Abstract
Abstract 51
Telomeres are ribonucleoprotein structures located at the end of linear chromosomes that serve to maintain genomic integrity and cellular proliferative capacity. In highly proliferative cells, the enzyme complex telomerase is responsible for the maintenance and elongation of telomeres, as the process of DNA replication inherently results in loss of terminal nucleotides. Critically short telomeres and deficiency in telomerase activity are etiologic contributors in the bone marrow failure syndromes, idiopathic pulmonary fibrosis, and liver cirrhosis. Although dysfunctional telomere machinery clearly is pathogenic in humans, important clinical features such as highly variable penetrance of phenotype and organ specificity are not well understood. A major obstacle in the investigation of these diseases is the lack of primary tissue, especially early in the clinical course. Direct reprogramming of somatic cells to a pluripotent phenotype (induced pluripotent stem cells, iPSc) by forced expression of a set of defined transcription factors may allow investigation of these phenotypes and specificity in a patient-specific manner. One hallmark of all pluripotent cells, embryonic stem cells and iPSc, is the maintenance of telomere length, most likely due to upregulation of telomerase. Thus, these cells are good candidates through which to directly investigate the effect of loss-of-function mutations within the telomerase complex.
Using forced expression of the reprogramming factors Oct4, Sox2, Klf4, and c-myc via retroviral (single transgenes) or lentiviral (polycistronic) gene transfer, we derived multiple iPSc lines from dermal fibroblasts of patients harboring heterozygous loss-of-function mutations in the telomerase genes TERT([R1084P], [R889X]) and TERC([-58C>G]), as well as from healthy subjects. These mutations were shown in vitro to reduce telomerase activity of the mutant allele. Generated iPSc lines morphologically resembled human ES cells, expressed endogenous pluripotency markers (such as TRA 1–60, TRA 1–81, SSEA4, NANOG, OCT4), and showed a similar mRNA expression profile as compared to embryonic stem cells in microarray analysis. Over multiple passages (currently up to 40), iPSc retained their self-renewal capacity and formed teratomas in immune-compromised NSG mice.
We randomly chose three iPSc lines from each patient and healthy controls to study telomere dynamics. Telomerase-mutant iPS cells elongated their telomeres during the first 10 passages compared to parental (telomerase negative) fibroblasts, as determined by quantitative real-time PCR and Southern blot. However, telomere elongation was significantly less than in iPSc derived from healthy individuals (p=0.003). Moreover, the pattern and extent of elongation varied among different iPSc lines harboring the same mutation. Telomerase mRNA expression was lower in telomerase-mutant iPS cells than in healthy controls. Additionally, telomerase activity, measured by standard TRAP assay, in early telomerase-mutant iPS cells was reduced relative to control iPSc, but later passage cells tended to have similar activity, suggesting a passage-effect on telomerase activity levels.
In conclusion, iPSc can be derived from human telomerase-deficient cells. These cells elongate telomeres to a lesser extent than iPSc from healthy controls, indicating that functional telomerase is the main mechanism of telomere elongation in iPSc. Therefore these cells could be valuable tools in the study of human telomerase deficiencies. Unlike previous studies (Agarwal et al., Nature 2010) investigating iPSc from patients with X-linked dyskeratosis congenita (in which a loss-of-function mutation within DKC1 results in short telomeres), we did not observe significant upregulation of TERC as a compensating mechanism during reprogramming in our telomerease-mutant iPSc. Furthermore, in contrast to the DKC1-mutant iPSc described by Batista et al. (Nature 2011), TERT/TERC mutant iPSc did not show signs of impaired proliferation or self-renewal capacity in long-term culture, likely reflecting clinical differences among patients with TERT/TERC and DKC1 mutations. However, elongation patterns and telomerase activity levels are heterogeneous among clones and passages, indicating the importance of kinetic studies and sample size when studying telomere dynamics in iPSc.
Disclosures:
Dunbar: ASH: Honoraria.
Peculiarities of Yeasts and Human Telomerase RNAs Processing
