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

Abstract

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. Graphical Abstract

Efficacy of Engraftment and Safety of Human Umbilical Di-Chimeric Cell (HUDC) Therapy after Systemic Intraosseous Administration in an Experimental Model

“…Our previous studies laid the groundwork for the development of novel human hematopoietic cell lines through an ex vivo PEG-mediated cell fusion procedure [16][17][18][19]. Utilizing this protocol, we successfully confirmed the creation of the human umbilical di-chimeric cell (HUDC) line by fusing human UCB cells derived from two unrelated human donors.…”

“…The human UCB cells, derived from two unrelated donors, were individually labeled using fluorescence dyes: PKH26 (red) and PKH6 7(green) (MiliporeSigma, Burlington, MA, USA). These labeled cells were mixed in a 1:1 ratio and fused using polyethylene glycol (PEG) 4000 solution (EMD, Burlington, MA, USA), as previously reported [17][18][19][20]. The fluorescence-activated cell sorting (FACS) was conducted employing a BD FACSAria TM II cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) to select the HUDC population based on double PKH26/PKH67 labeling.…”

Amelioration of Morphological Pathology in Cardiac, Respiratory, and Skeletal Muscles Following Intraosseous Administration of Human Dystrophin Expressing Chimeric (DEC) Cells in Duchenne Muscular Dystrophy Model

“…However, despite the efforts, the achievement of satisfactory therapeutic outcomes has become challenging [13,14,[24][25][26][31][32][33]. To address these challenges and meet the current needs, based on our experience in chimeric cell-based therapies and technology [37,50] we have introduced a new myoblast-based chimeric cell line of DEC cells that proved to be safe and efficacious when tested in the mdx mice models of DMD [38]. Specifically, to address the problem of myoblast engraftment, we compared different methods of delivering DEC cells, including the local-intramuscular [38] and systemicintraosseous [28,29] routes.…”

“…However, despite the efforts, the achievement of satisfactory therapeutic outcomes has become challenging [ 13 , 14 , 24 , 25 , 26 , 31 , 32 , 33 ]. To address these challenges and meet the current needs, based on our experience in chimeric cell-based therapies and technology [ 37 , 50 ] we have introduced a new myoblast-based chimeric cell line of DEC cells that proved to be safe and efficacious when tested in the mdx mice models of DMD [ 38 ].…”

Immune modulation in transplant medicine: a comprehensive review of cell therapy applications and future directions

“…Over the past three decades, cellular therapies have emerged as a novel approach to address the persisting challenges linked to multidrug and lifelong immunosuppression ( 26 , 27 ). The concept of cellular therapies is centered around the infusion of immunomodulating cells.…”