Banca de DEFESA: GIRLAINE CAFE SANTOS

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : GIRLAINE CAFE SANTOS
DATA : 03/08/2020
HORA: 14:00
LOCAL: Webconferência
TÍTULO:

Generation and characterization of a mesenchymal stem cell line genetically modified to overexpress Leukemia Inhibitory Factor (LIF) and investigation of its pro-angiogenic potential. 

PALAVRAS-CHAVES:

Mesenchymal stem/stromal cells; genetic modification; LIF; proangiogenic factors; angiogenesis

PÁGINAS: 64
GRANDE ÁREA: Ciências Biológicas
ÁREA: Biologia Geral
RESUMO:

ABSTRACT

INTRODUCTION: Angiogenesis is the process of blood vessel formation in adult tissues and its understanding can help in the development of therapies for ischemic diseases. Mesenchymal stem cells (MSCs) are cells capable of self-renewal, differentiation and secretion of molecules that exert multiple biological effects, such as tissue regeneration, reduction of inflammation and neovascularization, characterizing an important tool for regenerative medicine. The therapeutic potential of MSCs can be increased by genetic modification for overexpression of cytokines and growth factors. The hypothesis of this study considered that overexpression of the Leukemia Inhibitory Factor (LIF) in mouse MSCs increases its pro-angiogenic potential. OBJECTIVE: The present study aimed to generate a cell line of mesenchymal stem cells genetically modified to overexpress LIF (MSC_LIF) and to evaluate its angiogenic potential in vitro and in vivo models. METHODS: Mouse bone marrow-derived MSCs were transduced using a second generation lentiviral system to express human LIF. LIF expression was confirmed by RT-qPCR and ELISA. The characterization of generated cells was carried out by the trilineage differentiation assay and the immunophenotype was evaluated by flow cytometry. The immunosuppressive activity of MSC_LIF was confirmed using the lymphoproliferation assay. The analysis of gene expression of the main angiogenesis markers and perivascular cells was performed by RT-qPCR and immunofluorescence. To evaluate the effects of MSC_LIF on endothelial cells obtained from the umbilical vein (HUVECs), the wound healing test was performed. The ability of MSC_LIF to form microvessel sprouting in vitro was investigated in aortic ring cultures. Finally, an experiment was carried out to demonstrate the formation of new blood vessels in vivo with Matrigel's angioreactor. RESULTS: After transduction, MSC_LIF increased LIF expression and secretion, indicating the success of the genetic modification. Flow cytometry analysis and trilineage differentiation assay showed that MSC_LIF maintained the immunophenotype and multipotency characteristic of MSCs. The gene expression analysis demonstrated positive regulation of genes that encode strategic factors in the neovascularization process, such as angiogenin, IL-8, Mcp-1 and Vegf, and for the perivascular cellular markers ɑSma, Col4a1, Sm22 and Ng2. In the wound healing assay, the conditioned medium of MSC_LIF promoted a significant increase in cell migration compared to the conditioned medium of MSC control. The aortic ring culture sprouting assay demonstrated that MSC_LIF favored the appearance and elongation of microvessels in the tissue. The angioreactor trial showed that MSC_LIF was more potent in promoting tissue vascularization in vivo than control MSC. CONCLUSION: In conclusion, LIF overexpression is a promising strategy to increase the proangiogenic potential of MSCs and sets precedents for future investigations of their applications for the treatment of ischemic diseases and tissue repair

MEMBROS DA BANCA:
Presidente - 012.993.637-50 - MILENA BOTELHO PEREIRA SOARES - UFRJ
Externo à Instituição - SHEILLA ANDRADE DE OLIVEIRA - Fiocruz - PE
Interno - 1205225 - WASHINGTON LUIS CONRADO DOS SANTOS