Surface Proteomic Signature Of Rat Bone Marrow Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are a type of widely distributed adult stem cell, which are easy to isolate and could be long-term propagated. MSCs possess the ability to differentiate into many different cell lineages, including osteoblasts, adipocytes, chondrocytes, hepatocytes and neuron-like cells, therefore they have been widely applied in regenerative medicine and cell therapy.MSCs from different tissues were transplanted into different disease models on the same or different species, and were reported to have positive effects, but with highly different results. MSCs from different species behave differently with their differentiation ability and CD molecule expression. To date, no specific molecule was used for the identification of MSCs, which made it difficult for the quality control of MSCs, and limited the research and application of MSCs. The relationship between the molecule characterization and functional properties as well as the development origin and differentiation regularity of MSCs should be illustrated at the level of surface proteomic.In this study, rat bone marrow MSCs (rBMMSCs) of passage4-7were used for the analysis. Surface proteins of rBMMSCs were labelled with EZLink-Sulfo-NHS-SS-Biotin and then purified with streptavidin-conjugated Latex beads. The enriched proteins were separated by SDS-PAGE, the protein lane were dissected into8bands and subjected to in gel digestion. Digested fragments were characterised by LC-MS/MS. In this study,2637proteins were identified, of which674were annotated as transmembrane proteins, lipid anchored proteins or secreted proteins on Swissport, which we selected as putative surface proteins for further analysis. We randomly selected65cell surface proteins and examined their expression in rBMMSCs by RT-PCR. Of the65proteins,63were confirmed to be expressed in rBMMSCs, indicating low rate of false identification. Moreover, the expression of several surface proteins were also confirmed at protein level by WB and ICC. We performed a Gene Ontology (GO) surveys on Molecule Function (MF) with the DAVID software, and results showed that most proteins serve as transportation or ion channel activity. Furthermore, the analysis of signal transducers of rBMMSCs reveled that different from mouse ESCs (mESCs), rBMMSCs was highly enriched with neural associated proteins and which presented a first hint of the neural lineage bias of these cells. The results of tissue specificity according to Uniprot annotation indicated that, of the thirteen tissue types, ten were of neural or neural endocrine lineages or could be traced to neural crest as a developmental origin, which presented another hint of the neural lineage bias of rBMMSCs. We compared the proteomics of rBMMSCs with the existed datasets of mESCs, human ESCs (hESCs) and human bone marrow MSCs (hMSCs) surface proteome, which further proved rBMMSCs possessed a neural and neural-endocrine lineage bias surface proteomic pattern of rBMMSCs. The different surface proteomic pattern between rBMMSCs and hMSCs also indicated that MSCs isolated from different species might possess a different lineage bias and differentiation tendency. At last, we analysed the neural differentiation ability of rBMMSCs and hMCs, results showed that rBMMSCs were more sensitive to neural induction, which to some extent suggested rBMMSCs is easy to differentiate into neurons.Our research presented the most comprehensive cell surface proteomic of rBMMSCs available to date, which revealed the lineage expression pattern of rBMMSCs and provided some explanation into the varied differentiation bias of MSCs from different species and tissue types. The proteomic analysis of MSCs would shed light on the identification of MSCs.