Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Stem Cell Research & Therapy

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[1] 0000 0001 0657 3887, grid.410849.0, Division of Pediatrics, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan;0000 0001 1014 9130, grid.265073.5, Department of Child Health and Development, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan;0000 0001 1014 9130, grid.265073.5, Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan;0000 0001 1014 9130, grid.265073.5, Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan;0000 0001 1014 9130, grid.265073.5, Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan;0000 0004 0372 3116, grid.412764.2, Division of Rheumatology and Allergy, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Allergy and Rheumatology, Graduation School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Molecular Sciences on Diabetes, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Prevention of Diabetes and Life-style Related Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0000 9239 9995, grid.264706.1, Department of Metabolism and Nutrition, Mizonokuchi Hospital, Teikyo University, Kawasaki, Kanagawa, Japan;0000 0001 2151 536X, grid.26999.3d, Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;0000 0001 2151 536X, grid.26999.3d, Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108-8639, Tokyo, Japan;0000 0001 2230 7538, grid.208504.b, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan;TOKIWA-Bio Inc., Tsukuba, Ibaraki, Japan;0000 0001 2369 4728, grid.20515.33, Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan;

关键词: Human-induced pluripotent stem cells;    Sendai virus vector;    SeVdp-302L;    Feeder-free;    CD34 hematopoietic stem and progenitor cells;    Peripheral blood;    Disease modeling;    Biobank;   

DOI  :  10.1186/s13287-019-1273-2

来源: publisher

【 摘 要 】

BackgroundDisease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming.MethodsWe have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined.ResultsWe succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.ConclusionThis robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

【 授权许可】

CC BY