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Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein

Nature Biotechnology volume 30, pages 876–882 (2012)Cite this article

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Abstract

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which regulates chloride and water transport across all epithelia and affects multiple organs, including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction–coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof of concept, treatment of CF patient induced pluripotent stem cell–derived epithelial cells with a small-molecule compound to correct for the common CF processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.

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Figure 1: Low concentration of BMP4 upregulates genes associated with early proximal lung progenitors.
Figure 2: FGF18 promotes proximal airway epithelia formation.
Figure 3: Air liquid interface induces airway epithelial cell differentiation and promotes apical expression of CFTR.
Figure 4: Establishment of functional CFTR hESC-derived airway epithelia and correction of CF phenotype in CF-iPSC-derived epithelial cells with a small-molecule compound C18.

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Acknowledgements

We would like to thank S. Yamanaka (CiRA, Kyoto, Japan) and A. Nagy (Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada) for providing human iPSC lines (201B7, 253G1 and PB-4Fout, respectively). We would also like to thank R. Bridges (Rosalind Franklin University, Chicago) who provided the C18 analog. This work was funded by an Emerging Team grant from the Canadian Institutes of Health Research (GPG-102171) to C.E.B., F.R., J.E. and J.R. This work was supported (in part) by an Ontario Ministry of Economic Development and Innovation (MEDI) grant. A.P.W. was a recipient of the MEDI Post-doctoral Award. Monoclonal CFTR antibodies #450 and #660 were courtesy of J.R. Riordan (University of North Carolina, Chapel Hill, North Carolina, USA). CA1, CA2 hESC were obtained from A. Nagy (Mount Sinai Hospital, Toronto, Canada). H9 hESC were obtained from The WiCell Research Institute (Wisconsin, USA).

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Authors and Affiliations

  1. Program in Developmental & Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada

    Amy P Wong, Peter Pasceri, James Ellis & Janet Rossant

  2. Ontario Human Induced Pluripotent Stem Cell Facility, Toronto, Ontario, Canada

    Amy P Wong, Tadeo O Thompson & James Ellis

  3. Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada

    Christine E Bear, Stephanie Chin & Ling-Jun Huan

  4. Program in Physiology & Experimental Medicine, Hospital for Sick Children, Toronto, Ontario, Canada

    Felix Ratjen

  5. Division of Respiratory Medicine, Hospital for Sick Children, Toronto, Ontario, Canada

    Felix Ratjen

  6. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    James Ellis & Janet Rossant

  7. Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, Canada

    Janet Rossant

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  1. Amy P Wong
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Contributions

A.P.W., J.R., J.E., C.E.B. and F.R. conceived the study and experimental design. A.P.W. performed and analyzed experiments and wrote the manuscript. C.E.B., P.P., T.O.T., L.-J.H., S.C. and F.R. provided reagents, conceptual and/or technical support in generating iPSC lines, doing the teratoma assay and making iodide efflux measurements. All authors edited and approved the final manuscript.

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Correspondence to James Ellis or Janet Rossant.

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The authors declare no competing financial interests.

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Supplementary Figures 1–14 and Supplementary Tables 1–3 (PDF 2223 kb)

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Wong, A., Bear, C., Chin, S. et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol 30, 876–882 (2012). https://doi.org/10.1038/nbt.2328

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