Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector
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...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Okumura, Takashi [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2019 |
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| Schlagwörter: |
Human-induced pluripotent stem cells |
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| Anmerkung: |
© The Author(s). 2019 |
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| Übergeordnetes Werk: |
Enthalten in: Stem cell research & therapy - London : BioMed Central, 2010, 10(2019), 1 vom: 24. Juni |
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| Übergeordnetes Werk: |
volume:10 ; year:2019 ; number:1 ; day:24 ; month:06 |
| Links: |
|---|
| DOI / URN: |
10.1186/s13287-019-1273-2 |
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| Katalog-ID: |
SPR031227996 |
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| 100 | 1 | |a Okumura, Takashi |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector |
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| 520 | |a 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. | ||
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| 700 | 1 | |a Okano, Tsubasa |4 aut | |
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| 700 | 1 | |a Morio, Tomohiro |4 aut | |
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| 700 | 1 | |a Hasegawa, Hisanori |4 aut | |
| 700 | 1 | |a Mizoguchi, Fumitaka |4 aut | |
| 700 | 1 | |a Kawahata, Kimito |4 aut | |
| 700 | 1 | |a Kohsaka, Hitoshi |4 aut | |
| 700 | 1 | |a Moritake, Hiroshi |4 aut | |
| 700 | 1 | |a Nunoi, Hiroyuki |4 aut | |
| 700 | 1 | |a Waki, Hironori |4 aut | |
| 700 | 1 | |a Tamaru, Shin-ichi |4 aut | |
| 700 | 1 | |a Sasako, Takayoshi |4 aut | |
| 700 | 1 | |a Yamauchi, Toshimasa |4 aut | |
| 700 | 1 | |a Kadowaki, Takashi |4 aut | |
| 700 | 1 | |a Tanaka, Hiroyuki |4 aut | |
| 700 | 1 | |a Kitanaka, Sachiko |4 aut | |
| 700 | 1 | |a Nishimura, Ken |4 aut | |
| 700 | 1 | |a Ohtaka, Manami |4 aut | |
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| 700 | 1 | |a Otsu, Makoto |0 (orcid)0000-0002-9769-0217 |4 aut | |
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10.1186/s13287-019-1273-2 doi (DE-627)SPR031227996 (SPR)s13287-019-1273-2-e DE-627 ger DE-627 rakwb eng Okumura, Takashi verfasserin aut Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2019 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. Human-induced pluripotent stem cells (dpeaa)DE-He213 Sendai virus vector (dpeaa)DE-He213 SeVdp-302L (dpeaa)DE-He213 Feeder-free (dpeaa)DE-He213 CD34 (dpeaa)DE-He213 hematopoietic stem and progenitor cells (dpeaa)DE-He213 Peripheral blood (dpeaa)DE-He213 Disease modeling (dpeaa)DE-He213 Biobank (dpeaa)DE-He213 Horie, Yumi aut Lai, Chen-Yi aut Lin, Huan-Ting aut Shoda, Hirofumi aut Natsumoto, Bunki aut Fujio, Keishi aut Kumaki, Eri aut Okano, Tsubasa aut Ono, Shintaro aut Tanita, Kay aut Morio, Tomohiro aut Kanegane, Hirokazu aut Hasegawa, Hisanori aut Mizoguchi, Fumitaka aut Kawahata, Kimito aut Kohsaka, Hitoshi aut Moritake, Hiroshi aut Nunoi, Hiroyuki aut Waki, Hironori aut Tamaru, Shin-ichi aut Sasako, Takayoshi aut Yamauchi, Toshimasa aut Kadowaki, Takashi aut Tanaka, Hiroyuki aut Kitanaka, Sachiko aut Nishimura, Ken aut Ohtaka, Manami aut Nakanishi, Mahito aut Otsu, Makoto (orcid)0000-0002-9769-0217 aut Enthalten in Stem cell research & therapy London : BioMed Central, 2010 10(2019), 1 vom: 24. Juni (DE-627)624251047 (DE-600)2548671-8 1757-6512 nnns volume:10 year:2019 number:1 day:24 month:06 https://dx.doi.org/10.1186/s13287-019-1273-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2019 1 24 06 |
