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Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts
Journal of Bioscience and Bioengineering VOL. 109 No. 3, 310 – 313, 2010 www.elsevier.com/locate/jbiosc
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Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts Shiplu Roy Chowdhury,1 Yuichi Muneyuki,2 Yasunori Takezawa,2 Masahiro Kino-oka,1 Atsuhiro Saito,3 Yoshiki Sawa,3 and Masahito Taya1,2,⁎ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan 1 Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan 2 and Department of Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 3 Received 26 June 2009; accepted 8 September 2009
The transitional behaviors of myoblasts toward differentiation were investigated in the cultures at the low and high seeding densities (respectively, X0 = 1.0 × 103 and 2.0 × 105 cells/cm2). In the culture at the low seeding density, an increase in confluence degree accompanied a decrease in growth potential (Rp), being Rp = 0.85 and 0.11 at t = 48 and 672 h, respectively. Myoblasts seeded at the high density resulted in the immediate cessation of growth with keeping the low range of Rp = 0.02–0.09 throughout the culture. The reduction of Rp led to the generation of three subpopulations of cells in proliferative, quiescent and differentiated states. Close cell contacts in the confluent state of high seeding culture induced cell quiescence to a higher extent with suppressing differentiation. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Tissue engineering; Myoblasts; Confluent state; Quiescence; Differentiation]
Skeletal muscle myoblasts exhibit a remarkable capacity of selfrenewal, which leads to a practical concept of myoblast transplantation for the treatment of degenerative diseases such as muscular dystrophy (1). The myoblast transplantation makes the revolutionary development in recovering damaged myocardial tissue, which lacks self-renewal potential (2). The performance of myoblast transplantation by direct injection of cell suspension into a damaged site of cardiac muscle made significant improvement in its functionality accompanied by the enhancement of left ventricular ejection fraction and muscle contractility (3). However, in the direct injection method, the grafted cells may be easily dispersed, leading to low efficiency of engraftment on host cardiac tissue (4). To overcome this drawback, the employment of prepared myoblast sheet has been proposed as an alternative strategy for grafting (5). For the preparation of myoblast sheet, the cells are incubated in a confluent state on a thermo-responsive culture dish, and the sheet is recovered as a single layer with intact cell-cell junctions by temperature lowering to 20 °C (6). Such a confluent state of myoblasts allows the close contacts among cells, which is known to initiate the process towards myoblast differentiation (7). The process of myoblast differentiation is associated with the active fusion of mononuclear multiplying cells and the resultant formation of multinuclear myotubes that permanently loose the proliferative ability. Recent ⁎ Corresponding author. Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 5608531, Japan. Tel.: +81 0 6 6850 6251; fax: +81 0 6 6850 6254. E-mail address: [email protected] (M. Taya).
studies (8, 9) suggested that the functional efficacy of grafted myoblasts relies on the existing cells with proliferative potential due to their ability to secrete stimulators such as hepatocyte growth factor, vascular endothelial growth factor, pro-angiogenic growth factor and angiogenin. These factors are known to stimulate the angiogenesis in damaged cardiac tissue and enhance the survival of cardiomyocytes. In this context, understanding of populational profiles of myoblasts participating in the proliferation and differentiation in a confluent state is prerequisite for evaluating the quality of prepared cell sheet. In the present study, the cultivations of human skeletal muscle myoblasts were conducted at varied seeding densities, and the transitional cell behaviors were investigated in terms of cell growth potential and gene expressions of cellular state markers. Human skeletal muscle myoblasts (Lot no. 4F1618; Lonza Walkersville, Inc., Walkersville, MD, USA) were maintained through subculturing in a 75-cm2 T-flask (Nunclon Delta Flask; Nulgene Nunc International, Rochester, NY, USA) with 15 ml of Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA), as described elsewhere (10). The cells experiencing four passages were used as seeds, and the low and high seeding densities were set at X0 = 1.0 × 103 and 2.0 × 105 cells/cm2, respectively. The cultures were conducted in 8-square well plates (surface area of 10.5 cm2; Nulgene Nunc International) with a medium depth of 2 mm at 37 °C under a 5% CO2 atmosphere. The medium change was daily performed. The confluent degree (Cd), growth potential (Rp) and total cell concentration based on nucleus number (XT) were estimated with
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VOL. 109, 2010 respect to the adherent cells during the cultures. The Cd was determined using phase-contrast images, as described elsewhere (11). For determining the Rp and XT, the proliferative and total nucleus numbers were evaluated by fluorescent staining. The proliferative nuclei were immunostained for Ki67, a nuclear protein that is produced by the proliferating cells in all phases of active cell cycle and down-regulated in differentiated and quiescent states. For staining, the cells on the culture surface were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), followed by permeabilization with 0.05% Triton X-100 in PBS. After masking nonspecific proteins with Block Ace® (Dainippon Sumitomo Pharma, Osaka), the cells were kept overnight at 4 °C with a mouse monoclonal anti-Ki67 antibody (1:250 dilution; Abcam, Cambridge, UK). The cells were then immunolabeled with goat anti-mouse IgG (1:400 dilution; Alexa Fluor 488, Molecular probes, Eugene, OR, USA) accompanied by nuclear staining using DAPI (1:15000 dilution; Molecular probes). Images were captured at randomly selected five positions from each sample using a fluorescence microscope. The Ki67- and DAPI-positive nuclei were counted to calculated the Rp value, which is defined as a ratio of proliferative (Ki67-positive) nucleus number to the XT value (DAPI-positive nucleus number). According to the protocol in previous work (10), the cells were subjected to the quantitative real-time PCR to analyze the gene expressions of p130, myogenic factor 5 (Myf5), myogenin and skeletal muscle specific myosin heavy chain 2 (MYH2). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as a reference gene. The specific primers for the target genes (see supplementary Table S1) were designed using Primer3 software (http://primer3.sourceforge.net/). To evaluate growth properties after re-seeding, the suspended cells were plated at X0 = 1.0 × 103 cells/cm2. The number of adherent cells on the culture surface was determined at 24 h of incubation, and the efficiency of cell attachment (α) was obtained as a ratio of adherent cell concentration to the seeding density. The incubation lasted until 48 h to determine the growth potential after re-seeding (Rp′) according to the immunostaining procedure as mentioned above.
