Ex vivo expansion of human mobilized peripheral blood stem cells using epigenetic modifiers

BackgroundEpigenetic modifications likely control the fate of hematopoietic stem cells (HSCs). The chromatin-modifying agents (CMAs), 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA), have previously been shown to expand HSCs from cord blood and marrow. Here we assessed whether CMA can also expand HSCs present in growth factor-mobilized human peripheral blood (MPB). Study Design and Methods5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines, and the cells were cultured for 9days. ResultsAfter culture, a 3.60.5-fold expansion of CD34+CD90+ cells, a 10.1 +/- 0.5-fold expansion of primitive colony-forming unit (CFU)-mix, and a 2.2 +/- 0.5-fold expansion of long-term cobblestone-area-forming cells (CAFCs) was observed in 5azaD/TSA-expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather, a reduction in CD34+CD90+ cells (0.7 +/- 0.1-fold) and CAFCs (0.3 +/- 0.1-fold) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA-expanded MPB cells in contrast to controls. 5azaD/TSA-expanded MPB cells retained in vivo hematopoietic engraftment capacity. ConclusionMPB CD34+ cells from donors can be expanded using 5azaD/TSA, and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSC numbers for patients who fail to mobilize.