The lack of optimal number of repopulating hematopoietic stem cells (HSC) and graft failure following transplantation remain as obstacles to using cord blood (CB) as a true alternative graft for adult patients. Using chromatin modifying agents {5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA)}, we have developed a culture regimen that permits expansion of primitive CB CD34+ CD90+ cells resulting in a 10 fold expansion of in vivo repopulating cells without jeopardizing their multilineage differentiation capacity. Our results also indicate that the chromatin modifying agents likely mediate this function by inducing expression of endogenous genes (HoxB4, Bmi-1, GATA-2, etc.) previously implicated in self-renewal of HSC (

Araki H et al.
Blood
2007
:
109
:
3670
–3678
). In our current studies we have further characterized the allostimulatory function of these expanded CB to assess its potential as grafts to mount an immune response. The percentage of 5azaD/TSA treated expanded CB cells co-expressing HLA-DR and CD86, or CD11c was less than cells expanded in cytokines alone. The expression of HLA-DR/CD40 was not altered following 5azaD/TSA treatment. In a mixed lymphocyte reaction (MLR), the irradiated CB cells (stimulator) were co-cultured with allogeneic peripheral blood T cells (responder) at 1:2 ratios. The MLR results indicate that 5azaD/TSA treated expanded CB grafts possessed diminished allostimulatory response as revealed by their relatively lower stimulation index in comparison to cells expanded in cytokines alone. Interestingly, the stimulation index of 5azaD/TSA treated cells (day 9) was even less than unmanipulated primary CB CD34+ cells (day 0). The reduced allostimulatory capacity of 5azaD/TSA expanded CB cells may favor in the reduction of incidence of graft rejection / graft failure following infusion. Alterations in cell fate regulatory mechanisms raise concerns for tumorigenesis, so the safety of such grafts was further assessed by examining the telomere length and cytogenetic analysis. Both cytogenetic analysis and examination of cell-cycle showed normal karyotype and diploid nature of expanded grafts, respectively. The telomere length of ex vivo expanded grafts as a putative indicator for their proliferative potential did not reveal significant alteration in their length in comparison to unmanipulated CB grafts. Also, in vivo repopulation assays with xenogeneic NOD/SCID mouse model showed no apparent tumor development following transplantation of ex vivo expanded 5azaD/TSA treated CB cells. In summary, our results indicate that the chromatin modifying agents not only result in expansion of in vivo repopulating CB cells but are capable of suppressing immunostimulatory capacity of expanded grafts and these grafts retain their telomere length and normal ploidy and can be expanded from a single unit for potential clinical use.

Topics:
chromatin, tissue transplants, umbilical cord blood, cytogenetic analysis, cytokine, hla-dr antigens, transplantation, cd40 antigens, deoxycytidine, infusion procedures

Author notes

Disclosure: No relevant conflicts of interest to declare.

2007, The American Society of Hematology
2007
Sign in via your Institution