Figure 1. Dyskeratosis congenita induced pluripotent stem cell (iPSC)-derived type II alveolar epithelial cells (iAT2s) fail to form alveolospheres with successive passaging.
(A) Differentiation protocol used to probe the effects of the DKC1 A386T mutation on iAT2 cells. D.E., definitive endoderm specification; Ant., anteriorization. (B) Representative images of differentiating wild type and DKC1 A386T mutant bearing iAT2 alveolospheres (scale bars, 100 µm as indicated for iPS and NKX2.1+ progenitors; 1 mm for all alveolosphere images). (C) Quantifications of alveolosphere area and formation efficiency on D70 (n = 4; ** p<0.01, **** p<0.0001, and Student’s t-test). (D) Quantification of the percentage SFTPC+ cells and the number of SFTPC+ cells produced with passage of the iAT2 cells shows DKC1 A386T iAT2 alveolospheres accumulate fewer SFTPC+ cells (n = 4; ** p<0.01, and Student’s t-test). (E) RNA-seq of sorted SFTPC+ iAT2 cells at different passages shows AT2 cell genes are not grossly affected by the DKC1 A386T mutation (n = 4, W = wild type, M = mutant).
Figure 1—source data 1. Source Data for Figure 1C-E.
Figure 1C-SourceData-FormationEfficiency&OrganoidArea.pzfx – Raw counts of organoid counts normalized to number of cells input to calculate formation efficiency as well as area measurements along with statistical tests. Figure 1D-SourceData-SFTPC%&SFTPCCumYield.pzfx – Percentage SFTPC+ along the differentiation of the induced pluripotent stem cell-derived type II alveolar epithelial cells (iAT2s) from sort flow plots along with cumulative yield of surfactant protein C calculated as the number of SFTPC+ cells produced over the experiment over time. Figure 1E-SourceData-AT2Markers.pzfx – Raw counts from RNA-seq of iAT2s over the differentiation specifically looking at AT2 markers.
elife-64430-fig1-data1.zip (97.4KB, zip)
Figure 1—figure supplement 1. Introduction of the DKC1 A386T mutation into the BU3 NKX2.1::GFP SFTPC::TdTomato (NGST) induced pluripotent stem (iPS) cell line.
(A) CRISPR-based editing strategy for introducing the DKC1 A386T mutation into the BU3 NSGT iPS cell line. (B) Genotyping by RFLP of DKC1 A386T clonal cell lines and their wild-type (WT) counterparts that also were exposed to the CRISPR reagents using the introduced NdeI site. (C) Sanger sequence verification of the introduction of the mutation yielding 1156G>A. Note the WT sequence is marked in red. (D) Paired iPS cell clones maintain high expression of NANOG, a marker of pluripotency (scale bars, 100 µm). (E) Paired iPS cell clones show normal karyotypes after the introduction of the DKC1 A386T mutation.
Figure 1—figure supplement 1—source data 1. Source data for Figure 1—figure supplement 1B,C and E.
Figure 1—figure supplement 1B-SourceData-2020-07-17-FigureS1-NdeIGeno-CroppingLabeled.tiff – Genotyping agarose gel showing area that was cropped for the Figure 1—figure supplement 1B. Figure 1—figure supplement 1B-SourceData-2020-07-17-FigureS1-NdeIGeno.tiff – Raw agarose gel image for Figure 1—figure supplement 1B. Figure 1—figure supplement 1C-SourceData-DKC1A386T-34.2DA9_B05_045.ab1 – Sanger sequencing file for Figure 1—figure supplement 1C of BU3 NGST DKC1 A386T mutant iPS line. Figure 1—figure supplement 1C-SourceData-WT-33.3EF6_A05_047.ab1 – Sanger sequencing file for Figure 1—figure supplement 1C of BU3 NGST wild type (WT) control iPS line. Figure 1—figure supplement 1E-SourceData-DKC1A386T-KaryotypeImageCLG-35870.pdf – Image of a representative karyotype of BU3 NGST DKC1 A386T iPS line. Figure 1—figure supplement 1E-SourceData-DKC1A386T-REPORTCLG-35870.pdf – Report of karyotype analysis for BU3 NGST DKC1 A386T iPS line. Figure 1—figure supplement 1E-SourceData-WT-KaryotypeImageCLG-35869.pdf – Image of a representative karyotype of BU3 NGST WT control iPS line. Figure 1—figure supplement 1E-SourceData-WT-REPORTCLG-35869.pdf – Report of karyotype analysis for BU3 NGST WT control iPS line.
