|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2012) 36, 669675 (Printed in Great Britain)
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, and ‡Iranian Blood Transfusion Organization Research Center, Tehran, Iran
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.
Key words: immunocytochemistry, insulin-like growth factor-I, mechanical loading, myogenic differentiation, myogenic regulatory factor, stem cell
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
1These authors contributed equally to this article
2To whom correspondence should be addressed (email email@example.com).
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 Schöck, 2009). Cells adapt to biomechanical forces through changes in morphology, gene expression 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 Schöck, 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.
2.1. 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 had been obtained from the Iranian Blood Transfusion Organization. Bone marrow mononuclear cells separated using the Ficoll protocol were cultured in dishes incubated at 37°C in air plus 5% CO2. Culture medium was replaced after 48 h to remove haemopoietic cells, and subsequently every 3 days. Colonies of MSCs appeared 6–8 days after initial plating. Mononuclear cells were cultured in DMEM-LG (Dulbecco's modified Eagle's medium-LG) (Sigma) containing 10% FBS (Sigma) and 100 I.U. (international units)/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) (Ghazanfari et al., 2009; Gnecchi and Meloy, 2009).
2.2. Flow cytometric analysis
Bone marrow-derived cell expression of specific MSC surface markers, such as CD166, CD105, CD44 and CD90, was followed, as also the haemopoietic-specific markers, including CD45, CD34 and CD14, by flow cytometry protocol using the appropriate antibodies (Shahdadfar et al., 2005; Kolf et al., 2007; Vieira et al., 2008).
2.3. Mesodermal lineage differentiation
The ability of hMSCs to differentiate along adipogenic and osteogenic lineages was examined in passage 4 cells isolated from bone marrow. For adipogenic differentiation, confluent cultures were incubated in DMEM-LG containing 10% FBS, 0.5 μM 1-ethyl-3 isobutylxanthine (Sigma), 1 μM dexamethasone (Sigma), 10 μg/ml insulin (Sigma) and 100 μM indomethacin (Sigma), with the medium being replaced every 3 days. After 3 weeks, differentiated cells were fixed with 4% formalin (Sigma), washed in 50% propan-2-ol (Sigma), and subsequently incubated with Oil-Red O for 10 min (Sigma) to visualize lipid droplets. Cells were washed with propan-2-ol and the nuclei stained with haematoxylin and eosin (Sigma) (Shahdadfar et al., 2005; Vieira et al., 2008). For osteogenic differentiation, cells (3000 cells/cm2) were incubated in DMEM/F12 (Sigma) containing 10% FBS, 100 nM dexamethasone, 10 mM 2-glycerophosphate (Sigma), and 0.05 mM l-ascorbic acid-2-phosphate (Sigma), the medium being replaced every 3 days. After 3 weeks, differentiated cells were fixed with 4% formalin for 1 h and rinsed with Ca2+/Mg2+-free PBS (Sigma). To detect the mineralization of the extracellular matrix, cells were stained with 40 mM Alizarin Red S (Sigma), pH 4.2, for 5 min (Shahdadfar et al., 2005; Vieira et al., 2008).
2.4. Cell culture on type I collagen-coated silicon membrane
A medical grade silicon membrane was coated with 0.5 μg/ml collagen type I (Sigma) in 0.2% acetic acid. The cell suspension was transferred to this membrane and incubated for 12 h for l attachment (Ghazanfari et al., 2009).
2.5. Mechanical loading
A uniaxial cyclic strain device, designed in National Cell Bank of Iran (Haghighipour et al., 2007), was used to expose hMSCs cultured on type I collagen-coated silicon membrane 10% strain for 24 h at a frequency of 1 Hz.
2.6. Chemical differentiation of hMSCs into skeletal muscle cells
To induce chemical differentiation into the skeletal muscle lineage, hMSCs were incubated in differentiation medium (DMEM+5% FBS+9 ng/ml IGF-I) for 4 days. RT–PCR (reverse transcription–PCR) and ICC methods were used to assess myogenic differentiation (Sachek et al., 2004).
2.7. Staining of actin filaments
Actin staining was used to study the cellular cytoskeletal structure before and after loading. Briefly, cells were washed twice with PBS, and fixed in PFA (paraformaldehyde) for 10 min. They were rinsed several times with PBS and permeabilized with 0.1% Triton X-100 (Sigma) for 10 min. After washing cells with PBS, they were incubated with 4 μg/ml Phalloidin (Sigma) in PBS for 45 min at room temperature. The samples were examined by fluorescence microscopy at λex and λem of 495 and 513 nm respectively, actin filaments being visible in green (Ghazanfari et al., 2009).
After detaching cells from the silicon membrane using a lysis solution, total RNA of different test groups were isolated using innuPREP RNA Mini Kit (AJ ROBOSCREEN). RT–PCR involved one step with a RT–PCR kit (Qiagen). Thermal cycler primers (Rasta daroo) listed in Table 1 served as an international standard for RNA integrity and equal gel loading. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as internal control. B bands were quantified by Image Pro Plus program.
