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Published in final edited form as: Cell Stem Cell. 2016 Aug 18;19(3):397–405. doi: 10.1016/j.stem.2016.05.024

Enhancing a Wnt-telomere feedback loop restores intestinal stem cell function in a human organotypic model of dyskeratosis congenita

Dong-Hun Woo 1, Qijun Chen 2, Ting-Lin B Yang 2,3, M Rebecca Glineburg 2,3, Carla Hoge 1,4, Nicolae A Leu 1, F Brad Johnson 2,3,5,*, Christopher J Lengner 1,3,6,7,*
PMCID: PMC7900823  NIHMSID: NIHMS790812  PMID: 27545506

Summary

Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using CRISPR/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation. We show DC tissue has sub-optimal Wnt pathway activity causing intestinal stem cell failure, and that enhanced expression of the telomere capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. Thus, the isogenic DC cell model provides a platform for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients.

Graphical Abstract

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eTOC

Using a human iPS cell-derived dyskeratosis congenita (DC) organotypic model, Woo and colleagues show that telomere shortening triggers abrogation of canonical Wnt signaling resulting in defects in intestinal stem cell function. Restoring telomere capping and enhancing Wnt signaling both can restore the Wnt-telomere feedback loop and rescue DC-associated intestinal dysfunction.

Introduction

Dyskeratosis congenita (DC) is a debilitating disorder caused primarily by mutations in the DKC1 gene that encodes DYSKERIN, a protein critical for telomerase complex function, leading to failures in telomere maintenance (Armanios and Blackburn, 2012; Savage, 2014). In the context of DC, telomere defects manifest in highly proliferative tissues that normally require telomerase in their stem cell compartments, including the hematopoietic system, epidermis, and intestinal epithelium (Fernandez Garcia and Teruya-Feldstein, 2014; Jonassaint et al., 2013; Mason and Bessler, 2011). Despite the tissue degeneration resulting from telomere dysfunction in DC, treatment is aimed primarily at reversing hematopoietic failure, typically through bone marrow transplantation (Fernandez Garcia and Teruya-Feldstein, 2014). Aside from hematopoietic failure, the gastrointestinal (GI) manifestations of DC [which include enterocolitis, mucosal ulceration, malabsorption, and hematochezia (Berezin et al., 1996; Borggraefe et al., 2009; Brown et al., 1993; Dokal, 2000; Womer et al., 1983)] remain major clinical problems. Recently, the Johns Hopkins Telomere Syndrome Registry found that 16% of subjects showed significant GI defects (Jonassaint et al., 2013). Thus, there is significant need for developing therapies that can prevent and/or reverse DC degenerative pathologies, including the GI manifestations, and to this end a model system that recapitulates human DC pathology would provide a platform for such therapeutic development.

Murine models of telomerase deficiency, including mTR−/− mice lacking the RNA template for the enzyme complex, exhibit many of the same symptoms seen in human DC. mTR−/− mice exhibit stem cell failure (Hao et al., 2005; Tao et al., 2015) and down-regulation of Wnt pathway target genes, resulting in compromised intestinal barrier function (Tao et al., 2015). Consistently, mouse models with reduced telomerase and inactivating mutations in the shelterin complex component POT1b exhibit apoptosis in the crypt stem cell compartment known to rely upon Wnt pathway activity (Hockemeyer et al., 2008). Interestingly, the gene encoding another shelterin component, TRF2, is a direct β-CATENIN target (Diala et al., 2013). More recently, human induced pluripotent stem cells (iPSCs) cultures revealed decreased activity of the Wnt pathway in DC patient-derived iPSCs (Gu et al., 2015). Thus, these studies suggest the existence of a positive feedback loop between the Wnt pathway and telomere capping, the disruption of which may contribute to the GI defects in DC patients.

Here we describe the generation of DC patient-specific iPS cells followed by the correction of the disease allele with CRISPR/Cas9-mediated homologous recombination resulting in isogenic pairs of DC and control cell lines. We demonstrate that, upon intestinal differentiation of these cell lines, the failure of intestinal stem cell self-renewal observed in mouse DC models is recapitulated. Ultimately, we demonstrate using both in vitro and in vivo xenograft models that Wnt agonists can substantially rescue the DC phenotype. Our findings indicate that clinically approved Wnt agonists may represent a viable therapeutic intervention for DC patients.

