Effect of a Matrigel Sandwich on Endodermal Differentiation of Human Embryonic Stem Cells

Springer Science and Business Media LLC - Tập 9 - Trang 578-585 - 2013
Betty R. Lawton1,2, Julie Ann Sosa3, Sanziana Roman3, Diane S. Krause1,2
1Department of Laboratory Medicine, Yale University School of Medicine, New Haven, USA
2Yale Stem Cell Center Yale University School of Medicine, New Haven, USA
3Section of Endocrine Surgery, Department of Surgery, Duke University School of Medicine, Durham, USA

Tóm tắt

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition, including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients, we demonstrate that cell viability can be preserved throughout differentiation. In addition, modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique, as well as gene expression patterns, indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm.

Tài liệu tham khảo

Cerdan, C., Hong, S. H., & Bhatia, M. (2007). Formation and hematopoieitic differentiation of human embryoid bodies by suspension and hanging drop cultures. Current Protocols in Stem Cell Biology, 3, 1D.2.1–1D.2.16. Tesar, P. J., Chenoweth, J. G., Brook, F. A., et al. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature, 448, 196–199. Nowotschin, S., & Hadjantonakis, A.-K. (2010). Cellular dynamics in the early mouse embryo: from axis formation to gastrulation. Current Opinion in Genetics and Development, 20, 420–427. Zhang, J., Klos, M., Wilson, G. F., et al. (2012). Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method. Circulation Research, 111, 1125–1136. Wang, P., McKnight, K. D., Wong, D. J., Rodriguez, R. T., et al. (2012). A molecular signature for purified definitive endoderm guides differentiation and isolation of endoderm from mouse and human embryonic stem cells. Stem Cells and Development, 21(12), 2273–2287. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆C T method. Methods, 25, 402–408. Goni, R., Garcia, P., & Foissac, S. (2009). The qPCR data statistical analysis. Intergenomics SL, 1–9. D’Amour, K. A., Bang, A. G., Eliazer, S., et al. (2006). Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nature Biotechnology, 24(11), 1392–1401. Francis, G. L. (2010). Albumin and mammalian cell culture: implications for biotechnology applications. Cytotechnology, 62, 1–16. Borowiak, M., Maehr, R., Chen, A. E., et al. (2009). Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell, 4, 348–358. Barnes, D., & Sato, G. (1980). Methods for growth of cultured cells in serum-free medium. Analytical Biochemistry, 102, 255–270. Kwon, G. S., Viotti, M., & Hadjantonakis, A.-K. (2008). The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages. Developmental Cell, 15, 509–520. Tada, S., Era, T., Furusawa, C., et al. (2005). Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development, 132(19), 4363–4374. Eastham, A. M., Spencer, H., Soncin, F., et al. (2007). Epithelial–mesenchymal transition events during human embryonic stem cell differentiation. Cancer Research, 67(23), 11254–11262. Ullman, U., In’t Veld, P. I., Gilles, C., et al. (2007). Epithelial–mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Molecular Human Reproduction, 13(1), 21–32. Thiery, J. P., Acloque, H., Huang, R. Y., & Nieto, M. A. (2009). Epithelial–mesenchymal transitions in development and disease. Cell, 139(5), 871–890. Hasegawa, K., Fujioka, T., Nakamura, Y., Nakatsuji, N., & Suemori, H. (2006). A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells, 24, 2649–2660. Brafman, D. A., Phung, C., Kumar, N., & Willert, K. (2012). Regulation of endodermal differentiation of human embryonic stem cells through integrin–ECM interactions. Cell Death and Differentiation, 1–13. Kenny, A. P., Rankin, S. A., Allbee, A. W., et al. (2012). Developmental Cell, 23, 292–304.