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 eight 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, 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 (27% and 12% 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 (048%) 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-Î2) HHCC were present in the peripheral blood of the athymic nude rat recipients 24 hours, 72 hours, 7 and 14 days after their intraosseous injection Their presence at 7 and 14 days after intraosseous delivery was also confirmed in the bone marrow and spleen.
Conclusions
We successfully characterized the viability, phenotype, genotype, clonogenic properties, 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.