Results

To model human DC, we generated iPSCs from DKC1 mutant fibroblasts (A386T) (Agarwal et al., 2010; Wong and Collins, 2006) and an unaffected wildtype individual. Isogenic pairs of DC and control lines were generated using CRISPR/Cas9n (D10A) nickase (Ran et al., 2013) along with ssDNA donor templates for homology-directed repair (in the DC iPSCs) as well as introduction of the DKC1 mutation (in wildtype iPSCs) (Figure 1A, B and Table S1). We identified two corrected clones from DC iPSCs (5% A-to-G correction efficiency) and three DKC1 mutation (Mt)-introduced clones from sex-matched wildtype iPSCs (15% G-to-A mutation efficiency, Figure 1C). We verified pluripotency and developmental potential after genome editing in all iPSC lines by staining for OCT4 and TRA1–60 and teratoma formation assays (Figure 1D and not shown). To confirm the correction and introduction of DKC1 mutations, we measured telomerase activity in two pairs of isogenic iPSC lines and found that wildtype cells had the highest activity, which decreased upon DKC1 mutation introduction. Conversely, the DKC1 mutant line had the lowest telomerase activity, which was rescued upon correction (Figure 1E).

Figure 1. Generation of isogenic DC iPSC lines.

Figure 1.

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(A) Strategy for the correction and introduction of DKC1 mutation. Double-nicking 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=50mm. (E) Telomerase activity in iPS cells measured by qTRAP (n = 3). *p < 0.05, **p < 0.01. Error bars indicate mean ± SD.

To model the intestinal defects seen in DC patients, human intestinal organoids (HIOs) were generated by directed differentiation (Spence et al., 2011). During lineage specification, cultures derived from DKC1 patient or Mt-introduced iPSCs exhibited no defects in the activation of definitive endoderm markers (FOXA2 and SOX17) nor of mid/hind gut markers (CDX2 and KLF5) compared to cultures derived from wildtype or DKC1 mutation-corrected iPSCs (Figure S1A). All cultures formed spheroid structures 8 days after mid/hind gut specification (Figure 2A). Shortly thereafter (day 12–15) the spheroids began cavitating to form gut tube-like structures; however, cavitation was not maintained in DKC1 mutant cultures (Figure 2A, C and Figure S1B). DKC1-corrected and wildtype clones maintained for 30 days after mid/hindgut specification formed HIOs with robust crypt budding. In contrast, DKC1 mutant clones did not undergo further cavitation and failed to form budding crypts (Figure 2B and C). Histological analysis at day 35 showed well-developed intestinal epithelial structures in the corrected and wildtype HIOs with crypt budding along with expression of epithelial E-CADHERIN and robust β-CATENIN expression including cells exhibiting nuclear (transcriptionally active) β-CATENIN, consistent with the presence of intestinal stem cells with an active canonical Wnt pathway (Figure S1C and D). In contrast, DKC1 mutant HIOs exhibited incomplete and thin epithelia, overgrowth of mesenchymal cells, and poor E-CADHERIN and β-CATENIN expression (Figure S1C and D).

Figure 2. Directed differentiation of isogenic DC iPSC lines into intestinal organoids.

Figure 2.

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(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), Wildtype (n = 5), Introduced (clone 1: n=4, clone 2: n=5). *p < 0.05, **p < 0.01 versus DKC1 HIOs or wildtype HIOs. (D and E) Southern blot analysis for measurement of telomere length of 25 day-old HIOs from isogenic DC iPSC lines. (F) Quantitative RT-PCR analysis for 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 wildtype HIOs. Error bars indicate mean ± SD. See also Figure S1.