FIG. 1. Time profiles of myoblast concentration and growth potential durin cultures at low and high seeding densities. The open square and close diamond correspond to the cultures at the low and high seeding densities, X0 = 1.0 × 103 and 2.0 × 105 cells/cm2, respectively. The data were obtained from three independent experiments. The figures in the parentheses represent the values of Cd at the data points.
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The cultures of myoblasts were performed in subconfluent and confluent states at X0 = 1.0 × 103 and 2.0 × 105 cells/cm2, respectively. As shown in Fig. 1, in the low seeding density culture, the XT increased exponentially with time, accompanying the increment in Cd, and at t = 672 h XT = 1.0 × 105 nuclei/cm2 and Cd = 0.97 were achieved. In this culture, the Rp decreased gradually with time. In the high seeding density culture, on the other hand, the XT at t = 48 h was 1.1 × 105 nuclei/cm2 with Cd close to unity, and no significant variations in XT and Cd were observed with elapsed time, meaning the cessation of myoblast growth. In addition, the Rp was at a low value of 0.09 even at t = 48 h, which was one ninth of that in the culture at the low seeding density, and the low level was kept until the end of culture. These results suggest that cell–cell contacts in the culture play a pivotal role in reducing myoblast growth potential. To investigate the cellular states in the myoblast cultures, the mRNA expressions of p130, Myf5, myogenin and MYH2 were analyzed. As shown in Fig. 2A, in the low seeding density culture, the expression of p130, an indicator for suppression of cell cycle as well as entering into a quiescence state (12), was enhanced with time, and at t = 672 h the level was 1.8 times higher than that at t = 48 h. In the culture seeded at the high density, the p130 level was higher throughout the culture compared to that in the low seeding density culture. The expression level of Myf5, which is expressed in actively proliferating myoblasts (13), was almost unchanged until t = 336 h, and down-regulated at t = 672 h in the culture at low seeding density (Fig. 2B). In the high seeding density culture, the down-regulated expression of Myf5 was observed throughout the culture, giving the negligible expression at the end of culture. The expression of myogenin, which indicates myoblast commitment towards differentiation (13), increased with elapsed time in the culture at the low seeding density, being maximized at t = 336 h, which was 36 times that at t = 48 h (Fig. 2C). The expression of MYH2, an indicator for phenotypic differentiation of myoblasts (13), was up-regulated at t = 672 h in the culture at the low seeding density (Fig. 2D), corresponding to the down-regulation of myogenin. In the high seeding density culture, with culture proceeding, the up-regulation of myogenin was also observed with a maximum at t = 168 h, though was still lower than that in the low seeding density culture at t = 336 h. The expression of MYH2 showed a slight increase with elapsed time in the high seeding density culture, although the expression level at t = 672 h was fairly lower than that in the culture at the low seeding density. The down-regulated expressions of myogenin and MYH2 as well as the up-regulated expression of p130 in the high seeding density culture suggest the suppression of myoblast differentiation with entering into a quiescence state. To analyze the efficiency of cell attachment (α) and subsequent growth potential of the adhered cells (Rp′), the suspended cells were re-seeded after harvesting from the cultures indicated in Fig. 1. As seen in Fig. 3, for the cells from the low seeding density culture, the α decreased gradually with elapsed time. For the cells from the high seeding density culture, the moderate decrease in α was observed. On the other hand, the Rp′ of adhered cells after re-seeding was almost constant around Rp′ = 0.8 on an average irrespective of the cells from both the cultures. Cruci et al. (14) reported that focal adhesion disappeared along differentiation of C2C12 myoblasts, with loosing filopodia and stress fibers, and this cytoskeletal variations led to loss in the ability of cells to adhere to a substrate. In the present study, the cells attaching on the surface after re-seeding were considered to preserve the proliferative ability because of excluding the nonadhered cells arising from differentiation. In this context, in the culture at the low seeding density, the decrease in α, in accordance with decreasing Rp as seen in Fig. 1, indicated that the non-proliferative cells progressed towards the process of myoblast differentiation which was confirmed by the increments in myogenin and MYH-2 expressions with elapsed time (Figs. 2C and D). To contrast,
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FIG. 2. mRNA expression levels of p130 (A), Myf5 (B), myogenin (C) and MYH-2 (D) in cells during cultures at low and high seeding densities. The symbols are the same as those shown in the legend of Fig. 1. The data were obtained from three independent experiments.