Table 1 Primers used for RT–PCR analysis
Immunocytochemistry protocols were used to follow the differentiation of hMSCs into MSCs. Briefly cells were washed twice with PBS and fixed in 4% PFA for 10 min. They were permeabilized with 0.1% Triton X-100 for 10 min, rinsed thrice in PBS and incubated in 10% goat serum in PBS for 1 h at room temperature. For MyoD staining, samples were incubated overnight at 4°C with monoclonal antibodies against human MyoD1 (Sigma) at 1:100 dilution, followed by rinsing in 1% goat serum in PBS thrice for 10 min. Incubation was continued for 2 h with fluorophore-conjugated secondary antibody (mouse anti-human antibody FITC; Sigma) at 1:100 dilution. Samples were rinsed in 1% goat serum. Preparations were examined by fluorescence microscopy.
2.10. Experimental protocol
The study included three experimental groups (L, F and L+F) and a negative control one. F-group cells were cultured in differentiation medium (DMEM+5% FBS+9 ng/ml IGF-I). L-group cells were subjected to mechanical loading alone. In L+F-group, cells were subjected to mechanical loading (10% strain and 1 Hz) for 24 h before being incubated in differentiation medium for 4 days.
Results are expressed as means±S.D. The resultant data have been compared with those of the control group, with mean values being determined with the t test, with P<0.05 taken as statistically significant.
3.1. Characterization of MSCs
3.1.1. Flow cytometry
Adherent bone marrow cells expressed CD44, CD90 (Thy-1), CD105/SH2/endoglin and CD166. Neither haemopoietic lineage markers such as CD34 nor monocyte-macrophage antigens such as CD45 was expressed. The results indicate that the cell had the MSC phenotype (Figure 1).
3.1.2. Mesodermal lineage differentiation
The plasticity of MSCs was assessed 3 weeks after mesodermal lineage induction. Adipogenic and osteogenic differentiation was demonstrated by lipid vacuoles and mineralized colonies (Figure 2). After 3 weeks of mineralization, osteogenic colonies stained with Alizarin Red (Figure 2a). After 1 week culture in adipogenic medium, lipid droplets appeared and were clearly evident at day 12. Intracellular lipid vacuoles were seen as red spots after Oil Red O staining (Figure 2b). These results confirm the mesenchymal nature of isolated cells, as well as their multipotent potential.
3.2. Morphological assessment
Morphological characteristics of cells before loading showed random orientation of the cells (Figure 3a). After loading, the arrangement changed, with cells being elongated in the direction of minimal body deformation (Figure 3b) (Yamada et al., 2000; Wang et al., 2001). After loading for 96 h, convergence of cells was observed and their excrescences were connected to one another (Figures 3d and 3f). In the case of non-loaded cells, these morphological changes occurred with a delay (Figures 3c and 3e).
3.3. Actin fibres staining
The arrangement of actin filaments before chemical differentiation was random, but an increase in the number of actin filaments in chemically differentiated cells was evident. After loading, actin filaments tended to rearrange, join together and form thicker fibres (stress fibres) (Figure 4). This is an adaptation of actin fibres to minimize the amount of stress on cell structures (Kurpinski et al., 2006; Ghazanfari et al., 2009).
Myogenic differentiation of bone marrow-derived MSCs in the three experimental groups and the negative control group was measured by RT–PCR. Based on the results and quantitative assessment of mRNA levels of these genes using Image Pro Plus program on the first, third, fourth and seventh days after loading, the fourth day was selected as appropriate day for assessment of myogenic differentiation (Figure 5). mRNA levels of MyoD and MyoG in IGF-I-treated cells increased significantly on the third day; maximum expression of MyoG mRNA occurred in the first week, whereas MyoD mRNA rapidly subsided and disappeared by the end of the first week (Mizuno et al., 2002; Gang et al., 2004). Therefore the fourth day after loading was selected for evaluation of myogenic differentiation of these cells.
Figure 6 shows mRNA levels of Myf5, MyoD and early myogenic regulatory factors with their quantitative assessment using Image Pro Plus program in the experimental and negative control groups. mRNA levels of Myf5 and MyoD, in L+F-group has declined compared with F- and L-groups. None of these genes showed specific mRNA levels in negative control group.
mRNA levels of MyoG, Myf6, later myogenic regulatory factors and their quantitative assessment in experimental groups and negative control group are shown in Figure 7. None of these genes showed specific mRNA levels in negative control group. In comparison with other test groups, L+F-group had the highest mRNA levels of MyoG and Myf6 genes as later myogenic regulatory factors.
Expression of MyoD gene was investigated by the previously described method. In L+F, F- and L-groups, expression of MyoD on the fourth day was noted, whereas ctrl- group did not express this gene (Figure 8).
