Role of ctcfl genes in the differentiation of mouse embryonic stem cells into endothelial cells

Objective: CTCFL is a protein called as BORIS (Brother of the Regulator of Imprinted Sites), CCCTC-binding factor-like protein or CTCF-T in literature. Overexpression of Ctcfl has been shown to be associated with vascular disorders, multiple organ pathologies and neonatal death during embryogenesis. Thus, in the present work, we aimed to investigate whether or not overexpression of Ctcfl induced by doxycycline would affect in vitro endothelial differentiation in Ctcfl transgenic mouse embryonic stem cells (ESCs). Method: Ctcfl transgenic and non-transgenic mouse embryonic stem cells were used. Ctcfl gene induction was performed by adding 2 μg/ml doxycycline to the culture media. Ectopic gene expression was confirmed by qRT-PCR at mRNA and by Western Blot method at protein level after culture. The transgenic group with ectopic Ctcfl gene expression and control groups with no transgene and no gene induction were included in the study. Each group was also divided into two subgroups that cells differentiated into endothelial cells or not. Phase-contrast microscopic evaluation was performed between groups for endothelial cell differentiation. Expression of early markers for endothelial differentiation, Flk1 and VE-cadherin, were analyzed by flow cytometry. Besides, expression of Flk1, VE-kaderin, CD31, CD34, VEGF and Flt1 proteins were assessed by immunofluorescence staining. Results: The Ctcfl gene and protein expressions in the doxycycline-induced group were found to be significantly higher than control groups. There were no differences in the expression patterns for Flk1, VE-cadherin, CD31, CD34, VEGF and Flt1 proteins between experimental groups. Flow cytometry results demonstrated that Flk1 and VE-cadherin proteins showed similar mean fluorescence intensity values in each group. Conclusion: According to our results, ectopic Ctcfl expression has no obvious effects on in vitro endothelial cells differentiation by means of Flk1 and VE-cadherin expressions. However, we believe that elucidation of potential molecular mechanisms of this phenomenon with different markers and experimental models are needed. Key words: Ctcfl, vasculogenesis, endothelial differentiation, embryonic stem cell