Effects of substrate stiffness on cardiomyocyte differentiation of human mesenchymal stem cells

• Main Authors: Yun-Chu Lin, 林昀築
• Other Authors: Chun-Min Lo
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/86794288570383931924

碩士 === 國立陽明大學 === 生物醫學工程學系 === 103 === Extracellular matrix (ECM) provide physical and biochemical signals to the surrounding cells and tissues. In particular, the stiffness of ECM is known to have profound effects on cell behaviors including morphology, proliferation, migration, and differentiation. Human mesenchymal stem cells (hMSCs) have become one of the most intensively studied cell types since they can differentiate into a variety of mesodermal cell lineages such as adipocytes, chondrocytes, myocytes, and osteoblasts. However, the mechanism of how hMSCs sense and respond to substrate stiffness remains unclear. The aim of this study is to test the hypothesis that substrate stiffness affects the proliferation and cardiac differentiation of hMSCs. To address this possibility we cultured hMSCs on flexible polyacrylamide sheets coated with type-I collagen. The stiffness of polyacrylamide gel, ranging from 1 to 20 kPa, was controlled by varying the final concentrations of acrylamide and bisacrylamide. Proliferation, differentiation, and traction forces of hMSCs were determined by alarmaBlue® assay, quantitative PCR, and traction force microscopy respectively. Our results showed that hMSCs on stiffer polyacrylamide gels demonstrated larger spreading area and higher proliferation rate than those on softer gels. While the overall pattern of traction forces was similar for hMSCs on soft and hard substrates, cells on stiff gels generated stronger traction forces than those on soft gels. In addition, the effect of substrate stiffness on the differentiation of hMSCs into cardiomyocytes was observed by examining the expression of the specific proteins of cardiomyocytes such as cTnT and MLC2a. During the differentiation process, the cardiac gene expression increased and the values of 7.4 kPa group were considerably higher than the values of other groups as well as coverslip group. Taken together, it is indicated that hMSCs are sensitive to mechanical modulation and proper substrate stiffness can significantly alter their tendency to become cardiomyocytes.