| spelling |
10.1186/s13287-019-1273-2 doi (DE-627)SPR031227996 (SPR)s13287-019-1273-2-e DE-627 ger DE-627 rakwb eng Okumura, Takashi verfasserin aut Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2019 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. Human-induced pluripotent stem cells (dpeaa)DE-He213 Sendai virus vector (dpeaa)DE-He213 SeVdp-302L (dpeaa)DE-He213 Feeder-free (dpeaa)DE-He213 CD34 (dpeaa)DE-He213 hematopoietic stem and progenitor cells (dpeaa)DE-He213 Peripheral blood (dpeaa)DE-He213 Disease modeling (dpeaa)DE-He213 Biobank (dpeaa)DE-He213 Horie, Yumi aut Lai, Chen-Yi aut Lin, Huan-Ting aut Shoda, Hirofumi aut Natsumoto, Bunki aut Fujio, Keishi aut Kumaki, Eri aut Okano, Tsubasa aut Ono, Shintaro aut Tanita, Kay aut Morio, Tomohiro aut Kanegane, Hirokazu aut Hasegawa, Hisanori aut Mizoguchi, Fumitaka aut Kawahata, Kimito aut Kohsaka, Hitoshi aut Moritake, Hiroshi aut Nunoi, Hiroyuki aut Waki, Hironori aut Tamaru, Shin-ichi aut Sasako, Takayoshi aut Yamauchi, Toshimasa aut Kadowaki, Takashi aut Tanaka, Hiroyuki aut Kitanaka, Sachiko aut Nishimura, Ken aut Ohtaka, Manami aut Nakanishi, Mahito aut Otsu, Makoto (orcid)0000-0002-9769-0217 aut Enthalten in Stem cell research & therapy London : BioMed Central, 2010 10(2019), 1 vom: 24. Juni (DE-627)624251047 (DE-600)2548671-8 1757-6512 nnns volume:10 year:2019 number:1 day:24 month:06 https://dx.doi.org/10.1186/s13287-019-1273-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2019 1 24 06 |
| allfields_unstemmed |
10.1186/s13287-019-1273-2 doi (DE-627)SPR031227996 (SPR)s13287-019-1273-2-e DE-627 ger DE-627 rakwb eng Okumura, Takashi verfasserin aut Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2019 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. Human-induced pluripotent stem cells (dpeaa)DE-He213 Sendai virus vector (dpeaa)DE-He213 SeVdp-302L (dpeaa)DE-He213 Feeder-free (dpeaa)DE-He213 CD34 (dpeaa)DE-He213 hematopoietic stem and progenitor cells (dpeaa)DE-He213 Peripheral blood (dpeaa)DE-He213 Disease modeling (dpeaa)DE-He213 Biobank (dpeaa)DE-He213 Horie, Yumi aut Lai, Chen-Yi aut Lin, Huan-Ting aut Shoda, Hirofumi aut Natsumoto, Bunki aut Fujio, Keishi aut Kumaki, Eri aut Okano, Tsubasa aut Ono, Shintaro aut Tanita, Kay aut Morio, Tomohiro aut Kanegane, Hirokazu aut Hasegawa, Hisanori aut Mizoguchi, Fumitaka aut Kawahata, Kimito aut Kohsaka, Hitoshi aut Moritake, Hiroshi aut Nunoi, Hiroyuki aut Waki, Hironori aut Tamaru, Shin-ichi aut Sasako, Takayoshi aut Yamauchi, Toshimasa aut Kadowaki, Takashi aut Tanaka, Hiroyuki aut Kitanaka, Sachiko aut Nishimura, Ken aut Ohtaka, Manami aut Nakanishi, Mahito aut Otsu, Makoto (orcid)0000-0002-9769-0217 aut Enthalten in Stem cell research & therapy London : BioMed Central, 2010 10(2019), 1 vom: 24. Juni (DE-627)624251047 (DE-600)2548671-8 1757-6512 nnns volume:10 year:2019 number:1 day:24 month:06 https://dx.doi.org/10.1186/s13287-019-1273-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2019 1 24 06 |
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10.1186/s13287-019-1273-2 doi (DE-627)SPR031227996 (SPR)s13287-019-1273-2-e DE-627 ger DE-627 rakwb eng Okumura, Takashi verfasserin aut Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2019 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. Human-induced pluripotent stem cells (dpeaa)DE-He213 Sendai virus vector (dpeaa)DE-He213 SeVdp-302L (dpeaa)DE-He213 Feeder-free (dpeaa)DE-He213 CD34 (dpeaa)DE-He213 hematopoietic stem and progenitor cells (dpeaa)DE-He213 Peripheral blood (dpeaa)DE-He213 Disease modeling (dpeaa)DE-He213 Biobank (dpeaa)DE-He213 Horie, Yumi aut Lai, Chen-Yi aut Lin, Huan-Ting aut Shoda, Hirofumi