Secretome profile of TNF-α-induced human umbilical cord mesenchymal stem cells unveils biological processes relevant to skin wound healing.

Aim: To profile and study the proteins responsible for the beneficial effect of the TNF-α-induced human umbilical cord mesenchymal stem cells (hUCMSCs) secretome in wound healing. Methods: The hUCMSCs secretome was generated with (induced) or without (uninduced) TNF-α and was subsequently analyzed by liquid chromatography-mass spectrometry, immunoassay and in vitro scratch assay. Results: Proteomic analysis revealed approximately 260 proteins, including 51 and 55 unique proteins in the induced and uninduced secretomes, respectively. Gene ontology analysis disclosed that differential proteins in the induced secretome mainly involved inflammation-related terms. The induced secretome, consisting of higher levels of FGFb, VEGF, PDGF and IL-6, significantly accelerated wound closure and enhanced MMP-13 secretion in HaCaT keratinocytes. Conclusion: The secretome from induced hUCMSCs includes factors that promote wound closure.

An interference or delay in normal stages of the wound healing process, particularly in the elderly population and individuals with comorbid conditions, generally results in the development of chronic wounds with uncontrolled inflammation. Innovative therapies, such as stem cells and their secreted factors (the 'secretome') are potential tools in regulating wound repair. We used an inflammatory factor to precondition human umbilical cord stem cells to generate a secretome (induced secretome) that was beneficial in response to the inflammation environment. Approximately 260 proteins were detected. Further analysis identified that unique proteins in the induced secretome are mainly related to inflammation-related biological processes. We also demonstrated that the induced secretome enhanced the wound closure rate in human keratinocyte cells, as compared with the control and naive secretome. This is likely due to the higher levels of growth factors and cytokines in the induced secretome, which play significant roles in the regulation of the wound healing process. The present findings provide useful information to better understand the role of the human umbilical cord mesenchymal stem cell secretome, especially in an inflammatory niche, as well as the proteins that are important for clinical translation in wound repair.

Oxidative Stress Down-Regulates MiR-20b-5p, MiR-106a-5p and E2F1 Expression to Suppress the G1/S Transition of the Cell Cycle in Multipotent Stromal Cells.

Oxidative stress has been linked to senescence and tumorigenesis via modulation of the cell cycle. Using a hydrogen peroxide (H2O2)-induced oxidative stress-induced premature senescence (OSIPS) model previously reported by our group, this study aimed to investigate the effects of oxidative stress on microRNA (miRNA) expression in relation to the G1-to-S-phase (G1/S) transition of the cell cycle and cell proliferation. On global miRNA analysis of the OSIPS cells, twelve significantly up- or down-regulated miRNAs were identified, the target genes of which are frequently associated with cancers. Four down-regulated miR-17 family miRNAs are predicted to target key pro- and anti-proliferative proteins of the p21/cyclin D-dependent kinase (CDK)/E2F1 pathway to modulate G1/S transition. Two miR-17 miRNAs, miR-20-5p and miR-106-5p, were confirmed to be rapidly and stably down-regulated under oxidative stress. While H2O2 treatment hampered G1/S transition and suppressed DNA synthesis, miR-20b-5p/miR-106a-5p over-expression rescued cells from growth arrest in promoting G1/S transition and DNA synthesis. Direct miR-20b-5p/miR-106a-5p regulation of p21, CCND1 and E2F1 was demonstrated by an inverse expression relationship in miRNA mimic-transfected cells. However, under oxidative stress, E2F1 expression was down-regulated, consistent with hampered G1/S transition and suppressed DNA synthesis and cell proliferation. To explain the observed E2F1 down-regulation under oxidative stress, a scheme is proposed which includes miR-20b-5p/miR-106a-5p-dependent regulation, miRNA-E2F1 autoregulatory feedback and E2F1 response to repair oxidative stress-induced DNA damages. The oxidative stress-modulated expression of miR-17 miRNAs and E2F1 may be used to develop strategies to retard or reverse MSC senescence in culture, or senescence in general.

Oxidative stress-induced premature senescence in Wharton's jelly-derived mesenchymal stem cells.

BACKGROUND: On in vitro expansion for therapeutic purposes, the regenerative potentials of mesenchymal stem cells (MSCs) decline and rapidly enter pre-mature senescence probably involving oxidative stress. To develop strategies to prevent or slow down the decline of regenerative potentials in MSC culture, it is important to first address damages caused by oxidative stress-induced premature senescence (OSIPS). However, most existing OSIPS study models involve either long-term culture to achieve growth arrest or immediate growth arrest post oxidative agent treatment and are unsuitable for post-induction studies. METHODS: In this work, we aimed to establish an OSIPS model of MSCs derived from Wharton's Jelly by hydrogen peroxide (H2O2) treatment. RESULTS: The optimal H2O2 concentration was determined to be 200 µM to achieve OSIPS when MSC reached growth arrest in 3 to 4 passages post-H2O2 treatment. H2O2-treated cells became heterogeneous in morphology, and were irregularly enlarged and flattened with granular cytoplasm. The cells were stained positive for SA-ß-galactosidase, a senescence marker, and were shown to express elevated levels of other well-characterized senescence molecular markers, including p53, p21, p16 and lysosomal ß-galactosidase (GLB1) in real-time RT-PCR analysis. The OSIPS-like features were confirmed with three independent WJ-MSC lines. CONCLUSION: The establishment of an OSIPS model of WJ-MSC is a first step for subsequent investigation on molecular mechanisms of senescence and for screening potential anti-oxidative agents to delay or revert stressed-induced senescence.