Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation

Abstract

Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC) differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

Figure 1. Experimental system.

(A) The system consisted of an inlet reservoir (1000-μL pipette tip), a microfluidic channel where cells were cultured and exposed to shear flow, a coiled tube, and an osmotic pump dipped into a Petri dish containing 0.1 M PEG solution. (B) The coiled tube had a large capacity that served as an outlet reservoir providing clean water to the osmotic pump. An air bubble moved through the tube to prevent the mixing of medium and water. (C) The osmotic flow was generated at the membrane window by the concentration difference between water and PEG solution. (D) Cells in the main microchannel (200-μm height) were exposed to an extremely slow interstitial level of flow.

Figure 2. Computed shear stress distribution.

(A) The rectangular area (9 mm × 4 mm) of the main microchannel was considered the cell culture zone for experimental observation. Due to the tapered geometry near the inlet and outlet ports, higher shear stresses developed in these areas, whereas almost uniform shear stress developed in the cell culture zone. (B) Quantitative analysis showed that over 70% of the cell culture zone had a good uniformity of shear stress (approximately 7% variation) and over 80% of the area had an acceptable uniformity (approximately 20% variation).

Figure 3. Shear stress stimulates TAZ.

(A) Shear stress increases the nuclear localization of TAZ. MSCs were loaded onto the microfluidic chip, and osmotic pressure-driven shear stress was applied to the chip. Stationary states were used as controls. Media change was assessed every 12 hours for control cells. After 48 h, the cells were fixed and analyzed using immunocytochemical analysis. A TAZ-specific antibody was used to observe the location of TAZ. Scale bars indicate 20 μm size. (B) The cellular distribution of TAZ in (A) was quantitatively analyzed to determine whether it was higher in the nucleus (N > C), higher in the cytoplasm (N < C), or evenly distributed between the nucleus and cytoplasm (N  =  C). The percentage was scored after observing cells from three independent experiments of (A). * indicate p-value as determined by Student's t-test; * for p < 0.05, ** for p < 0.01. (C) Increased transcriptional activity of TAZ after shear stress. CTGF and Cyr61 mRNA were analyzed by qRT-PCR. Their relative expression was calculated after normalization to the GAPDH level. ** for p < 0.01, t-test.

Figure 4. Shear stress stimulates osteoblast differentiation.

(A) Increased alkaline phosphatase activity by shear stress. MSCs were loaded onto the chips and differentiated into osteoblasts in the presence of osteogenic differentiation media for 4 days. Alkaline phosphatase activity in the differentiated cells was stained according to the experimental method. Control cells were incubated in the osteogenic differentiation media without low shear stress. (B) Increased osteogenic marker gene expression by shear stress. Total RNA in (A) was harvested at 2 days after differentiation and analyzed by reverse transcription and qRT-PCR. The relative expression levels of TAZ, Runx2, DLX5, and Msx2 were determined after normalization to the GAPDH level. ** for p < 0.01, t-test.

Figure 5. Cells with decreased TAZ expression show defects in shear stress-induced osteogenic differentiation.

(A) Depletion of TAZ in MSCs was assessed by infecting cells with a retrovirus carrying TAZ small hairpin RNA. The expression of TAZ was analyzed by immunoblot analysis. Con and Ti indicate vector control cells and TAZ-depleted cells, respectively. (B) Decreased alkaline phosphatase activity in TAZ-depleted cells after shear stress. The Con and Ti cells were differentiated into osteoblasts in the presence of osteogenic differentiation media for 4 days with microfluidic shear stress. Alkaline phosphatase activity in the differentiated cells was stained according to the experimental method. (C) Decreased osteogenic marker gene expression in TAZ-depleted cells after shear stress. Total RNA in (B) was harvested at 2 days after differentiation and analyzed by reverse transcription and qRT-PCR. The relative expression levels of TAZ, Runx2, DLX5, and Msx2 were determined after normalization to the GAPDH level. ** for p < 0.01, t-test.

Figure 6. Rho GTPase is involved in TAZ localization after shear stress.

(A) MSCs were loaded onto microfluidic chips, and shear stress was applied to the chips in the presence of 50 μM Y27632 (Rock inhibitor) and 100 μM Rac1 inhibitor. After 24 h, the cells were fixed and analyzed by immunocytochemistry. A TAZ-specific antibody was used to observe the location of TAZ. Scale bars indicate 20 μm size. (B) Decreased alkaline phosphatase activity in Y27632-treated cells after shear stress. Control and Y27632-treated cells were differentiated into osteoblasts in the presence of osteogenic differentiation media for 4 days with microfluidic shear stress. Alkaline phosphatase activity in the differentiated cells was determined according to the experimental method. (C) Decreased osteogenic marker gene expression in Y27632-treated cells after shear stress. Total RNA in (B) was harvested at 2 days after differentiation and analyzed by reverse transcription and qRT-PCR. The relative expression levels of TAZ, Runx2, DLX5, and Msx2 were determined after normalization to the GAPDH level. * for p < 0.05, ** for p < 0.01, t-test.