The purpose of this work is to analyse the role of NOTCH receptors on the osteoblastic differentiation of mesenchymal C3H10T1/2 cells. To reach this objective cell populations stably overexpressing each of the four NOTCH receptors were generated by transfection with a plasmid. Expression levels of Notch1-4 genes and its
target genes, Hes1 and Hey1, were assessed by RT-qPCR. The level of each NOTCH receptor in its overexpressing population was determined by Western blot. NOTCH activity was measured by luciferase assays. The osteoblastic differentiation capacity of each population was evaluated by the alkaline phosphatase staining method of induced cell cultures and the measurement of the expression levels of osteogenic differentiation markers in RNA samples obtained from differentiation assays.
The overexpression of a single NOTCH receptor produces an increase in the global NOTCH activity and modifies the expression of the other Notch and their target genes, Hes1 and Hey1. The generated transfectants showed different levels of osteogenic differentiation in in vitro assays. Alkaline phosphatase staining was more intense in cells overexpressing NOTCH3 or NOTCH2 and less intense in cells overexpressing NOTCH1 or NOTCH4. Populations overexpressing NOTCH1, NOTCH2 or NOTCH3 exhibit an increase in mRNA expression of markers compared with the control. Cells overexpressing NOTCH4 only showed a significative increase in Alpl marker expression.
Results indicate that the expression of Notch, Hes1 and Hey1 is interrelated and that the overexpression of the NOTCH1-3 receptors seems to have an inducing effect on the osteoblastic differentiation of C3H10T1/2 cells, while NOTCH4 overexpressing cells does not seem to modify the osteoblastic process.
I pursued my bachelor’s degree in biotechnology in the Technical University of Madrid (2016/2020). During that period, I worked in plant molecular biology in the Centre of Plants’ Biotechnology and Genomics (CBGP). During 2020/2021 academic course I did my Maestry in Experimental Biomedicine. Since September 2020 I have been researching in cell differentiation in the Biochemistry Lab of the Medicine Faculty (UCLM).