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. Author manuscript; available in PMC: 2021 Sep 10.
Published in final edited form as: Cell Rep. 2021 Aug 31;36(9):109636. doi: 10.1016/j.celrep.2021.109636

Figure 2. Derivation of parallel self-renewing corrected (SFTPCtdT/WT) and mutant (SFTPCI73T/tdT) iAEC2s.

Figure 2.

(A) Representative flow cytometry dot plots of day 82 (p2), day 115 (p4), day 149 (p6), day 190 (p8), day 208 (p9), day 243 (p11), and day 285 (p13) SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s. MFI, mean fluorescence intensity.

(B) Representative live-cell imaging of SFTPCtdT/WT and SFTPCI73T/tdT alveolospheres (bright-field/tdTomato overlay; day 149). Scale bars, 500 μm.

(C) Graph showing yield per input tdT/WT or I73T/tdT SFTPCtdTomato+ cell sorted on day 51 and passaged without further sorting. **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired, two-tailed Student’s t test.

(D) Bar graph shows retention of the AEC2 cell fate in SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s maintained in culture for 302 days, measured by flow cytometry as the frequency of cells expressing the SFTPCtdTomato reporter.

(E) Histograms show higher proliferation rates in SFTPCtdT/WT compared to SFTPCI73T/tdT iAEC2s, measured by flow cytometry as the frequency of SFTPCtdTomato+ cells that incorporate EdU.

(F) Representative confocal immunofluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s stained for activated caspase 3 (green) and DNA (Hoechst, blue) shows absence of significant apoptosis. Scale bars, 50 μm.

(A and C–E) Mean ± SD is shown; n = 3 experimental replicates of independent wells of a differentiation.