Calvariae-Resident Osteomacs That Are Phenotypically and Functionally Distinct from Marrow-Derived Macrophages Interact with Megakaryocytes to Regulate Hematopoietic Stem Cell Function
Authors: Mohamad Safa F , Xu Linlin , Evan Himes R , Wu Hao , Alvarez Marta , Childress Paul , Davis Korbin , Kacena Melissa , Srour Edward F. 
Published: October 2019

Abstract
Maintenance of stem cell function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Precise networking between hematopoietic stem cells (HSC) and these cell types is critical for the maintenance of the stem cell pool. Evidence is accumulating that multiple cell types cooperate to collectively maintain HSC function in the hematopoietic niche. We report here a detailed characterization of calvariae-resident osteomacs (OM) and outline how these cells require cooperation from megakaryocytes (MK) to sustain HSC function. We also describe in detail discriminating phenotypic and functional properties that clearly distinguish OM from marrow-derived macrophages (Mφ).
Osteomacs, identified as CD45+F4/80+ cells, were easily detectable in calvarial cell (CC) preparations (3-5% of total CC) collected by the enzymatic digestion of calvariae from 2d-old pups. To assess the effect of MK, a known regulator of osteoblast (OB) proliferation and differentiation, on OM, we performed co-cultures using CC and MK prepared from fetal liver. In the absence of MK, OM did not increase in numbers over a period of 5 days in culture and remained approximately 5% of total cultured cells. However, in the presence of MK, OM significantly increased to become between 25% and 30% of total cells demonstrating that MK regulate OM proliferation. Clonogenic assays established that OM support hematopoiesis enhancing activity of OB and that this activity can be upregulated by MK. Interestingly, marrow-derived Mφ were unable to mediate the same hematopoiesis enhancing activity regardless of whether MK were present in the co-culture or not. These results were validated via primary and secondary transplantations in lethally irradiated hosts whereby the highest repopulating potential was observed among marrow-derived LSK cells co-cultured for 5 days with a mixture of OB, OM, and MK. Using eight surface markers and flow cytometric analysis, we established that although marrow-derived Mφ and OM share many phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and GR-1), only OM expressed MCSFR and CD166, thus providing a distinct and unique profile for these cells.
To assess changes in pathway activation between resting and MK-activated OM, we performed single cell genomic analysis. This approach detected the upregulation of several canonical pathways important in HSC maintenance such as Ephrin receptor signaling, PDGF signaling, and leukocyte extravasation signaling in MK-stimulated OM. Single cell genomic analysis between CC-derived OM and marrow-derived Mφ (isolated from each tissue as CD45+F4/80+ cells) revealed 39 genes to be significantly different between the two cell types. Strikingly, many genes such as IGF1, KITL and NOTCH2 that have previously been implicated in HSC regulation were upregulated in OM. MCSFR1 a known regulator of proliferation, differentiation and survival of Mφ was also upregulated in OM corroborating the data previously collected from flow cytometric analyses. However, OM did not respond to exogenous MCSF stimulation suggesting that MCSF alone is not sufficient to induce OM proliferation or that direct contact with MK is required for induction of proliferation. To investigate changes at the protein translational level, we examined both cell types using CyTOF and a panel of 24 surface and intracellular antibodies. The surface marker CD169 which was previously associated with HSC retention when present on cellular components of the hematopoietic niche was expressed on OM but not on Mφ. Intriguingly, OM expressed both CD86 and CD206 which are known M1 and M2 Mφ markers, respectively. TNF-α, TIMP2, FGF2 and MCP1 which are known HSC regulators were also upregulated in OM. Finally, the majority of OM expressed embigin and IL-18, both of which have been implicated in the maintenance of HSC function.
These data demonstrate that although bone-associated OM share many properties with marrow-derived Mφ, they are phenotypically and functionally distinct and are critical for the maintenance of HSC function. Furthermore, the function of OM, and consequently that of the two components of CC, namely OB and OM, is significantly augmented by interactions with MK demonstrating that the crosstalk between OM, OB and MK form a novel network in supporting HSC function.

Disclosures
No relevant conflicts of interest to declare.