Effect of the deletion of agrin in Runx2-expressing cells on osteoblast differentiation of mesenchymal stem cells derived from adipose tissue

Abstract

Osteoblasts are key cells for bone formation and repair and the process of osteoblast differentiation is regulated by the runt-related transcription factor 2 (Runx2), as well as several other proteins that act on cell signaling pathways. Results of our research group showed that the extracellular matrix protein agrin is expressed by mesenchymal stem cells (MSCs) and osteoblasts and that this protein favors the osteoblast differentiation of bone marrow-derived MSCs. However, up to now, there is no data in the literature about the participation of agrin in the osteoblast differentiation of MSCs derived from adipose tissue. Therefore, the aim of this study is to investigate the participation of agrin synthesized by cells that express Runx2 in the osteoblast differentiation of MSCs derived from adipose tissue, using transgenic mice. MSCs will be obtained from the inguinal adipose tissue of Runx2-CRE; Agrinfl/fl (Agrin) and Agrinfl/fl (Control) mice and will be cultured under osteogenic conditions for up to 21 days to evaluate: (1) cell proliferation by counting the number of cells and (2) the gene expression of agrin and its receptors, and of bone markers by real-time PCR at 3, 7 and 10 days, (3) alkaline phosphatase by Fast red at 10 days and (4) the formation of mineralized extracellular matrix using Alizarin red stain at 21 days. Data will be submitted to the adherence test to the normal curve and homogeneity of variances to determine the appropriate statistical analysis. The significance level will be 5% (p d 0.05). Considering that agrin is underexplored in the context of bone biology, the results generated by this project will be relevant, as agrin may be a target protein for the development of new therapeutic strategies that favor bone formation and regeneration, regulating events related to osteoblast differentiation.