先端医療技術開発センター 再生・細胞医薬研究ラボラトリー

原 弘真

Hiromasa Hara

基本情報

所属
自治医科大学 先端医療技術開発センター 再生・細胞医薬研究ラボラトリー 講師
学位
農学(信州大学)

J-GLOBAL ID
201601004312261838
researchmap会員ID
B000259287

学歴

 3

論文

 30
  • Suvd Byambaa, Hideki Uosaki, Tsukasa Ohmori, Hiromasa Hara, Hitoshi Endo, Osamu Nureki, Yutaka Hanazono
    Molecular therapy. Methods & clinical development 20 451-462 2021年3月12日  
    We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.
  • Tatsuya Anzai, Hiromasa Hara, Nawin Chanthra, Taketaro Sadahiro, Masaki Ieda, Yutaka Hanazono, Hideki Uosaki
    Methods in molecular biology (Clifton, N.J.) 2320 247-259 2021年  
    A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.
  • Tomoyuki Abe, Hideki Uosaki, Hiroaki Shibata, Hiromasa Hara, Borjigin Sarentonglaga, Yoshikazu Nagao, Yutaka Hanazono
    Experimental hematology 2021年1月1日  
    We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.
  • Byambaa S, Uosaki H, Hara H, Nagao Y, Abe T, Shibata H, Nureki O, Ohmori T, Hanazono Y
    Experimental animals 69(2) 189-198 2019年12月  査読有り
  • 阿部 朋行, 柴田 宏昭, 魚崎 英毅, 原 弘真, ボラジギン・サラントラガ, 長尾 慶和, 花園 豊
    Organ Biology 26(3) 87-87 2019年10月  

MISC

 10

講演・口頭発表等

 52

共同研究・競争的資金等の研究課題

 4