Abstract
Mammalian telomere lengths are primarily regulated by telomerase, consisting of a reverse transcriptase protein (TERT) and an RNA subunit (TERC). We previously reported the generation of mouse Terc+/− and Terc−/− embryonic stem cells (ntESCs) by somatic cell nuclear transfer. In the present work, we investigated the germ layer development competence of Terc−/−, Terc+/− and wild-type (Terc+/+) ntESCs. The telomere lengths are longest in wild-type but shortest in Terc−/− ntESCs, and correlate reversely with the population doubling time. Interestingly, while in vitro embryoid body (EB) differentiation assay reveals EB size difference among ntESCs of different genotypes, the more stringent in vivo teratoma assay demonstrates that Terc−/− ntESCs are severely defective in differentiating into the mesodermal lineage cartilage. Consistently, in a directed in vitro chondrocyte differentiation assay, the Terc−/− cells failed in forming Collagen II expressing cells. These findings underscore the significance in maintaining proper telomere lengths in stem cells and their derivatives for regenerative medicine.
Highlights
The ends of eukaryotic chromosomes are capped with telomeres, copies of a hexamer repeat sequence, and associated proteins, which play central roles in stabilizing the ends of chromosomes during replication
After establishing embryonic stem cells (ESCs) by using tail tip fibroblast cells for somatic cell nuclear transfer (SCNT), we demonstrated that Terc+/− nuclear transfer embryonic stem cells (ntESCs), whose telomeres were robustly elongated after SCNT and the ESC derivation processes, supported term pup development in the tetraploid complementation assay; whereas Terc−/− ntESCs, whose telomeres remained critically short, failed to do so [22]
The telomere lengths, as measured by the Southern blot (Figure 1G) and T/S ratio (Figure 1H), were Terc dependent; the longest in the wild-type, followed by heterozygous knockout, and the shortest in the homozygous knockout. These results show that the expression of conventional pluripotency markers was not sensitive to telomerase insufficiency, while telomere lengths and the cell grow rate were
Summary
The ends of eukaryotic chromosomes are capped with telomeres, copies of a hexamer repeat sequence, and associated proteins, which play central roles in stabilizing the ends of chromosomes during replication. Loss of function mutation in genes that encode telomerase components, such as TERT or TERC, cause premature aging and age-related diseases, including dyskeratosis congenital (DC), aplastic anemia, and idiopathic pulmonary fibrosis (IPF), which are collectively referred to as “telomere syndromes” to reflect the short and dysfunctional telomeres commonly found in these patients’ cells [6,7,8]. Tert−/− and Terc−/− mice displayed premature ageing phenotypes such as grey hair, wrinkled skins, as well as dramatically decreased fertility, indicating that a threshold short telomere length, once it is reached, would perturb normal functions of various types of cells [11,12,13,14,15,16]
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