B2M knockout for hypo-immunity alters the global profiles of membrane-proteins and cytokines and impairs therapeutic efficacy of human stem cells. B2M knockout for hypo-immunity alters the global profiles of membrane-proteins and cytokines and impairs therapeutic efficacy of human stem cells

β2-microglobulin (B2M) is the light chain subunit of type I human leukocyte antigen complexes that play a key role in the immune system. Genetic knockout (KO) of B2M in human pluripotent stem cells (hPSCs) has been used as a major approach together with many other strategies to generate hypo-immunogenic, universal stem cells for allogeneic cell transplantation without triggering immune rejection. However, this approach assumes that B2M is dispensable for stem cell differentiation and therapy, although it lacks evidence from thorough evaluation. Here, we conducted genome-wide analyses of isogenic pairs of wild-type (WT) and B2M-KO hESCs and their derived mesenchymal stem cells (E-MSCs), which revealed proteomic and transcriptomic alternations due to the KO. Over 200 proteins were mistargeted from the plasma membranes of B2M-KO hESCs and E-MSCs, suggesting that B2M is crucial for protein trafficking in the subcellular organelles. The expression of osteo-chondrogenic determinants and inflammation-related cytokines differed dramatically in B2M-KO E-MSCs from WT E-MSCs, consistent with the impaired osteogenic differentiation and wound-healing efficacy of B2M-KO E-MSCs. This study reveals previously unrecognized functions of B2M, which alerts cautions be used when applying B2M-KO cells to therapy. Overall design: RNA seq of human pluripotent stem cells (hPSCs) in control and experiment B2M group

β2-微球蛋白（β2-microglobulin, B2M）是I型人类白细胞抗原复合物的轻链亚基，该复合物在免疫系统中发挥核心调控作用。人类多能干细胞（human pluripotent stem cells, hPSCs）的B2M基因敲除（genetic knockout, KO）结合多种其他策略，是制备低免疫原性通用干细胞的主流手段，可用于同种异体细胞移植且不会引发免疫排斥反应。然而，该方法默认B2M对于干细胞分化与临床治疗是非必需的，但这一假设尚未获得全面评估的实验证据支持。本研究针对野生型（wild-type, WT）与B2M敲除的人类胚胎干细胞（human embryonic stem cells, hESCs）及其诱导的间充质干细胞（E-MSCs）的同基因配对样本开展全基因组分析，结果显示B2M敲除会引发蛋白质组与转录组的显著改变。在B2M敲除的hESCs与E-MSCs的质膜上，超过200种蛋白质出现定位异常，提示B2M对于亚细胞器内的蛋白质运输过程至关重要。在B2M敲除的E-MSCs中，成骨-成软骨相关决定因子以及炎症相关细胞因子的表达水平与野生型E-MSCs存在显著差异，这与B2M敲除E-MSCs的成骨分化能力受损、伤口愈合效能下降的表型一致。本研究揭示了此前未被认知的B2M生物学功能，提醒科研与临床工作者在将B2M敲除细胞应用于治疗时需保持谨慎。整体实验设计：对对照组与B2M敲除实验组的人类多能干细胞（hPSCs）进行RNA测序。