DKC1 correction resulted in telomere lengthening relative to DC patient HIOs after 25 days of culture (Figure 2D). Interestingly, while telomere shortening was detectable in Mt-introduced HIOs (Figure 2E), it was not as dramatic as the shortening seen in the DC patient HIOs (Figure 2D). Previous reports showed that telomere lengths in DC fibroblasts are shorter than in wildtype fibroblasts and that these differences are more pronounced when comparing wildtype iPSCs and DC iPSCs, with exacerbated telomere shortening in response to iPSC passaging (Batista et al., 2011). In light of this, our observation that DC patient HIOs exhibit shorter telomeres relative to DKC1 Mt-introduced HIOs might be due simply to genetic heterogeneity in the human population, or, more likely, reflects the years of telomerase deficiency (since the patient’s conception) in the DC patient fibroblasts that were used to generate iPSCs and HIOs, in contrast to the telomerase deficiency in the DKC1 Mt-introduced iPSCs/HIOs that existed only a few passages in culture. This highlights the importance of using isogenic controls when modeling human diseases in pluripotent culture models. We conclude that introduction of the DKC1 mutation in wildtype cells results in phenotypic defects similar to those seen in the DC patient-derived HIOs.

Examination of well-established Wnt pathway target genes normally expressed in, and important for intestinal stem cell self-renewal (ASCL2, LGR5, SOX9 and AXIN2) revealed a significant reduction of expression in DKC1 mutant HIOs versus their isogenic controls (Figure 2F). Non-phosphorylated (active) β-CATENIN (Ser33/37/Thr41) expression was also significantly reduced in DKC1 mutant HIOs relative to isogenic controls (Figure S1D). Expression of genes associated with intestinal epithelial cell types (VILLIN, MUCIN2, and LYSOZYME) was also moderately upregulated in DKC1-corrected HIOs and reduced in Mt-introduced HIOs (Figure 2F and S1D). Taken together, these findings indicate that telomere shortening results in sub-optimal activity of Wnt pathway and attenuated intestinal stem cell self-renewal.

Given the link between DKC1 mutations, Wnt signaling, and intestinal stem cell self-renewal, we asked whether Wnt pathway agonists could rescue DC defects. Treatment of two distinct DC patient iPSC-derived HIOs [A386T and ΔL37 (Agarwal et al., 2010; Gu et al., 2015)] and two DKC1 Mt-introduced HIOs with the GSK-3β inhibitor CHIR99021 (CHIR) was able to restore cavitation, crypt budding, and intestinal stem cell gene expression (Figure 3A–D, S2B–F). Withdrawal of CHIR resulted in a deterioration of the rescued HIOs, including loss of budding crypt structure along with decreased ASCL2 and LGR5 expression (Figure S2A and B).

Figure 3. Rescue of the DC phenotype with Wnt agonism and TRF2 expression in DC HIOs.

Figure 3.

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(A) Representative morphology of DKC1 HIOs 8 days after CHIR treatment. (B) E-CADHERIN staining in DKC1 HIOs w/ or w/o 5 mM CHIR. (C) Quantitative RT-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 mM 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 mM CHIR treatment. (E) Southern blot analysis for telomere length and quantification of mean telomere length in 25 days old HIOs. (F) Representative images for telomere-dysfunction induced foci (TIFs), and (G) Quantification of TIFs in HIOs (n=4 for non-treated, n=3 for CHIR treated). **p < 0.01. (H) Quantitative RT-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 mM CHIR treated DKC1 HIOs (I) Quantitative RT-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.

To test if this restored intestinal stem cell function was associated with improved telomere function, we measured telomerase activity and telomere length. CHIR treatment resulted in increased telomerase activity both in 25 day-old DKC1 mutant and corrected HIOs relative to untreated controls (Figure S3A). Telomere length in DKC1 mutant HIOs (assessed by TRF Southern, qPCR, and XpYp STELA) was also reproducibly increased after CHIR treatment, consistent with the Wnt-dependence of TERT expression (Hoffmeyer et al., 2012) (Figure 3E and S3B–E). Moreover, the telomere lengthening observed in CHIR-treated DKC1 mutant HIOs was again reduced upon CHIR withdrawal (Figure S3E). Taken together, these findings support the existence of a feedback loop between Wnt signaling and telomerase function in intestinal stem cells.

