Mapping the Secretome of Dental Pulp Stem Cells Under Variable Microenvironmental Conditions

There is substantial evidence supporting the anti-inflammatory and regenerative potential of dental pulp stem cells (DPSCs) through direct cell transplantation or paracrine action. However, DPSC secretome profile remains inadequately studied. This study provides proteomic profiling of the human DPSC secretome by comparatively analysising cell lysates and respective culture supernatants (i.e. conditioned media-CM) under variable oxygen tension conditions (normoxia-20% O-2/CM_Norm vs. hypoxia 2% O-2/CM_Hyp) and/or stimulation with Tumor Necrosis Factor alpha (TNF-alpha). DPSC-CM samples and respective crude lysates (DPSC-CL) were collected and subjected to SDS-PAGE, followed by LC-MS/MS analysis. The identified proteins were analyzed by Gene Ontology, Reactome, and String databases. The anti-inflammatory properties of DPSC-CMs were validated via an in vitro RAW_246.7 murine macrophages model through evaluation of the expression of pro-and anti-inflammatory markers by real-time PCR. Results showed a total of 2413 proteins identified in CM_Norm, 2479 in CM_Norm+TNF-alpha, 1642 in CM_Hyp, and 2002 in CM_Hyp + TNF-alpha samples. CM_Norm contained 122 proteins statistically significantly upregulated compared to the CM_Hyp and involved in pathways related to ECM organization, cellular response to hypoxia, and IL signaling. Functional network analysis showed that TGF beta 1, TIMP1 and TIMP2 were key nodes among proteins significantly upregulated in the CM_Norm compared to the CM_Hyp, interacting with more than 10 proteins, each. DPSC-CM application in the in vitro RAW_246.7 model decreased the expression of pro-inflammatory markers (MMP-3, MMP-9, MMP-13, MCP-1), while increasing anti-inflammatory markers (IL-10). Overall, DPSC-CM collected under normoxic conditions is enriched with anti-inflammatory, tissue repair and regenerative factors, which prompts further investigation on its therapeutic applications.

Automated summary

Human DPSC secretome collected under normoxic conditions is enriched with anti-inflammatory, tissue repair, and regenerative factors, showing potential therapeutic applications through downregulation of pro-inflammatory markers and upregulation of anti-inflammatory markers in vitro. Further investigation is needed to explore the therapeutic potential of these factors.