Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

Abstract

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.

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression

“…Inspired by the potential of the encouraging outcomes of the rat DRCCs, our Microsurgery Laboratory successfully created HHCC from the BM-derived CD34 + cells originating from two unrelated human donors, according to the previously described protocol ( 36 ). The properties of the HHCC originating from human CD34 + cells were characterized in vitro , confirming the tolerogenic properties of HHCC and the potential application of HHCC therapy for tolerance induction in BM, solid organs, and VCA transplantation ( 36 ).…”

“…There are several similarities between our two hematopoietic lines—the HHCC and HUDC cell line of created human chimeric cells. Both lines represent cells of the human hematopoietic lineage origin and both contain rich sources of the HSCs applicable for transplantation ( 36 ). When comparing the hematopoietic markers, the UCB cells have been shown to contain a high percentage of primitive stem cells, including very small embryonic-like stem cells, and a lower immunogenicity compared to other sources of stem cells, such as BM and PB ( 60 , 61 ).…”

“…The DNA samples from the UCB parent cells and HUDC cells were extracted using the DNeasy Blood and Tissue Isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The STR-PCR analysis (n=4) was performed as previously described ( 36 ). The extracted DNA was amplified using Biosystems ABI 3730 DNA Analyzer (Thermo Fisher Scientific) and AmpFLSTR TM Identifiler TM PCR Amplification kit (Thermo Fisher Scientific).…”

“…Based on our twenty years of experience in studies on chimerism and tolerance induction in transplantation ( 27 - 35 ), we have recently reported the new generation of BM-derived CD34 + human hematopoietic chimeric cell (HHCC) via ex vivo polyethylene glycol (PEG)-mediated fusion ( 36 ). Moreover, we confirmed HHCC safety by the confirmation of the viability by Trypan Blue staining and LIVE/DEAD ® assay, a low apoptosis profile by Annexin V staining and TUNEL assay, and the donor-specific genotype by lymphocytotoxicity test and STR-PCR analyses, as well as the proliferative properties by colony forming unit (CFU) assay and the immunogenicity by mixed lymphocyte reaction (MLR) of this new hematopoietic cell line.…”

Novel cell-based strategies for immunomodulation in vascularized composite allotransplantation

“…In a rat face transplant model, they found that DRCC up-regulated the gene expression of Il-10 which correlated with FoxP3 expression and an increased number of Treg. They compared different cell-based therapies and the longest allograft survival without continuous immunosuppression was observed in group supported by DRCC therapy [14,37,74] Siemionow et al also developed a human hematopoietic chimeric cell (HHCC) line from two unrelated bone marrow donors, demonstrating the feasibility and reproducibility of the technique [75 ▪▪ ]. They characterized the HHCC, confirming high viability and immunomodulatory effects highlighting HHCC application as a novel and effective cell-based therapeutic strategy for chimerism and tolerance induction in VCA and solid organs transplantation.…”