Next, we investigated whether Wnt agonism also rescued telomere capping by analyzing telomere dysfunction-induced foci (TIFs), measured by colocalization of telomeres with 53BP1 (Herbig et al., 2004). Consistent with the enhanced telomerase activity and telomere lengthening, CHIR treatment also significantly decreased in the number of TIFs in DKC1 mutant HIOs (Figure 3F and G). Further, the β-CATENIN target and shelterin component TRF2 showed lower expression in DKC1 mutant HIOs relative to isogenic controls, and this was restored by CHIR treatment (Figure 3H). We thus asked whether forced expression of TRF2 is sufficient to restore the phenotypic abnormalities of DKC1 mutant HIOs. TRF2 overexpression restored gut tube cavitation during early stages of intestinal differentiation (day 10), and rescued crypt budding defects at later stages (day 25) (Figure 3I–K). The phenotypic rescue was accompanied by significant increases in Wnt target gene activation both during early stages (ASCL2, SOX9) and later stages (LGR5, ASCL2) of differentiation (Figure S3F). In addition, expression of another shelterin component, POT1, was significantly increased (Figure S3G) and TIFs were reduced in DKC1 mutant HIOs after TRF2 overexpression (Figure S3H and I). Mean telomere length was essentially unaffected (though perhaps slightly elongated) after TRF2 overexpression (Figure S3J), consistent with prior studies demonstrating that TRF2 overexpression does not alter the length of shortened telomeres (Nera et al., 2015). These findings provide further support for a feedback loop in which critically shortened telomeres attenuate Wnt signaling, which in turn inhibits telomere capping via loss of shelterin complex component expression.

Ultimately, we asked whether DKC1 mutant HIOs would respond to systemic administration of a clinically utilized Wnt agonist (lithium salts) at a clinically relevant dosage in vivo (Leroy et al., 2010; O’Brien et al., 2004). We cultured DKC1 mutant and corrected HIOs for 40 days in vitro under rescue conditions (2 M CHIR), and these cultures exhibited similar size and crypt budding (Figure 4A). We then xenografted DKC1 mutant HIOs and DKC1-corrected HIOs into the kidney capsule of immune-deficient (NSG) mice (Watson et al., 2014). Mice engrafted with DKC1 mutant HIOs were kept either on control diet or on LiCl chow diet (Figure 4B). DKC1-corrected HIOs grafted mice were maintained on control diet.

Figure 4. Systemic lithium treatment reverses DC phenotypes in vivo.

Figure 4.

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(A) Representative morphology of DKC1 and DKC1-corrected HIOs treated with 2mM CHIR prior to transplantation. (B) Schematic representation of HIO transplantation and lithium feeding (Li). (C) HE staining and ECADHERIN/Ki67 staining in control mouse small intestine and lithium fed mouse small intestine. (D) Quantification of Ki67+ cells per crypt and quantitative RT-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) Quantitative RT-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.

Four weeks post-transplantation, we initially examined the host intestinal epithelium. LiCl-treated host intestinal epithelium exhibited clear evidence of Wnt agonism including expansion of the crypt proliferative zone and upregulation of Wnt target genes (Axin2 and Lgr5) (Figure 4C and D). In the grafted HIOs (Figure 4E), the intestinal architecture of DKC1 mutant grafts had lost structural integrity: poor E-CADHERIN and β-CATENIN staining with no Ki-67+ proliferative cells were observed relative to the DKC1-corrected grafts (Figure 4F and G). In contrast, LiCl treated DKC1 mutant grafts maintained epithelial structure with high E-CADHERIN expression, proliferation, and high β-CATENIN expression (both in adherens junctions and rare cells with active, nuclear localized β-CATENIN), as in the DKC1-corrected grafts (Figure 4F and G). Proliferative cells were clearly detectable in these grafts, but not as frequently as in DKC1-corrected grafts. In addition, Wnt target/intestinal stem cell genes (AXIN2, ASCL2, LGR5, and SOX9) were highly upregulated in DKC1-corrected grafts relative to DKC1 mutant grafts. Strikingly, LiCl-treated DKC1 mutant grafts had significantly increased expression of these genes, with some (SOX9 and AXIN2) being expressed as highly as in the genetically corrected isogenic controls (Figure 4H). Further, marker genes for the major intestinal differentiated cell types (VILLIN: enterocytes, LYZ: Paneth cells, and CHGA: enteroendocrine cells) were also increased in DKC1-corrected grafts and LiCl treated DKC1 mutant grafts (Figure 4H). Expression of the goblet cell marker MUC2 was not significantly up-regulated in DKC1-corrected grafts or LiCl treated DKC1 mutant grafts, however, MUC2+ goblet cells were observable in the DKC1-corrected and LiCl treated DKC1 mutant grafts, but not in untreated DKC1 mutant grafts (Figure 4G).

