Association of telomere instability with senescence of porcine cells

Abstract Background Telomeres are essential for the maintenance of genomic stability, and telomere dysfunction leads to cellular senescence, carcinogenesis, aging, and age-related diseases in humans. Pigs have become increasingly important large animal models for preclinical tests and study of human diseases, and also may provide xeno-transplantation sources. Thus far, Southern blot analysis has been used to estimate average telomere lengths in pigs. Telomere quantitative fluorescence in situ hybridization (Q-FISH), however, can reveal status of individual telomeres in fewer cells, in addition to quantifying relative telomere lengths, and has been commonly used for study of telomere function of mouse and human cells. We attempted to investigate telomere characteristics of porcine cells using telomere Q-FISH method. Results The average telomere lengths in porcine cells measured by Q-FISH correlated with those of quantitative real-time PCR method (qPCR) or telomere restriction fragments (TRFs) by Southern blot analysis. Unexpectedly, we found that porcine cells exhibited high incidence of telomere doublets revealed by Q-FISH method, coincided with increased frequency of cellular senescence. Also, telomeres shortened during subculture of various porcine primary cell types. Interestingly, the high frequency of porcine telomere doublets and telomere loss was associated with telomere dysfunction-induced foci (TIFs). The incidence of TIFs, telomere doublets and telomere loss increased with telomere shortening and cellular senescence during subculture. Conclusion Q-FISH method using telomere PNA probe is particularly useful for characterization of porcine telomeres. Porcine cells exhibit high frequency of telomere instability and are susceptible to telomere damage and replicative senescence.</p

Dual-sized hollow particle incorporated fibroin thermal insulating coatings on catheter for cerebral therapeutic hypothermia

Selective endovascular hypothermia has been used to provide cooling-induced cerebral neuroprotection, but current catheters do not support thermally-insulated transfer of cold infusate, which results in an increased exit temperature, causes hemodilution, and limits its cooling efficiency. Herein, air-sprayed fibroin/silica-based coatings combined with chemical vapor deposited parylene-C capping film was prepared on catheter. This coating features in dual-sized-hollow-microparticle incorporated structures with low thermal conductivity. The infusate exit temperature is tunable by adjusting the coating thickness and infusion rate. No peeling or cracking was observed on the coatings under bending and rotational scenarios in the vascular models. Its efficiency was verified in a swine model, and the outlet temperature of coated catheter (75 μm thickness) was 1.8–2.0 °C lower than that of the uncoated one. This pioneering work on catheter thermal insulation coatings may facilitate the clinical translation of selective endovascular hypothermia for neuroprotection in patients with acute ischemic stroke

Telomere Reprogramming and Maintenance in Porcine iPS Cells

<div><p>Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate <i>in vivo</i> by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, 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, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.</p></div

Telomeric circles (t-circles) detected in porcine cells or tissues by neutral-neutral two dimension (2D) gel-electrophoresis.

<p>Human embryonic fibroblasts (HEF) and U2OS served as controls. White arrows, t-circles. Percentage of t-circles is shown at the right hand corner on top of each image for relevant cells or tissues. P, passage.</p

Telomeric doublets following porcine iPS generation.

<p>(A–C) Incidence of telomeric doublets increased in iPS cell lines compared with primary cells, PFXP4, NMP4 and SWFP8 respectively. *, p<0.05; **, p<0.01. (D) Frequency of telomere doublets decreased in 68P9, 102P10 and KSR4P9 with exo-genes silencing or partial silencing, compared with progenitor LFFP5. (E) Frequency of telomere doublets increased in LPPD2P10 induced by culture under low oxygen and small molecules, compared with PEF. ** p<0.01. (F) Representative images of telomere FISH and telomere doublets (exampled by red arrows). Blue, chromosomes stained with DAPI; Green, telomeres labeled with PNA probes.</p