despite the low Rp level in the high seeding density culture, α was kept at a relatively higher level after t = 336 h than that in the low seeding density culture. Considering together with the data showing the lower expression of myogenin after t = 336 h (Fig. 2C), these results suggested the existence of cells in a growth-arrest state without initiating the process towards differentiation in the high seeding density culture. It is likely that such high confluence causes the contact inhibition to introduce a portion of the cells into a quiescent state. From the above-mentioned considerations, the myoblasts in the cultures were expected to consist of three subpopulations in proliferative, quiescent, and differentiated states,
FIG. 3. Myoblast efficiency of attachment and growth potential at t = 48 h after re-seeding of cells from cultures at low and high seeding densities. The symbols are the same as those shown in the legend of Fig. 1. The data were obtained from three independent experiments. The asterisks mean the statistically significance between the respective data sets, which were analyzed by Student's t-test (p b 0.001).
and the α was suggested to reflect a population balance of the cells in proliferative and quiescent states against the whole existing cells. It was reported that the progression of myoblast differentiation by serum deprivation was associated with the generation of quiescent cells (15). The terminal differentiation of myoblasts is followed by a series of biological events including the cellular commitment to differentiation by up-regulation of myogenin, and the phenotypic differentiation by expression of muscle contractile protein such as MYH2 (13). To contrast, the quiescent myoblasts exhibit the downregulated expression of muscle specific genes in place of the elevated level of p130 (12). The present study described the populational profiles of myoblasts during the contact-dependent induction of differentiation process. In the culture at the low seeding density, the cells in a proliferative state were found to commit to the process toward the differentiation, as supported by the decreases in Rp (Fig. 1) and α (Fig. 3). This phenomenon was associated with the consistent expression of Myf5 and up-regulation of myogenin (Figs. 2B and C). Lasting the culture to confluent state promoted the phenotypic differentiation, causing the up-regulation of MYH2 in accordance with the down-regulations of Myf5 and myogenin (Figs. 2B–D). In our previous study (16), it was reported that myoblasts made the cell-cell contacts during cell division that attributed to the induction of differentiation process along with progressing confluent state of culture. Theil et al. (17) also pointed out that high frequency of cellcell contacts promoted the differentiation process in myoblast culture. However, in the high seeding density culture as examined in the current work, the higher confluence was already created from the beginning of culture, and this condition made the sudden quiescence of cells due to contact inhibition before committing to the process toward differentiation, resulting in the up-regulation of p130 (Fig. 2A). Consequently, although the α decreased at t = 168 h, which corresponded to the up-regulation of myogenin (Fig. 2C), the relatively high level of α was kept (Fig. 3), in spite of low Rp in the culture (Fig. 1). Under this condition, in addition, the down-regulated expression of Myf5 was observed with the suppressive expressions of myogenin and MYH2 (Figs. 2B–D). Previous report demonstrated that the expression of myogenin was recruited by the enhancement of Myf5 expression (18). Furthermore, Andrés et al. (19) reported that myoblast differentiation by serum deprivation in a medium occurred
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with the up-regulation of myogenin prior to entering into a growtharrest state. These considerations suggest that the sudden growth arrest caused the Myf5 depression in the high seeding density culture, leading the cells into a quiescent state with inhibiting process towards myotube formation. The similar phenomenon was observed in the monolayer and multilayer myoblast sheets prepared by keeping the cells in a confluent state (data not shown). In this context, the confluent condition of culture achieved by dense cell seeding is considered to be an effective way to maintain the proliferative potential in the myoblast sheet to be grafted. In conclusion, the contact-dependent inhibition of myoblast proliferation resulted in generating a mixture of subpopulations containing proliferative, quiescent and differentiated cells. The cellular efficiency of attachment after re-seeding was presented as an important measure for the estimation of cell population that preserved the proliferative potency. In the high seeding density culture, during which the cells were in a confluent state, it was found that the confluency brought the cells into quiescence due to the contact inhibition, which can allow them to maintain the proliferative potential. ACKNOWLEDGMENTS We are grateful to Drs. S. Miyagawa and Y. Imanishi, Osaka University, for their invaluable advices. This study was supported in part by the New Energy and Industrial Technology Development Organization of Japan and a Grant-in-Aid for Scientific Research on Priority Areas in the Ministry of Education, Culture, Sports, Science and Technology, Japan. APPENDIX A. SUPPLEMENTARY DATA Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbiosc.2009.09.042. References 1. Gussoni, E., Pavlath, G. K., Lanctot, A. M., Sharma, K. R., Miller, R. G., Steinman, L., and Blau, H. M.: Normal dystrophin transcripts detected in duchenne muscular dystrophy patients after myoblast transplantation, Nature, 365, 435–437 (1992). 2. Menasché, P., Hagège, A. A., Scorsin, M., Pouzet, B., Desnos, M., Duboc, D., Schwartz, K., Vilquin, J., and Marolleau, J.: Myoblast transplantation for heart failure, Lancet, 357, 279–280 (2001).
