Project description:The efficient clearance of dead and dying cells, also known as efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs in the BMME and on their function. To investigate this, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including osteoporosis in aging.

Project description:Multipotent stromal cells (MSCs) are known for their distinctive ability to differentiate into different cell lineages such as adipocytes, chondrocytes and osteocytes. They can be isolated from numerous tissue sources including bone marrow, adipose tissue, skeletal muscle and others. Because of their differentiation potential and their secretion of growth factors, MSCs are believed to have an inherent quality of regeneration and immune suppression, which are considered advantageous in treating multiple disorders such as graft-versus-host disease. Since the number of MSCs derived from a tissue source is low, cellular expansion is necessary to obtain sufficient numbers for a desired cell therapy. However, after several rounds of passaging, our previous results have shown that MSCs exhibit reduced capacity for proliferation and differentiation. In this study, gene markers of MSC proliferation were identified and evaluated for their ability to predict the cell population proliferative quality. Microarray data of human bone marrow-derived MSCs were correlated with two proliferation assays. A collection of 24 genes were observed to significantly correlate with both proliferation assays (|r| > 0.70) for 8 MSC donors at multiple passages. These 24 identified genes were then confirmed using an additional set of MSCs from 8 new donors using RT-qPCR. The proliferative potential of the second set of MSCs was measured for each donor/passage by three proliferation assays for confluency fraction, fraction of EdU+ cells and population doubling time. The second set of MSCs exhibited a greater proliferative potential at passage 4 in comparison to passage 8, which was distinguishable by 15 genes; however, only 7 of the genes (BIRC5, CCNA2, CDC20, CDK1, PBK, PLK1, SPC25) demonstrated significant correlation (FDR: q < 0.05 and |r| > 0.62) with MSC proliferation regardless of passage. These 7 genes and the proliferative capacity of different non-MSC cell lines were assessed for comparison. Our analyses revealed that correlation between gene expression and proliferation was consistently reduced with the introduction of non-MSC cell lines; therefore this set of 7 genes may be more strongly associated with MSC proliferative quality. These correlative methods may be further used to identify additional markers that exhibit strong correlation with a particular MSC quality such as differentiation or immune suppression potential. Our results pave the way toward the identification of specific gene markers that could rapidly determine the quality of an MSC population for a particular cellular therapy in lieu of an extended in vitro or in vivo assay. Microarray gene expression analysis with confirmation of genes by RT-qPCR

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Expression data from primary sqWAT-MSC cells from mouse

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