Differential effects of cyclic uniaxial stretch on human mesenchymal stem cell into skeletal muscle cell Nooshin Haghighipour 1 *, Saeide Heidarian 1 * ,{ , Mohammad Ali Shokrgozar 2 * and Naser Amirizadeh { * National Cell Bank of Iran, Pasteur Institute of Iran, National Cell Bank of Iran, Tehran, Iran { Department of Molecular Cell Biology, Khatam University, Tehran, Iran { Iranian Blood Transfusion Organization Research Center, Tehran, Iran Abstract Both fetal and adult skeletal muscle cells are continually being subjected to biomechanical forces. Biomechanical stimulation during cell growth affects proliferation, differentiation and maturation of skeletal muscle cells. Bone marrow-derived hMSCs [human MSCs (mesenchymal stem cells)] can differentiate into a variety of cell types, including skeletal muscle cells that are potentially a source for muscle regeneration. Our investigations involved a 10% cyclic uniaxial strain at 1 Hz being applied to hMSCs grown on collagen-coated silicon membranes with or without IGF-I (insulin-like growth factor-I) for 24 h. Results obtained from morphological studies confirmed the rearrangement of cells after loading. Comparison of MyoD and MyoG mRNA levels between test groups showed that mechanical loading alone can initiate myogenic differentiation. Furthermore, comparison of Myf5, MyoD, MyoG and Myf6 mRNA levels between test groups showed that a combination of mechanical loading and growth factor results in the highest expression of myogenic genes. These results indicate that cyclic strain may be useful in myogenic differentiation of stem cells, and can accelerate the differentiation of hMSCs into MSCs in the presence of growth factor. Keywords: immunocytochemistry; insulin-like growth factor-I; mechanical loading; myogenic differentiation; myogenic regulatory factor; stem cell 1. Introduction Non-haemopoietic stem cells isolated from bone marrow can self- renew, undergo clonal expansion and differentiate into different phenotypes (Majumdar et al., 2000; Bianco et al., 2001; Hwang et al., 2009). MSCs (mesenchymal stem cell) as multipotent cells capable of differentiating into multiple cell types, such as osteocytes, chondrocytes, adipocytes, hepatocytes, myocytes, neurons and cardiomyocytes, can be used as autologous cell source for cell therapy and tissue engineering (Horwitz, 2003; Park et al., 2004; Gnecchi and Meloy, 2009). All tissues and cells of the body are continuously influenced by chemical and mechanical parameters. Environmental factors can affect the growth, proliferation and differentiation of stem cells (Park et al., 2004; Ju et al., 2007; Jani and Scho ¨ ck, 2009). Cells adapt to biomechanical forces through changes in morphology, gene expre- ssion and phenotype (Kurpinski et al., 2006a, 2006b; Cohen and Chen, 2008). Since skeletal muscles are constantly exposed to mechanical stimulation, these forces play major roles in muscle development and function (Cheema et al., 2005; Tidball, 2005; Jani and Scho ¨ ck, 2009). There are various diseases and damaging agents that affect skeletal muscle tissue. Since muscle is a post-mitotic tissue, cell replacement and an effective local cellular repair system are required for regeneration (Goldspink, 2005). Cell therapy and tissue engineering offer promising treatment. The role of MSCs in regeneration has been studied in different in vivo models (Ferrari et al., 1998; De Bari et al., 2003). The MRF (myogenic regulatory factor) family, including MyoD, Myf5, MyoG, Myf6 and the MEF-2 factors, play crucial roles in the differentiation and specification of skeletal muscle cells (Charvet et al., 2006). Chemical differentiation of stem cells into skeletal muscle cells has been well investigated, but few have looked at the effects of mechanical forces on myogenic differentiation in vitro. In vitro experiments on the effects of applying mechanical loading on cultured stem cells show that expression of certain genes changes during myogenic differentiation (Zhan et al., 2006; Bullard et al., 2007). The first authors also showed that skeletal muscle responded to mechanical stimulation by activating p38 MAPK (mitogen-activated protein kinase), a key signal for myogenesis. Uniaxial cyclic stretch affects the orientation of C2C12 myoblasts and induces further differentiation (Pennisi et al., 2011). We have assumed that biomechanical forces induce myogenic differentiation process in bone marrow-derived hMSCs (human MSCs) and control the expression of MRFs that ultimately lead to myotube formation. Therefore mechanical stimulation has been used to induce myogenic differentiation of hMSCs in the presence and absence of IGF-I. 2. Methods 2.1. hMSCs isolation and culture hMSCs were isolated from bone marrow aspirates of 10–20 ml taken from the iliac crest of patients. Ethical approval for this study 1 These authors contributed equally to this article 2 To whom correspondence should be addressed (email mashokrgozar@pasteur.ac.ir). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; MSC, mesenchymal stem cell; hMSC, human MSC; IGF-I, insulin-like growth factor-I; MRF, myogenic regulatory factor; PFA, paraformaldehyde; RT–PCR, reverse transcription–PCR. Cell Biol. Int. (2012) 36, 669–675 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2012 International Federation for Cell Biology Volume 36 (7) N pages 669–675 N doi:10.1042/CBI20110400 N www.cellbiolint.org 669