Prostaglandin E2 Is a Potent Regulator of Vertebrate Hematopoietic Stem Cell Homeostasis.

Hematopoietic stem cells (HSCs) give rise to each of the blood lineages found in the adult vertebrate. Definitive HSCs are derived during embryogenesis in the aorta-gonad-mesonephros (AGM) region and subsequently colonize the niche in fetal and adult hematopoietic organs. To identify regulators of this process, a library of biologically active compounds was screened for effects on zebrafish HSC development. Embryos were exposed to individual chemicals (2498) from the 5-somite stage until 36 hpf, then processed by whole-mount in situ hybridization for the expression of HSC markers cmyb and runx1. Exposure to 47 of the compounds reduced the number of runx1+cmyb+ HSCs, while 35 increased this cell population. Several chemicals isolated in the screen were known to affect the prostaglandin (PG) synthesis pathway. Prostaglandin (PG) precursors, mead acid and linoleic acid, expanded HSCs, while fenbufen and celecoxib, inhibitors of cyclooxygenase (cox) activity, reduced HSC number. An examination of blood and vessel specific molecular markers demonstrated that angiogenesis was not altered significantly in treated embryos, illustrating the specific effect on HSCs. Exposure to exogenous PGE2 modestly increased HSCs, while use of a long-acting version of PGE2, 16,16-dimethylPGE2 (dmPGE2), markedly enhanced HSC number. Incubation of zebrafish embryos with known cox inhibitors confirmed the observation that inhibition of PG synthesis reduces HSCs. Injection of morpholino oligonucleotides designed against cox1, cox2 and PGE synthase inhibited the formation of HSCs in a dose dependent fashion, verifying that signaling through PGE2 is regulating AGM HSC number. Using CD41:GFP;LMO2:DsRed bigenic zebrafish, the modulation of HSCs following exposure to dmPGE2 or indomethacin was documented in vivo by confocal microscopy. Exposure to dmPGE2 following irradiation of adult fish induced HSC gene expression and enhanced hematopoietic recovery, indicating that PGE2 also functions in regulating adult marrow HSC homeostasis. Incubation of murine embryonic stem cells with PGE2 during embryoid body differentiation resulted in a dose-dependent increase of hematopoietic colonies in methylcellulose, while indomethacin exposure inhibited colony formation, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Furthermore, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in CFU-S number and enhanced the frequency of multi-lineage engrafting HSCs more than 3-fold in a limiting dilution competitive repopulation assay. The identification of the prostaglandin synthesis pathway as a potent regulator of vertebrate hematopoietic stem cell homeostasis has major implications for new treatments of diseases such as anemia and leukemia.