Abstract：
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.
摘要：
从基因操纵的多能干细胞中衍生低免疫原性人细胞对于未来的移植医学和过继性免疫疗法具有巨大的希望。破坏多能干细胞中的β-2-微球蛋白（B2M），然后分化为专门的细胞类型是一种有希望的方法来获得低免疫原性细胞。鉴于基于CRISPR/Cas9的基因编辑工具和杆状病毒递送系统的有吸引力的特征，杆状病毒可以提供CRISPR/Cas9组件用于B2M的位点特异性基因编辑。在这里，我们报道了一种杆状病毒CRISPR/Cas9载体系统的开发，用于人细胞中B2M基因座的破坏。在人类胚胎干细胞（hESCs）中进行测试时，成功实现了B2M基因敲除/敲除，导致人白细胞抗原I类在细胞表面表达的稳定下调。然后将源自B2M基因破坏的hESC的成纤维细胞用作与人外周血单核细胞共培养的刺激细胞。如通过敏感的Elispot测定所评估的,这些成纤维细胞触发的同种免疫应答显著降低。B2M阴性hESC在体外和体内保持多能性和分化成三个胚芽谱系的能力。这些发现证明了使用杆状病毒-CRISPR/Cas9系统建立B2M破坏的多能干细胞的可行性。B2M敲低/敲除足以导致低免疫原性条件，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。
