Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents

Objective. We investigated whether the addition of two chromatin-modifying agents, 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA), to cord blood (CB) CD34⁺ cells in culture results in expansion of the numbers of severe combined immunodeficient (SCID) repopulating cells (SRC). Materials and Methods. Human CB CD34⁺ cells were cultured with cytokines in the presence or absence of 5azaD/TSA. After 9 days of culture, the fold expansion of CD34⁺ and CD34⁺CD90⁺ cell numbers, colony-forming unit (CFU)-mix, cobblestone area-forming cell (CAFC), and SRC numbers were determined. Results. A 12.5-fold expansion of CD34 ⁺CD90⁺ cells was observed in the 5azaD/TSA-treated cultures in comparison to the input cell numbers. Expansion of CD34 ⁺CD90⁺ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold increase in CAFC. 5azaD/TSA treatment of the CB CD34⁺ cells resulted in a 9.6-fold expansion of the absolute number of SRC following 9 days of culture as determined by limiting dilution analysis. Expansion of cells maintaining CD34⁺CD90⁺ phenotype was not due to the retention of a quiescent population of cells because all of the CD34⁺CD90⁺ cells in the culture had undergone cellular division. 5azaD/TSA-treated CD34⁺CD90⁺ cells, but not CD34⁺CD90^- cells were responsible for in vivo hematopoietic repopulation potential of nonobese diabetic/SCID mice. Conclusion. Ex vivo expansion strategy using chromatin-modifying agents provides a potential avenue by which to expand the number of hematopoietic stem cells (HSC) with a single CB unit for use as an alternative source of HSC grafts for adult recipients.