Prostaglandin E₂ Enhances Survival, Proliferation, Homing and Engraftment of Mouse Hematopoietic Stem Cells

Adult hematopoietic stem cells (HSC) are used to reconstitute patients after myeloablative therapy. Hematopoietic reconstitution is a multi-step process and efficacy can be limited by inadequate stem cell number, inability to migrate/home to appropriate marrow niches and poor engrafting efficiency and self-renewal. A number of studies have suggested that prostaglandin E₂ (PGE₂) can increase the proliferation of early hematopoietic cells, although activity on repopulating cells was not addressed. Recently, it was reported that short-term exposure (2 hrs) to a stabilized derivative of PGE₂, 16, 16-dimethyl PGE₂ (dmPGE₂) increased HSC in murine marrow following irradiation and transplantation; however, the mechanism of action was undefined. Enhancement of HSC frequency induced by dmPGE₂ was validated using a limiting dilution competitive transplantation model that compared engraftment of control and dmPGE₂-treated cells in direct head-to-head analysis within the same animal. We demonstrated ~4-fold competitive advantage of PGE₂-pulsed HSC based upon calculation of HSC frequency by Poisson statistics and analysis of competitive repopulating units (CRU). Frequency analysis demonstrates equivalent reconstitution using one-fourth the number of PGE₂ treated cells vs control cells. Enhanced engraftment of PGE₂-treated cells was stable over 28 weeks. Analysis in secondary transplanted animals 90 days post-transplant demonstrated full multilineage reconstitution and continued higher HSC frequency, indicating a stable effect of short-term PGE₂-treatment on long-term repopulating HSC. Flow cytometry and QRT-PCR confirm expression of all 4 PGE₂ receptors (EP1-EP4) on Sca-1⁺, c-kit⁺, Lineage^neg (SKL) murine marrow cells and on CD34⁺ human cord blood cells (UCB) with no overt differences in receptor subtype expression. To define mechanisms responsible for enhanced engraftment of PGE₂-pulsed cells, we analyzed several functional properties relevant to HSC function. A significant increase in CXCR4 expression on both SKL (26.8%) and CD34⁺ UCB (17.3%) was seen after PGE₂ exposure, with significant upregulation of CXCR4 mRNA at ~6 hours post-exposure. Increased CXCR4 was coincident with an ~2-fold increase in in vivo marrow homing efficiency of PGE₂-treated grafts and was observed with unmanipulated bone marrow (p<0.001, 3 expts, n=6 mice/group/expt, assayed individually) and with purified SKL cells in head-to-head competition in the same animal (p<0.001, 2 expts, n=5 mice/group/expt, assayed individually), indicating a direct effect of PGE₂ on HSC. The increase in homing efficiency was significantly reduced by treatment with the selective CXCR4 antagonist AMD3100. In vitro, PGE₂ treatment results in an increase in the proportion of SKL cells actively in cell cycle within 24 hours post-treatment. In addition, transplantation of PGE₂-treated cells in BrdU treated recipient mice showed ~2-fold more donor SKL cells in S+G²/M phase of the cell cycle compared to transplanted cells pulsed with vehicle only. Survival assays indicated that PGE₂ dose-dependently decreased apoptosis of SKL cells in vitro, coincident with a 1.7 fold increase in Survivin protein expression and a decrease in active caspase-3 (23–59% decrease; 24–72 hours post exposure). These studies suggest that the ~4-fold increase in HSC frequency observed after PGE₂ treatment results from ~2-fold more HSC homing to recipient marrow and with ~2-fold more HSC undergoing self-renewal. Our results define novel mechanisms of action whereby PGE₂ enhances HSC function and suggest a possible therapeutic strategy to facilitate hematopoietic transplantation, particularly for hematopoietic grafts with limiting cell number or poor engraftment potential.