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 2

AT2 phenotype after expansion is largely similar to freshly isolated AT2s.

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AT2 phenotype after expansion is largely similar to freshly isolated AT2...
(A) Bright-field images comparing isoAT2s to P2 AT2s. Scale bar: 100 μm. (B) Immunostaining for NKX2-1 (red) in P2 AT2s. Scale bar: 50 μm (C) Immunostaining for HT2-280 (green) and pSP-C (red) in isoAT2s and P2 AT2s. Scale bar: 50 μm. (D) Immunostaining for pSP-C (gray) in isoAT2s and P2 AT2s at higher magnification. Scale bar: 25 μm. (E) Quantitation of HT2-280 and pSP-C as a percentage of cells expressing both, none, or either protein above secondary-only control (isoAT2, n = 3 donors; P2 AT2, n = 6 donors; mean of n = 3 wells per bank; unpaired t test of HT2-280+/pSP-C+ population in P2 AT2 compared with isoAT2, P = 0.22). (F) Immunostaining for LAMP3 (green) and mSP-C (red) in isoAT2s and P2 AT2s. Scale bar: 25 μm. (G) Western blot of pSP-C and mSP-C in P2 AT2 lysates (n = 3 donors). (H) Quantitation of bulk mRNA transcripts in isoAT2s and expanded AT2s (n = 6 donors per passage; mean of log2 normalized counts; repeated measures 1-way ANOVA, Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with isoAT2s). (I) Immunostaining for SP-D (gray) in isoAT2s and P2 AT2s. Scale bar: 50 μm. (J) Quantitation of HT2-280 and SP-D immunostaining as a percentage of cells expressing both, none, or either protein above secondary-only control (n = 3 AT2 banks/passage; mean of n = 6 wells per bank; unpaired t test of HT2-280/SPD dual-positive population in P2 AT2 compared with isoAT2, P = 0.38). (K) HPLC analysis of SP-B in AT2-conditioned media collected from bioreactors after day 5 media exchange (P3; n = 3 donors). Data represent mean ± SD. (L) Transmission electron microscopy images showing structures resembling tubular myelin in conditioned media. Scale bar: 0.5 μm (main image); 0.1 μm (top left). DAPI (cyan) was used as a counterstain in B, C, D, F and I. Scale bar: 50 μm, unless otherwise noted.