Background: Cell-based and chimerism-based therapies represent a promising approach for tolerance induction in transplantation. We propose a new cell therapy of the ex vivo created human hematopoietic chimeric cells (HHCC) as an alternative approach to bone marrow (BM)-based therapies in support of solid organ and vascularized composite allotransplantation (VCA). This study aimed to characterize in vitro the phenotype, genotype, clonogenic, and tolerogenic properties of HHCC.Methods: Thirty ex vivo fusions of CD34 + cells from two unrelated human BM donors were performed. CD34 + cells were stained separately with PKH26 and PKH67 membrane dyes and fused using polyethylene glycol (PEG). Creation of human HHCC and chimeric state was confirmed by flow cytometry (FC), confocal microscopy (CM) and electron microscopy (EM). HHCC's phenotype (CD34, CD133, CD117, CD4, CD19, CD4/CD25) was assessed by FC, viability by Trypan Blue, LIVE/DEAD and apoptosis by AnnexinV/Sytox Blue and TUNEL assay, while mixed lymphocyte reaction (MLR) assay assessed HHCC's immunogenicity and tolerogenic properties. HHCC differentiation, proliferation and clonogenic potential were assessed by the colony forming unit (CFU). Polyploidy was evaluated by fluorescence in situ hybridization (FISH), whereas polymerase chain reaction-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) and short tandem repeats-polymerase chain reaction (STR-PCR) assessed HHCC's genotype, and chimerism. Reverse transcription polymerase chain reaction (RT-PCR) analyzed cytokines secretion [interleukin (IL)-10, transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α)] up to 14 days post-fusion.Results: FC and CM confirmed creation of HHCC by fusion of CD34 + cells from two unrelated human donors. After fusion, maintenance of hematopoietic markers and increased expression of Treg-cells (CD4/ CD25) was confirmed. Moreover, high HHCC viability (99%) and a low apoptosis rate (1.2%) were revealed HHCC presented decreased immunogenicity by MLR, and significant, 40-fold increase of IL-10 the protolerogenic cytokine at 21 days after fusion (RT-PCR) P<0.0001. The number of polyploid cells was negligible (0.48%). PCR-rSSOP of HHCC after fusion confirmed presence of human leukocyte antigen (HLA) class I and class II-alleles and presence of the loci specific for both CD34 + cells BM donors by STR-PCR. Conclusions:We have created a new hematopoietic cell line of HHCC from two unrelated human donors, and have successfully characterized in vitro, viability, phenotype, genotype, clonogenic, and tolerogenic properties of HHCC. These unique immunomodulatory and tolerogenic properties introduce HHCC as a novel therapeutic approach for tolerance induction in VCA and solid organ transplantation.

Background Different types of nerve conduits are used to bridge peripheral nerve gaps when a tension-free repair is unattainable. To best support nerve regeneration, naturally occurring conduits have been tested. Since allografts offer an unlimited source of epineurium, we have developed human epineural conduit (hEC) as a novel technology to bridge nerve gaps. Considering acellular properties, and lack of immunogenic response, epineurium-derived conduits represent an attractive material, when compared with nerve allografts that require systemic immunosuppression. In this study, we introduce the hEC as a novel naturally occurring material applied for repair of nerve gaps after trauma. Methods We tested the application of hEC created from human sciatic nerve in the restoration of 20 mm sciatic nerve defects in the nude rat model. Four experimental groups were studied: group 1: no repair control (n = 6), group 2: autograft control (n = 6), group 3: matched diameter hEC (n = 6), and group 4: large diameter hEC (n = 6). Functional tests of toe-spread and pin prick were performed at 1, 3, 6, 9, 12 weeks after repair. At 12 weeks, nerve samples were collected for immunostaining of Laminin B, S-100, glial fibrillary acidic protein (GFAP), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), von Willebrand factor, and histomorphometric analysis of myelin thickness, axonal density, fiber diameter, and percentage of the myelinated nerve fibers. Muscle samples were gathered for gastrocnemius muscle index (GMI) and muscle fiber area ratio measurements. Results Best functional recovery, as well as GMI, was revealed for the autograft group, and was comparable to the matched hEC group. Significant differences were revealed between matched and large hEC groups in expression of S100 (p = 0.0423), NGF (p = 0.269), VEGF (p = 0.0003) as well as in percentage of myelinated fibers (p < 0.001) and axonal density (p = 0.0003). Conclusion We established the feasibility of hEC creation. The innovative method introduces an alternative technique to autograft repair of nerve defects.