Comparative study of the immunophenotypic profile, differentiation potential, capacity of production of cytokines and cryopreservation of endometrial stromal cells of cattle during the oestrous cycle

Author(s)

Carolina Nogueira de Moraes Maia

• Document type: Doctoral Thesis
• Institution: Universidade Estadual Paulista (Unesp)
• Defense date: 2017-08-31
• Advisor: Eunice Oba

Abstract

Mesenchymal stem cells from the endometrial tissue (MSCsE) and their conditioned medium (CM) have important therapeutic properties and are alternatives for studies in veterinary medicine. Studies involving MSCsE and its CM are still considered scarce in the bovine species until now. Thus, the aim of this study was to evaluate evaluate the potential of differentiation, immunophenotypic profile, chromosomal stability, clonicity efficiency and cryopreservation response of MSCsE from bovines collected in two phases of the estrous cycle. Additionally, to evaluate the secretoma production of cytokine and prostaglandin E2 (PGE2) of MSCsE stimulated or not with bacterial lipopolysaccharide (LPS). For this, the uterus of healthy females (Phase II n = 6 / Phase III n = 6) was collected for isolation of MSCsE by enzymatic digestion. MSCsE in first passage were evaluated for the number of chromosomes and the second passage was conducted the clonicity assay. The MSCsE in third passage (P3) were differentiated into the adipogenic, chondrogenic and osteogenic lineages and characterized by the immunophenotypic profile by flow cytometry (FC) (vimentin, CD29, CD44, MHC-II, CD34) and immunocytochemistry (vimentin, CD44). Additionally, MSCsE in P3 were cryopreserved using two cryopreservation medium: and evaluated by FC before and after cryopreservation. For evaluation of cytokine, PGE2 production and analysis of the secretome, CM was collected after 2, 6, 12 and 24 hours of challenge (treated group - TG) or not (control group - CG) with LPS. The identified proteins were classified according to the biological processes, molecular function, cellular component and protein class. According to the results, was observed a homogeneous cell population, with a fibroblastoid morphology and adherent to the plastic. In FC analysis, the MSCsE expressed high labeling for CD29, CD44 and vimentin, low labeling for CD34 and absence of expression for the MHC-II marker. Still, they presented chromosomal stability, high efficiency of clonicity and differences (P<0.05) in the production of cytokines and PGE2 after stimulus or not with LPS. Differences were not observed (P>0.05) between the medium or phase after thawing. 397 groups of proteins were identified in GT and 302 in CG. There was a positive enrichment for proteins related to antibacterial response, macrophages activation, hydrolase activity and inhibitory enzymes in TG, and structural activity molecules and intermediate filaments in CG. It is possible to conclude that bovine MSCsE present in the experimental conditions clonicity, multipotentiality, chromosomal stability and satisfactory cryopreservation response, which corroborates for the establishment of cell banks for therapeutic use or new in vitro studies. Additionally, these cells respond to LPS at the concentration used, by the production of cytokines, and this model can be used to evaluate the inflammatory response. (AU)

• FAPESP's process: 14/20447-2 - Comparative study of the immunophenotypic profile, differentiation potential, capacity of production of cytocines and cryopreservation of endometrial stromal cells of cattle during the oestrous cycle
• Grantee: Carolina Nogueira de Moraes Maia
• Support Opportunities: Scholarships in Brazil - Doctorate