Characterization of Ex Vivo Created Human Multi-Chimeric Cells - A Novel Approach for Tolerance Induction in Vascularized Composite Allotransplantation

Abstract

Introduction Various stem cell-based therapies have been proposed as supportive treatments for patients following solid organ and vascularized composite allotransplantation (VCA) to achieve donor-specific tolerance and eliminate the toxic, systemic, life-long, multi-drug immunosuppression. We introduce a novel cellular therapy of ex vivo created umbilical cord blood derived multi-chimeric cells (mCC) as an alternative approach to bone marrow based therapies in support of VCA. The aim of this study was to establish the ex vivo fusion protocol and characterize in vitro the phenotype, genotype, viability and proliferative potential of fused human mCC. Methods Twenty ex vivo fusions of human umbilical cord blood (UCB) cells were performed. Mononuclear cells (MNC) were isolated from UCB originating from three unrelated donors. Next, MNC were stained separately by PKH26, PKH67 and eFluor670 proliferation dye and fused using polyethylene glycol (PEG). Triple PKH26/PKH67/eFluor670 stained mCC were sorted and assessed by confocal microscopy and flow cytometry for the efficacy of the cell fusion procedure. The viability of mCC (Trypan blue and LIVE/DEAD cell viability assay) and distribution of hematopoietic surface markers (CD4, CD8, CD19, CD45 and CD90) were performed by flow cytometry. PCR-rSSOP (Antigens: A, B, C, Bw, DRB1, DQB1, DR51, DR52 and DR53), and STR-PCR (Loci: TH01, D21S11, D5S818, D13S317, D7S820, D16S539, vWA, TPOX) characterized the genotype of mCC. Proliferative potential of mCC was assessed by colony forming unit (CFU) assay. Results Flow cytometry and confocal microscopy analysis confirmed UCB fusion and creation of human mCC. Using PCR-rSSOP and STR-PCR assays, we determined that human mCC are sharing HLA class I and class II antigens, as well as selected loci specific for all three UCB donors used for fusion. After fusion 90-95% of cells were viable. Phenotype characterization showed similar percentage and pattern of hematopoietic markers distribution on the surface of mCC and UCB donors. Maintenance of proliferative properties of mCC was confirmed by CFU assay. Conclusions We have successfully confirmed the feasibility of ex vivo fusion procedure and creation of human mCC. We characterized the phenotype, genotype, viability and proliferative potential of mCC. This unique concept of mCC introduces a novel universal therapy for tolerance induction in solid organ and VCA transplantation. DOD AFIRM W81XWH-13-2-0053.