Volume 19, Issue 3, Pages 397-405 (September 2016) Enhancing a Wnt-Telomere Feedback Loop Restores Intestinal Stem Cell Function in a Human Organotypic Model of Dyskeratosis Congenita  Dong-Hun Woo, Qijun Chen, Ting-Lin B. Yang, M. Rebecca Glineburg, Carla Hoge, Nicolae A. Leu, F. Brad Johnson, Christopher J. Lengner  Cell Stem Cell  Volume 19, Issue 3, Pages 397-405 (September 2016) DOI: 10.1016/j.stem.2016.05.024 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2016 19, 397-405DOI: (10.1016/j.stem.2016.05.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Generation of Isogenic DC iPSC Lines (A) Strategy for the correction and introduction of DKC1 mutation. Double-nicking guide RNAs (gRNAs) targeting exon 12 of the DKC1 gene were used with ssDNA donor templates for correction (A to G) and introduction (G to A) of DKC1 mutation. (B) Indel detection by SURVEYOR assay in HEK293 cells, demonstrating cutting only in the presence of both gRNAs (asterisks). (C) DNA sequences showing DKC1 mutation correction and introduction. (D) Immunofluorescence staining of OCT4 and TRA-1-60 in isogenic DC iPSC lines. Scale bar, 50 μm. (E) Telomerase activity in iPSCs measured by quantitative telomeric repeat amplification protocol (qTRAP) (n = 3). ∗p < 0.05, ∗∗p < 0.01. Error bars indicate mean ± SD. Cell Stem Cell 2016 19, 397-405DOI: (10.1016/j.stem.2016.05.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Directed Differentiation of Isogenic DC iPSC Lines into Intestinal Organoids (A and B) Representative morphological changes in HIOs during differentiation in intestinal growth medium. (C) Quantification of cavitation at 15 days (n = 3) and budding at day 30; DC (n = 4), corrected (clone 1: n = 7, clone 2: n = 5), wild-type (n = 5), introduced (clone 1: n = 4, clone 2: n = 5). ∗p < 0.05, ∗∗p < 0.01 versus DKC1 HIOs or wild-type HIOs. (D and E) Southern blot analysis for measurement of telomere length of 25-day-old HIOs from isogenic DC iPSC lines. (F) qRT-PCR analysis of expression of Wnt target intestinal stem cell and differentiated intestinal cell genes in 15-day-old HIOs (n = 3). ∗p < 0.05, ∗∗p < 0.01 versus DKC1 HIOs or wild-type HIOs. Error bars indicate mean ± SD. See also Figure S1. Cell Stem Cell 2016 19, 397-405DOI: (10.1016/j.stem.2016.05.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Rescue of the DC Phenotype with Wnt Agonism and TRF2 Expression in DC HIOs (A) Representative morphology of DKC1 HIOs 8 days after CHIR treatment. (B) E-CADHERIN staining in DKC1 HIOs with or without 5 μM CHIR. (C) qRT-PCR analysis of Wnt target intestinal stem cell gene expression in DKC1 HIOs after CHIR treatment (n = 3). ∗p < 0.05, ∗∗p < 0.01 versus 0 μM CHIR treatment. (D) Quantification of cavitation and budding in DKC1 HIOs after CHIR treatment (A386T and ΔL37, n = 7 for each group). ∗p < 0.05, ∗∗p < 0.01 versus 0 μM CHIR treatment. (E) Southern blot analysis for telomere length and quantification of mean telomere length in 25-day-old HIOs. (F and G) Representative images for telomere-dysfunction induced foci (TIFs) (F) and quantification of TIFs in HIOs (n = 4 for non-treated, n = 3 for CHIR treated) (G). ∗∗p < 0.01. (H) qRT-PCR analysis of TRF2 expression in DKC1-corrected HIOs, DKC1 HIOs, and CHIR-treated DKC1 HIOs (n = 3 for each group). ∗p < 0.05 versus 0 μM CHIR-treated DKC1 HIOs. (I) qRT-PCR analysis of TRF2 expression in control and TRF2-overexpressed DKC1 HIOs. (J) Representative morphology of control and TRF2-overexpressed DKC1 HIOs at 10 and 25 days. (K) Quantification of cavitation in HIOs at day 10 and budding HIOs at day 25 in control and TRF2-overexpressing DKC1 HIOs (n = 5 for each group). ∗p < 0.05, ∗∗p < 0.01 versus DKC1 HIOs. Error bars indicate mean ± SD. See also Figures S2 and S3. Cell Stem Cell 2016 19, 397-405DOI: (10.1016/j.stem.2016.05.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Systemic Lithium Treatment Reverses DC Phenotypes In Vivo (A) Representative morphology of DKC1 and DKC1-corrected HIOs treated with 2 μM CHIR prior to transplantation. (B) Schematic representation of HIO transplantation and lithium feeding (Li). (C) HE staining and E-CADHERIN/Ki67 staining in control mouse small intestine and lithium-fed mouse small intestine. (D) Quantification of Ki67+ cells per crypt and qRT-PCR analysis of Wnt target genes in control mouse small intestine and lithium-fed mouse small intestine. ∗∗p < 0.01 versus control. (E) Kidneys from HIO-grafted mice. Arrows indicate grafted HIOs. (F) H&E in paraffin sections from HIO grafts 4 weeks after transplantation. (G) Staining of E-CADHERIN/Ki67, β-CATENIN, and MUC2 in HIO grafts. Red arrows in insets indicate nuclear-located β-CATENIN. (H) qRT-PCR analysis of Wnt target intestinal stem cell and differentiated intestinal cell gene expression in HIO grafts. ∗p < 0.05, ∗∗p < 0.01 versus DKC1 HIO grafts. Error bars indicate mean ± SD. See also Figure S4. Cell Stem Cell 2016 19, 397-405DOI: (10.1016/j.stem.2016.05.024) Copyright © 2016 Elsevier Inc. Terms and Conditions