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. 2011 May 22;474(7351):399-402.
doi: 10.1038/nature10084.

Telomere Shortening and Loss of Self-Renewal in Dyskeratosis Congenita Induced Pluripotent Stem Cells

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Free PMC article

Telomere Shortening and Loss of Self-Renewal in Dyskeratosis Congenita Induced Pluripotent Stem Cells

Luis F Z Batista et al. Nature. .
Free PMC article

Abstract

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.

Conflict of interest statement

Competing interests statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. DC iPSCs with heterozygous TERT mutations show reduced telomerase levels
a, Telomere lengths by Flow-FISH in peripheral blood lymphocytes from DC patients and their first-degree relatives. Squares, TERT_P704S family; Diamonds, TERT_R979W family; Triangles, TCAB1_H*Y/G*R family; Circles, DKC1_L54V family. Symbols: solid, probands; grey, carriers; Open, first-degree relatives. b, Direct telomerase assays on wild-type TERT, or TERT mutants, assembled with TERC + or − recombinant Pot1 (P), TPP1 (T) or T+P. c, Expression of TERT, TERC, DKC1, and TCAB1 with reprogramming. RT, RT-PCR; IB, immunoblot. GAPDH, U6 and Tubulin, loading controls. d, Telomerase activity by TRAP in wild-type, TERT_P704S and TERT_R979W iPSCs. Range of concentrations represent 4-fold serial dilutions. NP40, buffer control.
Figure 2
Figure 2. Preserved activity, but pronounced mislocalization of telomerase in TCAB1-mutant iPSCs
a, Expression of TERT, TERC, DKC1, and TCAB1 with reprogramming. RT, RT-PCR; NB, northern blot; IB, immunoblot. GAPDH, U6 and Tubulin, loading controls. b, Telomerase activity by TRAP in wild-type and TCAB1_H*Y/G*R iPSCs. Range of concentrations represent 4-fold serial dilutions. NP40, buffer control. c, Immunofluorescence for TCAB1 (red) and p80-coilin (green) in wild-type and TCAB1_H*Y/G*R iPSCs. d, Co-staining for dyskerin (red) and p80-coilin (green) in wild-type and TCAB1_H*Y/G*R iPSCs. e, RNA FISH analysis for TERC (red) in wild-type and TCAB1_H*Y/G*R iPSCs. White arrows, Cajal bodies. Blue, DAPI.
Figure 3
Figure 3. Diminished TERC levels, reduced activity and impaired assembly of mature telomerase in X-linked DC iPSCs
a, Expression of TERT, TERC, DKC1, and TCAB1 with reprogramming. RT, RT-PCR; NB, northern blot; IB, immunoblot. GAPDH, U6 and Tubulin, loading controls. b–c, Telomerase activity by TRAP in (b) DKC1_L54V and (c) DKC1_ ΔL37 iPSCs. Range of concentrations represent 4-fold serial dilutions. NP40, buffer control. Internal PCR control band at bottom of gel. d, Analysis of mature telomerase in iPSCs. Immunoprecipitation of 1 mg of whole-cell extracts with IgG, anti-dyskerin antibodies, or anti-TCAB1 antibodies. Purified complexes were analyzed for the indicated proteins by IB and for TERC by NB. Recovery control, TERC fragment control for differential recovery of RNA.
Figure 4
Figure 4. Impaired telomere maintenance and loss of self-renewal in DC iPSCs
a–d, Telomere lengths by Southern blot using genomic DNA from fibroblasts and iPSCs. (a) TERT_P704S and TERT_R979W iPSCs (b) TCAB1_H*Y/G*R (c) DKC1_L54V and (d) DKC1_ΔL37 iPSCs and DKC1_ΔL37_TT iPSCs at indicated passages after reprogramming. Molecular weight, kilobases. Black line in a, membrane cut for hybridization. e–h, Telomere FISH on metaphase chromosomes from: e, wild-type iPSCs f, DKC1_ΔL37 iPSC clone 1 and g, DKC1_ΔL37_TT iPSC at p22. White arrows, SFEs. High magnification, inset. h, Quantification of SFEs per metaphase. i, Western blot for p53 and p21 at p9 and p36 in DKC1_ΔL37 iPSCs. DOX, doxorubicin treated. Tubulin, loading control.

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