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3. Haider, H. K., Tan, A. C. K., Aziz, S., Chachques, J. C., and Sim, E. K. W.: Myoblast transplantation for cardiac repair: a clinical perspective, Mol. Ther., 9, 14–23 (2004). 4. Zammaretti, P. and Jaconi, M.: Cardiac tissue engineering: regeneration of the wounded heart, Curr. Opin. Biotechnol., 15, 430–437 (2004). 5. Sawa, Y.: Surgical and autologous transplant regeneration therapy using myoblast sheets for severe heart failure, Kokyu to Junkan, 55, 743–747 (2007) (in Japanese). 6. Shimizu, T., Yamato, M., Kikuchi, A., and Okano, T.: Cell sheet engineering for myocardial tissue reconstruction, Biomaterials, 24, 2309–2316 (2003). 7. Krauss, R. S., Cole, F., Gaio, U., Takaesu, G., Zhang, W., and Kang, J.-S.: Close encounters: regulation of vertebrate skeletal myogenesis by cell–cell contact, J. Cell. Sci., 118, 2355–2362 (2005). 8. Menasché, P.: Skeletal myoblasts and cardiac repair, J. Mol. Cell. Cardiol., 45, 545–553 (2008). 9. Perez-Ilzarbe, M., Agbulut, O., Pelacho, B., Ciorba, C., San Jose-Eneriz, E., Desnos, M., Hagège, A. A., Aranda, P., Andreu, E. J., Menasché, P., and Prósper, F.: Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium, Eur. J. Heart Fail., 10, 1065–1072 (2008). 10. Kino-oka, M., Chowdhury, S. R., Muneyuki, Y., Manabe, M., Saito, A., Sawa, Y., and Taya, M.: Automating the expansion process of human skeletal muscle myoblasts with suppression of myotube formation, Tissue Eng.: Part C, 15 (2009) (DOI: 10.1089/ten.TEC.2008.0429). 11. Kino-oka, M., Agatahama, Y., Hata, N., and Taya, M.: Evaluation of growth potential of human epithelial cells by motion analysis of pairwise rotation under glucose-limited condition, Biochem. Eng. J., 19, 109–117 (2004). 12. Carnac, G., Fajas, L., L ' honor é, A., Sardet, C., Lamb, N. J. C., and Fernandez, A.: The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts, Curr. Biol., 10, 543–546 (2000). 13. Kitzmann, M., Carnac, G., Vandrommme, M., Primig, M., Lamb, N. J. C., and Fernandez, A.: The muscle regulatory factors MyoD and Myf5 undergo distinct cell cycle-specific expression in muscle cells, J. Cell Biol., 142, 1447–1459 (1998). 14. Curci, R., Battistelli, M., Burattini, S., D 'Emilio, A., Ferri, P., Lattanzi, D., Ciuffoli, S., Ambrogini, P., Cuppini, R., and Falcieri, E.: Surface and inner cell behavior along skeletal muscle cell in vitro differentiation, Micron, 39, 843–851 (2008). 15. Yoshida, N., Yoshida, S., Koishi, K., Masuda, K., and Nabeshima, Y.: Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates “reserve cells”, J. Cell. Sci., 111, 769–779 (1998). 16. Chowdhury, S. R., Muneyuki, Y., Takezawa, Y., Kino-oka, M., Saito, A., Sawa, Y., and Taya, M.: Synergic stimulation of laminin and epidermal growth factor facilitates the myoblast growth through promoting migration, J. Biosci. Bioeng., 108, 174–177 (2009). 17. Theil, P. K., Sørensen, I. L., Nissen, P. M., and Oksbjerg, N.: Temporal expression of growth factor genes of primary porcine satellite cells during myogenesis, Anim. Sci. J., 77, 330–337 (2006). 18. Lindon, C., Albagli, O., Pinset, C., and Montarras, Didier: Cell density-dependent induction of endogenous myogenin (myf4) gene expression by Myf5, Dev. Biol., 240, 574–584 (2001). 19. Andrés, V. and Walsh, K.: Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis, J. Cell Biol., 132, 657–666 (1996).