As with DC patient HIOs, pairs of Mt-introduced and wildtype HIO xenografts demonstrated that systemic Wnt agonism with LiCl rescued the DKC1 Mt-introduced grafts (including epithelial architecture and intestinal gene expression) (Figure S4). Thus, systemic administration of lithium salts at clinically relevant concentrations effectively stimulates the canonical Wnt signaling pathway, rescuing human DKC1-mutant intestinal xenografts in vivo. These findings suggest that DC patients may benefit from the use of these clinically approved compounds already vetted for safety and in use for other indications.

Discussion

In the current study, we demonstrate that isogenic DC iPSC lines can serve as a platform for interrogating the mechanisms underlying the stem cell failure observed in DC patients as well as for testing the efficacy of potential therapeutic interventions in a human in vitro (or xenograft) model.

We took advantage of in vitro directed differentiation protocols (Spence et al., 2011) and in vivo xenograft models (Watson et al., 2014) to demonstrate that the human iPSC-based platform reflects the phenotypic and molecular manifestations of critical telomere shortening. Indeed, DKC1 mutant organoids suffer from sub-optimal activity of the canonical Wnt signaling pathway required to maintain intestinal stem cell self-renewal and epithelial barrier integrity. Studies in murine systems suggested that canonical Wnt signaling lies in a feedback loop with the shelterin telomere capping complex, as genes encoding components of this complex (e.g. Trf2) are known to be direct β-catenin binding targets. This too was recapitulated in the HIO system as DKC1 mutant HIOs exhibited attenuated TRF2 expression relative to their isogenic control counterparts. Further, forced expression of TRF2 in DKC1 mutant cultures was sufficient to restore Wnt target gene expression and HIO morphology, suggesting that TRF2 overexpression is capable of promoting the capping of shortened telomeres in DKC1 mutant cells, thus relieving the inhibition of canonical Wnt signaling, although the precise mechanisms through which the Wnt pathway might sense telomere shortening and uncapping are not well understood.

The link between telomere capping and the canonical Wnt signaling pathway observed in the murine model prompted us to ask whether Wnt pathway agonists, including clinically approved lithium salts (Oruch et al., 2014), could reverse the phenotypes seen in DKC1 HIOs and restore activation of shelterin component gene expression, and, subsequently telomere capping and length (as the DKC1 mutations are hypomorphic, exhibiting reduced telomerase activity rather than complete loss of function). In fact, this was the case: Wnt pathway agonism restored the molecular, phenotypic, and telomeric defects of DKC1 HIOs to a state nearly indistinguishable from their mutation-corrected isogenic controls. Taken together, we have developed a platform for understanding the molecular mechanisms underlying DC-associated phenotypes, as well as for screening potential therapies aimed at slowing or reversing these phenotypes, and, most importantly, provide a proof-of-principle that Wnt pathway agonists may be clinically relevant therapeutic compounds effective at treating the gastrointestinal and possibly other phenotypes seen in DC patients.

