Establishment of Immune-Evasive iPSCs from PBMCs Using B2M Knockout and CD47/HLA-E Overexpression
Establishment of Immune-Evasive iPSCs from PBMCs Using B2M Knockout and CD47/HLA-E Overexpression
Kim Cha Yeon(Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center); Jeong Cholong(Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center); Jeong Yeon-Ju(Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine); Sung Young Hoon(Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine); Han Youngjin(Department of Vascular Surgery, Asan Medical Center, University of Ulsan College of Medicine); 황창모(울산대학교)
22권 7호, 1005~1017쪽
초록
Background: Induced pluripotent stem cells (iPSCs) represent a promising source for regenerative therapies, yet allogeneic transplantation is limited by immune rejection. While strategies for generating hypoimmune iPSCs have been proposed, their efficacy after differentiation into lineage-specific cell types remains underexplored. Methods: A human iPSC line (36A) from peripheral blood mononuclear cells using a Sendai virus-based reprogramming protocol. Hypoimmune properties were conferred via CRISPR-Cpf1-mediated B2M knockout, combined with lentiviral overexpression of HLA-E and CD47. Immune evasion was validated using NK cell cytotoxicity assays. Endothelial differentiation was induced using a defined, stepwise protocol, and in vivo functionality was evaluated in humanized NSG mice. Results: The hypoimmune iPSCs retained pluripotency, exhibited stable karyotype, and demonstrated > 99% expression of HLA-E/CD47. NK cell-mediated lysis was significantly reduced in edited cells, although IFN-γ levels remained elevated. Upon differentiation, the hypoimmune iPSCs yielded > 98% CD31+CD144+ endothelial cells, which showed enhanced survival in vivo compared to wild-type controls. Conclusion: Multiplex gene editing successfully conferred durable immune evasion in both undifferentiated and endothelial-differentiated iPSCs. These findings support the clinical potential of hypoimmune iPSC-derived cell therapies for allogeneic transplantation without immunosuppression.
Abstract
Background: Induced pluripotent stem cells (iPSCs) represent a promising source for regenerative therapies, yet allogeneic transplantation is limited by immune rejection. While strategies for generating hypoimmune iPSCs have been proposed, their efficacy after differentiation into lineage-specific cell types remains underexplored. Methods: A human iPSC line (36A) from peripheral blood mononuclear cells using a Sendai virus-based reprogramming protocol. Hypoimmune properties were conferred via CRISPR-Cpf1-mediated B2M knockout, combined with lentiviral overexpression of HLA-E and CD47. Immune evasion was validated using NK cell cytotoxicity assays. Endothelial differentiation was induced using a defined, stepwise protocol, and in vivo functionality was evaluated in humanized NSG mice. Results: The hypoimmune iPSCs retained pluripotency, exhibited stable karyotype, and demonstrated > 99% expression of HLA-E/CD47. NK cell-mediated lysis was significantly reduced in edited cells, although IFN-γ levels remained elevated. Upon differentiation, the hypoimmune iPSCs yielded > 98% CD31+CD144+ endothelial cells, which showed enhanced survival in vivo compared to wild-type controls. Conclusion: Multiplex gene editing successfully conferred durable immune evasion in both undifferentiated and endothelial-differentiated iPSCs. These findings support the clinical potential of hypoimmune iPSC-derived cell therapies for allogeneic transplantation without immunosuppression.
- 발행기관:
- 한국조직공학과 재생의학회
- 분류:
- 기타의공학