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

ObjectiveWe 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 MethodsHuman 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.ResultsA 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.ConclusionEx 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.