Experimental Procedures

Generation of isogenic DC induced pluripotent stem cells (iPSCs)

1 × 106 of dyskeratosis congenita (DC) fibroblasts (AG04646, DKC1 A386T, Coriell) were seeded onto gelatin-coated plates and infected with STEMCCA virus and rtTA virus. iPSC colonies were picked 25 – 30 days post-infection. Sex matched wildtype male iPSCs were used as control. To correct and introduce the DKC1 mutation, guide RNAs were cloned in humanized Cas9n vector (px335, D10A, Addgene 42335), and transfected into iPSCs along with 197 bp ssDNA donor templates. Subcloning was performed after puromycin selection. Correction and introduction of DKC1 mutations was confirmed by sequencing.

Differentiation of human intestinal organoids (HIOs)

HIOs were differentiated from isogenic DC iPSC lines and maintained as previously described (Spence et al., 2011). To activate Wnt pathway in differentiating DKC1 mutant and DKC1 mutation introduced HIOs, 0, 1, 2, 5 μM of CHIR 99021 (Tocris) was added to intestinal growth medium after mid-/hind gut specification.

Kidney capsule xenograft of HIOs

HIOs were cultured for 40 days under rescue condition with 2 μM of CHIR before transplantation, and grafted into kidney capsule as described previously (Watson et al., 2014). For Wnt activation, DKC1 mutant and Mt-introduced HIO grafted mice were kept on LiCl chow (Harlan). 0.2 % LiCl chow was fed to mice for 3 weeks, then mice were switched to 0.4 % Lithium chow for one additional week.

Supplementary Material

Highlights.

  • CRISPR/Cas9-mediated gene targeting used to model human dyskeratosis congenita

  • Human iPS cell-derived intestinal organoids recapitulate DC disease phenotypes

  • Wnt agonists are potential therapeutics for intestinal regeneration in DC patients

  • Wnt signaling and telomere function form a feedback loop in intestinal stem cells

Acknowledgments

We thank members of the Lynch, Anguera, and Rustgi labs at the University of Pennsylvania for fruitful discussions, and Peter Nicholas at the Children’s Hospital of Philadelphia for kind providing DC iPS cells (A353V and ΔL37). C.J.L. and F.B.J. were supported by R21 AG054209 from the NIA and a pilot award from the Penn Institute on Ageing. D.H.W. was supported by a fellowship from the Penn Institute for Regenerative Medicine. This work was supported in part by the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases (P30DK050306) and its core facilities. C.J.L. and FBJ were further supported by R01 CA16865 from the NCI and P01 AG031862 from the NIA, respectively.

Footnotes

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See the Supplemental Experimental Procedure for more details.

References

  1. Agarwal S, Loh YH, McLoughlin EM, Huang J, Park IH, Miller JD, Huo H, Okuka M, Dos Reis RM, Loewer S, et al. (2010). Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature 464, 292–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armanios M, and Blackburn EH (2012). The telomere syndromes. Nature reviews Genetics 13, 693–704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Batista LF, Pech MF, Zhong FL, Nguyen HN, Xie KT, Zaug AJ, Crary SM, Choi J, Sebastiano V, Cherry A, et al. (2011). Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells. Nature 474, 399–402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berezin S, Schwarz SM, Slim MS, Beneck D, Brudnicki AR, and Medow MS (1996). Gastrointestinal problems in a child with dyskeratosis congenita. The American journal of gastroenterology 91, 1271–1272. [PubMed] [Google Scholar]
  5. Borggraefe I, Koletzko S, Arenz T, Fuehrer M, Hoffmann F, Dokal I, Vulliamy T, Weiler V, Griese M, Belohradsky BH, et al. (2009). Severe variant of x-linked dyskeratosis congenita (Hoyeraal-Hreidarsson Syndrome) causes significant enterocolitis in early infancy. Journal of pediatric gastroenterology and nutrition 49, 359–363. [DOI] [PubMed] [Google Scholar]
  6. Brown KE, Kelly TE, and Myers BM (1993). Gastrointestinal involvement in a woman with dyskeratosis congenita. Digestive diseases and sciences 38, 181–184. [DOI] [PubMed] [Google Scholar]
  7. Diala I, Wagner N, Magdinier F, Shkreli M, Sirakov M, Bauwens S, Schluth-Bolard C, Simonet T, Renault VM, Ye J, et al. (2013). Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway. EMBO reports 14, 356–363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dokal I (2000). Dyskeratosis congenita in all its forms. British journal of haematology 110, 768–779. [DOI] [PubMed] [Google Scholar]
  9. Fernandez Garcia MS, and Teruya-Feldstein J (2014). The diagnosis and treatment of dyskeratosis congenita: a review. Journal of blood medicine 5, 157–167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gu BW, Apicella M, Mills J, Fan JM, Reeves DA, French D, Podsakoff GM, Bessler M, and Mason PJ (2015). Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients. PloS one 10, e0127414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hao LY, Armanios M, Strong MA, Karim B, Feldser DM, Huso D, and Greider CW (2005). Short telomeres, even in the presence of telomerase, limit tissue renewal capacity. Cell 123, 1121–1131. [DOI] [PubMed] [Google Scholar]
  12. Herbig U, Jobling WA, Chen BP, Chen DJ, and Sedivy JM (2004). Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Molecular cell 14, 501–513. [DOI] [PubMed] [Google Scholar]
  13. Hockemeyer D, Palm W, Wang RC, Couto SS, and de Lange T (2008). Engineered telomere degradation models dyskeratosis congenita. Genes & development 22, 1773–1785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoffmeyer K, Raggioli A, Rudloff S, Anton R, Hierholzer A, Del Valle I, Hein K, Vogt R, and Kemler R (2012). Wnt/beta-catenin signaling regulates telomerase in stem cells and cancer cells. Science 336, 1549–1554. [DOI] [PubMed] [Google Scholar]
  15. Jonassaint NL, Guo N, Califano JA, Montgomery EA, and Armanios M (2013). The gastrointestinal manifestations of telomere-mediated disease. Aging cell 12, 319–323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leroy K, Ando K, Heraud C, Yilmaz Z, Authelet M, Boeynaems JM, Buee L, De Decker R, and Brion JP (2010). Lithium treatment arrests the development of neurofibrillary tangles in mutant tau transgenic mice with advanced neurofibrillary pathology. Journal of Alzheimer’s disease : JAD 19, 705–719. [DOI] [PubMed] [Google Scholar]
  17. Mason PJ, and Bessler M (2011). The genetics of dyskeratosis congenita. Cancer genetics 204, 635–645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nera B, Huang HS, Lai T, and Xu L (2015). Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions. Nature communications 6, 10132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O’Brien WT, Harper AD, Jove F, Woodgett JR, Maretto S, Piccolo S, and Klein PS (2004). Glycogen synthase kinase-3beta haploinsufficiency mimics the behavioral and molecular effects of lithium. The Journal of neuroscience : the official journal of the Society for Neuroscience 24, 6791–6798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Oruch R, Elderbi MA, Khattab HA, Pryme IF, and Lund A (2014). Lithium: a review of pharmacology, clinical uses, and toxicity. European journal of pharmacology 740, 464–473. [DOI] [PubMed] [Google Scholar]
  21. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, et al. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Savage SA (2014). Human telomeres and telomere biology disorders. Progress in molecular biology and translational science 125, 41–66. [DOI] [PubMed] [Google Scholar]
  23. Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, et al. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tao S, Tang D, Morita Y, Sperka T, Omrani O, Lechel A, Sakk V, Kraus J, Kestler HA, Kuhl M, et al. (2015). Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage. The EMBO journal. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Watson CL, Mahe MM, Munera J, Howell JC, Sundaram N, Poling HM, Schweitzer JI, Vallance JE, Mayhew CN, Sun Y, et al. (2014). An in vivo model of human small intestine using pluripotent stem cells. Nature medicine 20, 1310–1314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Womer R, Clark JE, Wood P, Sabio H, and Kelly TE (1983). Dyskeratosis congenita: two examples of this multisystem disorder. Pediatrics 71, 603–609. [PubMed] [Google Scholar]
  27. Wong JM, and Collins K (2006). Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes & development 20, 2848–2858. [DOI] [PMC free article] [PubMed] [Google Scholar]

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NIHMS790812-supplement-1.pdf (3.2MB, pdf)

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