Background: Cell-based therapies represent a new promising approach for tolerance induction in transplantation. One of the methods for immune response modulation in solid organ and vascularized composite allograft (VCA) recipients is donor bone marrow (BM) transplantation. We propose a new cellular therapy based on application of the 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 further characterization of human hematopoietic chimeric cells (HHCC).

Methods: Twenty five ex vivo fusions were performed to create HHCC. Briefly, CD34+ cells isolated from two unrelated bone marrow donors were stained separately by PKH26 and PKH67 and fused with polyethylene glycol. Double PKH26 and PKH67 stained cells were sorted out and subjected to further analysis. Flow cytometry (FC), confocal microscopy (CM),

PCR-rSSOP, FISH, CFU, MLR assays and real-time PCR were used to characterize phenotype, genotype, clonogenic properties and tolerogenic potential of created HHCC. Migratory pathways of HHCC were assessed in vivo in athymic nude rat model by CM.

Results: FC and CM analysis confirmed CD34+ cell fusion. PCR-rSSOP results showed that HHCC share HLA class I and II specific for both fusion donors. After fusion ~99% of HHCC were viable with low level of apoptosis (2.7% and 1.2% of HHCC in early and late stages of apoptosis, respectively). HHCC number increased 6-fold after 7-days of culturing. HHCC expressed markers CD34, CD133, CD117, CD4 and CD19 markers. The percentage of polyploid cells within HHCC was low (0.48%). HHCC differentiated into all classes of myeloid and erythroid progenitor cells. HHCC have tolerogenic potential as they express pro-tolerogenic cytokines (IL-10 and TGF-β) and decrease the response of immune system. HHCC were present in the peripheral blood the athymic nude rat recipients 24 hours, 72 hours and 7 days after their intraosseous injection. Their presence after 7 days of intraosseous delivery was also confirmed in the liver.

Conclusions: We successfully characterized the viability, phenotype, genotype, clonogenic properties and tolerogenic and migratory potential of HHCC. Application of HHCC as a supportive therapy represents a novel approach for tolerance induction in solid organ and VCA transplantation.

CITATION INFORMATION: Siemionow M, Tfaily E, Cyran M, Gendek R, Strojny M, Szilagyi E, Cwykiel J. In Vitro and In Vivo Characterization of Human Hematopoietic Chimeric Cells – A Novel Approach for Tolerance Induction in Transplantation. Am J Transplant. 2016;16 (suppl 3).

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