title: Evaluating protocols for embryonic stem cell differentiation into insulin-secreting beta-cells using insulin II-GFP as a specific and noninvasive reporter.
abstract: Stable and full differentiation of pluripotent stem cells into functional beta-cells offers the potential to treat type I diabetes with a theoretically inexhaustible source of replacement cells. In addition to the difficulties in directed differentiation, progress toward an optimized and reliable protocol has been hampered by the complication that cultured cells will concentrate insulin from the media, thus making it difficult to tell which, if any, cells are producing insulin. To address this, we utilized a novel murine embryonic stem cell (mESC) research model, in which the green fluorescent protein (GFP) has been inserted within the C-peptide of the mouse insulinII gene (InsulinII-GFP). Using this method, cells producing insulin are easily identified. We then compared four published protocols for differentiating mESCs into beta-cells to evaluate their relative efficiency by assaying intrinsic insulin production. Cells differentiated using each protocol were easily distinguished based on culture conditions and morphology. This comparison is strengthened because all testing is performed within the same laboratory by the same researchers, thereby removing interlaboratory variability in culture, cells, or analysis. Differentiated cells were analyzed and sorted based on GFP fluorescence as compared to wild type cells. Each differentiation protocol increased GFP fluorescence but only modestly. None of these protocols yielded more than 3% of cells capable of insulin biosynthesis indicating the relative inefficiency of all analyzed protocols. Therefore, improved beta-cells differentiation protocols are needed, and these insulin II GFP cells may prove to be an important tool to accelerate this process.
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, Pittsburgh Development Center, Magee-Womens Research Institute and Foundation, University of Pittsburgh School of Medicine , Pittsburgh, PA, USA. benyehudaha@upmc.edu
authors.forename: Ahmi
authors.initials: A
authors.lastname: Ben-Yehudah
authors.forename: Carlie
authors.initials: C
authors.lastname: White
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Navara
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Castro
authors.forename: Diego
authors.initials: D
authors.lastname: Ize-Ludlow
authors.forename: Benjamin
authors.initials: B
authors.lastname: Shaffer
authors.forename: Meena
authors.initials: M
authors.lastname: Sukhwani
authors.forename: Clayton E
authors.initials: CE
authors.lastname: Mathews
authors.forename: J Richard
authors.initials: JR
authors.lastname: Chaillet
authors.forename: Selma F
authors.initials: SF
authors.lastname: Witchel
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000502:physiology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D007328:Insulin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins