Abstract
The differentiation process of murine embryonic stem cells into cardiomyocytes was investigated with a compliant microfluidic platform which allows for versatile cell seeding arrangements, optical observation access, long-term cell viability, and programmable uniaxial cyclic stretch. Specifically, two environmental cues were examined with this platform—culture dimensions and uniaxial cyclic stretch. First, the cardiomyogenic differentiation process, assessed by a GFP reporter driven by the α-MHC promoter, was enhanced in microfluidic devices (µFDs) compared with conventional well-plates. The addition of BMP-2 neutralizing antibody reduced the enhancement observed in the µFDs and the addition of exogenous BMP-2 augmented the cardiomyogenic differentiation in well plates. Second, 24 h of uniaxial cyclic stretch at 1 Hz and 10% strain on day 9 of differentiation was found to have a negative impact on cardiomyogenic differentiation. This microfluidic platform builds upon an existing design and extends its capability to test cellular responses to mechanical strain. It provides capabilities not found in other systems for studying differentiation, such as seeding embryoid bodies in 2D or 3D in combination with cyclic strain. This study demonstrates that the microfluidic system contributes to enhanced cardiomyogenic differentiation and may be a superior platform compared with conventional well plates. In addition to studying the effect of cyclic stretch on cardiomyogenic differentiation, this compliant platform can also be applied to investigate other biological mechanisms.
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References
Alsan, B. H., and T. M. Schultheiss. Regulation of avian cardiogenesis by Fgf8 signaling. Development 129:1935–1943, 2002.
Barron, M., M. Gao, and J. Lough. Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev. Dyn. 218:383–393, 2000.
Boheler, K. R., J. Czyz, D. Tweedie, H. T. Yang, S. V. Anisimov, and A. M. Wobus. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ. Res. 91:189–201, 2002.
Chen, K., L. Wu, and Z. Z. Wang. Extrinsic regulation of cardiomyocyte differentiation of embryonic stem cells. J. Cell. Biochem. 104:119–128, 2008.
Chung, S., R. Sudo, P. J. Mack, C. Wan, V. Vickerman, and R. D. Kamm. Cell migration into scaffolds under co-culture conditions in a microfluidic platform. Lab Chip 9:269–275, 2009.
Dickman, E. D., and S. M. Smith. Selective regulation of cardiomyocyte gene expression and cardiac morphogenesis by retinoic acid. Dev. Dyn. 206:39–48, 1996.
Gallo, P., and G. Condorelli. Human embryonic stem cell-derived cardiomyocytes: inducing strategies. Regen. Med. 1:183–194, 2006.
Heng, B. C., H. K. Haider, E. K. W. Sim, T. Cao, and S. C. Ng. Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovasc. Res. 62:34–42, 2004.
Hwang, Y. S., B. G. Chung, D. Ortmann, N. Hattori, H. C. Moeller, and A. Khademhosseini. Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11. Proc. Natl. Acad. Sci. USA 106:16978–16983, 2009.
Jacot, J. G., J. C. Martin, and D. L. Hunt. Mechanobiology of cardiomyocyte development. J. Biomech. 43:93–98, 2010.
Kawai, T., T. Takahashi, M. Esaki, H. Ushikoshi, S. Nagano, H. Fujiwara, and K. Kosai. Efficient cardiomyogenic differentiation of embryonic stem cell by fibroblast growth factor 2 and bone morphogenetic protein 2. Circ. J. 68:691–702, 2004.
Ke, Q., Y. Yang, J. S. Rana, Y. Chen, J. P. Morgan, and Y. F. Xiao. Embryonic stem cells cultured in biodegradable scaffold repair infarcted myocardium in mice. Sheng Li Xue Bao. 57:673–681, 2005.
Kellar, R. S., L. K. Landeen, B. R. Shepherd, G. K. Naughton, A. Ratcliffe, and S. K. Williams. Scaffold-based three-dimensional human fibroblast culture provides a structural matrix that supports angiogenesis in infarcted heart tissue. Circulation 104:2063–2068, 2001.
Kim, Y. Y., S. Y. Ku, J. Jang, S. K. Oh, H. S. Kim, S. H. Kim, Y. M. Choi, and S. Y. Moon. Use of long-term cultured embryoid bodies may enhance cardiomyocyte differentiation by BMP2. Yonsei Med. J. 49:819–827, 2008.
Laflamme, M. A., and C. E. Murry. Regenerating the heart. Nat. Biotechnol. 23:845–856, 2005.
Leor, J., S. Aboulafia-Etzion, A. Dar, L. Shapiro, I. M. Barbash, A. Battler, Y. Granot, and S. Cohen. Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 102:III56–III61, 2000.
Mammoto, T., and D. E. Ingber. Mechanical control of tissue and organ development. Development 137:1407–1420, 2010.
Matsumoto, T., Y. C. Yung, C. Fischbach, H. J. Kong, R. Nakaoka, and D. J. Mooney. Mechanical strain regulates endothelial cell patterning in vitro. Tissue Eng. 13:207–217, 2007.
Meyvantsson, I., and D. J. Beebe. Cell culture models in microfluidic systems. Annu. Rev. Anal. Chem. 1:423–449, 2008.
Monzen, K., I. Shiojima, Y. Hiroi, S. Kudoh, T. Oka, E. Takimoto, D. Hayashi, T. Hosoda, A. Habara-Ohkubo, T. Nakaoka, T. Fujita, Y. Yazaki, and I. Komuro. Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4 Mol. Cell Biol. 19:7096–7105, 1999.
Paquin, J., B. A. Danalache, M. Jankowski, S. M. McCann, and J. Gutkowska. Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes. Proc. Natl. Acad. Sci. USA 99:9550–9555, 2002.
Patwari, P., and R. T. Lee. Mechanical control of tissue morphogenesis. Circ. Res. 103:234–243, 2008.
Rathjen, J., and P. D. Rathjen. Mouse ES cells: experimental exploitation of pluripotent differentiation potential. Curr. Opin. Genet. Dev. 11:587–594, 2001.
Raty, S., E. M. Walters, J. Davis, H. Zeringue, D. J. Beebe, S. L. Rodriguez-Zas, and M. B. Wheeler. Embryonic development in the mouse is enhanced via microchannel culture. Lab Chip 4:186–190, 2004.
Saha, S., L. Ji, J. J. de Pablo, and S. P. Palecek. TGF beta/activin. Biophys. J. 94:4123–4133, 2008.
Saha, S., J. Lin, J. J. De Pablo, and S. P. Palecek. Inhibition of human embryonic stem cell differentiation by mechanical strain. J. Cell. Physiol. 206:126–137, 2006.
Samuelson, L. C., and J. M. Metzger. Differentiation of embryonic stem (ES) cells using the hanging drop method. Cold Spring Harb. Protoc. 2006. doi:10.1101/pdb.prot4485.
Sauer, H. Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells. FEBS Lett. 476:218–223, 2000.
Sauer, H., G. Rahimi, J. Hescheler, and M. Wartenberg. Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells. J. Cell. Biochem. 75:710–723, 1999.
Schmelter, M., B. Ateghang, S. Helmig, M. Wartenberg, and H. Sauer. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation. FASEB J. 20:1182–1184, 2006.
Schultheiss, T. M., J. B. Burch, and A. B. Lassar. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11:451–462, 1997.
Segers, V. F. M., and R. T. Lee. Stem-cell therapy for cardiac disease. Nature 451:937–942, 2008.
Serena, E., E. Figallo, N. Tandon, C. Cannizzaro, S. Gerecht, N. Elvassore, and G. Vunjak-Novakovic. Electrical stimulation of human embryonic stem cells: cardiac differentiation and the generation of reactive oxygen species. Exp. Cell Res. 315:3611–3619, 2009.
Shimko, V. F., and W. C. Claycomb. Effect of mechanical loading on three-dimensional cultures of embryonic stem cell-derived cardiomyocytes. Tissue Eng Part A. 14(1):49–58, 2008.
Sudo, R., S. Chung, I. K. Zervantonakis, V. Vickerman, Y. Toshimitsu, L. G. Griffith, and R. D. Kamm. Transport-mediated angiogenesis in 3D epithelial coculture. FASEB J. 23(7):2155–2164, 2009.
Takahashi, T., B. Lord, P. C. Schulze, R. M. Fryer, S. S. Sarang, S. R. Gullans, and R. T. Lee. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107:1912–1916, 2003.
van Laake, L. W., R. Passier, J. Monshouwer-Kloots, A. J. Verkleij, D. J. Lips, C. Freund, K. den Ouden, D. van Ward-Oostwaard, J. Korving, L. G. Tertoolen, C. J. van Echteld, P. A. Doevendans, and C. L. Mummery. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Res. 1:9–24, 2007.
Walker, G. M., H. C. Zeringue, and D. J. Beebe. Microenvironment design considerations for cellular scale studies. Lab Chip 4:91–97, 2004.
Yamada, M., J. P. Revelli, G. Eichele, M. Barron, and R. J. Schwartz. Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev. Biol. 228:95–105, 2000.
Yu, H., I. Meyvantsson, I. A. Shkel, and D. J. Beebe. Diffusion dependent cell behavior in microenvironments. Lab Chip 5:1089–1095, 2005.
Acknowledgments
The authors would like to acknowledge the contribution of Dr. Richard Lee for his invaluable suggestions on this work. Seok Chung was supported by the International Research & Development Program (Grant number: 2009-00631). We acknowledge support from the Singapore-MIT Alliance for Research and Technology and an American Heart Association Predoctoral Fellowship for Chen-rei Wan. This work was also supported by National Science Foundation (Science and Technology Center (EBICS) Emergent Behaviors of Integrated Cellular Systems Grant CBET-0939511).
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Associate Editor Laura Suggs oversaw the review of this article.
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Wan, Cr., Chung, S. & Kamm, R.D. Differentiation of Embryonic Stem Cells into Cardiomyocytes in a Compliant Microfluidic System. Ann Biomed Eng 39, 1840–1847 (2011). https://doi.org/10.1007/s10439-011-0275-8
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DOI: https://doi.org/10.1007/s10439-011-0275-8