Chromatin-Modifying Agent-Expanded Human Cord Blood Cells Display Reduced Allostimulatory Capacity.

ABSTRACT

The limited number of hematopoietic stem cells (HSC) within a single unit of human cord blood currently limits its use as an alternate graft source. However, we have developed a strategy using 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), which expands transplantable HSC 7- to 10-fold. In our current studies, we have assessed the allostimulatory capacity of the 5azaD/TSA-expanded grafts. The coexpression of immunophenotypic dendritic cell (DC) markers, such as HLA-DR/CD86 and HLA-DR/CD11c as determined by flow cytometry, and the allostimulatory capacity of 5azaD/TSA-expanded cells as determined by MLC were both significantly lower than control. It has been previously demonstrated that STAT3 is indispensable for the differentiation of DC from HSC. Real-time quantitative PCR analysis revealed that 5azaD/TSA-expanded cells expressed more STAT3 transcript than control while also expressing increased transcripts for STAT3 inhibitors including SHP1, p21, and GATA1. Western blot analysis indicates that chromatin-modifying agent-expanded grafts displayed a reduced ratio of p-STAT3 to total STAT3 than control cultures, which is likely indicative of STAT3 inactivity in 5azD/TSA-expanded grafts. Culturing 5azaD/TSA-expanded cord blood cells in extended cultures reveals that they are still capable of generating DC. Notably, STAT3 inactivity was transient because the transcript levels of STAT3 and its inhibitors, including SHP1, were comparable between 5azaD/TSA and control cultures following extended culture. Taken together, our studies indicate that the reduced allostimulatory capacity of 5azaD/TSA-expanded cells is likely because of reversible inhibition of STAT3-dependent DC differentiation. These results suggest that a graft composed of 5azaD/TSA-expanded cells possesses relatively less allostimulatory response but is still capable of generating DC in permissive conditions.