Indiana University-Purdue University Indianapolis (IUPUI) / 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. / 2020-09-10

Basigin and Embigin are members of the immunoglobulin superfamily that function as cell adhesion molecules. Studies of Basigin null mice revealed reproductive sterility, increased pain sensitivity, and blindness. It is thought that the mechanism causing blindness involves misexpression of monocarboxylate transporter 1 (MCT1) in the absence of Basigin. It is known that the transmembrane domain of Basigin interacts with MCT1. In the absence of Basigin, MCT1 does not localize to the plasma membrane of expressing cells and photoreceptor function is disrupted. Studies of the Basigin null mouse brain suggest that MCT1 is properly expressed, which suggests a separate mechanism causes the increased pain sensitivity in these animals, and also that a different protein directs MCT1 to the plasma membrane of expressing cells in mouse brain. Embigin is known to interact with MCT2 in neurons and with MCT1 in erythrocytes. It is not known, however, if Embigin normally interacts with MCT1 in the mouse brain or if Embigin acts to compensate for the lack of Basigin in the Basigin null animals. Therefore, the purpose of this study was to determine if Embigin normally interacts with MCT1, 2, or 4 in the mouse brain and if so, whether the interaction is similar to that between Basigin and MCT1. Expression of Basigin, Embigin, MCT1, MCT2, and MCT4 in mouse brain was assessed via immunoblotting and immunohistochemical analyses. In addition, recombinant protein probes corresponding to the Embigin transmembrane domain were generated for ELISA binding assays using endogenous mouse brain MCTs. It was determined that the proteins in question are rather ubiquitously expressed throughout the mouse brain, and that the cell adhesion molecules Basigin and Embigin may be co-expressed in the same cells as the MCT2 and MCT4 transporter proteins. In addition, it was determined that the Embigin transmembrane domain does not interact with the MCTs. The data therefore suggest that MCTs do not require Basigin or Embigin for plasma membrane expression in mouse brain.