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Background: <p>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.</p> Methods: <p>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 <i>in vivo</i> functionality was evaluated in humanized NSG mice.</p> Results: <p>The hypoimmune iPSCs retained pluripotency, exhibited stable karyotype, and demonstrated &gt; 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 &gt; 98% CD31<sup>+</sup>CD144<sup>+</sup> endothelial cells, which showed enhanced survival <i>in vivo</i> compared to wild-type controls.</p> Conclusion: <p>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.</p>

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Establishment of Immune-Evasive iPSCs from PBMCs Using B2M Knockout and CD47/HLA-E Overexpression

  • 期刊论文

  • 发表日期：

    2025年7月19日

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  • Cha Yeon Kim,
  • Cholong Jeong,
  • Yeon-Ju Jeong,
  • Young Hoon Sung,
  • Youngjin Han,
  • Changmo Hwang

摘要

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.