Stem Cell Core: iPSC Technology and Therapeutic implications SCHOOL of MEDICINE 1 2 3 The Office of Research Core Arazdordi Toumadje , Michael McConnell , Kenneth Brayman Administration 1Department of Cell Biology, Director of Stem cell Core; 2 Department of Biochemistry and Molecular Genetics, Co-director of Stem Cell Core; 3Department of Surgery, Co-director of Stem Cell Core; University of Virginia, Charlottesville, VA
ABSTRACT: The human Stem Cell Core is the latest addition to the UVA School of Medicine Core METHODS: Facility family that is missioned to provide research grade induced pluripotent stem cells (iPSCs) and 1) Plate 150-200,000 fibroblasts in 2 wells of 6-well plate their downstream derivatives to investigators to facilitate the generation of clinical quality cells for 2) Transfect with Sendai virus carrying Oct4, Sox2, KLF4, c-MYC human cell therapy. Overall, the core will provide three categories of services: Production and Cell Banking (organ-and disease-specific derived iPSCs); Product Development (individualized projects - 3) After 7 days, harvest transfected cells and plate 200000, 100000, 50000 cells in each of 6 10-cm generation of specific cell types from iPSCs such as neurons, cardiomyocytes, insulin producing β dishes coated with mouse embryonic fibroblasts (MEF) cells cells); and Education and Training workshops to students and faculties. 4) After 3-4 weeks, pick “good” colonies and re-plate in MEF coated 12-well plates (one colony in each well) Human pluripotent stem cells including embryonic stem cells (hESC) and induced stem cells (hiPSC) have unlimited ability for self-renewal and can differentiate to all cell types of the three germ layers, 5) After few days when the colonies are large, pick “good” colonies from 12-well plates and re- which make them well suited for generating autologous and custom-tailored cells for regenerative plate into 6-wellplates coated with MEF cells medicine and to produce in vitro disease models to study pathogenesis, treatments, drug screening, 6) Harvest “good colonies” from each of 6-well plates into 10- cm dishes using collagenase and toxicology. While hESCs are derived from inner-cell mass of early embryos, hiPSCs are generated enzyme (25-35 minutes). Collagenase concentration and time of incubation need to be by artificially converting non-pluripotent somatic cells to pluripotency by forced expression of specific optimized to detach only colonies leaving behind MEF cells. key pluripotent stem cell transcription factor genes including Oct-4, Sox2, c-MYC, and KLF4. Here, we show a step-by-step production of iPSC from an established human foreskin fibroblast (ATCC # CRL 7) Collect colonies and cryo-store. 2522) using non-integrating Sendai virus reprogramming Kit (LifeTechnology # A13780-01).
Reprogramming of Somatic Cells to Induced Pluripotent Stem Cells
Stem Cell Core services Differentiation of iPSCs to insulin producing cell
Stem Cell 400 Core 300
200 Insulin PDX-1 100
0 S0Day0 S1 Day2 S2 Day4 Day8S3 Day11 S4 Day14 S5 Day19 S6 S7 50000 Production Education and Cell Product and Training 40000 Banking Development
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3000 20000 Glut-2 2000 MafA
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0 0 Day0 Day2 Day4 Day8 Day11 Day14 Day19 Generate S0Day0 S1 Day2 S2Day4 S3Day8 S4Day11 S5Day14 S6Day19 iPSCs for Cell Banking Individualized S7 Multistage differentiation of iPSCS0 to insulin S1 producingS2 S3 cells S4 S5 S6 Workshops Consultations S7 distribution projects
ADVISORY COMMITTEE: Kenneth L. Brayman, Michael J. McConnell, Jay W. ACKNOWLEDGEMENT: This project is funded by Focus to Cure Diabetes Foundation and UVA Fox, Jeffery J. Saucerman, Brent A. French, Mazhar Adli, Bryce M. Pascal, George Launchpad grant. J. Christ, Shayn Peirce-Cottler, Quanjun Cui, Charles R. Farber, Erin P. Foff