aut Natsumoto, Bunki aut Fujio, Keishi aut Kumaki, Eri aut Okano, Tsubasa aut Ono, Shintaro aut Tanita, Kay aut Morio, Tomohiro aut Kanegane, Hirokazu aut Hasegawa, Hisanori aut Mizoguchi, Fumitaka aut Kawahata, Kimito aut Kohsaka, Hitoshi aut Moritake, Hiroshi aut Nunoi, Hiroyuki aut Waki, Hironori aut Tamaru, Shin-ichi aut Sasako, Takayoshi aut Yamauchi, Toshimasa aut Kadowaki, Takashi aut Tanaka, Hiroyuki aut Kitanaka, Sachiko aut Nishimura, Ken aut Ohtaka, Manami aut Nakanishi, Mahito aut Otsu, Makoto (orcid)0000-0002-9769-0217 aut Enthalten in Stem cell research & therapy London : BioMed Central, 2010 10(2019), 1 vom: 24. Juni (DE-627)624251047 (DE-600)2548671-8 1757-6512 nnns volume:10 year:2019 number:1 day:24 month:06 https://dx.doi.org/10.1186/s13287-019-1273-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2019 1 24 06 |
| allfieldsSound |
10.1186/s13287-019-1273-2 doi (DE-627)SPR031227996 (SPR)s13287-019-1273-2-e DE-627 ger DE-627 rakwb eng Okumura, Takashi verfasserin aut Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2019 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. Human-induced pluripotent stem cells (dpeaa)DE-He213 Sendai virus vector (dpeaa)DE-He213 SeVdp-302L (dpeaa)DE-He213 Feeder-free (dpeaa)DE-He213 CD34 (dpeaa)DE-He213 hematopoietic stem and progenitor cells (dpeaa)DE-He213 Peripheral blood (dpeaa)DE-He213 Disease modeling (dpeaa)DE-He213 Biobank (dpeaa)DE-He213 Horie, Yumi aut Lai, Chen-Yi aut Lin, Huan-Ting aut Shoda, Hirofumi aut Natsumoto, Bunki aut Fujio, Keishi aut Kumaki, Eri aut Okano, Tsubasa aut Ono, Shintaro aut Tanita, Kay aut Morio, Tomohiro aut Kanegane, Hirokazu aut Hasegawa, Hisanori aut Mizoguchi, Fumitaka aut Kawahata, Kimito aut Kohsaka, Hitoshi aut Moritake, Hiroshi aut Nunoi, Hiroyuki aut Waki, Hironori aut Tamaru, Shin-ichi aut Sasako, Takayoshi aut Yamauchi, Toshimasa aut Kadowaki, Takashi aut Tanaka, Hiroyuki aut Kitanaka, Sachiko aut Nishimura, Ken aut Ohtaka, Manami aut Nakanishi, Mahito aut Otsu, Makoto (orcid)0000-0002-9769-0217 aut Enthalten in Stem cell research & therapy London : BioMed Central, 2010 10(2019), 1 vom: 24. Juni (DE-627)624251047 (DE-600)2548671-8 1757-6512 nnns volume:10 year:2019 number:1 day:24 month:06 https://dx.doi.org/10.1186/s13287-019-1273-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2019 1 24 06 |
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Enthalten in Stem cell research & therapy 10(2019), 1 vom: 24. Juni volume:10 year:2019 number:1 day:24 month:06 |
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Enthalten in Stem cell research & therapy 10(2019), 1 vom: 24. Juni volume:10 year:2019 number:1 day:24 month:06 |
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Human-induced pluripotent stem cells Sendai virus vector SeVdp-302L Feeder-free CD34 hematopoietic stem and progenitor cells Peripheral blood Disease modeling Biobank |
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Okumura, Takashi @@aut@@ Horie, Yumi @@aut@@ Lai, Chen-Yi @@aut@@ Lin, Huan-Ting @@aut@@ Shoda, Hirofumi @@aut@@ Natsumoto, Bunki @@aut@@ Fujio, Keishi @@aut@@ Kumaki, Eri @@aut@@ Okano, Tsubasa @@aut@@ Ono, Shintaro @@aut@@ Tanita, Kay @@aut@@ Morio, Tomohiro @@aut@@ Kanegane, Hirokazu @@aut@@ Hasegawa, Hisanori @@aut@@ Mizoguchi, Fumitaka @@aut@@ Kawahata, Kimito @@aut@@ Kohsaka, Hitoshi @@aut@@ Moritake, Hiroshi @@aut@@ Nunoi, Hiroyuki @@aut@@ Waki, Hironori @@aut@@ Tamaru, Shin-ichi @@aut@@ Sasako, Takayoshi @@aut@@ Yamauchi, Toshimasa @@aut@@ Kadowaki, Takashi @@aut@@ Tanaka, Hiroyuki @@aut@@ Kitanaka, Sachiko @@aut@@ Nishimura, Ken @@aut@@ Ohtaka, Manami @@aut@@ Nakanishi, Mahito @@aut@@ Otsu, Makoto @@aut@@ |