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Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts Shiplu Roy Chowdhury,1 Yuichi Muneyuki,2 Yasunori Takezawa,2 Masahiro Kino-oka,1 Atsuhiro Saito,3 Yoshiki Sawa,3 and Masahito Taya1,2,⁎ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan 1 Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan 2 and Department of Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 3 Received 26 June 2009; accepted 8 September 2009
The transitional behaviors of myoblasts toward differentiation were investigated in the cultures at the low and high seeding densities (respectively, X0 = 1.0 × 103 and 2.0 × 105 cells/cm2). In the culture at the low seeding density, an increase in confluence degree accompanied a decrease in growth potential (Rp), being Rp = 0.85 and 0.11 at t = 48 and 672 h, respectively. Myoblasts seeded at the high density resulted in the immediate cessation of growth with keeping the low range of Rp = 0.02–0.09 throughout the culture. The reduction of Rp led to the generation of three subpopulations of cells in proliferative, quiescent and differentiated states. Close cell contacts in the confluent state of high seeding culture induced cell quiescence to a higher extent with suppressing differentiation. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Tissue engineering; Myoblasts; Confluent state; Quiescence; Differentiation]
Skeletal muscle myoblasts exhibit a remarkable capacity of selfrenewal, which leads to a practical concept of myoblast transplantation for the treatment of degenerative diseases such as muscular dystrophy (1). The myoblast transplantation makes the revolutionary development in recovering damaged myocardial tissue, which lacks self-renewal potential (2). The performance of myoblast transplantation by direct injection of cell suspension into a damaged site of cardiac muscle made significant improvement in its functionality accompanied by the enhancement of left ventricular ejection fraction and muscle contractility (3). However, in the direct injection method, the grafted cells may be easily dispersed, leading to low efficiency of engraftment on host cardiac tissue (4). To overcome this drawback, the employment of prepared myoblast sheet has been proposed as an alternative strategy for grafting (5). For the preparation of myoblast sheet, the cells are incubated in a confluent state on a thermo-responsive culture dish, and the sheet is recovered as a single layer with intact cell-cell junctions by temperature lowering to 20 °C (6). Such a confluent state of myoblasts allows the close contacts among cells, which is known to initiate the process towards myoblast differentiation (7). The process of myoblast differentiation is associated with the active fusion of mononuclear multiplying cells and the resultant formation of multinuclear myotubes that permanently loose the proliferative ability. Recent ⁎ Corresponding author. Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 5608531, Japan. Tel.: +81 0 6 6850 6251; fax: +81 0 6 6850 6254. E-mail address: [email protected] (M. Taya).
studies (8, 9) suggested that the functional efficacy of grafted myoblasts relies on the existing cells with proliferative potential due to their ability to secrete stimulators such as hepatocyte growth factor, vascular endothelial growth factor, pro-angiogenic growth factor and angiogenin. These factors are known to stimulate the angiogenesis in damaged cardiac tissue and enhance the survival of cardiomyocytes. In this context, understanding of populational profiles of myoblasts participating in the proliferation and differentiation in a confluent state is prerequisite for evaluating the quality of prepared cell sheet. In the present study, the cultivations of human skeletal muscle myoblasts were conducted at varied seeding densities, and the transitional cell behaviors were investigated in terms of cell growth potential and gene expressions of cellular state markers. Human skeletal muscle myoblasts (Lot no. 4F1618; Lonza Walkersville, Inc., Walkersville, MD, USA) were maintained through subculturing in a 75-cm2 T-flask (Nunclon Delta Flask; Nulgene Nunc International, Rochester, NY, USA) with 15 ml of Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA), as described elsewhere (10). The cells experiencing four passages were used as seeds, and the low and high seeding densities were set at X0 = 1.0 × 103 and 2.0 × 105 cells/cm2, respectively. The cultures were conducted in 8-square well plates (surface area of 10.5 cm2; Nulgene Nunc International) with a medium depth of 2 mm at 37 °C under a 5% CO2 atmosphere. The medium change was daily performed. The confluent degree (Cd), growth potential (Rp) and total cell concentration based on nucleus number (XT) were estimated with
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.09.042
VOL. 109, 2010 respect to the adherent cells during the cultures. The Cd was determined using phase-contrast images, as described elsewhere (11). For determining the Rp and XT, the proliferative and total nucleus numbers were evaluated by fluorescent staining. The proliferative nuclei were immunostained for Ki67, a nuclear protein that is produced by the proliferating cells in all phases of active cell cycle and down-regulated in differentiated and quiescent states. For staining, the cells on the culture surface were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), followed by permeabilization with 0.05% Triton X-100 in PBS. After masking nonspecific proteins with Block Ace® (Dainippon Sumitomo Pharma, Osaka), the cells were kept overnight at 4 °C with a mouse monoclonal anti-Ki67 antibody (1:250 dilution; Abcam, Cambridge, UK). The cells were then immunolabeled with goat anti-mouse IgG (1:400 dilution; Alexa Fluor 488, Molecular probes, Eugene, OR, USA) accompanied by nuclear staining using DAPI (1:15000 dilution; Molecular probes). Images were captured at randomly selected five positions from each sample using a fluorescence microscope. The Ki67- and DAPI-positive nuclei were counted to calculated the Rp value, which is defined as a ratio of proliferative (Ki67-positive) nucleus number to the XT value (DAPI-positive nucleus number). According to the protocol in previous work (10), the cells were subjected to the quantitative real-time PCR to analyze the gene expressions of p130, myogenic factor 5 (Myf5), myogenin and skeletal muscle specific myosin heavy chain 2 (MYH2). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as a reference gene. The specific primers for the target genes (see supplementary Table S1) were designed using Primer3 software (http://primer3.sourceforge.net/). To evaluate growth properties after re-seeding, the suspended cells were plated at X0 = 1.0 × 103 cells/cm2. The number of adherent cells on the culture surface was determined at 24 h of incubation, and the efficiency of cell attachment (α) was obtained as a ratio of adherent cell concentration to the seeding density. The incubation lasted until 48 h to determine the growth potential after re-seeding (Rp′) according to the immunostaining procedure as mentioned above.
