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.