Department of Biology, College of Science, Shantou University, Shantou, Guangdong, China.
Department of Dermatology and Venereology, Second Clinical Medical College of Guangdong Medical University, Dongguan, China.
Mol Genet Genomics. 2024 Aug 1;299(1):74. doi: 10.1007/s00438-024-02167-w.
Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy.
从基因改造的多能干细胞中衍生出低免疫原性的人类细胞，为未来的移植医学和过继免疫疗法带来了巨大的希望。在多能干细胞中破坏β-2-微球蛋白（B2M），然后分化为专门的细胞类型，是一种衍生低免疫原性细胞的有前途的方法。鉴于 CRISPR/Cas9 基因编辑工具和杆状病毒传递系统的诱人特点，杆状病毒可以传递 CRISPR/Cas9 组件，用于 B2M 的特异性基因编辑。在此，我们报告了一种用于人类细胞 B2M 基因座敲除的杆状病毒 CRISPR/Cas9 载体系统的开发。在人胚胎干细胞（hESC）中进行测试时，成功实现了 B2M 基因敲低/敲除，导致细胞表面人白细胞抗原 I 类表达的稳定下调。然后，将源自 B2M 基因敲除 hESC 的成纤维细胞用作与人类外周血单核细胞共培养的刺激细胞。这些成纤维细胞在敏感的 Elispot 测定中引发了显著降低的同种免疫反应。B2M 阴性 hESC 保持了多能性，并具有在体外和体内分化为三个生殖谱系的能力。这些发现证明了使用杆状病毒-CRISPR/Cas9 系统建立 B2M 敲除多能干细胞的可行性。B2M 的敲低/敲除足以导致低免疫原性状态，从而支持 B2M 阴性细胞作为同种异体细胞治疗的通用供体细胞的潜在用途。
