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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Institute of Bioengineering, School of Medicine, Miguel Hernández University, San Juan Campus, Alicante, Spain.
T
T
León-Quinto
J
J
Jones
A
A
Skoudy
M
M
Burcin
B
B
Soria
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018398:Homeodomain Proteins / Q000235:genetics; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000196:embryology / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection
D018398:Homeodomain Proteins; D007328:Insulin; C491328:Nkx6-1 protein, mouse