Stem Cell Cytoskeletal Responses to Pulsatile Flow in Heart Valve Tissue Engineering Studies

Figures

• 

  Figure 1. Shear stress profiles of the three flow conditions utilized in the HBMSC culture experiments: Pulsatile shear stress (PSS), Steady state (SS), and no flow (control group).

• 

  Figure 2. Procedure for quantification of actin filament changes in HBMSCs using multiple metrics: First, the number and length of actin filaments/cell was determined (ImageJ). Actin filament angles were subsequently evaluated with respect to a horizontal axis as shown (ImageJ; 0°). The angle measurements were spatially-averaged for each cell for the first 5 h of each day, and 48 h time points were recorded. The actin filament density was computed as a function of the cell area (ImageJ; number of filaments/cm2). Finally, the nuclear eccentricity (circularity) was quantified, where a circle has an eccentricity of 1, whereas a more elongated shape would be associated with a lower number (minimum is 0).

• 

  Figure 3. (A) Transfected HBMSCs with actin GFP were assessed for 2 days after electroporation using flow cytometry; 77.43% of the cells were shown to express high GFP intensity as seen in the first quadrant. (B) Transfected HBMSCs with actin GFP assessed for 2 days after transfection were incubated in propidium iodide (PI) solution to evaluate cellular viability; 50.07% of cells were efficiently transfected and viable after electroporation, as seen in the first quadrant.

• 

  Figure 4. (A) The effect of HBMSCs when exposed to PSS, SS, and No Flow (static) groups on the average number of filaments/cell (n = 8 wells/group). There was a significantly higher number of actin filaments (p < 0.05) found in HBMSCs exposed to PSS compared to SS and no flow groups; however, no significant difference (p > 0.05) was observed between SS and no flow groups. (B) The PSS, SS, and No Flow (static) groups on average HBMSCs average length of filaments/cell (n = 8 wells/group). There was a significant decrease (p < 0.05) in comparing the average number of filaments in cells exposed to PSS compared to SS and no flow groups. (C) Average nuclear eccentricity/cell (circularity) in HBMSCs while being exposed to PSS, SS, and No Flow (static) groups (n = 8 wells/group). Noticeable changes in nuclear shape (unpublished observations) in the first 5 h of exposure were seen. However, at 48 h, cells in all groups seemed to have reverted to their original configuration (at 0 h), and thus no significant differences (p > 0.05) were observed among the three groups. (D) Average angle of filaments/cell (n = 8 wells/group) for the exposure of PSS, SS, and No Flow (static) groups. Actin filament…

• 

  Figure 5. Gene expression follows 48 h of HBMSCs exposure to PSS, SS, and no flow groups. Error bars are displayed as ± SEM; (n = 4). Of particular importance is the significantly greater (p < 0.05) expression of klf2a found in the PSS-treated cells compared to SS and no flow groups; klf2a, a critical gene regulated by PSS, is required for normal valve development and whose absence results in valvular defects. *indicates a significant difference (p < 0.05) between the flow groups (SS and/or PSS) and the no flow controls. +indicates the PSS flow group is significantly different (p < 0.05) compared to the SS flow group.