IADR Abstract Archives
The Role of Focal Adhesion Kinase in Mouse Embryonic Stem Cell Differentiation
Objectives: Embryonic stem cell (ESC) differentiation is vital for proper metazoan development. Understanding how the process is controlled informs regenerative medicine approaches, including applications for clinical dentistry. A first step in ESC differentiation involves transition from naïve ESC to primed epiblasts. While the transcriptional regulation of early ESC differentiation is well characterized, the role of cell-substrate adhesion remains largely unknown. We tested the importance of cell-substrate adhesion in ESC differentiation by focusing on focal adhesion kinase (FAK), an integrin-linked regulator of adhesion.
Methods: To determine whether FAK is necessary for clonal mouse ESC differentiation, we inhibited FAK using siRNA knockdown to decrease total FAK abundance and a pharmacological inhibitor PND-1186 to block FAK activity. To determine effects of decreased FAK abundance and activity on differentiation we used a dual reporter (DR) ESC line that expresses mCherry when in the naive state and GFP when in the primed state. We then used Fluorescence Activated Cell Sorting (FACS) to quantify the number of cells in naïve (red), primed (green) and intermediate (double-positive cells being yellow) states after 3 days of differentiation.
Results: I confirmed FAK siRNA knockdown and decreased FAK activity with PND-1186 by immunoblotting for total FAK and active FAK-pY397, respectively. We also confirmed specificity of FAK inhibition by showing no effect on abundance of the closely related kinase PYK2. FACS analysis with siRNA-treated cells showed decreased ESC differentiation, with 29% of cells remaining naïve (red) compared with 15% of control cells. PND-1186 showed dose-dependent inhibition of differentiation, with maximal inhibition at 1 μM resulting in 82% of the cells remaining in the naïve state.
Conclusions: Our results suggest that FAK activity is necessary for efficient mouse ESC. Future directions include 1) confirming a role for cell-substrate adhesion by scoring for adhesions during ESC differentiation in the absence and presence of inhibiting FAK and targeting another cell adhesion molecule to inhibit, such as paxillin, and 2) testing cell-substrate adhesion molecules in therapeutic approaches for regenerative medicine.
Methods: To determine whether FAK is necessary for clonal mouse ESC differentiation, we inhibited FAK using siRNA knockdown to decrease total FAK abundance and a pharmacological inhibitor PND-1186 to block FAK activity. To determine effects of decreased FAK abundance and activity on differentiation we used a dual reporter (DR) ESC line that expresses mCherry when in the naive state and GFP when in the primed state. We then used Fluorescence Activated Cell Sorting (FACS) to quantify the number of cells in naïve (red), primed (green) and intermediate (double-positive cells being yellow) states after 3 days of differentiation.
Results: I confirmed FAK siRNA knockdown and decreased FAK activity with PND-1186 by immunoblotting for total FAK and active FAK-pY397, respectively. We also confirmed specificity of FAK inhibition by showing no effect on abundance of the closely related kinase PYK2. FACS analysis with siRNA-treated cells showed decreased ESC differentiation, with 29% of cells remaining naïve (red) compared with 15% of control cells. PND-1186 showed dose-dependent inhibition of differentiation, with maximal inhibition at 1 μM resulting in 82% of the cells remaining in the naïve state.
Conclusions: Our results suggest that FAK activity is necessary for efficient mouse ESC. Future directions include 1) confirming a role for cell-substrate adhesion by scoring for adhesions during ESC differentiation in the absence and presence of inhibiting FAK and targeting another cell adhesion molecule to inhibit, such as paxillin, and 2) testing cell-substrate adhesion molecules in therapeutic approaches for regenerative medicine.