Development of Donor Recipient Chimeric Cells of bone marrow origin as a novel approach for tolerance induction in transplantation.

Abstract

Cell therapies and chimerism-based strategies are currently the most successful approach for tolerance induction in transplantation. This study aimed to establish and characterize novel Donor Recipient Chimeric Ccell (DRCC) therapy of bone marrow (BM) origin presenting donor-recipient phenotype to support tolerance induction. Ex vivo fusions of fully MHC-mismatched BM cells from ACI (RT1a) and Lewis (RT1l) rats were performed using polyethylene-glycol (PEG). The creation of rat DRCC was tested by flow cytometry (FC), confocal microscopy and PCR. FC characterized DRCC's phenotype (CD3, CD4, CD8, CD45, CD90, CD11b/c, CD45RA, OX-82, or CD4/CD25) and apoptosis, while mixed lymphocyte reaction assessed DRCC's immunogenicity and colony forming unit assay tested DRCC's differentiation and proliferation. DRCC's polyploidy was evaluated using Hoechst33342 staining and COMET assay tested genotoxicity of fusion procedure. ELISA analyzed the secretion of IL-2, IL-4, IL-10, TGFß1, IFNγ and TNFα by DRCC at day 1, 5 and 14 post-fusion. The DRCC's phenotype after long-term culturing was assessed by reverse-transcription PCR. The chimeric state of DRCC was confirmed. Fusion did not change the expression of hematopoietic markers compared to BM controls. Although an increased number of early and late apoptotic (Annexin V+/Sytox blue- and Annexin V+/Sytox blue+, respectively) DRCC was detected at 24h post-fusion, the number significantly decreased at day 5 (38.4%±3.1% and 22.6%±2.5%, vs. 28.3%±2.5% and 13.9%±2.6%, respectively, P<0.05). DRCC presented decreased immunogenicity, increased expression of IL-10 and TGFβ1 and proliferative potential comparable to BM controls. The average percentage of tetraploid DRCC was 3.1%±0.2% compared to 0.96%±0.1% in BM controls. The lack of damage to the DRCC's DNA content supported the DRCC's safety. In culture, DRCC maintained proliferation for up to 28 days while preserving hematopoietic profile. This study confirmed feasibility of DRCC creation via ex vivo PEG mediated fusion. The created DRCC revealed pro-tolerogenic properties indicating potential immunomodulatory effect of DRCC therapy when applied in vivo to support tolerance induction in solid organ and vascularized composite allograft transplantation.