Telomere Reprogramming Mechanism During Induced Pluripotent Stem Cell Generation

Induced pluripotent stem cells are derived from somatic cells with defined transcriptional factors. Safty is critical for iPS application. Pigs have become increasingly important large animal models for preclinical tests and the study of human disease, and also may provide xeno-organ transplantation in humans. We attempt to derive procine iPS without exo-factors. These iPS clonies showed falt colony morphology with high nuclear-palsma ratio and enhanced pluripotent gene expression, though they showed abbrent karyotypes. iPS with Oct-4transfection was no teratoma formation thouth there was pluripotent gene expression and normal karyotype. Pig somatic cells with Yannaka factors could form teratoma with three embryonic layers, however, they failed to get any chimaeras. Further experiments are needed to find out the reasons.Telomeres are essential to the maintenance of genomic stability, and their dysfunction is associated with cellular senescence, carcinogenesis, aging, and age-related diseases in humans, however, little is known about pig telomere biology, and only Southern blot analysis that estimates average telomere length has been reported for measurement of pig telomeres thus far. We thought to study pig telomeres using quantitative fluorescence in situ hybridization (Q-FISH) with telomeric peptide nucleic acid (PNA) probe, which can reveal status of individual telomeres, in addition to the average telomere lengths, and this method has been commonly used for study of telomere function in mouse and human cells. Pig telomeres exhibit unique structure and instability, co-incident with increased cellular senescence. Telomere lengths measured by Q-FISH or quantitative real-time PCR methods (qPCR) are correlated with Telomere Restriction Fragments (TRFs) by the Southern blot method. As expected, telomeres shorten during the subculture of various pig primary cells. Notably, telomere doublets identified by Q-FISH, in association with telomere dysfunction-induced foci (TIFs), are found at high frequency in pig primary cells, and their frequency increases with cellular senescence during subculture. The incidence of telomere loss and the expression levels of genes associated with senescence, p53/p21, elevate with telomere damage and shortening. These data show that Q-FISH method using telomere PNA probe is particularly useful for studies of porcine cells, as it can estimate telomere lengths as well as telomere function of individual chromosomes, and suggest that porcine cells are susceptible to telomere damage and replicative senescence. These findings may have implications in potential preclinical tests and xeno-transplantation using pigs.Telomeres generally are composed of various TTAGGG repeats at the chromosome ends, protecting chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences also are found at intrachromosomal sites, named as interstitial telomeric sequences (ITSs). Increased ITSs are implicated in chromosomal instability and found in cancer cells. We attempted to further characterize ITSs and their regulation using pig cells, because vertebrate telomere sequences TTAGGG (vITSs) are defined to localize at the centromeric region of only one chromosome6in pigs, to minimize complexity. Surprisingly, plant telomere sequences of TTTAGGG (bITSs) also are found to localize with vertebrate telomere sequences TTAGGG (vITSs) at the pericentromeric regions of chromosome6by telomere fluorescence in situ hybridization (FISH) and comparison analysis of several species. Further, the length of ITSs is not fixed, but rather very dynamic in different cells and during passages. Average lengths of vITS are highly correlated with those of terminal telomeres (TTS). Notably, lengths of pig ITSs are closely associated with expression levels of telomerase gene TERT. Also, pig ITSs show high incidence of telomere doublets and telomere sister chromatid exchange, suggesting that a telomerase-independent mechanism may involve in ITS maintenance and dynamics. Together, pig cells maintain conservatory telomere sequences at ITSs from plants to vertebrate animals. Further understanding of function and regulation of ITSs may provide new clues for evolution, chromosomal instability, tumorigenesis and anti-cancer therapy.Sufficient telomere reprogramming and silencing of exogenous genes are critical for induction and self-renewal of induced pluripotent stem cells (iPS cells). Porcine iPS cells have been generated, but shown to sustain active exogenes in general, unlike mouse and human iPS cells. We compared activity of exogenes, telomerase levels, telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. We show that insufficient telomerase and incomplete telomere reprogramming and/or telomere maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, few porcine iPS cells with exogenous gene silencing or partial silence exhibit telomere shortening with increasing passages, coincided with reactivation of the exogenes. These iPS cells also show insufficient activation of endogenous pluripotent genes and telomerase genes. Moreover, frequencies of telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells. Notably, the iPS cells with sustained activation of exogenes and maintenance of telomeres show higher pluripotency and reduced telomere damage, whereas low expression of exogenes and telomerase genes increased DNA damage, telomere shortening, and chromosomal instability. We suggest that failed telomere maintenance may contribute to reactivation of exogenes and DNA and telomere damage in porcine iPS cells. Further dissection of these pathways may help deeper understanding of telomere reprogramming and maintenance in porcine iPS cells, and improve the quality of porcine iPS cells.