Novel ex vivo culture of purified mouse mesenchymal stromal cells reveals tissue-dependent niche heterogeneity in bone marrow and functional interaction with macrophages

Mesenchymal stem/stromal cells (MSCs) are skeletal stem cells capable of regenerating bone, cartilage, and adipose tissues, while also serving as an essential immunosupportive cell for the hematopoietic stem cell pool. Therefore, the isolation and purification of murine MSCs is essential to understanding the differentiation capabilities necessary for skeletal regeneration, hematopoietic support, and immunomodulatory roles of MSCs within the bone marrow microenvironment (BMME). While many protocols on isolation from bone marrow (BM-MSCs) and bone associated cells (BAC-MSCs) have been published, none achive purity from macropahge contamination and many do not address heterogeneity of MSCs between these two compartments. To ensure a pure MSC isolation, we established a new strategy to exclude macrophages and their precursors by targeting F4/80/Ly6C/CD45. By analyzing MSC transcriptional profiles from both compartments with and without our purification strategy, we demonstrated distinct functional characteristics of MSCs by compartment, as well as functional impacts on the MSC transcriptional profile as a result of macrophage interaction. Four populations were collected in total from two different compartments: Bone marrow derived (BM) and bone associated cells (BAC). Bone marrow derived mesenchymal cells (MSC) were harvested from bilateral hind limbs and pelvis whole bone marrow and plated on collagen-I coated plates. After the first passage, samples were either used directly (BM-nonS) or sorted to purely isolate CD31-/CD45-/Ly6C-/F4/80-/CD51+/Sca-1+ cells (BM-S) for bulk RNAseq. BAC derived MSCs were isolated from collagenase-I treated bone fragments from bilateral hind limbs. After harvest these cells were either used directly without depletion (BAC-nonD) or depleted by magnetic separation to remove CD45+/Ly6C+/F4/80+ cells (BAC-D) for bulk RNAseq.