FIG. 1. Time profiles of myoblast concentration and growth potential durin cultures at low and high seeding densities. The open square and close diamond correspond to the cultures at the low and high seeding densities, X0 = 1.0 × 103 and 2.0 × 105 cells/cm2, respectively. The data were obtained from three independent experiments. The figures in the parentheses represent the values of Cd at the data points.
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The cultures of myoblasts were performed in subconfluent and confluent states at X0 = 1.0 × 103 and 2.0 × 105 cells/cm2, respectively. As shown in Fig. 1, in the low seeding density culture, the XT increased exponentially with time, accompanying the increment in Cd, and at t = 672 h XT = 1.0 × 105 nuclei/cm2 and Cd = 0.97 were achieved. In this culture, the Rp decreased gradually with time. In the high seeding density culture, on the other hand, the XT at t = 48 h was 1.1 × 105 nuclei/cm2 with Cd close to unity, and no significant variations in XT and Cd were observed with elapsed time, meaning the cessation of myoblast growth. In addition, the Rp was at a low value of 0.09 even at t = 48 h, which was one ninth of that in the culture at the low seeding density, and the low level was kept until the end of culture. These results suggest that cell–cell contacts in the culture play a pivotal role in reducing myoblast growth potential. To investigate the cellular states in the myoblast cultures, the mRNA expressions of p130, Myf5, myogenin and MYH2 were analyzed. As shown in Fig. 2A, in the low seeding density culture, the expression of p130, an indicator for suppression of cell cycle as well as entering into a quiescence state (12), was enhanced with time, and at t = 672 h the level was 1.8 times higher than that at t = 48 h. In the culture seeded at the high density, the p130 level was higher throughout the culture compared to that in the low seeding density culture. The expression level of Myf5, which is expressed in actively proliferating myoblasts (13), was almost unchanged until t = 336 h, and down-regulated at t = 672 h in the culture at low seeding density (Fig. 2B). In the high seeding density culture, the down-regulated expression of Myf5 was observed throughout the culture, giving the negligible expression at the end of culture. The expression of myogenin, which indicates myoblast commitment towards differentiation (13), increased with elapsed time in the culture at the low seeding density, being maximized at t = 336 h, which was 36 times that at t = 48 h (Fig. 2C). The expression of MYH2, an indicator for phenotypic differentiation of myoblasts (13), was up-regulated at t = 672 h in the culture at the low seeding density (Fig. 2D), corresponding to the down-regulation of myogenin. In the high seeding density culture, with culture proceeding, the up-regulation of myogenin was also observed with a maximum at t = 168 h, though was still lower than that in the low seeding density culture at t = 336 h. The expression of MYH2 showed a slight increase with elapsed time in the high seeding density culture, although the expression level at t = 672 h was fairly lower than that in the culture at the low seeding density. The down-regulated expressions of myogenin and MYH2 as well as the up-regulated expression of p130 in the high seeding density culture suggest the suppression of myoblast differentiation with entering into a quiescence state. To analyze the efficiency of cell attachment (α) and subsequent growth potential of the adhered cells (Rp′), the suspended cells were re-seeded after harvesting from the cultures indicated in Fig. 1. As seen in Fig. 3, for the cells from the low seeding density culture, the α decreased gradually with elapsed time. For the cells from the high seeding density culture, the moderate decrease in α was observed. On the other hand, the Rp′ of adhered cells after re-seeding was almost constant around Rp′ = 0.8 on an average irrespective of the cells from both the cultures. Cruci et al. (14) reported that focal adhesion disappeared along differentiation of C2C12 myoblasts, with loosing filopodia and stress fibers, and this cytoskeletal variations led to loss in the ability of cells to adhere to a substrate. In the present study, the cells attaching on the surface after re-seeding were considered to preserve the proliferative ability because of excluding the nonadhered cells arising from differentiation. In this context, in the culture at the low seeding density, the decrease in α, in accordance with decreasing Rp as seen in Fig. 1, indicated that the non-proliferative cells progressed towards the process of myoblast differentiation which was confirmed by the increments in myogenin and MYH-2 expressions with elapsed time (Figs. 2C and D). To contrast,
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FIG. 2. mRNA expression levels of p130 (A), Myf5 (B), myogenin (C) and MYH-2 (D) in cells during cultures at low and high seeding densities. The symbols are the same as those shown in the legend of Fig. 1. The data were obtained from three independent experiments.
