Human Di-Chimeric Cells a New Approach for Tolerance Inducing Protocols in Transplantation: A Preliminary Study

Background: Successful vascularized composite allograft (VCA) transplantation requires life-long treatment combining more than two immunosuppressive agents and pose significant risks and side effects. Cell based therapies are a new promising approach for tolerance induction that could prevent or reduce negative impact of life-long immunosuppression. Bone marrow transplantation has already been tested for solid organs, face and upper extremity transplants for modulation of immune responses. We propose a new cellular therapy based on the ex vivo created donor-recipient chimeric cells as an alternative approach to bone marrow based therapies in support of VCA. The aim of this preclinical study was to create and characterize in vitro the phenotype, genotype and viability of fused human di-chimeric cells (dCC).

Materials and Methods: Fourteen ex vivo fusions of human umbilical cord blood (UCB) cells were performed. Mononuclear cells (MNCs) were isolated from UCB originating from 2 different donors. Next MNCs were stained separately by PKH26 and PKH67. Fusion procedure was performed using polyethylene glycol (PEG) technique. Double PKH26 and PKH67 stained cells were sorted out and subjected to further assessments. Flow cytometry (FC), (CD3, CD4, CD8, CD19, CD34 and CD90, viability test), confocal microscopy (CM), fluorescent lymphocytotoxicity assay (LCT), PCR-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) and short tandem repeat- PCR (STR-PCR) and colony- forming unit (CFU) assay were assessed to characterize the phenotype and genotype of fused human chimeric cells.

Results: FC and CM analysis confirmed UCB fusion and creation of human dCC. Using LCT assay we determined that human dCC are sharing HLA class I and class II antigens specific for both types of UCB donors used for fusion. Results of the LCT test were confirmed by rSSOP and STR assay which revealed that fused dCC were in fact originating (39-51%) from each of the UCB donors. After fusion 96-99% of cells were viable. Preliminary phenotype characterization showed expression of all assessed markers on the surface of dCC. CFU assay confirmed the presence and functionality of dCC mature progenitor comparable to untreated cord blood progenitor cells.

Conclusions: We successfully confirmed feasibility of ex vivo fusion of UBC cells leading to creation of human fused dCC. We characterized cell phenotype and viability. This unique concept of di-chimeric cell therapy introduces new applications in transplant surgery. The ultimate goal is to induce tolerance in VCA transplants.