Hypoimmunogenic Human Pluripotent Stem Cells are Valid Cell Sources for Cell Therapeutics with Normal Self-Renewal and Multi-Lineage Differentiation Capacity

Abstract Hypoimmunogenic human pluripotent stem cells (hPSCs) are expected to serve as an unlimited cell source for generating universally compatible “off-the-shelf” cell grafts. However, whether the engineered hypoimmunogenic hPSCs still preserve their advantages of unlimited self-renewal and multilineage differentiation to yield functional tissue cells remains unclear. Here, we systematically studied the self-renewal and differentiation potency into functional tissue cells of three types of hypoimmunogenic hPSCs, established through biallelic lesion of B2M gene to remove all surface expression of classical and nonclassical HLA class I molecules (B2Mnull), biallelic homologous recombination of nonclassical HLA-G1 to the B2M loci to knockout B2M while express membrane-bound β2m-HLA-G1 fusion proteins (B2MmHLAG), and ectopic expression of soluble and secreted β2m-HLA-G5 fusion proteins in B2MmHLAG hPSCs (B2Mm/sHLAG). Our results show that hypoimmunogenic hPSCs with variable expression patterns of HLA molecules and immune compromising spectrums retain their normal self-renewal capacity and three germ-layer differentiation potency. More importantly, as exemplified by neurons, cardiomyocytes and hepatocytes, hypoimmunogenic hPSC-derived tissue cells are fully functional as of their morphology, electrophysiological properties, macromolecule transportation and metabolic regulation. Taken together, our findings indicate that engineered hypoimmunogenic hPSCs hold great promise of serving as an unlimited universal cell source for cell therapeutics.