Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs

The Journal of clinical investigation (Impact Factor: 13.22). 05/2013; 123(5). DOI: 10.1172/JCI67146


Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations' impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

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    • "Telomere lengths of peripheral blood leukocytes in the patient and his parents were measured by qPCR as described by Cawthon [24,25], with several modifications [26], using the Qiagility robot and the Rotor-Gene Q (Qiagen). Telomere lengths of peripheral blood leukocytes in the MDS patient, his father, son and siblings, as well as of the patient’s sperm cells were also measured by PCR at Umeå University as described previously [27]. "
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    ABSTRACT: Background Telomeres are repeated sequences (the hexanucleotide TTAGGG in vertebrates) located at chromosome ends of eukaryotes, protecting DNA from end joining or degradation. Telomeres become shorter with each cell cycle, but telomerase, a ribonucleoprotein complex, alleviates this attrition. The telomerase RNA component (TERC) is an essential element of telomerase, serving as a template for telomere elongation. The H/ACA domain of TERC is indispensable for telomere biogenesis. Mutations in the telomerase components allow accelerated telomere loss, resulting in various disease manifestations, including bone marrow failure. To date, this is the first detailed report of an H-box mutation in TERC that is related to human disease. Case presentation A 26-year-old man with myelodysplastic syndrome (MDS) had very short telomeres. Sequencing identified a single heterozygous mutation in the H box of the patient’s TERC gene. The same mutation was also present in his father and his son, demonstrating that it was germline in origin. The telomere length in the father’s blood was shorter compared to age-matched healthy controls, while it was normal in the son and also in the sperm cells of the patient. In vitro experiments suggested that the mutation was responsible for the telomere shortening in the patient’s leukocytes and contributed to the pathogenesis of bone marrow failure in our patient. Conclusion We analyzed a mutation (A377G) in the H box of TERC in a young MDS patient who had significantly short-for-age telomeres. As telomeres protect chromosomes from instability, it is highly plausible that this genetic lesion was responsible for the patient’s hematological manifestations, including marrow failure and aneuploidy in the hematopoietic stem cell compartment.
    BMC Medical Genetics 06/2014; 15(1):68. DOI:10.1186/1471-2350-15-68 · 2.08 Impact Factor
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    • "Moreover, we were able to observe various behavior of the Ph+ iPSCs obtained from the same CML patients, in terms of BCR-ABL1 pattern, sensitivity to imatinib and hematopoietic differentiation. We cannot rule out that these variations could result from heterogeneity of iPSCs reprogramming, as recently published by Winkler et al [22]. To assess specific heterogeneity of hematopoietic differentiation from the CML-iPSC obtained from the same CML patient, it will be necessary to study more control iPSC and CML-derived iPSC clones. "
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    ABSTRACT: Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34(+) blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.
    PLoS ONE 08/2013; 8(8):e71596. DOI:10.1371/journal.pone.0071596 · 3.23 Impact Factor
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    • "The iPSCs from dyskeratosis congenita patients displayed restored telomere elongation, suggesting that reprogramming may provide a beneficial therapeutic strategy in the future (Agarwal et al. 2010). On the other hand, limited telomere elongation and impaired hematopoietic differentiation have been observed in iPSCs harboring TERC and TERT mutations, thus reflecting the clinical phenotypes in patients, and supporting the utility of the iPSCs for disease modeling, at least for dyskeratosis congenita (Winkler et al. 2013). "
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    ABSTRACT: The discovery of induced pluripotent stem cells (iPSCs) has the potential to revolutionize the field of regenerative medicine. In the past few years, iPSCs have been the subject of intensive research towards their application in disease modeling and drug screening. In the future, these cells may be applied in cell therapy to replace or regenerate tissues by autologous transplantation. However, two major hurdles need to be resolved in order to reach the later goal: the low reprogramming efficiency and the safety risks, such as the integration of foreign DNA into the genome of the cells and the tumor formation potential arising from transplantation of residual undifferentiated cells. Recently, aging emerged as one of the barriers that accounts, at least in part, for the low reprogramming efficiency of bona fide iPSCs. Here, we review the molecular pathways linking aging and reprogramming along with the unanswered questions in the field. We discuss whether reprogramming rejuvenates the molecular and cellular features associated with age, and present the recent advances with iPSC-based models, contributing to our understanding of physiological and premature aging.
    Biogerontology 08/2013; 14(6). DOI:10.1007/s10522-013-9455-2 · 3.29 Impact Factor
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