MSCs can differentiate into different cell types, such as osteoblasts, chondroblasts, fibroblasts, cardiomyocytes and myoblasts. Understanding the differentiation pathways of stem cells and related effective factors can have significant impact on the success of regeneration mechanisms in tissue engineering. Since cells in the body are constantly exposed to mechanical environment, using them in tissue engineering involves the dynamics of the environmental stimuli (Vogel and Sheetz, 2006). Mechanical signals are important in the function, differentiation and proliferation of these cells.
Our RT–PCR results showed that, in comparison with cells treated with growth factor alone, mRNA levels of Myof5 and MyoD taken as early myogenic differentiation genes declined in stem cells subjected to combined mechanical loading and differentiation medium. From the mRNA level of these genes, acceleration in early myogenic differentiation trend is due to transcription being sooner into the later phase of differentiation. Therefore expression of MyoD and Myof5 genes must have begun earlier and declined faster, as confirmed by RT–PCR. The proliferative phase of the myogenic programme was associated with Myf5 and MyoD expression (Cooper et al., 1999; McKay et al., 2008). Similar to muscle development, the two muscle-specific transcription factors were expressed during myogenic differentiation of bone marrow-derived MSCs (Zuk et al., 2002; Gang et al., 2004). Down-regulation of Myf5 and MyoD genes was associated with the induction of differentiation, whereas the up-regulation of MyoG and MRF4 genes directs terminal differentiation (Holterman and Rudnicki, 2005; McKay et al., 2008).
Significant levels of Myf5 and MyoD mRNA observed in mechanical loading group showed that uniaxial cyclic loading can initiate myogenic differentiation without using growth factor. Studies have confirmed that mechanical stimulations are one of the factors affecting stem cell differentiation and thus have effects on repairing muscle damages in tissue engineering (Bayati et al., 2011; Pennisi et al., 2011). The effect of mechanical loading on differentiation depends on mechanical parameters such as stretching amplitude as well as frequency and duration of loading (Ghazanfari et al., 2009; Bayati et al., 2011).
The mRNA levels of Myf5 and MyoD in chemical differentiation confirmed the role of IGF-I in initiation of myogenic differentiation (Sachek et al., 2004). In comparison with cells treated only with growth factor, stem cells subjected to a combination of mechanical loading and differentiation medium had increased mRNA levels of MyoG and Myf6 genes that are involved in late stages of differentiation. This increase is related to the acceleration of myogenic differentiation and the role of mechanical cyclic loading. Consequently, mechanical stimulation has an accelerating effect on all processes involved in myogenic differentiation of stem cells (Bayati et al., 2011; Pennisi et al., 2011).
Significant mRNA levels of these genes in mechanical loading group showed that this stress can also promote myogenic differentiation in the absence of growth factor. Therefore, despite much less mRNA levels of these genes being present compared with the chemical and chemical+mechanical groups, mechanical loading alone, it can still be one of the differentiation factors that accelerates the differentiation process using growth factor.
mRNA level of genes in F-group also indicate the role of growth factor IGF-I in myogenic differentiation (Sachek et al., 2004; Kurpinski et al., 2006a, 2006b). The higher the mRNA levels of myogenic genes in the chemical compared with the mechanical group indicates that, in comparison with mechanical loading alone, the growth factor is more effective in further myogenic differentiation.
From immunocytochemistry studies, surface expression of MyoD and MyoG in the mechanical loading group also showed that it alone can result in the activation of quiescent cells and lead them to myogenic differentiation process.
Examination of morphological and cytoskeletal changes showed that hMSCs can sense mechanical loading and adapt to such conditions through rearrangement and reorientation of their actin fibres, seen in previous studies. However, the amount and direction of these morphological changes depends on the level of stress of the mechanical loading (Ghazanfari et al., 2009; Ahmed et al., 2010; Pennisi et al., 2011). Since cyclic uniaxial strain can accelerate the differentiation process of hMSCs into skeletal muscle cells and engineered cells also adapt to mechanical loading, these differentiated cells could be used in cell therapy to repair damaged skeletal tissues in animals, but this requires further investigation.
Nooshin Haghighipour and Saeide Heidarian performed all of the research. Mohammad Ali Shokrgozar supervised the researched and Naser Amirizadeh contributed by isolating and performing flow cytometry on stem cells.
This work was supported by the
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Received 22 July 2011/29 November 2011; accepted 13 March 2012
Published online 8 June 2012, doi:10.1042/CBI20110400
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 6 mRNA levels of Myf5 and MyoD (early myogenic differentiation genes) on the fourth day after loading in L+F, F, L and ctrl- groups
Figure 7 mRNA levels of MyoG, Myf6 (genes related to late stages of differentiation) on the fourth day after loading in L+F, F, L and ctrl groups