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 5

Expanded AT2s express AT1 markers after culture in AT1 medium.

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Expanded AT2s express AT1 markers after culture in AT1 medium. (A) Schem...
(A) Schematic representation of AT2-to-AT1 differentiation. Expanded AT2s were cultured in T2-Max for 2 days, T1-Diff for 3 days, and T1-Base for 3 days to generate AT1s. (B) Immunostaining time course from the AT1 differentiation paradigm described in A. Scale bars: 50 μm. (C) Immunostaining quantification of AT1 differentiation. Mean fluorescent intensity (MFI) of cells expressing each target was quantified for cells from each donor on days 1, 2, 5, and 8 (nuclear [nuc]; n = 3 donors; mean ± SD). (D) Immunostaining quantification of AT1 differentiation. Sum fluorescent intensity (SFI) of cells expressing each target was quantified for cells from each donor on days 1, 2, 5, and 8 (n = 3 donors; mean ± SD). (E) Immunostaining quantification of AT1 differentiation. Quantification of proportion of cells expressing each target above the day 1 MFI+1 SD (n = 3 donors; mean ± SD). (F) Gene expression quantification using qRT-PCR of AT1 differentiation over time (log2 fold change normalized to donor-specific P2 AT2 controls; n = 3 donors). (G) Gene expression quantification using qRT-PCR of AT1-specific markers on day 8 (2-ΔΔCT normalized to donor-specific P2 AT2 controls (red dotted line); n = 5 donors; mean ± SD).