Tolerance Inducing Therapy of Human Multi-Chimeric Cells for Vascularized Composite Allotransplantation.
Joanna Cwykiel, MS, George Rafidi, BS, Maria Siemionow, MD, PhD, DSc.
University of Illinois at Chicago, Chicago, IL, USA.
PURPOSE: Cellular therapies, a promising approach for tolerance induction in vascularized composite allotransplantation (VCA) patients, could reduce the toxicity of life-long immunosuppression. We propose a new therapy of ex-vivo created umbilical cord blood-derived multi-chimeric cell (mCC) as an alternative approach to support VCA. The aim of this study was to develop a protocol and characterize the phenotype, genotype and viability of human mCC in-vitro.
METHODS: Sixteen ex-vivo fusions of human umbilical cord blood (UCB) cells were performed. Mononuclear cells were isolated from UCB originating from three unrelated donors and stained with PKH26, PKH67 and eFluor670 dye then fused using polyethylene-glycol. Triple PKH26/PKH67/eFluor670 stained mCC were sorted and assessed by confocal microscopy (CM) and flow cytometry (FC) for efficacy of the cell fusion. Phenotype (CD4, CD8, CD19, CD45 and CD90) and viability of mCC were assessed by FC. PCR-rSSOP, and STR-PCR characterized the genotype of mCC and colony forming unit (CFU) assay assessed proliferative potential.
RESULTS: FC and CM confirmed creation of mCC. PCR-rSSOP and STR-PCR determined that human mCC share HLA class I and class II antigens, and other donor specific DNA sequences of all three UCB donors. After fusion 90% of cells were viable. Phenotype characterization showed expression of all assessed markers on the surface of mCC.
CONCLUSION: The creation of human mCC via ex-vivo fusion was confirmed and phenotype, genotype, viability and proliferative properties of mCC characterized. The unique concept of mCC introduces a new universal therapy for tolerance induction in solid organ and VCA transplantation.
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