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+, Lineageneg (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.