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. 2022 May 13;11:e64430. doi: 10.7554/eLife.64430

Figure 4. CHIR99021 rescues growth and telomere defects in dyskeratosis congentia (DC) induced pluripotent stem cell-derived type II alveolar epithelial (iAT2) cell alveolospheres.

(A) Differentiation protocol used to test how CHIR99021 affects growth of DC iAT2s. (B) Representative images of differentiating wild type (WT) and DKC1 A386T mutant bearing cells with increasing amounts of CHIR99021 (scale bars, 1 mm for all alveolosphere images). (C) Quantifications of alveolosphere formation efficiency after treatment with differing concentrations of CHIR99021 (n = 4, * p<0.05, **** p<0.0001, Student’s t-test). (D) When D70 alveolospheres are cultured with 3 µM CHIR99021, DKC1 A386T mutant iAT2 cells have a lower fraction of cells with 53 BP1 foci. Note, data for no CHIR99021 bars are from Figure 2 (n = 3, * p<0.05, Student’s t-test; scale bars, 10 µm). (E) When D70 alveolospheres are cultured with 3 µM CHIR99021, DKC1 A386T mutant iAT2 cells have a lower fraction of p21 positive cells. Note, data for no CHIR99021 bars are from Figure 2 (n = 3, * p<0.05, Student’s t-test; scale bars, 10 µm). (F) When D70 alveolospheres are cultured with 3 µM CHIR99021, DKC1 A386T mutant iAT2 cells have a lower fraction of telomere dysfunction induced foci (TIF) positive cells. Note, data for no CHIR99021 bars are from Figure 2 (n = 3, * p<0.05, Student’s t-test; scale bars, 10 µm; insets highlight cells with TIFs, each one noted by the white arrowheads). (G) Telomeric repeat amplification protocol assay for telomerase activity in iAT2 cells using fivefold extract dilutions (n = 3).

Figure 4—source data 1. Source data for Figure 4C-G.
Figure 4C-SourceData-OrganoidFormationEfficiency.pzfx – Raw counts of organoid counts normalized to number of cells input to calculate formation efficiency along with statistical tests. Figure 4D-SourceData-53BP1Rescue.pzfx – Raw counts of 53BP1 positive cells after treatment with CHIR99021 along with statistics. Figure 4E-SourceData-p21Rescue.pzfx – Raw counts of p21 positive cells after treatment with CHIR99021 along with statistics. Figure 4F-SourceData-TIFRescue.pzfx – Raw counts of telomere dysfunction induced foci positive cells after treatment with CHIR99021 along with statistics.Figure 4G-SourceData-2021-05-04-109-CK-Cropped.tiff – Raw blot of telomeric repeat amplification protocol (TRAP) assay of iPSC-derived type II alveolar epithelial (iAT2) cells treated with CHIR99021 showing area that was cropped. Figure 4G-SourceData-2021-05-04-109-CK.tif – Raw blot of TRAP assay of iAT2 cells treated with CHIR99021. Figure 4G-SourceData-2021-05-04-109-K-Cropped.tiff – Raw blot of TRAP assay of untreated iAT2 showing area that was cropped. Figure 4G-SourceData-2021-05-04-109-K.tif – Raw blot of TRAP assay of untreated iAT2. Figure 4G-SourceData-2021-05-04-109-QuantificationTRAP.xlsx – Quantifications of TRAP assay in Figure 4G.

Figure 4.

Figure 4—figure supplement 1. The growth defect in dyskeratosis congenita (DC) induced pluripotent stem cell-derived type II alveolar epithelial (iAT2) cells is rescued by continuous treatment of CHIR99021 from D28 to D70 but not if withdrawn at D56.

Figure 4—figure supplement 1.

(A) Differentiation protocol used to test how withdrawal of CHIR99021 at D56 affects growth of DC iAT2s at D70. (B) Representative images of iAT2 alveolospheres at D70 when CHIR99021 (present up to D56) was either continued or removed after D56 and the cells analyzed at D70. If CHIR99021 is maintained, the growth defect is suppressed, while if CHIR99021 is removed the growth defect emerges (scale bars, 1 mm).
Figure 4—figure supplement 2. CHIR98014 can rescue dyskeratosis congenita (DC) induced pluripotent stem cell-derived type II alveolar epithelial (iAT2) cell growth.

Figure 4—figure supplement 2.

(A) Differentiation protocol used to test whether CHIR98014 can affect the growth of DC iAT2s at D70. (B) Rescue of DC iAT2 alveolosphere growth with CHIR98014. These alveolospheres grew from cells plated at 400 cells/µL (scale bars, 1 mm).
Figure 4—figure supplement 3. TeSLA revealed a modest increase in average telomere length but no apparent decrease in the frequency of shortest detectable telomeres.

Figure 4—figure supplement 3.

(A) Differentiation protocol used to test how CHIR99021 affects growth of dyakeratosis congenita induced pluripotent stem cell-derived type II alveolar epithelial cells (iAT2s), same as in Figure 4. (B) TeSLA of DKC1 A386T iAT2 alveolospheres treated with 3 µM CHIR99021 as per differentiation protocol in A. (C) Quantification of TeSLA blot of DKC1 A386T iAT2 alveolospheres (n = 1, ‘shortest 20%’ reports the 20th percentile of telomere length, in Kb).
Figure 4—figure supplement 3—source data 1. Source data for Figure 4—figure supplement 3.
Figure 4—figure supplement 3B-SourceData-2020-08-31-MergedTeslaDKC1A386T-Cropped – Uncropped raw image of TeSLA blot when DKC1 A386T iPSC-derived type II alveolar epithelial cells (iAT2s) were treated with CHIR99021 showing where the crop was placed for Figure 4—figure supplement 3B. This is the same blot as used in Figure 2E. Figure 4, Figure 4—figure supplement 3-SourceData-2020-08-31-MergedTeslaDKC1A386T – Uncropped raw image of TeSLA blot when DKC1 A386T iAT2s were treated with CHIR99021. This is the same blot as used in Figure 2E. Figure 4—figure supplement 3C-SourceData-DKC1A386TTesLARescue – Quantification of TeSLA using TeSLA quant software for DKC1 A386T mutant cells treated with CHIR99021.