despite the low Rp level in the high seeding density culture, α was kept at a relatively higher level after t = 336 h than that in the low seeding density culture. Considering together with the data showing the lower expression of myogenin after t = 336 h (Fig. 2C), these results suggested the existence of cells in a growth-arrest state without initiating the process towards differentiation in the high seeding density culture. It is likely that such high confluence causes the contact inhibition to introduce a portion of the cells into a quiescent state. From the above-mentioned considerations, the myoblasts in the cultures were expected to consist of three subpopulations in proliferative, quiescent, and differentiated states,
FIG. 3. Myoblast efficiency of attachment and growth potential at t = 48 h after re-seeding of cells from cultures at low and high seeding densities. The symbols are the same as those shown in the legend of Fig. 1. The data were obtained from three independent experiments. The asterisks mean the statistically significance between the respective data sets, which were analyzed by Student's t-test (p b 0.001).
and the α was suggested to reflect a population balance of the cells in proliferative and quiescent states against the whole existing cells. It was reported that the progression of myoblast differentiation by serum deprivation was associated with the generation of quiescent cells (15). The terminal differentiation of myoblasts is followed by a series of biological events including the cellular commitment to differentiation by up-regulation of myogenin, and the phenotypic differentiation by expression of muscle contractile protein such as MYH2 (13). To contrast, the quiescent myoblasts exhibit the downregulated expression of muscle specific genes in place of the elevated level of p130 (12). The present study described the populational profiles of myoblasts during the contact-dependent induction of differentiation process. In the culture at the low seeding density, the cells in a proliferative state were found to commit to the process toward the differentiation, as supported by the decreases in Rp (Fig. 1) and α (Fig. 3). This phenomenon was associated with the consistent expression of Myf5 and up-regulation of myogenin (Figs. 2B and C). Lasting the culture to confluent state promoted the phenotypic differentiation, causing the up-regulation of MYH2 in accordance with the down-regulations of Myf5 and myogenin (Figs. 2B–D). In our previous study (16), it was reported that myoblasts made the cell-cell contacts during cell division that attributed to the induction of differentiation process along with progressing confluent state of culture. Theil et al. (17) also pointed out that high frequency of cellcell contacts promoted the differentiation process in myoblast culture. However, in the high seeding density culture as examined in the current work, the higher confluence was already created from the beginning of culture, and this condition made the sudden quiescence of cells due to contact inhibition before committing to the process toward differentiation, resulting in the up-regulation of p130 (Fig. 2A). Consequently, although the α decreased at t = 168 h, which corresponded to the up-regulation of myogenin (Fig. 2C), the relatively high level of α was kept (Fig. 3), in spite of low Rp in the culture (Fig. 1). Under this condition, in addition, the down-regulated expression of Myf5 was observed with the suppressive expressions of myogenin and MYH2 (Figs. 2B–D). Previous report demonstrated that the expression of myogenin was recruited by the enhancement of Myf5 expression (18). Furthermore, Andrés et al. (19) reported that myoblast differentiation by serum deprivation in a medium occurred
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with the up-regulation of myogenin prior to entering into a growtharrest state. These considerations suggest that the sudden growth arrest caused the Myf5 depression in the high seeding density culture, leading the cells into a quiescent state with inhibiting process towards myotube formation. The similar phenomenon was observed in the monolayer and multilayer myoblast sheets prepared by keeping the cells in a confluent state (data not shown). In this context, the confluent condition of culture achieved by dense cell seeding is considered to be an effective way to maintain the proliferative potential in the myoblast sheet to be grafted. In conclusion, the contact-dependent inhibition of myoblast proliferation resulted in generating a mixture of subpopulations containing proliferative, quiescent and differentiated cells. The cellular efficiency of attachment after re-seeding was presented as an important measure for the estimation of cell population that preserved the proliferative potency. In the high seeding density culture, during which the cells were in a confluent state, it was found that the confluency brought the cells into quiescence due to the contact inhibition, which can allow them to maintain the proliferative potential. ACKNOWLEDGMENTS We are grateful to Drs. S. Miyagawa and Y. Imanishi, Osaka University, for their invaluable advices. This study was supported in part by the New Energy and Industrial Technology Development Organization of Japan and a Grant-in-Aid for Scientific Research on Priority Areas in the Ministry of Education, Culture, Sports, Science and Technology, Japan. APPENDIX A. SUPPLEMENTARY DATA Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbiosc.2009.09.042. References 1. Gussoni, E., Pavlath, G. K., Lanctot, A. M., Sharma, K. R., Miller, R. G., Steinman, L., and Blau, H. M.: Normal dystrophin transcripts detected in duchenne muscular dystrophy patients after myoblast transplantation, Nature, 365, 435–437 (1992). 2. Menasché, P., Hagège, A. A., Scorsin, M., Pouzet, B., Desnos, M., Duboc, D., Schwartz, K., Vilquin, J., and Marolleau, J.: Myoblast transplantation for heart failure, Lancet, 357, 279–280 (2001).
