The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance.

Telomeres are linear guanine-rich DNA structures at the ends of chromosomes. The length of telomeric DNA is actively regulated by a number of mechanisms in highly proliferative cells such as germ cells, cancer cells, and pluripotent stem cells. Telomeric DNA is synthesized by way of the ribonucleoprotein called telomerase containing a reverse transcriptase (TERT) subunit and RNA component (TERC). TERT is highly conserved across species and ubiquitously present in their respective pluripotent cells. Recent studies have uncovered intricate associations between telomeres and the self-renewal and differentiation properties of pluripotent stem cells. Interestingly, the past decade's work indicates that the TERT subunit also has the capacity to modulate mitochondrial function, to remodel chromatin structure, and to participate in key signaling pathways such as the Wnt/ß-catenin pathway. Many of these non-canonical functions do not require TERT's catalytic activity, which hints at possible functions for the extensive number of alternatively spliced TERT isoforms that are highly expressed in pluripotent stem cells. In this review, some of the established and potential routes of pluripotency induction and maintenance are highlighted from the perspectives of telomere maintenance, known TERT isoform functions and their complex regulation.

Protein deposition on a lathe-cut silicone hydrogel contact lens material.

PURPOSE: To determine the quantity of total protein, total lysozyme, and the conformational state of lysozyme deposited on a novel, lathe-cut silicone hydrogel (SiHy) contact lens material (sifilcon A) after 3 months of wear. METHODS: Twenty-four subjects completed a prospective, bilateral, daily-wear, 9-month clinical evaluation in which the subjects were fitted with a novel, custom-made, lathe-cut SiHy lens material. The lenses were worn for three consecutive 3-month periods, with lenses being replaced after each period of wear. After 3 months of wear, the lenses from the left eye were collected and assessed for protein analysis. The total protein deposited on the lenses was determined by a modified Bradford assay, total lysozyme using Western blotting and the lysozyme activity was determined using a modified micrococcal assay. RESULTS: The total protein recovered from the custom-made lenses was 5.3 +/- 2.3 microg/lens and the total lysozyme was 2.4 +/- 1.2 microg/lens. The denatured lysozyme found on the lenses was 1.9 +/- 1.0 microg/lens and the percentage of lysozyme denatured was 80 +/- 10%. CONCLUSIONS: Even after 3 months of wear, the quantity of protein and the conformational state of lysozyme deposited on these novel lens materials was very similar to that found on similar surface-coated SiHy lenses after 2 to 4 weeks of wear. These results indicate that extended use of the sifilcon A material is not deleterious in terms of the quantity and quality of protein deposited on the lens.

Imaging protein deposits on contact lens materials.

PURPOSE: The majority of studies investigating protein deposition on contact lens materials require that the deposit of interest be removed, potentially resulting in erroneous results if some proteins are not removed adequately. The purpose of this study was to investigate the use of in situ imaging methods to examine protein deposition on conventional poly(2-hydroxyethyl methacrylate) (polyHEMA)-based and silicone hydrogel contact lens materials. METHODS: Six silicone hydrogel and five polyHEMA-based hydrogel contact lens materials were examined by Atomic Force Microscopy (AFM) and/or Scanning Electron Microscopy (SEM) techniques, after being deposited with proteins in an in vitro model. AFM studies examined lenses deposited solely with lysozyme at approximate physiological concentrations and SEM studies were conducted on lenses exposed to a dilute mixture of lysozyme and albumin-conjugated gold spheres. RESULTS: AFM studies demonstrated that the lens materials had markedly differing surface topographies. SEM results showed that galyfilcon A and balafilcon A lenses deposited both lysozyme and albumin in relatively large aggregates, as compared with lotrafilcon A and B, in which the proteins were deposited in a more evenly spread, monolayer formation. Polymacon lenses deposited more protein than any of the silicone hydrogel materials and much of the protein was aggregated together. AFM data indicated that balafilcon A, lotrafilcon A and polymacon deposited lysozyme in a similar manner, with very little lysozyme being deposited in discrete areas. Galyfilcon A behaved very differently, with the lysozyme exhibiting both aggregates as well as string-like formations over the lens surface. CONCLUSIONS: Imaging techniques that allow proteins to be examined in situ show much promise for determining the extent and physical characterization of protein on contact lens materials. These techniques indicate that the pattern of deposition of proteins onto silicone hydrogel contact lens materials differs between materials, depending upon their bulk and surface composition.

Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δß splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naïve Pluripotency.

Naïve and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naïve counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naïve pluripotent state using LIF and inhibitors of glycogen synthase kinase 3ß and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naïve pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIF-FGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naïve PSCs.

Microenvironmental regulation of telomerase isoforms in human embryonic stem cells.

Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δß hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival, self-renewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms.

Derivation and culture of canine embryonic stem cells.

The derivation of canine embryonic stem cells (cESCs) represents a significant achievement and opens the door to further stem cell research and therapies in the dog. Canines share a common environment with humans and exhibit a host of genetic diseases, many of which have human parallels. Thus, the canine model presents unique advantages over other currently used organisms to help develop stem cell therapies in humans. To reveal the therapeutic potential of cESCs further basic research on the molecular mechanisms controlling their pluripotency and self-renewal characteristics is needed. Herein, we present the methods for derivation and culture of cESCs. Following collection of the canine blastocyst, two derivation methods are presented; immunodissection and whole blastocyst explant. These two methods lead to cESCs differing in morphology and subculture techniques. Additional protocols for subculture of established lines, feeder-free culture, and cryopreservation protocols are also described.

Suppression of the imprinted gene NNAT and X-chromosome gene activation in isogenic human iPS cells.

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem, we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102-154 genes (>2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore, a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs, isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes.