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

Abstract

Background Cell-based therapies are promising for tolerance induction in bone marrow (BM), solid organs, and vascularized composite allotransplantation (VCA). The toxicity of bone marrow transplantation (BMT) protocols precludes this approach from routine clinical applications. To address this problem, we developed a new therapy of Human Umbilical Di-Chimeric (HUDC) cells for tolerance induction in transplantation. This study established in vitro characterization of the created HUDC cells. Methods We performed sixteen ex vivo polyethylene glycol (PEG)-mediated fusions of human umbilical cord blood (UCB) cells from two unrelated donors. Fusion feasibility was confirmed in vitro by flow cytometry (FC) and confocal microscopy (CM). The HUDC cells’ genotype was assessed by lymphocytotoxicity test and short tandem repeat-polymerase chain reaction (STR-PCR) analysis, phenotype by FC, viability by LIVE/DEAD ® assay, and apoptosis level by Annexin V staining. We used COMET assay to assess HUDC cells’ genotoxicity after the fusion procedure. Clonogenic properties of HUDC cells were evaluated by colony forming unit (CFU) assay. Mixed lymphocyte reaction (MLR) assay assessed immunogenic and tolerogenic properties of HUDC cells. Results We confirmed the creation of HUDC cells from two unrelated human donors of UCB cells by FC and CM. Human leukocyte antigen (HLA) class I and II typing, and STR-PCR analysis of HUDC cells confirmed the presence of alleles and loci from both unrelated UCB donors (donor chimerism: 49%±8.3%, n=4). FC confirmed the hematopoietic phenotype of HUDC cells. We confirmed high HUDC cells’ viability (0.47% of dead cells) and a low apoptosis level of fused HUDC cells (15.9%) compared to positive control of PKH-stained UCB cells (20.4%) before fusion. COMET assay of HUDC cells revealed a lack of DNA damage. CFU assay confirmed clonogenic properties of HUDC cells, and MLR assay revealed a low immunogenicity of HUDC cells. Conclusions This study confirmed creation of a novel HUDC cell line by ex vivo PEG-mediated fusion of UCB cells from two unrelated donors. The unique concept of creating a HUDC cell line, representing the genotype and phenotype of both, transplant donor and the recipient, introduces a promising approach for tolerance induction in BM, solid organs, and VCA transplantation.

Efficacy of Engraftment and Safety of Human Umbilical Di-Chimeric Cell (HUDC) Therapy after Systemic Intraosseous Administration in an Experimental Model

“…Our previous studies laid the groundwork for the development of novel human hematopoietic cell lines through an ex vivo PEG-mediated cell fusion procedure [16][17][18][19]. Utilizing this protocol, we successfully confirmed the creation of the human umbilical di-chimeric cell (HUDC) line by fusing human UCB cells derived from two unrelated human donors.…”

“…Based on these encouraging findings in rodent models, we translated cell fusion technology to human hematopoietic progenitor stem cells, including both bone marrow and umbilical cord blood (UCB) cells. Recently, we reported the creation of two novel hematopoietic cell lines: human hematopoietic CD34 + chimeric cells (HHCCs) [17] and human umbilical di-chimeric cell (HUDCs) [18]. Through in vitro characterization, we successfully confirmed the viability, genotype, phenotype, safety, and clonogenic properties of both HHCC and HUDC lines, thereby establishing their reliability for future therapeutic approaches in regenerative medicine applications.…”

“…Due to the nature of the study, no ethical approval or informed consent were required. As previously reported [18], the HUDC cell line was created from human UCB cells derived from two unrelated donors (Figure 1). Briefly, density gradient centrifugation (Lymphoprep™, StemCell™ Technologies, Vancouver, BC, Canada) and anti-human CD235a (Glycophorin A) MicroBeads in conjunction with magnetic-activated cell sorting (MACS ® , Miltenyi Biotec, Bergisch Gladbach, Germany) were used to isolate and purify UCB-derived cells.…”

“…The human UCB cells, derived from two unrelated donors, were individually labeled using fluorescence dyes: PKH26 (red) and PKH6 7(green) (MiliporeSigma, Burlington, MA, USA). These labeled cells were mixed in a 1:1 ratio and fused using polyethylene glycol (PEG) 4000 solution (EMD, Burlington, MA, USA), as previously reported [17][18][19][20]. The fluorescence-activated cell sorting (FACS) was conducted employing a BD FACSAria TM II cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) to select the HUDC population based on double PKH26/PKH67 labeling.…”

“…The fluorescence-activated cell sorting (FACS) was conducted employing a BD FACSAria TM II cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) to select the HUDC population based on double PKH26/PKH67 labeling. A total of sixteen fusions were performed, and the average fusion efficacy was at 67.4% ± 3.4% [18]. (green) and PKH26 (red), respectively.…”