Abstract
Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1+ lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.
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Data availability
This article includes weblinks to publicly available datasets, such as bioinformatics files deposited at the Gene Expression Ombinus (GEO accession number: GSE103918). There are no restrictions on data sharing, and any raw data, cell lines, or protocol downloads are available through the corresponding author by email request or through his website portal at www.kottonlab.com.
References
Mason, R. J. & Williams, M. C. Type II alveolar cell. Defender of the alveolus. Am. Rev. Respir. Dis. 115, 81–91 (1977).
Whitsett, J. A., Wert, S. E. & Weaver, T. E. Diseases of pulmonary surfactant homeostasis. Annu. Rev. Pathol. 10, 371–393 (2015).
Jacob, A. et al. Differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells. Cell Stem Cell 21, 472–488.e10 (2017).
Serra, M. et al. Pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung- versus thyroid-lineage specification. Development 144, 3879–3893 (2017).
Green, M. D. et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat. Biotechnol. 29, 267–272 (2011).
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).
Longmire, T. A. et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10, 398–411 (2012).
Hawkins, F. et al. Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells. J. Clin. Invest. 127, 2277–2294 (2017).
Kurmann, A. A. et al. Regeneration of thyroid function by transplantation of differentiated pluripotent stem cells. Cell Stem Cell 17, 527–542 (2015).
Gotoh, S. et al. Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Rep. 3, 394–403 (2014).
McCauley, K. B., Hawkins, F. & Kotton, D. N. Derivation of epithelial-only airway organoids from human pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 45, e51 (2018).
McCauley, K. B. et al. Efficient derivation of functional human airway epithelium from pluripotent stem cells via temporal regulation of Wnt signaling. Cell Stem Cell 20, 844–857.e6 (2017).
Yamamoto, Y. et al. Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat. Methods 14, 1097–1106 (2017).
McCauley, K. B. et al. Single-cell transcriptomic profiling of pluripotent stem cell-derived SCGB3A2+ airway epithelium. Stem Cell Rep. 10, 1579–1595 (2018).
Minoo, P., Su, G., Drum, H., Bringas, P. & Kimura, S. Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(−/−) mouse embryos. Dev. Biol. 209, 60–71 (1999).
Mucenski, M. L. et al. Beta-catenin regulates differentiation of respiratory epithelial cells in vivo. Am. J. Physiol. Lung Cell Mol. Physiol. 289, L971–L979 (2005).
Liu, Y. & Hogan, B. L. M. Differential gene expression in the distal tip endoderm of the embryonic mouse lung. Gene Expr. Patterns 2, 229–233 (2002).
Gonzales, L. W., Guttentag, S. H., Wade, K. C., Postle, A. D. & Ballard, P. L. Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP. Am. J. Physiol. Lung Cell Mol. Physiol. 283, L940–L951 (2002).
Frank, D. B. et al. Emergence of a wave of Wnt signaling that regulates lung alveologenesis by controlling epithelial self-renewal and differentiation. Cell Rep. 17, 2312–2325 (2016).
Huang, S. X. L. et al. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells. Nat. Protoc. 10, 413–425 (2015).
Chen, Y.-W. et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat. Cell Biol. 19, 542–549 (2017).
Dye, B. R. et al. In vitro generation of human pluripotent stem cell derived lung organoids. Elife 4, 1999 (2015).
Miller, A. J. et al. In vitro induction and in vivo engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells. Stem Cell Rep. 10, 101–119 (2018).
Korogi, Y. et al. In vitro disease modeling of Hermansky-Pudlak syndrome type 2 using human induced pluripotent stem cell-derived alveolar organoids. Stem Cell Rep. 12, 431–440 (2019).
Zacharias, W. J. et al. Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor. Nature 555, 251–255 (2018).
Barkauskas, C. E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Invest. 123, 3025–3036 (2013).
D’Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).
Gouon-Evans, V. et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat. Biotechnol. 24, 1402–1411 (2006).
Ogawa, S. et al. Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development 140, 3285–3296 (2013).
Christodoulou, C. et al. Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes. J. Clin. Invest. 121, 2313–2325 (2011).
Loh, K. M. et al. Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell Stem Cell 14, 237–252 (2014).
Acknowledgements
We wish to thank all members of the Kotton and Hawkins laboratories for helpful discussions and optimization of the presented protocol. We are grateful to B.R. Tilton of the BUSM Flow Cytometry Core, supported by NIH grant 1UL1TR001430, for technical assistance; and G. Miller, A. Iyer, and M. James of the CReM, supported by grants R24HL123828 and U01TR001810. This work was supported by grants TL1TR001410 and F31HL134274 (A.J.), HAWKIN15XX0 and R01HL139799 (F.H.), the I.M. Rosenzweig Junior Investigator Award from the Pulmonary Fibrosis Foundation (K.-D.A.), and grants U01HL099997, U01HL134745, R01HL095993, R01HL122442, R01HL128172, and U01HL134766 (D.N.K.). We thank A. Brivanlou, Rockefeller University, for the generous gift of RUES2, an embryonic stem cell line.
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A.J., M.V., and D.N.K. designed and performed experiments and wrote the manuscript. D.C.T. and F.H. performed serial-dilution experiments. C.V.-M. performed bioinformatics single-cell RNA sequencing analysis. D.A.R. and K.-D.A. performed experiments demonstrating clonality.
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Peer review information Nature Protocols thanks Amy Ryan (Firth) and other anonymous reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Jacob, A. et al. Cell Stem Cell 21, 472–488.e10 (2017): https://doi.org/10.1016/j.stem.2017.08.014
McCauley, K. B. et al. Stem Cell Rep. 10, 1579–1595 (2018): https://doi.org/10.1016/j.stemcr.2018.03.013
Hawkins, F. et al. J. Clin. Invest. 127, 2277–2294 (2017): https://doi.org/10.1172/JCI89950
Integrated supplementary information
Supplementary Fig. 1 Genomic stability of passaged alveolospheres.
All panels show array-based Comparative Genomic Hybridization (aCGH) analysis of alveolospheres at various points in long term maintenance. a) SPC2-ST-B2 iPSC line at alveolosphere passage 2, day 100 of differentiation. b) SPC2-ST-B2 at alveolosphere passage 12, day 214 of differentiation. c) BU3-NGST iPSC line at alveolosphere passage 3 post SFTPCtdTomato sort without prior CHIR withdrawal. d) BU3-NGST at alveolosphere passage 12 post SFTPCtdTomato sort without prior CHIR withdrawal. Note: Changes in BU3-NGST passage 3 alveolospheres, as well as the CNV in the SPC2-ST passage 3 alveolospheres, were present in the starting iPSCs in both cases.
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Jacob, A., Vedaie, M., Roberts, D.A. et al. Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells. Nat Protoc 14, 3303–3332 (2019). https://doi.org/10.1038/s41596-019-0220-0
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DOI: https://doi.org/10.1038/s41596-019-0220-0
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