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3. Haider, H. K., Tan, A. C. K., Aziz, S., Chachques, J. C., and Sim, E. K. W.: Myoblast transplantation for cardiac repair: a clinical perspective, Mol. Ther., 9, 14–23 (2004). 4. Zammaretti, P. and Jaconi, M.: Cardiac tissue engineering: regeneration of the wounded heart, Curr. Opin. Biotechnol., 15, 430–437 (2004). 5. Sawa, Y.: Surgical and autologous transplant regeneration therapy using myoblast sheets for severe heart failure, Kokyu to Junkan, 55, 743–747 (2007) (in Japanese). 6. Shimizu, T., Yamato, M., Kikuchi, A., and Okano, T.: Cell sheet engineering for myocardial tissue reconstruction, Biomaterials, 24, 2309–2316 (2003). 7. Krauss, R. S., Cole, F., Gaio, U., Takaesu, G., Zhang, W., and Kang, J.-S.: Close encounters: regulation of vertebrate skeletal myogenesis by cell–cell contact, J. Cell. Sci., 118, 2355–2362 (2005). 8. Menasché, P.: Skeletal myoblasts and cardiac repair, J. Mol. Cell. Cardiol., 45, 545–553 (2008). 9. Perez-Ilzarbe, M., Agbulut, O., Pelacho, B., Ciorba, C., San Jose-Eneriz, E., Desnos, M., Hagège, A. A., Aranda, P., Andreu, E. J., Menasché, P., and Prósper, F.: Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium, Eur. J. Heart Fail., 10, 1065–1072 (2008). 10. Kino-oka, M., Chowdhury, S. R., Muneyuki, Y., Manabe, M., Saito, A., Sawa, Y., and Taya, M.: Automating the expansion process of human skeletal muscle myoblasts with suppression of myotube formation, Tissue Eng.: Part C, 15 (2009) (DOI: 10.1089/ten.TEC.2008.0429). 11. Kino-oka, M., Agatahama, Y., Hata, N., and Taya, M.: Evaluation of growth potential of human epithelial cells by motion analysis of pairwise rotation under glucose-limited condition, Biochem. Eng. J., 19, 109–117 (2004). 12. Carnac, G., Fajas, L., L ' honor é, A., Sardet, C., Lamb, N. J. C., and Fernandez, A.: The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts, Curr. Biol., 10, 543–546 (2000). 13. Kitzmann, M., Carnac, G., Vandrommme, M., Primig, M., Lamb, N. J. C., and Fernandez, A.: The muscle regulatory factors MyoD and Myf5 undergo distinct cell cycle-specific expression in muscle cells, J. Cell Biol., 142, 1447–1459 (1998). 14. Curci, R., Battistelli, M., Burattini, S., D 'Emilio, A., Ferri, P., Lattanzi, D., Ciuffoli, S., Ambrogini, P., Cuppini, R., and Falcieri, E.: Surface and inner cell behavior along skeletal muscle cell in vitro differentiation, Micron, 39, 843–851 (2008). 15. Yoshida, N., Yoshida, S., Koishi, K., Masuda, K., and Nabeshima, Y.: Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates “reserve cells”, J. Cell. Sci., 111, 769–779 (1998). 16. Chowdhury, S. R., Muneyuki, Y., Takezawa, Y., Kino-oka, M., Saito, A., Sawa, Y., and Taya, M.: Synergic stimulation of laminin and epidermal growth factor facilitates the myoblast growth through promoting migration, J. Biosci. Bioeng., 108, 174–177 (2009). 17. Theil, P. K., Sørensen, I. L., Nissen, P. M., and Oksbjerg, N.: Temporal expression of growth factor genes of primary porcine satellite cells during myogenesis, Anim. Sci. J., 77, 330–337 (2006). 18. Lindon, C., Albagli, O., Pinset, C., and Montarras, Didier: Cell density-dependent induction of endogenous myogenin (myf4) gene expression by Myf5, Dev. Biol., 240, 574–584 (2001). 19. Andrés, V. and Walsh, K.: Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis, J. Cell Biol., 132, 657–666 (1996).