Persistent Contamination By Bone and Bone Marrow-Derived Macrophages Obscures Functional Assessment of Tissue-Dependent Heterogeneity in Mesenchymal Stromal Cells
Authors: Kawano Yuko , Kawano Hiroki , Busch Stephanie , Li Allison , Zhang Jane , Salama Noah A , Quarato Emily R , Chen Chunmo , Georger Mary , Azadniv Mitra , Byun Daniel , Lamere Elizabeth A , Lamere Mark W , Liesveld Jane L. , Becker Michael W. , Calvi Laura 
Published: December 2023

Mesenchymal stem/stromal cells (MSCs) in the bone marrow microenvironment (BMME) serve as an essential support for the hematopoietic stem cell pool and are enriched for skeletal stem cells capable of regenerating bone, cartilage and adipose tissues. Purification of murine MSCs is essential to explore the functions of MSCs not only in skeletal generation but also in the regulation of the BMME. However, conventional protocols have not sufficed to purify murine MSCs, limiting the functional assessment of MSCs ex vivo. Careful evaluation of macrophage contribution in conventional ex vivo cultures of MSCs obtained from either bone marrow (BM-MSCs) or bone associated cells (BAC-MSCs) revealed abundant and persistent contamination with macrophages (BM mean 53.35%, BAC: mean 43.52% at passage3). Therefore, we developed a novel culture system by targeting three immunological markers; F4/80, Ly6C, and CD45. The functional and transcriptional analysis with this isolation procedure ensured clear dissection of macrophages and MSCs. MSCs were isolated from bone marrow (BM-MSCs) and bone (bone associated cells: BAC-MSCs).BM-MSCs display more proliferative progenitor like characteristics while the BAC-MSCs display dormant stem cell like characteristics. Previously Sox9 was reported to be a marker of murine undifferentiated mesenchymal precursors that possess tri-lineage differentiation capacity and are equivalent to CXCL12-expressing stromal cells from adult mice. Interestingly, Sox9 was significantly expressed in BACs (mean 4.75% in MSC (Lin-/CD45-/CD31-/Sca-1+/CD51+)) but not in BM cells (mean 0.733% in MSC (Lin-/CD45-/CD31-/Sca-1+/CD51+)) in vivo by flow cytometric analysis. In our ex vivo MSC cultures, we confirmed that BM-MSCs are enriched for Sox9 expressing cells, similar to that of BAC-MSCs indicating that we successfully purified a very rare multipotent stromal cell population from both BM and BACs. Comparing MSCs from culture that included macrophages to those that successfully purified MSCs, we learned that the coexistence of macrophages significantly reduced SOX9 and PDGFα (CD140a) expression by MSCs, regardless of whether they were derived from bone or bone marrow. This suggested that the presence of macrophages limits MSCs stem and progenitor activity. We also evaluated the functional aspects of these MSCs using our coculture system of MSCs and BM derived LSK (Lineage-/Sca-1+/c-Kit+) without any support of growth factors mainly to elucidate the hematopoietic cell support capacity of these MSCs. Interestingly, purified MSCs significantly enhanced reconstitution capacity of LSK cells in comparison to macrophage contaminated MSCs, especially for BM-MSCs. Macrophages also disrupted the reconstitution of stem/progenitor cells with BAC-MSC coculture even though mature blood cell reconstitution did not seem to be affected. Collectively, our novel ex vivo culture of primary MSCs can be useful to eliminate the artifacts of macrophage contributions to MSCs, which was observed in the conventional ex vivo cultures where high percentages of macrophage can persistently exist with MSCs possibly from CD45 negative macrophage progenitors. Moreover, the presence of macrophages and tissue source significantly alters the cell characteristics and fate of cultured MSCs, which could provide further understanding for MSC-macrophage crosstalk. In summary, our study highlights the importance of assessing the purity of primary MSC culture systems and heterogeneity based on tissue source, and they enable the isolation of highly purified MSCs to further study their role and macrophage crosstalk at homeostasis of BMME and in disease.