Comparative proteomics analysis of transforming growth factor-beta1-overexpressed human dental pulp stem cell-derived secretome on CD44-mediated fibroblast activation via canonical smad signal pathway.

PURPOSE: The aim of this study investigates whether the secretome collected from human dental pulp stem cells (hDPSCs) transfected with transforming growth factor-beta1 (TGF-ß1) is related to CD44 expression of fibroblasts and canonical smad signaling pathway via proteomic analyzes. MATERIALS AND METHODS: In order to obtain secretome, hDPSCs were conditioned with serum-free alpha-MEM in an incubator containing 37°C, 5% CO2, and humidity for 18-24 h. Proteins in control and TGF-ß1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. CD44 expressions in fibroblasts were evaluated by real time-PCR, western blot, and immunofluorescent staining. The relationship of canonical smad pathway and CD44 was analyzed by western blot and LC-MS/MS. Cell cycle, proliferation and wound healing tests were performed in the secretome groups. RESULTS: Venn diagram was showed 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-ß1 secretome. CD44 gene and protein expressions were increased in fibroblasts treated with TGF-ß1 secretome. Relationship between targeted protein data showed that activation of the canonical TGF-ß1/Smad pathway was up-regulated CD44 expression in fibroblasts. The canonical smad pathway-mediated upregulation of CD44 may increase the mitotic activity, proliferation, and wound healing potential in fibroblasts. CONCLUSION: While TGF-ß1-transfected hDPSC secretome may be a potential therapeutic candidate in regenerative connective tissue therapies as it induces fibroblast activation, anti-TGF-ß1-based therapies would be considered in histopathological conditions such as pulmonary fibrosis or hepatic fibrosis.

Transforming growth factor ß1-enriched secretome up-regulate osteogenic differentiation of dental pulp stem cells, and a potential therapeutic for gingival wound healing: A comparative proteomics study.

OBJECTIVES: Current study aimed at comparing the human dental pulp-derived stem cell (hDPSC) secretome (Control secretome) and transforming growth factor beta1 (TGF-ß1)-transfected hDPSC secretome (TGF-ß1 Secretome), which have the potential to be therapeutic in terms of regenerative dentistry, in terms of osteogenesis, adipogenesis and gingival wound healing with proteomic analyses. MATERIALS AND METHODS: pCMV-TGF-ß1 plasmid was transfected into hDPSCs by electroporation. hDPSC and TGF-ß1 transfected hDPSC secretomes were collected for LC-MS/MS. Protein contents in control secretome and TGF-ß1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations for canonical pathways, upstream regulators and networks were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. Surface marker expressions between groups, treated secretome were measured by flow cytometry. To support the proteomic data morphologically, we performed osteogenic-adipogenic differentiation in hDPSCs treated with control secretome and TGF-ß1 secretome, and scratch wound healing assay in gingival fibroblasts. Statistical analyses were performed by GraphPad Prism 8.02. RESULTS: Venn diagram classification showed us 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-ß1 secretome. TGF-ß1 secretome was found to have therapeutic effect on MSC-specific immunophenotypes. TGF-ß1 secretome was determined to up-regulate osteogenesis-related molecules and pathways while down-regulating adipogenesis-related pathways. Analysis of canonical pathways showed that TGF-ß1 secretome is associated with the wound healing pathway. CONCLUSION: Our study provided the first evidence that proteins identified in TGF-ß1-transfected hDPSC secretomes are potential regulators of osteogenic/adipogenic differentiation and fibroblast wound healing. CLINICAL SIGNIFICANCE: Based on these results, TGF-ß1 secretome may have a therapeutic effect in repairing osteoporosis-related bone injuries, wound healing of oral mucosa and gingival tissue. TGF-ß1 secretome may be a potential cell-free therapeutic in orthopedics and regenerative dentistry.