Banca de DEFESA: STHEFANY DA CONCEIÇÃO PIRES

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : STHEFANY DA CONCEIÇÃO PIRES
DATE: 31/10/2022
TIME: 09:00
LOCAL: Auditório Aluízio Prata
TITLE:

In vitro characterization of the immunomodulatory potential of extracellular vesicles derived from Wharton's jelly stem cells

KEY WORDS:

Therapy. Stem-cell. Mesenchymal stem cell. extracellular vesicles.

PAGES: 66
BIG AREA: Ciências Biológicas
AREA: Microbiologia
SUMMARY:

INTRODUCTION: MSCs have become targets of clinical and preclinical studies due to their anti-inflammatory and immunoregulatory potential. It is known that part of the effects of MSCs are related to their secretome, which includes soluble factors such as cytokines, proteins and the release of extracellular vesicles (EVs). EVs are small cellular components, which have bioactive molecules (small RNAs, proteins, among others) that were inherited from the cells of origin and develop immunomodulatory and trophic actions. The analysis of parameters involving the immunomodulatory profile of MSC-derived EVs (MSC-EV) is essential for important assays for clinical development. OBJECTIVE: To characterize the in vitro immunomodulatory activity of hucMSC-EVs in a lymphoproliferation assay. MATERIAL AND METHODS: Human Wharton's jelly-derived MSCs (hucMSCs) were used for the isolation and purification of hucMSC-EVs. The hucMSCs were characterized according to ISCT by analyzing morphology, immunophenotyping and differentiation assays. By RT-PCR we evaluated the expression of genes related to EVs biogenesis (CD81, CD63) and immunomodulatory potential (IDO1 and TNFAIP6). HucMSC-EVs were isolated in passage 5 and characterized following ISEV recommendations,by transmission electron microscopy (TEM), nanoflow analysis and nanoparticle tracking analysis (NTA). Blood mononuclear cells (PBMC) from healthy donors were stimulated with anti-CD3/ anti-CD28 beads and incubated with different concentrations of hucMSC-EVs (10,25 and 50 μg/ml) for 72 hours at 37 °C with 5% CO2, lymphoproliferation analysis was performed by luminescence ATP assay. Regulatory T cells (Treg-CD4+CD25+FoxP3+) were evaluated by flow cytometry. Cytokines in the supernatant were quantified by ELISA. RESULTS: Cultured hucMSCs showed fibroblastoid morphology, expression > 95% for CD90, CD105, CD44, and CD73, with <0.05% of hematopoietic markers, and the ability to differentiate into adipocytes, osteocytes, and chondroblasts. Deprivation of human platelet lysate was associated with a peak in gene expression of CD81, CD63 and TNFAIP6 after 24h, 48h and 72h. IDO1 expression was not detected. We observed that CM stability remains when stored at -20°C for 7 days. The hucMSC-EVs showed an average diameter <200 nm, showed typical morphology in TEM and phenotypic analysis demonstrated positive markers for EVs (Annexin, CD81, CD63) and for hucMSC origin (CD90). Treatment with hucMSC-EVs inhibited lymphoproliferation in vitro at all concentrations tested. Stimulation of Tregs was slightly increased after treatment with 50 μg/ml MSC-EVs. The production of TNF-a, IFN-y, and IL-10 was inhibited by incubation with MSC-EVs, whereas IL-6 showed no significant changes. CONCLUSION: HucMSC-EVs showed immunoregulatory effects through in-vitro lymphocyte inhibition and moderate Treg cell induction, but additional tests need to be performed to elucidate further effects on large-scale application of hucMSC-EVs.

COMMITTEE MEMBERS:
Presidente - ***.601.955-** - BRUNO SOLANO DE FREITAS SOUZA - UFBA
Externa à Instituição - ELISALVA TEIXEIRA GUIMARÃES - UNEB
Externa ao Programa - 1237842 - VIVIANE ALINE OLIVEIRA SILVA SAITO - null