TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

Endothelial to mesenchymal transition is a cellular process in which endothelial cells change their cobblestone morphology into a fibroblast-like phenotype. And the cytokine TGF-beta, transforming growth factor beta, is a main driver of this transition. Analysis of the changes in cell morphology and the expression levels of endothelial and mesenchymal markers allows us to assess the occurrence of endothelial to mesenchymal transition.

Emerging data link the endothelial to mesenchymal transition process to various diseases including cancer, cardiovascular disease, and fibrosis. And the data suggests that the intervention may be of therapeutic benefit. By stimulating the endothelial to mesenchymal transition process, which occurs during embryogenesis and organ development, we may possibly be able to regenerate mesenchymal-derived tissues such as cartilage, bone, and muscle.

To detect the intracellular protein by immunoflourescence staining, cell permeabilization using 0.1%Triton X-100 is necessary to allow the antibodies to pass through the cell membranes. With this video demonstration, researchers will get a good grasp of how to investigate the role of cytokines such as TGF-beta in endothelial to mesenchymal transition. First, seed 1 x 10^5 mirroring pancreatic islet endothelial MS1 cells in DMEM supplemented with 10%FBS and 100 units per milliliter of penicillin and streptomycin onto a 0.1%gelatin-coated culture plate.

After overnight incubation in the cell culture incubator, wash the cells with PBS before treating with two milliliters of 0.25%trypsin, 0.02%EDTA solution for two minutes at 37 degrees Celsius. When the cells have begun to detach, quench the reaction with five milliliters of complete culture medium and transfer the resulting cell suspension to a 15-milliliter tube for centrifugation. Resuspend the pellet in four milliliters of fresh complete medium for counting.

And seed 1 X 10^3 cells per cubic centimeter into a new cell culture container. After overnight culture, treat two wells with five micromolar TGF-beta receptor kinase inhibitor SB-431542 and two wells with DMSO solvent control. And place the cells in cell culture incubator for 30 minutes.

At the end of the incubation, treat two wells with vehicle control and two wells with TGF-beta 2. After three days, use an inverted microscope to examine the cell morphology of each cell treatment group under brightfield imaging. To assess endMT-related marker changes by immunofluorescent staining, detach the cells from an appropriate MS1 cell culture, as demonstrated.

And seed the cells at a 1.9 X 10^3 cells concentration on 0.1%gelatin-coated 12 millimeter round cover glasses placed within individual wells of a 24-well plate. After overnight culture, treat the cells with one nanogram milliliter of TGF-beta 2 for three days. At the end of the incubation, wash the cells with PBS and fix the treated cultures with 300 microliters of 4%formaldehyde per well for 10 minutes at room temperature.

At the end of the fixation, wash the cells three times with PBS before permeabilizing the cells with 300 microliters of 0.1%Triton X-100 and PBS per well for 10 minutes at room temperature. At the end of the incubation, wash the cells three times in PBS and block the cells with 3%BSA in PBS. After 45 minutes at room temperature, label the cells with primary PECAM-1 and SM22-alpha antibodies for 45 minutes at room temperature, followed by three washes in PBS.

After the last wash, incubate the cells with the appropriate secondary antibodies for 45 minutes at room temperature, and wash the cells three times with PBS before mounting the cells with a DAPI-supplemented mounting medium and a cover slip. Then seal the edges of the cover slip with clear nail polish and image the cell marker expression by fluorescence confocal microscopy using the appropriate filters according to standard imaging protocols. After three days of treatment with TGF-beta 2, endothelial MS1 cells lose their cobblestone-like structure and differentiate into spindle-shaped mesenchymal-like cells.

This TGF-beta 2 induced differentiation is suppressed when the cells are exposed to the TGF-beta receptor kinase inhibitor SB-431542. The expression of endothelial protein PECAM-1 is robustly decreased after TGF-beta 2 stimulation while the mesenchymal factor SM-22 alpha is profoundly upregulated. In addition, Snail is markedly upregulated by TGF-beta 2 exposure while Slug expression is not affected.

As observed CRISPR-Cas9 gene editing can be used to facilitate the introduction of two independent Snail single guide RNAs into Cas9-expressing MS1 cells to disrupt Snail expression. The knockout of Snail is sufficient to inhibit the fibroblast-like cell morphology driven by TGF-beta 2 in MS1 cells and to block the TGF-beta 2 mediated PECAM-1 decline and SM-22 alpha enhancement. The assessment of changes in cell morphology and expression of endothelial and mesenchymal markers by immunofluorescence can be further validated by measuring the expression of these markers on quantitative PCR in Western blot analysis.

This technique for detecting endothelial to mesenchymal transition has allowed the researchers to investigate this emerging significance as a physiological process that occurs in multiple diseases.