In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

• Published in: Diabetologia 2004-08, Vol.47 (8), p.1442-1451
• Main Authors: LEON-QUINTO, T, JONES, J, SKOUDY, A, BURCIN, M, SORIA, B
• Format: Article
• Language: English
• Subjects: Animals; Bioengineering; Biological and medical sciences; Cell Differentiation - physiology; Cells, Cultured; Diabetes; Diabetes. Impaired glucose tolerance; Embryo, Mammalian; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Genes; Homeodomain Proteins - genetics; Hyperglycemia; Insulin; Insulin - metabolism; Insulin Secretion; Islets of Langerhans - embryology; Islets of Langerhans - metabolism; Medical sciences; Mice; Promoter Regions, Genetic; Stem cells; Stem Cells - cytology; Transcription factors; Transfection
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s00125-004-1458-8

We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice. The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols. These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.