Synergistic Effect of Laminin and Epidermal Growth Factor on Biological and Morphological Properties of Co-Cultured Myoblasts and Fibroblasts

Abstract

Background: One of the long-standing problems of myoblasts in vitro expansion is slow cell migration and this causes fibroblast population to exceed myoblasts. In this study, we investigated the synergistic effect of laminin and epidermal growth factor (EGF) on co-cultured myoblasts and fibroblasts for cell attachment, proliferation and migration.

Methods: Skeletal human muscle cells were cultured in four different conditions; control, EGF, laminin (Lam) and laminin EGF (Lam + EGF). Using live imaging system, their cellular properties; attachment, migration and growth were exposed to Rho kinase inhibitor, Y-27632, and EGF-receptor (EGF-R) inhibitor, gefitinib were measured.

Results: Myoblast migration and proliferation was enhanced significantly by synergistic stimulation of laminin and EGF (0.61 ± 0.14 µm/min, 0.008 ± 0.001 h^-1) compare to that by EGF alone (0.26 ± 0.13 µm/min, 0.004 ± 0.0009 h^-1). However, no changes in proliferation and migration were observed for fibroblasts among the culture conditions. Inhibition of Rho kinase resulted in the increase of the myoblast migration on the laminin-coated surface with EGF condition (0.64 ± 0.18 µm/min). Compared to the untreated conditions, myoblasts cultured on the laminin-coated surface and EGF demonstrated elongated morphology, and average cell length increase significantly. In contrast, inhibition of EGF-R resulted in the decrease of myoblast migration on the laminin coated surface with EGF supplemented condition (0.43 ± 0.05 µm/min) in comparison to the untreated control (0.53 ± 0.05 µm/min).

Conclusion: Laminin and EGF preferentially enhance the proliferation and migration of myoblasts, and Rho kinase and EGF-R play a role in this synergistic effect. These results will be beneficial for the propagation of skeletal muscle cells for clinical applications.

Keywords: Epidermal growth factor; Gefitinib; Laminin; Myoblast.

Fig. 1

Effect of laminin and EGF on myoblasts and fibroblast at different time interval in control, EGF, Lam and Lam + EGF conditions. A Myoblasts on laminin-coated surface with or without EGF showed small and rounded at the very early min, then an enlarged contact area with spreading initiated in the laminin coated with EGF. B Fibroblasts on the plain and laminin-coated surfaces with or without EGF showed similar attachment morphology and pattern across the conditions. (Scale bar: 50 µm)

Fig. 2

Quantitative analysis of morphoplogical and cell concentration of myoblasts and fibroblasts in laminin and EGF. A Immunocytochemistry of cells after 2 h plating in different conditions with anti-desmin mouse monoclonal antibody (red), phalloidin (green) and nuclei staining, DAPI (blue). Cells expressing desmin were identified as myoblasts; unstained cells were identified as fibroblasts. (Scale bar: 100 µm) B The cell concentration of myoblasts and fibroblasts (n = 3) at 0, 30, 60 and 120 min for attachment analysis. Myoblast attachment was significantly higher on laminin coated with or without EGF at all intervals compared to that on the plain surface with or without EGF. (At least 30 cells were measured per image). *Significant difference (p < 0.05). C Average cell area (µm²) and average cell circularity of myoblasts and fibroblasts in the plain, plain EGF, laminin and laminin EGF conditions at 0, 30, 60 and 120 min (n = 3). Average cell area and cell circularity were measured using NIS Element AR 4.2.0 (Nikon). Myoblasts on the laminin-coated surface with or without EGF had higher average cell area and lower average cell circularity compared to myoblasts on the plain surface with or without EGF. The fibroblasts showed similar trends for the average cell area and average cell circularity in all conditions

Fig. 3

Cellular behaviour analysis of myoblasts and fibroblasts in laminin and EGF. A Confocal images of myoblasts and fibroblasts. Cells were fixed and stained on day 1 and day 3. Cell expressing anti-desmin mouse monoclonal antibody (green) were identified as myoblasts via immunostaining; unstained cells were identified as fibroblasts with nuclei staining only, DAPI (blue). Lam + EGF shows higher number of myoblasts compared to myoblast in control and EGF at Day 3. (Scale bar: 100 µm) B Graphs show the myoblast and fibroblast growth rates and migration rates in the control, EGF, Lam and Lam + EGF conditions (n = 3). *Significant difference (p < 0.05). Myoblast in Lam + EGF has higher growth and migration rate compared to myoblast in control and EGF. Fibroblast demonstrate similar growth and migration rate for all conditions

Fig. 4

Effect of inhibitor on myoblasts morphology and cellular behaviour in laminin and EGF synergy. A Immunocytochemistry of cells with anti-EGF-R (red), Alexa Fluor™ 488 Phalloidin (green) and nuclei staining, DAPI (blue). Cells in Lam + EGF shows higher expression of EGF-R compared to that in Lam, EGF and control. (Scale bar: 10 µm). B Immunocytochemistry of the cells with staining for anti-desmin mouse monoclonal antibody (red), Alexa Fluor™ 488 Phalloidin (green) and DAPI (blue) treated with gefinitib and Y-27632. In treated with gefinitib, myoblasts and fibroblasts demonstrate more flattened, spread and bigger cell morphology Whilst, myoblasts treated with Y-27632 in Lam and Lam + EGF showed thin and elongated cell body with the longest tail formation (Scale bar: 100 µm). C Migration rates of myoblasts and fibroblasts treated and untreated with gefitinib and Y-27632 in the four different conditions. No significance difference between myoblast and fibroblast in all conditions treated and untreated with gefitinib. Myoblast migration rate were significantly higher in groups treated with Y-27632 for Lam + EGF and Lam as compared to untreated with Y-27632 in Lam + EGF and Lam (n = 3). *Significant difference (p < 0.05)

Fig. 5

A Quantitative evaluation of myoblast area, length, circularity and width before and after treatment of Y-26732 in plain, plain EGF, laminin and laminin EGF conditions (n = 3) untreated or treated with Y-27632. Myoblasts length in the laminin coated with EGF conditions was increased compared to that before treatment. B Time analysis of 12 h of length and width before and after treatment of Y-27632 myoblasts in plain, plain EGF, laminin and laminin EGF conditions (n = 3) with and without Y-27632. After adding Y-27632, myoblast in laminin-coated surface with and without EGF increase from 0 to 12 h