Institute of Clinical Oncology, Research Center of Cancer Diagnosis and Therapy, and Department of Clinical Oncology, The First Affiliated Hospital of Jinan University, Guangzhou, China.
Guangzhou Future Homo Sapiens Institute of Biomedicine and Health (GFBH), Guangzhou, China.
Stem Cell Res Ther. 2022 Feb 2;13(1):48. doi: 10.1186/s13287-022-02720-7.
Endothelial cells (ECs) derived from human-induced pluripotent stem cell (iPSC) are a valuable cell resource for cardiovascular regeneration. To avoid time-consuming preparation from primary autologous cells, the allogeneic iPSC-ECs are being expected to become "off-the-shelf" cell products. However, allorejection caused by HLA mismatching is a major barrier for this strategy. Although the "hypoimmunogenic" iPSCs could be simply generated by inhibition of HLA-I expression via β-2 microglobulin knockout (B2M KO), the deletion of HLA-I expression will activate natural killer (NK) cells, which kill the HLA-I negative cells. To inhibit NK activation, we proposed to generate HLA-matched iPSCs based on patient's HLA genotyping by HLA exchanging approach to express the required HLA allele.
To establish a prototype of HLA exchanging system, the expression of HLA-I molecules of iPSCs was inhibited by CRISPR/Cas9-mediated B2M KO, and then HLA-A*11:01 allele, as a model molecule, was introduced into B2M KO iPSCs by lentiviral gene transfer. HLA-I-modified iPSCs were tested for their pluripotency and ability to differentiate into ECs. The stimulation of iPSC-EC to allogeneic T and NK cells was detected by respective co-culture of PBMC-EC and NK-EC. Finally, the iPSC-ECs were used as the seeding cells to re-endothelialize the decellularized valves.
We generated the iPSCs only expressed one HLA-A allele (HLA-A 11:01) by B2M KO plus HLA gene transfer. These HLA-I-modified iPSCs maintained pluripotency and furthermore were successfully differentiated into functional ECs assessed by tube formation assay. Single HLA-A11:01-matched iPSC-ECs significantly less induced the allogeneic response of CD8+ T cell and NK cells expressing matched HLA-A*11:01 and other HLA-A,-B and -C alleles. These cells were successfully used to re-endothelialize the decellularized valves.
In summary, a simple HLA-I exchanging system has been created by efficient HLA engineering of iPSCs to evade both of the alloresponse of CD8+ T cells and the activation of NK cells. This technology has been applied to generate iPSC-ECs for the engineering of cellular heart valves. Our strategy should be extremely useful if the "off-the-shelf" and "non-immunogenic" allogeneic iPSCs were created for the common HLA alleles.
源自人诱导多能干细胞（iPSC）的内皮细胞（ECs）是心血管再生的有价值的细胞资源。为避免从原代自体细胞中进行耗时的制备，异体 iPSC-EC 有望成为“现成”的细胞产品。然而，HLA 错配引起的同种异体排斥是该策略的主要障碍。虽然通过β-2 微球蛋白敲除（B2M KO）抑制 HLA-I 表达可以简单地生成“低免疫原性”iPSC，但 HLA-I 表达的缺失会激活自然杀伤（NK）细胞，从而杀死 HLA-I 阴性细胞。为了抑制 NK 激活，我们建议通过 HLA 交换方法根据患者的 HLA 基因分型生成 HLA 匹配的 iPSC，以表达所需的 HLA 等位基因。
为了建立 HLA 交换系统的原型，通过 CRISPR/Cas9 介导的 B2M KO 抑制 iPSC 中 HLA-I 分子的表达，然后通过慢病毒基因转移将 HLA-A*11:01 等位基因作为模型分子引入 B2M KO iPSC。测试了 HLA-I 修饰的 iPSC 的多能性和分化为 EC 的能力。通过 PBMC-EC 和 NK-EC 的各自共培养检测 iPSC-EC 对同种异体 T 和 NK 细胞的刺激。最后，将 iPSC-EC 用作种细胞，以使脱细胞瓣膜再内皮化。
我们通过 B2M KO 加 HLA 基因转移生成了仅表达一个 HLA-A 等位基因（HLA-A11:01）的 iPSC。这些 HLA-I 修饰的 iPSC 保持多能性，并通过管形成测定进一步成功分化为功能性 EC。单 HLA-A11:01 匹配的 iPSC-EC 显著减少了表达匹配 HLA-A*11:01 和其他 HLA-A、-B 和 -C 等位基因的同种异体 CD8+T 细胞和 NK 细胞的同种异体反应。这些细胞成功地用于使脱细胞瓣膜再内皮化。
总之，通过高效的 HLA 工程改造 iPSC，创建了一个简单的 HLA-I 交换系统，以逃避 CD8+T 细胞的同种异体反应和 NK 细胞的激活。该技术已用于生成用于工程心脏瓣膜的 iPSC-EC。如果为常见 HLA 等位基因创建“现成”和“非免疫原性”异体 iPSC，我们的策略应该非常有用。