elife-64430-fig1-figsupp1-data1.zip (795.6KB, zip)
Figure 1—figure supplement 2. DKC1 A386T induced pluripotent stem (iPS) cells show decreased telomerase activity and telomeres shorten with passage.
(A) Quantitative telomeric repeat amplification protocol assay for telomerase activity in iPS cells shows a reduction in relative telomerase activity in DKC1 A386T introduced line (n = 3). AG04646 is an iPSC line derived from a dyskeratosis congenita patient with the A386T mutation, and the wild-type (WT) line is an unedited clone derived along with the A386T-introduced clone from the BU3 parental iPSC line. (B) Telomeric repeat amplification protocol assay for telomerase activity in iPS cells using a fivefold dilution series of the cellular extracts shows a reduction in relative telomerase activity in DKC1 A386T introduced lines. (C) Terminal restriction fragment telomere length analyses in the WT and DKC1 A386T paired iPS cells compared to the parental iPS line (BU3 NGST) and the patient derived iPS line (AG04646). Above each lane is the number of passages from the introduction of the mutation.
Figure 1—figure supplement 2—source data 1. Source data for Figure 1—figure supplement 2A-C.
Figure 1—figure supplement 2A-SourceData-qTRAPiPS-2018-09-06-007_qTRAP_BU3_Paired_Cell_Line.xlsx – Raw Cq (quantification cycle) data for quantitative telomeric repeat amplification protocol of different iPS lines. Figure 1—figure supplement 2B-SourceData-iPSWTDKC1A386TTRAP-2020-07-24-102Intense.tiff – Uncropped image of telomeric repeat amplification protocol (TRAP) gel for Figure 1—figure supplement 2B. Figure 1—figure supplement 2C-SourceData-2019-03-29-055TRFBU3A386T_60_60_60-Cropped.tif – Uncropped image of TRAP gel for Figure 1—figure supplement 2B showing where cropping occurred. Figure 1—figure supplement 2C-SourceData-2019-03-29-055TRFBU3A386T_60_60_60.tif – Uncropped raw image of Terminal restriction fragment (TRF blot for Figure 1—figure supplement 2C). Figure 1—figure supplement 2C-SourceData-2019-03-29-055TRFBU3A386T_60_60_60-Cropped.tiff – Uncropped raw image of TRF blot for Figure 1—figure supplement 2C showing how cropping removed the central ladder to aid in observation of iPS lines.
elife-64430-fig1-figsupp2-data1.zip (3MB, zip)
Figure 1—figure supplement 3. Representative sorting strategies for NKX2.1+ progenitors and SFTPC+ induced pluripotent stem cell-derived type II alveolar epithelial (iAT2) cells.
(A) Representative sorting strategy for NKX2.1+ progenitors. (B) Representative sorting strategy for SFTPC+ cells from iAT2 alveolospheres.
Figure 1—figure supplement 4. Differentiation of induced pluripotent stem cell (iPSC)-derived type II alveolar epithelial cell (iAT2) alveolospheres from early passage iPS cells yields no growth defect.
(A) Differentiation protocol used to probe the effects of the DKC1 A386T mutation on iAT2 cells. D.E., definitive endoderm specification; Ant., anteriorization. (B) Representative images of differentiating wild type (WT) and DKC1 A386T mutant bearing iAT2 alveolospheres derived from iPS cells that have undergone five passages after introduction of the mutation. (scale bars, 1 mm). (C) Quantifications of the formation efficiency on D70 of iAT2 alveolospheres derived from iPS cells that have undergone five passages after introduction of the mutation (n=3, p=0.81, and Student’s t-test).