CD34⁺CD90⁺ Cord Blood Cells Which Have Undergone Multiple Cell Divisions Retain Marrow Repopulating Potential.

We have previously shown that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase inhibitor, trichostatin A (TSA) is capable of changing the fate of adult bone marrow CD34⁺ cells (Milhem M et al Blood 2004). We have now studied whether these drugs could alter the behavior of dividing CD34⁺CD90⁺ cord blood (CB) cells. The 5azaD/TSA treated cultures yielded 10 times greater numbers of CD34⁺CD90⁺ cells as compared to the cultures containing cytokines alone after 9 days of culture. The 5azaD/TSA treated cultures contained 2 fold greater numbers of colony forming cells (CFC) and 14 fold greater numbers of long-term (5wk) cobblestone area forming cells (CAFC) in comparison to culture containing cytokines alone. Although the CFC/CAFC plating efficiency of cells in cultures exposed to cytokines alone declined during the time of incubation, the cloning efficiency of cells exposed to 5azaD/TSA was equivalent to that of primary CD34⁺ cells. In order to determine the effects of cell division on the behavior of CD34⁺CD90⁺ cells in the 5azaD/TSA treated cultures, we utilized the cytoplasmic dye, CFSE. All of the CD34⁺CD90⁺ cells within the 5azaD/TSA pre-treated cultures divided at least once after 9 days of culture. 60% of the 5azaD/TSA treated CD34⁺CD90⁺ cells divided 5 times or more while 40% divided 1–4 times. The CD34⁺CD90⁺ cells lacking 5azaD/TSA pre-treatment underwent more cell divisions (90%, 5 or more divisions). The CD34⁺CD90⁺ cells pre-treated with 5azaD/TSA which had undergone 1-2 cell divisions had 11 fold greater numbers of CFU-Mix and 9 fold greater number of CAFC as compared to CD34⁺CD90⁺ cell population cultured in presence of cytokines alone. Furthermore CD34⁺CD90⁺ cells having 5 and more divisions had 4 fold more CFU-mix and 6.5 fold more CAFC in comparison to cells lacking 5azaD/TSA exposure. The CD34⁺CD90⁺ cells experiencing 1–4 divisions had 60 fold greater number of CFU-mix and 54 fold more CAFC in contrast to culture treated with cytokines alone. The in vivo SCID repopulating potential of CD34⁺CD90⁺ cells generated in presence or absence of 5azaD/TSA was then evaluated. When 5x10⁴ CD34⁺CD90⁺ cells having undergone 1-2 cell divisions were re-isolated from 5azaD/TSA pre-treated cultures, all mice contained human hematopoietic cells. In addition, 1 of 3 mice transplanted with CD34⁺CD90⁺ cells (5x10⁴) having undergone 3 and more cell divisions isolated from cultures pre-treated with 5azaD/TSA also displayed human hematopoietic engraftment. Furthermore 1 of 3 mice transplanted with equal numbers of the 5azaD/TSA pre-treated CD34⁺CD90⁺ cells having undergone 5 and more cell divisions also had evidence of human multilineage hematopoietic engraftment. By contrast, an equivalent number of CD34⁺CD90⁺ cells which had undergone more than 3 or more than 5 cell divisions from the cultures containing cytokines alone were incapable of engrafting NOD/SCID mice. These data suggest that the sequential addition of 5azaD and TSA ex vivo is not only capable of expanding the numbers of CD34⁺CD90⁺ cells and assayable progenitor cells but also capable of preserving their SCID repopulating potential. We conclude that 5azaD/TSA treatment of CD34⁺CD90⁺ cells results in their retention of the cellular program required to maintain their marrow repopulating potential despite their undergoing multiple cell divisions.