Differentiation of mouse embryonic stem cells to insulin-producing cells.
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.
Schroeder
Insa S
IS
In Vitro Differentiation Group, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstr.3, D-06466 Germany.
Rolletschek
Alexandra
A
Blyszczuk
Przemyslaw
P
Kania
Gabriela
G
Wobus
Anna M
AM
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D007328:Insulin / Q000096:biosynthesis* / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D051761:Paired Box Transcription Factors / Q000378:metabolism
D018398:Homeodomain Proteins; D007328:Insulin; D051761:Paired Box Transcription Factors; C110218:Pax4 protein, mouse