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 6

AT1s derived from expanded AT2s exhibit morphological changes.

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AT1s derived from expanded AT2s exhibit morphological changes. (A) Violi...
(A) Violin plot of cell area quantification during AT2-to-AT1 differentiation. Analysis was performed on 3 donors after 17 hours in T2-Max (day 1, n = 534 cells), prior to switching to T1-Diff on day 2 (n = 1091 cells), prior to switching to T1-Base on day 5 (n = 1647 cells), and at completion of culture on day 8 (n = 936 cells; 1-way ANOVA with Tukey’s multiple comparison test; *P = 0.0219, ****P < 0.0001). Data represent mean ± SD. AT1s were derived from expanded AT2s. (B) Bright-field images with insets of AT1s derived from isoAT2s and expanded AT2s. Scale bar: 100 μm. (C) Scanning electron microscopy images of AT1s cultured on Transwell inserts alone (AT1 only) or with a second late seed of expanded AT2s for comparison (AT1+AT2). Images were taken from above (coronal) and at an angle (transverse). AT1s were derived from expanded AT2s.