Assistant Professor University of New Haven West Haven, Connecticut, United States
Introduction: Stem cell differentiation is impacted by the environmental conditions on the cells, but the specific conditions for differentiation are still unknown. Stem cells differentiate to provide the body with appropriate cell type for growth or regenerative processes. The shear stress encountered by human cells, such as the stresses applied by blood flow within veins may impact differentiation. Specific effects of shear stress on adipogenesis are unknown. Cell properties, such as viability, morphology or quantity differentiated may be impacted. Understanding the impact of shear stress may allow for more effective production of a large quantity of fat cells and may help develop the understanding of the effects of shear stress within the human body. We investigated the impacts of constant shear stress on stem cells and adipogenesis.
Materials and
Methods: Bone derived human mesenchymal stem cells (hMSC) were cultured with mesenchymal stem cell basal medium. The cell medium was changed every three days. Cells were incubated in a humidified incubator with 5% CO2 at 37°C. At 80-90% confluency, cells were passaged into four or six wells in two separate 24 well plates. Each well was prepared with 5*104 cells. These cells were cultured until 70-80% confluency before changing to differentiation medium. One of the two well plates was moved on top of a shaker to imitate shear stress. Images were taken on days 5, 7 and 10. The cells were stained on day 10 with phalloidin and Hoechst, to identify actin structure and cell nuclei. Images were taken with Echo Revolution inverted microscope. Number of differentiated cells were counted from images on days 5, 7 and 10 and the cell morphology was analyzed from day 10 stained images. Changes in cell quantity or morphology were analyzed between cells under the effects of shear stress and control wells.
Results, Conclusions, and Discussions: Resultss: Shear stress impacts the quantity and quality of differentiated cells during adipogenesis. A change in differentiated cells can be seen after 7 days of constants shear stress. Figure 1 includes graphs showing the average cell count from shaker and non-shaker wells after 5, 7 and 10 days.
Figure 1 images are 10x, but the cell counts are taken from 4x. Each data point is the average across several images. The cell morphology was analyzed after 10 days. Cells perimeters, aspect ratios and areas were compared between shaker and non-shaker wells. Figure 2 displays cell images and morphology data comparisons. Five trials have been performed. Cell quantity has been analyzed for all five and morphology has been analyzed for two.
Discussion: Shear stress reduces the quality and quantity of fat cells produced from adipogenesis. Control cells had as aspect ratio closer to one and a higher area, but lower perimeter. This is indicative of the formation of circles, squares or equilateral triangles. Experimental wells displayed a greater aspect ratio and perimeter, but a lesser area. This is indicative of longer shapes, such as ellipses or rectangles. The elongation of cells caused by shear stress may be a cause for the reduction of cell count.
Conclusion: Shear stress causes an elongation of fat cells during adipogenesis. This stress also results in a reduction in cell count. Induction of shear stress may be valuable in producing large quantity of non-adipocytes. Analyzing additional trials and analyzing the effects shear stress on cell viability will be crucial for verifying the validity of these results.
Acknowledgements (Optional): I would like to acknowledge the University of New Haven.
