Human Hematopoietic Stem Cell Homing and Engraftment Promoted By Glucocorticoid Induced Chromatin Remodeling

Hematopoietic stem cells (HSC) which give rise to cells of all the blood lineages throughout the lifetime are key cells in hematopoietic cell transplantation (HCT). HCT has been used to treat patients with malignant and non-malignant hematological disorders. Cord blood (CB) is an increasingly used source of HSC for HCT, but one considered limitation to its greater use is the limited numbers of HSC in single CB collections. Thus, in this situation, the homing efficiency of HSC can be a pivotal factor for a successful HCT based clinical therapy. One means to enhance single CB unit HCT is to enhance the homing efficiency of CB HSC. The SDF-1/CXCR4 chemotactic axis is a major pathway directing migration and homing of HSC from peripheral blood to bone marrow niches. To get enhanced insight into achieving better homing efficiency of human CB HSC, we performed a compound screen to search for small molecules which could enhance surface expression of CXCR4 and SDF-1/CXCR4 axis mediated chemotaxis. From a nuclear hormone ligand library including 74 chemical compounds, we found that treatment of CB CD34⁺ cells with Dexamethasone, a synthetic glucocorticoid (GC), greatly promoted surface expression of CXCR4. Expression of CXCR4 on CB CD34⁺ cells was also increased after treating cells with other glucocorticoids (not existing in the library) including Flonase, Cortisol, and Medrol. We focused on use of Flonase, the compound which forms the most stable activated complex with glucocorticoid receptor (GR) (Högger, P. & Rohdewald, P. Steriods, 1994), and which enhanced CXCR4 expression at concentrations as low as 10 nM. Pretreament of CB CD34⁺ cells with Flonase significantly increased surface CXCR4 expression and promoted SDF-1/CXCR4 axis mediated chemotaxis of human CB HSC. Further more, Flonase pretreatment rigorously enhanced homing of phenotypically-defined human CB HSC and progenitor cells (HPC) (by ~5 fold, p<0.01). To assess long-term reconstituting ability of GC pretreated human CB CD34⁺ cells, we performed limiting dilution analysis to calculate the frequency of in vivo SCID Repopulating Cells (SRC). Poisson distribution analysis revealed an SRC frequency of 1/5237 in Vehicle-treated CB CD34⁺ cells and an enhanced SRC frequency of 1/1078 in Flonase-treated CB CD34⁺ cells, suggesting the respective presence of 191 SRCs and 928 SRCs in Vehicle- and Flonase-pretreated and administered cells (~4.8 fold, p<0.001). GC pretreatment significantly increased mRNA and protein levels of CXCR4 in human CB CD34⁺ cells. A search for the consensus GR binding sequence AGAACAnnnTGTNCN (Watson, L.C. et al. Nat. Struct. Mol. Biol. 2013), found that a glucocorticoid response element (GRE), AGAACATTCTGTGCA existed in the CXCR4 promoter region (from -1662 to -1648). Chromatin immunoprecipitation analysis suggested that GR was remarkably enriched in this region of the CXCR4 promoter upon administration of GC. CXCR4 promoter activity was enhanced (by ~2.5 fold, p<0.001) by Flonase stimulation, and this was abrogated by deletion of the GRE. Co-immuoprecipitation analysis demonstrated that the SRC1/p300 complex which has histone acetyltransferase activity was recruited to the CXCR4 promoter by GR upon Flonase treatment. Acetylation levels of CXCR4 associated Histone 4 on Lysine 5 and Lysine 16 (H4K5 and H4K16) were dramatically enhanced by Flonase treatment, while acetylation levels of Histone 3 (H3K9 and H3K14) remained unchanged. Enhanced chemotaxis and homing of Flonase treated CB HSC and HPC was totally blocked by administration of p300 inhibitor (c646). Our findings indicate that short-term pretreatment with glucocorticoid hormone dramatically improves CB HSC homing and engraftment by selective chromatin remodeling. This suggests a simple, new, and easily adaptable means to enhance clinical efficacy of CB HCT.