Prostaglandin I2 in the Endosteal Bone Marrow Niche As a Novel Regulator of Hematopoietic Stem Cells

Abstract

Haematopoietic stem cells (HSCs) are regulated by their immediate microenvironment or niche. The most potent functional HSCs are enriched at the endosteum near the bone, which comprises ~10% of total bone marrow (BM). To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes that were >2-fold overexpressed in the endosteal BM relative to the central BM. In this screen, we uncovered known essential HSC niche factors overexpressed in the endosteal BM such as Scf, Cxcl12, and Angpt1, which validated our approach.Among the genes overexpressed in the endosteal BM, prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteum (>10-fold by qRT-PCR, p<0.001). Within the endosteal BM, Ptgis was most abundantly expressed by osteoblasts, followed by mesenchymal stem/progenitor cells and endothelial cells. Ptgis is the sole enzyme responsible for biosynthesis of PGI2, originally discovered as a potent anti-thrombotic and vasodilating agent. PGI2 has no reported roles in HSC regulation in the BM and was chosen as candidate niche factor for further investigation. Due to the extreme lability of PGI2, we employed a clinically approved PGI2 analogue iloprost for all subsequent experiments.In initial experiments where we cultured fluorescence activated cell sorted (FACS) BM lineage- Kit+ Sca-1- (LKS+) haematopoietic stem and progenitor cells (HSPCs) for 7 days in serum free conditions, we found iloprost treatment potently reduced proliferation and differentiation compared to vehicle controls, assessed by flow cytometry phenotyping (p<0.0001). To determine whether iloprost treatment alters HSC function, we pulse treated whole BM cells with iloprost for 1 hour before competitive transplant and found iloprost pulse-treatment resulted in 14-fold increased multilineage reconstitution potential (p<0.05). This suggests that PGI2 enhances HSC function by dampening differentiation.We next sought to determine whether iloprost affects HSC function in vivo. In steady state, low dose 0.1mg/kg iloprost administration to mice for 15 days did not alter BM HSPC phenotypes by flow cytometry nor HSC reconstitution potential in competitive transplants compared to vehicle controls suggesting PGI2 does not alter HSC function in homeostasis. To further test the effect of PGI2 following stress, mice were sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. We found low dose iloprost administration partially rescued BM HSC reconstitution potential 21 days following irradiation (p<0.05) or 3 days following G-CSF administration (p<0.01). These data suggest that PGI2 protects HSC from stress.To understand the extrinsic mechanisms through which PGI2 regulates HSC in the BM following stress, we performed a HSC homing assay using naïve donors transplanted into 2-day G-CSF administered recipients. Analysis of HSPC homing to the BM at 4 hours post-transplantation revealed ~4-fold greater proportion of LKS+ HSPC homing to the BM of iloprost co-administered recipients compared to vehicle controls (p<0.01). We assessed mRNA expression of essential HSC niche factors in the BM of 2-day G-CSF-administered mice and found that G-CSF administration decreased Scf and Cxcl12 expression, which were both partially rescued with iloprost co-administration (p<0.05). Together the data suggest that preservation of the BM niche function is one of the mechanisms through which PGI2 protects HSC during stress.In summary, we have identified PGI2 as a novel HSC niche factor abundant in the endosteal BM. PGI2 analogues like Iloprost are well-tolerated and used clinically to treat vascular diseases such as pulmonary arterial hypertension and Raynaud's phenomenon. Our research suggests that PGI2 analogues can be rapidly repurposed in the clinic to improve HSC transplant outcomes and protect against BM failure following acute stressors such as accidental irradiation or inflammation. DisclosuresLevesque:GlycoMimetics: Equity Ownership, Patents & Royalties.