Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector
Abstract Background Disease 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 ca...
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BMC
2019-06-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | http://link.springer.com/article/10.1186/s13287-019-1273-2 |
| _version_ | 1819115534931722240 |
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| author | Takashi Okumura Yumi Horie Chen-Yi Lai Huan-Ting Lin Hirofumi Shoda Bunki Natsumoto Keishi Fujio Eri Kumaki Tsubasa Okano Shintaro Ono Kay Tanita Tomohiro Morio Hirokazu Kanegane Hisanori Hasegawa Fumitaka Mizoguchi Kimito Kawahata Hitoshi Kohsaka Hiroshi Moritake Hiroyuki Nunoi Hironori Waki Shin-ichi Tamaru Takayoshi Sasako Toshimasa Yamauchi Takashi Kadowaki Hiroyuki Tanaka Sachiko Kitanaka Ken Nishimura Manami Ohtaka Mahito Nakanishi Makoto Otsu |
| author_facet | Takashi Okumura Yumi Horie Chen-Yi Lai Huan-Ting Lin Hirofumi Shoda Bunki Natsumoto Keishi Fujio Eri Kumaki Tsubasa Okano Shintaro Ono Kay Tanita Tomohiro Morio Hirokazu Kanegane Hisanori Hasegawa Fumitaka Mizoguchi Kimito Kawahata Hitoshi Kohsaka Hiroshi Moritake Hiroyuki Nunoi Hironori Waki Shin-ichi Tamaru Takayoshi Sasako Toshimasa Yamauchi Takashi Kadowaki Hiroyuki Tanaka Sachiko Kitanaka Ken Nishimura Manami Ohtaka Mahito Nakanishi Makoto Otsu |
| author_sort | Takashi Okumura |
| collection | DOAJ |
| description | Abstract Background Disease 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. Methods We 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. Results We 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. Conclusion This 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. |
| first_indexed | 2024-12-22T05:02:44Z |
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| id | doaj.art-e9a9e96b9aae4d00878ec8608b4ef191 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-22T05:02:44Z |
| publishDate | 2019-06-01 |
| publisher | BMC |
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| series | Stem Cell Research & Therapy |
| spelling | doaj.art-e9a9e96b9aae4d00878ec8608b4ef1912022-12-21T18:38:13ZengBMCStem Cell Research & Therapy1757-65122019-06-0110111610.1186/s13287-019-1273-2Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vectorTakashi Okumura0Yumi Horie1Chen-Yi Lai2Huan-Ting Lin3Hirofumi Shoda4Bunki Natsumoto5Keishi Fujio6Eri Kumaki7Tsubasa Okano8Shintaro Ono9Kay Tanita10Tomohiro Morio11Hirokazu Kanegane12Hisanori Hasegawa13Fumitaka Mizoguchi14Kimito Kawahata15Hitoshi Kohsaka16Hiroshi Moritake17Hiroyuki Nunoi18Hironori Waki19Shin-ichi Tamaru20Takayoshi Sasako21Toshimasa Yamauchi22Takashi Kadowaki23Hiroyuki Tanaka24Sachiko Kitanaka25Ken Nishimura26Manami Ohtaka27Mahito Nakanishi28Makoto Otsu29Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of TokyoDivision of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of TokyoDivision of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of TokyoDivision of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of TokyoDepartment of Allergy and Rheumatology, Graduation School of Medicine, The University of TokyoDepartment of Allergy and Rheumatology, Graduation School of Medicine, The University of TokyoDepartment of Allergy and Rheumatology, Graduation School of Medicine, The University of TokyoDepartment of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Child Health and Development, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDepartment of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental UniversityDivision of Pediatrics, Faculty of Medicine, University of MiyazakiDivision of Pediatrics, Faculty of Medicine, University of MiyazakiDepartment of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of TokyoDepartment of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of TokyoDepartment of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of TokyoDepartment of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of TokyoDepartment of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of TokyoDepartment of Pediatrics, Graduate School of Medicine, The University of TokyoDepartment of Pediatrics, Graduate School of Medicine, The University of TokyoLaboratory of Gene Regulation, Faculty of Medicine, University of TsukubaBiotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and TechnologyBiotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and TechnologyDivision of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of TokyoAbstract Background Disease 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. Methods We 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. Results We 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. Conclusion This 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.http://link.springer.com/article/10.1186/s13287-019-1273-2Human-induced pluripotent stem cellsSendai virus vectorSeVdp-302LFeeder-freeCD34+ hematopoietic stem and progenitor cellsPeripheral blood |
| spellingShingle | Takashi Okumura Yumi Horie Chen-Yi Lai Huan-Ting Lin Hirofumi Shoda Bunki Natsumoto Keishi Fujio Eri Kumaki Tsubasa Okano Shintaro Ono Kay Tanita Tomohiro Morio Hirokazu Kanegane Hisanori Hasegawa Fumitaka Mizoguchi Kimito Kawahata Hitoshi Kohsaka Hiroshi Moritake Hiroyuki Nunoi Hironori Waki Shin-ichi Tamaru Takayoshi Sasako Toshimasa Yamauchi Takashi Kadowaki Hiroyuki Tanaka Sachiko Kitanaka Ken Nishimura Manami Ohtaka Mahito Nakanishi Makoto Otsu 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 Human-induced pluripotent stem cells Sendai virus vector SeVdp-302L Feeder-free CD34+ hematopoietic stem and progenitor cells Peripheral blood |
| title | Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector |
| title_full | Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector |
| title_fullStr | Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector |
| title_full_unstemmed | Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector |
| title_short | Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector |
| title_sort | robust and highly efficient hipsc generation from patient non mobilized peripheral blood derived cd34 cells using the auto erasable sendai virus vector |
| topic | Human-induced pluripotent stem cells Sendai virus vector SeVdp-302L Feeder-free CD34+ hematopoietic stem and progenitor cells Peripheral blood |
| url | http://link.springer.com/article/10.1186/s13287-019-1273-2 |
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