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2019-06-24T00:00:00Z |
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624251047 |
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SPR031227996 |
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englisch |
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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. 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Okumura, Takashi |
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Okumura, Takashi misc Human-induced pluripotent stem cells misc Sendai virus vector misc SeVdp-302L misc Feeder-free misc CD34 misc hematopoietic stem and progenitor cells misc Peripheral blood misc Disease modeling misc Biobank Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector |
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Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector Human-induced pluripotent stem cells (dpeaa)DE-He213 Sendai virus vector (dpeaa)DE-He213 SeVdp-302L (dpeaa)DE-He213 Feeder-free (dpeaa)DE-He213 CD34 (dpeaa)DE-He213 hematopoietic stem and progenitor cells (dpeaa)DE-He213 Peripheral blood (dpeaa)DE-He213 Disease modeling (dpeaa)DE-He213 Biobank (dpeaa)DE-He213 |
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Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived $ CD34^{+} $ cells using the auto-erasable Sendai virus vector |
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Okumura, Takashi Horie, Yumi Lai, Chen-Yi Lin, Huan-Ting Shoda, Hirofumi Natsumoto, Bunki Fujio, Keishi Kumaki, Eri Okano, Tsubasa Ono, Shintaro Tanita, Kay Morio, Tomohiro Kanegane, Hirokazu Hasegawa, Hisanori Mizoguchi, Fumitaka Kawahata, Kimito Kohsaka, Hitoshi Moritake, Hiroshi Nunoi, Hiroyuki Waki, Hironori Tamaru, Shin-ichi Sasako, Takayoshi Yamauchi, Toshimasa Kadowaki, Takashi Tanaka, Hiroyuki Kitanaka, Sachiko Nishimura, Ken Ohtaka, Manami Nakanishi, Mahito Otsu, Makoto |
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Okumura, Takashi |
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10.1186/s13287-019-1273-2 |
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robust and highly efficient hipsc generation from patient non-mobilized peripheral blood-derived $ cd34^{+} $ cells using the auto-erasable sendai virus vector |
| title_auth |
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 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. © The Author(s). 2019 |
| abstractGer |
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. © The Author(s). 2019 |
| abstract_unstemmed |
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. © The Author(s). 2019 |
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Horie, Yumi Lai, Chen-Yi Lin, Huan-Ting Shoda, Hirofumi Natsumoto, Bunki Fujio, Keishi Kumaki, Eri Okano, Tsubasa Ono, Shintaro Tanita, Kay Morio, Tomohiro Kanegane, Hirokazu Hasegawa, Hisanori Mizoguchi, Fumitaka Kawahata, Kimito Kohsaka, Hitoshi Moritake, Hiroshi Nunoi, Hiroyuki Waki, Hironori Tamaru, Shin-ichi Sasako, Takayoshi Yamauchi, Toshimasa Kadowaki, Takashi Tanaka, Hiroyuki Kitanaka, Sachiko Nishimura, Ken Ohtaka, Manami Nakanishi, Mahito Otsu, Makoto |
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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.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Human-induced pluripotent stem cells</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Sendai virus vector</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">SeVdp-302L</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Feeder-free</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">CD34</subfield><subfield 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