title: A new experimental protocol for preferential differentiation of mouse embryonic stem cells into insulin-producing cells.
abstract: Mouse embryonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with beta-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohormonal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Ortwin
authors.initials: O
authors.lastname: Naujok
authors.forename: Flavio
authors.initials: F
authors.lastname: Francini
authors.forename: Sally
authors.initials: S
authors.lastname: Picton
authors.forename: Anne
authors.initials: A
authors.lastname: Jörns
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Bailey
authors.forename: Sigurd
authors.initials: S
authors.lastname: Lenzen
mesh_terms: D000465:Algorithms; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D003921:Diabetes Mellitus, Experimental / Q000628:therapy; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000637:transplantation; D005786:Gene Expression Regulation / Q000187:drug effects; D006728:Hormones / Q000378:metabolism; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000502:physiology*; D008297:Male; D051379:Mice; D033581:Stem Cell Transplantation / Q000379:methods; D013311:Streptozocin; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D015415:Biomarkers; D003470:Culture Media; D006728:Hormones; D007328:Insulin; D014157:Transcription Factors; D013311:Streptozocin