Background: Cellular therapies represent a new promising approach for transplant tolerance induction. One of the methods for immune response modulation in solid organ and vascularized composite allograft (VCA) recipients is bone marrow (BM) transplantation. We propose a new cellular therapy based on ex vivo created donor-recipient chimeric cells as an alternative approach to BM-based therapies in support of solid organ and VCA transplantation. The aim of this study was creation and preliminary characterization of the fused human BM-derived CD34+ hematopoietic chimeric cells.

Materials and Methods: Twelve ex vivo fusions were performed to create human hematopoietic chimeric cells (HHCC). Briefly, mononuclear cells (MNCs) isolated from two unrelated donors (female and male) were sorted out by MACS to obtain CD34+ cells. Next, CD34+ cells from each donor were stained separately by PKH26 (red) and PKH67 (green) and fused with polyethylene glycol (PEG). Double PKH26 and PKH67 stained cells were sorted out and subjected to further assessments. Flow cytometry (FC), (CD34, CD133, CD117, CD90, CD4, CD19, CD14 and CD45RA markers, viability tests), confocal microscopy (CM), PCR-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) were used to characterize the properties of fused HHCC.

Results: FC and CM analysis confirmed CD34+ cell fusion and creation of HHCC. Using PCR-rSSOP we determined that HHCC share HLA class I and class II antigens specific for both BM donors used for fusion. The viability tests (trypan blue and LIVE/DEAD staining) showed that 99% of HHCC were viable 3 hours after fusion. The viability of HHCC at 1, 4 and 7 days after fusion ranged between 97-98%. The number of apoptotic cells was determined by FC with Annexin V conjugates and was the highest at day 4 after fusion (3.02%). Preliminary phenotype characterization showed expression of all assessed markers on the surface of HHCC.

Conclusions: We successfully confirmed feasibility of ex vivo fusion of human BM-derived CD34+ hematopoietic cells leading to creation of HHCC. We characterized the phenotype and viability of HHCC. This unique concept of HHCC-based cell therapy introduces new applications in transplant surgery. The ultimate goal is to induce tolerance in solid organ and VCA transplants using novel approach with chimeric cell-based therapy.

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