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In Vitro Characterization of Phenotype, Genotype, Clonogenic Properties and Tolerogenic Potential of Human Hematopoietic Chimeric Cells - a Novel Strategy for Tolerance Induction in Transplantation
Ewa Bryndza Tfaily, PhD, Joanna Cwykiel, MSc, Maria Siemionow, MD PhD DSc.
UIC, Chicago, IL, USA.

PURPOSE:
Cellular therapies represent a new approach for tolerance induction that could reduce negative impact of life-long immunosuppression. The aim of this study was to create and characterize human hematopoietic chimeric cells (HHCC) as novel tolerogenic approach for VCA transplantation.
METHODS:
Twenty ex vivo fusions were performed to create HHCC. Briefly, CD34+ cells isolated from two unrelated bone marrow donors were stained separately by PKH26 and PKH67 and fused with polyethylene glycol. Double PKH26 and PKH67 stained cells were sorted out and subjected to further analysis. Flow cytometry (FC), (CD34, CD133, CD117, CD90, CD4, CD19, CD14 markers, proliferation and viability assays), confocal microscopy (CM), PCR-rSSOP, STR, FISH, CFU assays and real-time PCR were used to characterize HHCC.
RESULTS:
FC and CM analysis confirmed CD34+ cell fusion. PCR-rSSOP and STR results showed that HHCC share HLA and STR alleles specific for both fusion donors. After fusion ~99% of HHCC were viable with low level of apoptosis (2.7% and 1.2% of HHCC in early and late stages of apoptosis, respectively). HHCC increased 6-fold after 7-day culturing. HHCC expressed all assessed markers. The percentage of polyploid cells within HHCC was low (0.48%). HHCC differentiated into all classes of myeloid and erythroid progenitor cells. HHCC have tolerogenic potential as they express pro-tolerogenic Th2 cytokines.
CONCLUSION:
We confirmed feasibility of ex vivo HHCC creation. We characterized phenotype, genotype, clonogenic properties and tolerogenic potential of HHCC. Application of HHCC as a supportive therapy represents a novel approach for tolerance induction in VCA transplantation


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