Analysis of the agrin expression during osteoblast and adipocyte differentiation of mesenchymal stem cells derived from adipose tissue

• Grant number: 20/14553-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2021
• End date: January 31, 2022
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Márcio Mateus Beloti
• Grantee: Luana da Silva Pereira
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The extracellular matrix protein agrin acts on the regulation of neuromuscular synapses, signaling in immunological synapses and cardiac tissue regeneration. Furthermore, agrin plays a relevant role in skeletal development; however, it has been underexplored in the field of bone biology. Considering agrin as a therapeutic target for bone formation and repair, our research group identified its expression in osteoblasts and detected a positive correlation between the level of expression of agrin and the stage of osteoblast differentiation. As there are no data on agrin expression in mesenchymal stem cells (MSCs) derived from adipose tissue and considering that the balance between osteoblast and adipocyte differentiation of MCSs is relevant to the bone homeostasis, the aim of this project is to evaluate the gene and protein expression of agrin during the process of osteoblast and adipocyte differentiation of MSCs derived from adipose tissue of mice. MSCs will be harvested from the inguinal region of mice and cultured under osteogenic and adipogenic conditions. The gene expression of agrin, its receptors, and osteoblast and adipocyte markers will be evaluated by real-time PCR on days 3, 5, 7, 10, and 14. Agrin protein expression will be evaluated by immunofluorescence assay on days 3, 5, and 7. Additionally, osteoblast differentiation will be evaluated by in situ alkaline phosphatase activity using Fast red assay, on days 10 and 14, and the extracellular matrix mineralization using alizarin red stain, on day 21. Adipocyte differentiation will be evaluated by detection of lipid accumulation using Oil Red O staining, on day 21. The numerical data will be submitted to the appropriate statistical test. The results of this study may generate strategies for future investigations on the regulation of agrin expression in the development of new therapies for bone regeneration.