Leveraging factors that control alveolar epithelial cell fate enables large-scale expansion for lung tissue engineering
Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari
Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari
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Research Article Cell biology Pulmonology

Leveraging factors that control alveolar epithelial cell fate enables large-scale expansion for lung tissue engineering

Abstract

Alveolar type 2 cells (AT2s) are critical to lung regeneration, and the absence of large-scale methods to expand AT2s has hindered regenerative medicine efforts. We report a microcarrier-based, large-scale expansion method that was used to generate hundreds of billions of human AT2s. Through our process, expanded AT2s largely retained their phenotype. Furthermore, we showed that culture medium, substrate composition, and stiffness are all critical to the maintenance of AT2s. Finally, we showed that expanded AT2s can differentiate into alveolar type 1–like cells, both in vitro and in a decellularized porcine lung, demonstrating the utility of these cells for lung tissue engineering.

Authors

Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari

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Figure 7

AT1s derived from expanded AT2s exhibit key characteristics.

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AT1s derived from expanded AT2s exhibit key characteristics. (A) Gene ex...
(A) Gene expression quantification using qRT-PCR of alveolar-specific claudins from AT1s (log2 fold change normalized to donor-specific p2 AT2s controls [red dotted line]; n = 4 donors). (B) Immunostaining of AT1s for claudin-18 (white), ZO-1 (fuchsia), and DAPI (cyan). Scale bars: 50 μm. (C) Gene expression quantification using qRT-PCR of secretory ligands from AT1s (2^-ΔΔCT normalized to donor-specific p2 AT2 controls (orange dotted line); n = 3 donors). (D) Total VEGF-A protein quantitation in conditioned media samples collected from AT1s on days 2, 5, and 8 (n = 3 donors; mean ± SD). (E) VEGF-A protein quantitation in conditioned media samples collected from day 8 AT1s. Concentrations normalized to nuclear cell counts from a satellite culture at day 8 (n = 3 donors; 3 independent experiments). (F and G) Gene expression quantification using qRT-PCR of ECM genes from AT1s (2-ΔΔCT normalized to donor-specific p2 AT2s controls (orange dotted line); n = 3 donors). All cells analyzed were expanded AT2-derived AT1s. Data represent mean ± SD.