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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2019-06-24

AUTHORS

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

ABSTRACT

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. More... »

PAGES

185

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  • Journal

    TITLE

    Stem Cell Research & Therapy

    ISSUE

    1

    VOLUME

    10

    Author Affiliations

  • 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
  • Department of Allergy and Rheumatology, Graduation School of Medicine, The University of Tokyo, Tokyo, Japan
  • Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
  • Department of Child Health and Development, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
  • Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
  • Division of Rheumatology and Allergy, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
  • Division of Pediatrics, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
  • Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
  • Department of Molecular Sciences on Diabetes, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
  • Department of Metabolism and Nutrition, Mizonokuchi Hospital, Teikyo University, Kawasaki, Kanagawa, Japan
  • Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
  • Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan
  • TOKIWA-Bio Inc., Tsukuba, Ibaraki, Japan
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1186/s13287-019-1273-2

    DOI

    http://dx.doi.org/10.1186/s13287-019-1273-2

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1117484301

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/31234949


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    46 schema:description 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.
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    53 CD34
    54 ConclusionThis
    55 HSPC mobilization
    56 HSPCs
    57 KLF4
    58 MethodsWe
    59 PB mononuclear cells
    60 PB-derived CD34
    61 Pb
    62 ResultsWe
    63 SOX2
    64 Sendai virus vector
    65 T-cell receptor gene rearrangements
    66 appearance
    67 assays
    68 beads
    69 biobanking
    70 blood cells
    71 blood-derived CD34
    72 c-Myc
    73 capability
    74 cases
    75 cell origin
    76 cell recovery
    77 cells
    78 colonies
    79 conditions
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    97 factors
    98 feeder-free conditions
    99 free iPSC lines
    100 frozen stocks
    101 gene rearrangements
    102 generation
    103 generation series
    104 healthy donors
    105 hematopoietic stem
    106 hiPSC generation
    107 iPSC generation
    108 iPSC lines
    109 immunomagnetic beads
    110 induced pluripotent stem cells
    111 invasiveness
    112 karyotype
    113 less workload
    114 line establishment
    115 lines
    116 majority
    117 markers
    118 mechanism
    119 method
    120 minimum invasiveness
    121 mobilization
    122 modeling
    123 mononuclear cells
    124 non-T-cell origin
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    126 origin
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    129 patient's peripheral blood cells
    130 patient-derived induced pluripotent stem cells
    131 patients
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    134 pluripotency
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    144 rate
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