Human Multi-Chimeric Cell (HMCC) Therapy as a Novel Approach for Tolerance Induction in Transplantation.

Cellular therapies are regarded as the most promising approach for inducing transplant tolerance without life-long immunosuppression in solid organ and vascularized composite allotransplantation (VCA). Currently, no therapies are achieving this goal. This study introduces a novel Human Multi-Chimeric Cell (HMCC) line created by fusion of umbilical cord blood (UCB) cells, from three unrelated donors as an alternative therapeutic approach to bone marrow transplantation and tolerance induction in solid organ and VCA transplants. We performed eighteen ex vivo polyethylene glycol mediated fusions of human UCB cells from three unrelated donors to create HMCC. Mononuclear cells labeled with PKH26, PKH67, and eFluor™ 670 fluorescent dyes were fused and sorted creating a new population of triple-labeled (PKH26/PKH67/eFluor™ 670) HMCC. The creation of HMCC from three unrelated human UCB donors was confirmed by flow cytometry and confocal microscopy. Genotyping analyses determined the tri-chimeric state of HMCC by presence of parent alleles and selected loci specific for each of three UCB donors. Phenotype characterization confirmed hematopoietic markers distribution, comparable to UCB donors. HMCC maintained viability and displayed a low apoptosis level. The COMET assay revealed absence of genotoxicity, confirming fusion safety. Colony forming units assay showed clonogenic properties of HMCC. This study confirmed the feasibility of HMCC creation from three unrelated human UCB donors and characterized tri-chimeric state, hematopoietic phenotype, viability, safety, and clonogenic properties of HMCC. The created HMCC line, representing genotype characteristics of three unrelated human UCB donors, introduces a novel therapeutic approach for bone marrow, solid organ, and VCA transplants.