Abstract
Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in <5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.
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References
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Zwi, L. et al. Cardiomyocyte differentiation of human induced pluripotent stem cells. Circulation 120, 1513–1523 (2009).
Keirstead, H.S. Human embryonic stem cell–derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25, 4694–4705 (2005).
Soldner, F. et al. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977 (2009).
Phillips, M.J. et al. Modeling human retinal development with patient-specific induced pluripotent stem cells reveals multiple roles for visual system homeobox 2. Stem Cells 32, 1480–1492 (2014).
Cai, J. et al. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 45, 1229–1239 (2007).
Spence, J.R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2010).
Nostro, M.C. et al. Stage-specific signaling through TGF family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861–871 (2011).
Selekman, J.A., Grundl, N.J., Kolz, J.M. & Palecek, S.P. Efficient generation of functional epithelial and epidermal cells from human pluripotent stem cells under defined conditions. Tissue Eng. Part C Methods 19, 949–960 (2013).
Rezania, A. et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes 61, 2016–2029 (2012).
Orlova, V.V. et al. Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat. Protoc. 9, 1514–1531 (2014).
Shcheglovitov, A. et al. SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients. Nature 503, 267–271 (2013).
Yaghi, A., Zaman, A. & Dolovich, M. Primary human bronchial epithelial cells grown from explants. J. Vis. Exp. doi.org/10.3791/1789 (2010).
de Jong, P.M., van Sterkenburg, M.A., Kempenaar, J.A., Dijkman, J.H. & Ponec, M. Serial culturing of human bronchial epithelial cells derived from biopsies. In vitro Cell. Dev. Biol. Anim. 29A, 379–387 (1993).
Forrest, I.A. et al. Primary airway epithelial cell culture from lung transplant recipients. Eur. Resp. J. 26, 1080–1085 (2005).
Zhao, L., Yee, M. & O'Reilly, M.A. Transdifferentiation of alveolar epithelial type II to type I cells is controlled by opposing TGF-β and BMP signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 305, L409–L418 (2013).
Kasai, H., Allen, J.T., Mason, R.M., Kamimura, T. & Zhang, Z. TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Resp. Res. 6, 56 (2005).
Warshamana, G.S., Corti, M. & Brody, A.R. TNF-α, PDGF, and TGF-β1 expression by primary mouse bronchiolar-alveolar epithelial and mesenchymal cells: TNF-α induces TGF-β1. Exp. Mol. Pathol. 71, 13–33 (2001).
Tanjore, H. et al. Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress. J. Biol. Chem. 286, 30972–30980 (2011).
Vaughan, M.B., Ramirez, R.D., Wright, W.E., Minna, J.D. & Shay, J.W. A three-dimensional model of differentiation of immortalized human bronchial epithelial cells. Differentiation 74, 141–148 (2006).
Delgado, O. et al. Multipotent capacity of immortalized human bronchial epithelial cells. PLoS ONE 6, e22023 (2011).
Ott, H.C. et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat. Med. 16, 927–933 (2010).
Lam, E., Ramke, M., Groos, S., Warnecke, G. & Heim, A. A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence. J. Virol. Methods 178, 117–123 (2011).
Dupuis, A., Hamilton, D., Cole, D.E.C. & Corey, M. Cystic fibrosis birth rates in Canada: a decreasing trend since the onset of genetic testing. J. Pediatr. 147, 312–315 (2005).
Riordan, J.R. et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073 (1989).
Vanscoy, L.L. et al. Heritability of lung disease severity in cystic fibrosis. Am. J. Respir. Crit. Care Med. 175, 1036–1043 (2007).
Li, W. et al. Understanding the population structure of North American patients with cystic fibrosis. Clin. Genet. 79, 136–146 (2011).
Samadikuchaksaraei, A. & Bishop, A.E. Effects of growth factors on the differentiation of murine ESC into type II pneumocytes. Cloning Stem Cells 9, 407–416 (2007).
Samadikuchaksaraei, A. et al. Derivation of distal airway epithelium from human embryonic stem cells. Tissue Eng. 12, 867–875 (2006).
Van Vranken, B.E. et al. Coculture of embryonic stem cells with pulmonary mesenchyme: a microenvironment that promotes differentiation of pulmonary epithelium. Tissue Eng. 11, 1177–1187 (2005).
Ali, N.N. et al. Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng. 8, 541–550 (2002).
Roszell, B. et al. Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application. Tissue Eng. Part A 15, 3351–3365 (2009).
Rippon, H.J. et al. Embryonic stem cells as a source of pulmonary epithelium in vitro and in vivo. Proc. Am. Thorac. Soc. 5, 717–722 (2008).
Coraux, C. Embryonic stem cells generate airway epithelial tissue. Am. J. Respir. Cell Mol. Biol. 32, 87–92 (2004).
Ameri, J. et al. FGF2 specifies hESC-derived definitive endoderm into foregut/midgut cell lineages in a concentration-dependent manner. Stem Cells 28, 45–56 (2009).
Van Haute, L., De Block, G., Liebaers, I., Sermon, K. & De Rycke, M. Generation of lung epithelial-like tissue from human embryonic stem cells. Resp. Res. 10, 105 (2009).
Wang, D., Haviland, D.L., Burns, A.R., Zsigmond, E. & Wetsel, R.A. A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 4449–4454 (2007).
Wong, A.P. et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat. Biotechnol. 30, 876–882 (2012).
Huang, S.X.L. et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat. Biotechnol. 32, 84–91 (2014).
Firth, A.L. et al. Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 111, E1723–E1730 (2014).
Ghaedi, M. et al. Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix. J. Clin. Invest. 123, 4950–4962 (2013).
Serls, A.E., Doherty, S., Parvatiyar, P., Wells, J.M. & Deutsch, G.H. Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Development 132, 35–47 (2005).
Bellusci, S. et al. Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 124, 53–63 (1997).
Kim, S.K. & Melton, D.A. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proc. Natl. Acad. Sci. USA 95, 13036–13041 (1998).
Green, M.D. et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat. Biotechnol. 29, 267–272 (2011).
Acknowledgements
We thank J.R. Riordan (University of North Carolina) for providing the monoclonal antibodies specific to CFTR (nos. 450, 596 and 660). This work was funded by the Canadian Institutes of Health Research (GPG-102171) to C.E.B. and J.R. A.P.W. was the recipient of the Cystic Fibrosis Canada postdoctoral fellowship. The CA1 hESC line was obtained from A. Nagy (Mount Sinai Hospital). H9 hESCs were obtained from The WiCell Research Institute. CF iPSC line GM00997 and GM04320 were obtained from J. Ellis (Hospital for Sick Children).
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A.P.W., J.R. and C.E.B. conceived the study and the experimental design. A.P.W. performed and analyzed the experiments and wrote the manuscript. J.G., S.C. and S.X. performed the experiments and helped in preparing manuscript figures. All authors edited and approved the final manuscript.
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Wong, A., Chin, S., Xia, S. et al. Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells. Nat Protoc 10, 363–381 (2015). https://doi.org/10.1038/nprot.2015.021
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DOI: https://doi.org/10.1038/nprot.2015.021
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