Obtaining and proteomic analysis of human dental pulp stem cell conditioned medium

Introduction: Experiments indicate that the application of the stem cells conditioned medium (CM) promotes therapeutic efficacy similar to that exerted by direct administration of cells. Therefore, an important step in this context is the study of the composition of the CM, in order to characterize it in terms of the presence of specific soluble factors. The oral cavity has been an important source of stem cells, among which human dental pulp-derived stem cells (hDPSCs) stand out, which show promise in the context of regenerative medicine. Objective: Obtain and characterize the hDPSCs CM. Materials and Methods: To obtain the CM, hDPSCs were obtained from donors and cultured in mesenchymal stem cell expansion medium supplemented with 8% human serum. The obtained CM was lyophilized and, initially, the expression of the anti-inflammatory protein Annexin-A1 (ANXA1) was verified by Western Blotting, using the expansion medium as a control. Briefly, the total protein content of the CM and the control medium was measured by the Bradford assay, and equal amounts of proteins were electrophoresed in 12% SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was blocked in a 3% dry milk solution and incubated with the primary anti-ANXA1 antibody (1:1000). Subsequently, the membrane was incubated with the HRP-conjugated secondary antibody (1:1000) and the bands were detected with DAB. Results: The initial analyzes indicate the presence of ANXA1 only in the conditioned medium. In the continuity of the work, proteomic analyzes will be performed through liquid chromatography coupled to mass spectrometry, while other proteins related to immunomodulation will be investigated by Western blotting. Conclusions: The presence of ANXA1 in CM indicates future application potential of CM in inflammatory conditions.