemia of streptozotocin-treated diabetic mice.

*Note: In the version of this article originally published online, several citations of refs. 37 and 38 should have referred to ref. 35; "CRG8" should have read "CGR8" in the Materials section; some information was omitted from the Reagent Setup listings for ES-cell culture medium and Solution A for ELISA; the heading for Step 16B should have read "(B) 10 μg cm^–2 collagin I–coated dishes" (not 10 μg cm^–2); Step 46 should have referred to a total volume of 588.75 μl for the PCR master mix (not 587.5 μl); a query to the author at the end of the first subsection of Anticipated Results should have been removed; and the labels in Figure 3 should have been aligned more precisely with respect to the panels. These errors have been corrected in all versions of the article.

*Note: In the version of this article originally published online, the Reagent Setup listings for ES-cell culture medium should have included the following additional ingredients: “100 μM β-mercaptoethanol, 0.05 mg ml^-1 streptomycin, 0.03 mg ml^-1 penicillin, 15 % FCS, and 1000 units ml^-1 LIF.” In the HTML version, the ingredients were erroneously inserted in the wrong location. These errors have been corrected in the HTML and PDF versions of the article.