Background
The aim of this study was to evaluate the phenotype, engraftment and safety of ex-vivo fused human cord blood derived di-chimeric cell therapy (DCC) in the NOD SCID mouse model.
Methods
A total of 36 fusions of human umbilical cord blood (UCB) mononuclear clls were performed UCB from 2 unrelated donors were separately stained with PKH26 and PKH67 dyes Fused with polyethylene glycol, double (PKH26/PKH67) stained DCC were sorted and subjected to the in vitro evaluations (15 fusions): lymphocytotoxicity (LCT) test, PCR-rSSOP, STR-PCR, viability, apoptosis, colony forming unit (CFU) assay and COMET assay DCC (3–5 × 106 cells) from 18 fusions (n = 6/Group) were delivered: Group 1: intraosseous, Group 2: intramuscular, and Group 3: subcutaneous to the NOD SCID recipient mice Control mice in Groups 4, 5, and 6 (n = 6) received 3 × 106UCB utilizing the same 3 delivery sites Mice were observed daily for 90 days for changes in weight, activity, posture and hair loss Moreover, mice were evaluated bi-weekly by palpation and at 90 days by magnetic resonance imaging (MRI) The presence of DCC in the blood was determined by complete blood count (CBC) and flow cytometry (FC) The migratory pathways of DCC, peripheral blood, bone marrow and lymphoid organs were assessed at 3 months after delivery using immunofluorescent staining Furthermore, H&E staining was performed to assess harvested tissues for tumor growth.
Results
The presence of HLA class I and II from both UCB donors was confirmed by LCT, PCR-rSSOP, and STR COMET assay showed no damage to the DNA of DCC following fusion procedure Migratory properties of DCC were confirmed at 24 hours after cell delivery Human derived cells (CD45+, CD19+, HLA class I and CD4+) were detected at a level up to 2% in the peripheral blood as tested by CBC and flow cytometry at 90 days following delivery No DCC derived tumor-like growth was observed.
Conclusions
Phenotype, engraftment and safety of DCC were characterized The unique concept of supportive therapy of DCC introducing cells presenting phenotype characteristics of both transplant donor and recipient is a new promising approach for tolerance induction and prolonging VCA survival.