An unbiased proteomics approach to identify the senescence-associated secretory phenotype of human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from bone marrow can support skeletal tissue repair and regeneration owing to their self-renewing capacity, differentiation ability, and trophic functions. Bone marrow-derived MSCs undergo dramatic changes with aging, including the senescence-associated secretory phenotype (SASP) which may largely contribute to age-related changes in bone tissue leading to osteoporosis. A mass spectrometry-based proteomics approach was used to investigate the MSC SASP. Replicative senescence was achieved by exhaustive in vitro sub-cultivation and confirmed by standard proliferation criteria. Conditioned media from non-senescent and senescent MSCs underwent mass spectrometry. Proteomics and bioinformatics analyses enabled the identification of 95 proteins expressed uniquely in senescent MSCs. Protein ontology analysis revealed the enrichment of proteins linked to the extracellular matrix, exosomes, cell adhesion, and calcium ion binding. The proteomic analysis was independently validated by taking ten identified proteins with relevance to bone aging and confirming their increased abundance in conditioned media from replicatively senescent versus non-senescent MSCs (ACTα2, LTF, SOD1, IL-6, LTBP2, PXDN, SERPINE 1, COL1α1, THBS1, OPG). These target proteins were used to further investigate changes in the MSC SASP profile in response to other inducers of senescence, ionizing radiation (IR) and H₂O₂. Similar secreted protein expression profiles with replicatively senescent cells were seen with H₂O₂ treatment except for LTF and PXDN, which were increased by IR treatment. With both IR and H₂O₂ treatment there was a decrease in THBS1. In vivo investigation of these secreted proteins with aging was shown by significant changes in the abundance of OPG, COL1α1, IL-6, ACTα2, SERPINE 1, and THBS1 in the plasma of aged rats. This unbiased, comprehensive analysis of the changes in the MSC secretome with senescence defines the unique protein signature of the SASP in these cells and provides a better understanding of the aging bone microenvironment.