Hematopoietic Stem Cells Regulate Niche Development.

Events localized to endosteal surfaces are critical for the maintenance of hematopoetic stem cells (HSCs). Here we explored whether HSCs themselves regulate their microenvironment directly by regulating cytokine expression by osteoblasts (OBs) in response to physiologic demands, or influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression. To test the these possibilities, marrow was isolated from mice 48h after stressing the animals with a single acute bleed (removing 30% of the calculated blood volume by jugular vein venipucture) and in a second control group of non-stressed (puncture only) animals. The Sca-1(+) hematopoietic cells were co-cultured with confluent murine bone marrow stromal (BMSC) and calvarial digested OBs. The presence of HSCs stimulated the basal production of IL-6, SDF-1 and osteoclacin by OBs and BMSCs as determined by ELISA. Co-cultures of HSCs derived from the stressed group produced more IL-6, SDF-1 and osteocalcin relative to the non-stressed group. Newborn dermal fibroblasts did not respond in a similar fashion. To determine if HSCs influence the developmental pattern of the marrow, HSCs derived from stressed and non-stressed animals were separated from either murine OBs or BMSCs using Transwell^R membranes and the ability of target cells to differentiate along the osteoblastic lineage was evaluated. A significant proportion of the colonies established from calvarial-derived OB cultures were able to mineralize their extracellular matrix relative whole BMSC population (CFU-OB). In the presence of HSCs, the proportion of non-mineralized (CFU-F) and mineralized colonies from the OB populations significantly increased. Further enhancement of both colony types were induced by the HSCs derived from stressed and non-stressed animals. Adherent cells derived from mixed BMSCs also responded to the presence of HSCs by increasing the generation of CFU-F and OBs relative to the No HSC groups. HSCs derived from stressed vs. the non-stressed groups of animals were better able to induce CFU-OB differentiation. Microarray of HSCs derived from stressed vs. the non-stressed groups, and QRT-PCR of highly purified HSCs (CD150(+), CD48(−), CD41(−), Sca-1(+), cKit(+)) suggested that BMP-2 and BMP-6 were responsible for the activities. Antibody neutralization studies confirmed these observations that BMP-2 and BMP-6 derived from the HSCs themselves alters the developmental pattern of the marrow microenvironment. In conclusion, cross-talk between HSCs-OBs is essential for the development of both cellular populations. These studies demonstrate that at least 2 mechanisms whereby HSCs might set up a paracrine loop with OBs to establish the HSC niche; (i) HSCs directly regulate cytokine expression by OBs in response to physiologic demands or, (ii) HSCs may influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression in the marrow.