Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM), are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single cell mRNAseq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM, confirmed that loss of these molecules significantly reduced OM ability to augment the osteoblast-mediated hematopoietic enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells. 1x105 neonatal calvarial cells (NCC) were cultured for 16hrs with or without 50,000 megakaryocytes were stained with CD45, F4/80, and CD41. Sorted CD45+F4/80+CD41- cells were suspended at 3x105 cells/ml PBS then dispensed into medium sized IFC chips (Fluidigm C1 Single-Cell Auto Prep System)(Zhang et al., 2019). mRNA was isolated followed by cDNA synthesis (Clontech SMART-Se v4 Ultra Low Input RNA Kit). Up to 0.4ng cDNA was used for library preparation and indexing with Nextera XT DNA Library Prep Kit (Illumina, Inc., San Diego, CA). Pooled libraries (4nM, quality checked) were used for 150b paired-end sequencing (NextSeq 500). Cells that passed quality testing were analyzed.