Quadruple HLA-I/II gene homo/heterozygous knockout hESC line: generation, multi-omics profiling, pluripotency, and...

Human embryonic stem cells (hESCs) exhibit pluripotency, self-renewal, and broad therapeutic potential in diverse disease contexts. Meanwhile, allogeneic grafts derived from hESCs are confronted with the formidable challenge of immune rejection. Specific human leukocyte antigens (HLAs) are critically involved in mediating transplant rejection, thus establishing HLA-deficient stem cells can be an important step toward achieving immune evasion. However, there is currently no consensus on which gene editing strategy can reduce hESC immunogenicity to a level suitable for broad clinical applications. This study performed HLA-A/-B gene hetero/homozygous knockout in an HLA double-knockout hESC line using a CRISPR/Cas9 system, established HLA-DRA^-/-/-DQA1^-/-/-A^+/-/-B^-/- knockout monoclonal cell lines, and revealed the multi-omics profiles, along with preserved pluripotency and trends to reduced immunogenicity of the hESC lines, through multi-omics analyses, in vitro assessment of pluripotency-related gene expression, in vivo teratoma formation assays and xenotransplantation experiments in mouse models. This study presents two novel monoclonal HLA genes homo/heterozygous knockout hESC lines, along with their multi-omics landscapes, pluripotency and immunogenicity. The findings exhibit crucial application potential and reference value in the development of hypoimmunogenic hESCs for their universal clinical use.