The Role of Osteomacs in Regulating Stem Cell Function and the Hematopoietic Niche

Abstract

Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). Reported here is a detailed characterization of OM and description of discriminating phenotypic and functional properties that clearly distinguish OM from bone marrow-derived macrophages (BM Mφ). Furthermore, it was established that OM support hematopoiesis enhancing activity of osteoblasts and that this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing OM, osteoblasts and megakaryocytes. Interestingly, BM Mφ were unable to mediate the same hematopoiesis enhancing activity regardless of whether megakaryocytes were present in co-culture or not. Subsequently, to understand the importance of networking between the residents of the niche, 3D tissue cytometry was performed on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, osteoblasts, megakaryocytes and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. In addition, single cell mRNAseq and CyTOF was performed to assess genetic and proteomic expression changes in OM following their interaction with megakaryocytes. These studies revealed the upregulation of CD166 and embigin on OM via osteoblast and megakaryocyte interactions. Clonogenic assays were conducted to examine the impact of these molecules in hematopoietic function. When these assays were initiated with CD166 KO OM or shRNA-mediated embigin knockdown OM, it was established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. This data, for the first time, reveal intimate spatial interactions between OM, osteoblasts, megakaryocytes and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, osteoblasts and megakaryocytes and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function.