Canine pluripotent stem cells: Are they ready for clinical applications?

The derivation of canine embryonic stem cells and generation of canine-induced pluripotent stem cells are significant achievements that have unlocked the potential for developing novel cell-based disease models, drug discovery platforms, and transplantation therapies in the dog. A progression from concept to cure in this clinically relevant companion animal will not only help our canine patients but also help advance human regenerative medicine. Nevertheless, many issues remain to be resolved before pluripotent cells can be used clinically in a safe and reproducible manner

Canine pluripotent stem cells: Are they ready for clinical applications?

The derivation of canine embryonic stem cells and generation of canine-induced pluripotent stem cells are significant achievements that have unlocked the potential for developing novel cell-based disease models, drug discovery platforms, and transplantation therapies in the dog. A progression from concept to cure in this clinically relevant companion animal will not only help our canine patients but also help advance human regenerative medicine. Nevertheless, many issues remain to be resolved before pluripotent cells can be used clinically in a safe and reproducible manner

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\u27s 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/β-catenin pathway. Many of these non-canonical functions do not require TERT\u27s 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

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 (\u3e2 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. © 2011 Teichroeb et al

The p66\u3csup\u3eShc\u3c/sup\u3e adaptor protein controls oxidative stress response in early bovine embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos. © 2014 Betts et al

The long and short of it: The role of telomeres in fetal origins of adult disease

Placental insufficiency, maternal malnutrition, and other causes of intrauterine growth restriction (IUGR) can significantly affect short-term growth and long-term health. Following IUGR, there is an increased risk for cardiovascular disease and Type 2 Diabetes. The etiology of these diseases is beginning to be elucidated, and premature aging or cellular senescence through increased oxidative stress and DNA damage to telomeric ends may be initiators of these disease processes. This paper will explore the areas where telomere and telomerase biology can have significant effects on various tissues in the body in IUGR outcomes. © 2012 Stephanie E. Hallows et al

Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent states

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways, including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate, however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg Effect. Both isoforms catalyze the final, rate limiting step of glycolysis, generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm, but also accumulate in differential subnuclear regions of mESC, mEpiLCs, and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 changes during the transition from mESCs, mEpiLCs, and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson\u27s correlation coefficient and Manders\u27s overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency

P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture.

STUDY QUESTION: Do high oxygen tension and high glucose concentrations dysregulate p66Shc (Src homologous-collagen homologue adaptor protein) expression during mouse preimplantation embryo culture? SUMMARY ANSWER: Compared with mouse blastocysts in vivo, P66Shc mRNA and protein levels in blastocysts maintained in vitro increased under high oxygen tension (21%), but not high glucose concentration. WHAT IS KNOWN ALREADY: Growth in culture adversely impacts preimplantation embryo development and alters the expression levels of the oxidative stress adaptor protein p66Shc, but it is not known if p66Shc expression is linked to metabolic changes observed in cultured embryos. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We used a standard wild-type CD1 mouse model of preimplantation embryo development and embryo culture with different atmospheric oxygen tension and glucose media concentrations. Changes to p66Shc expression in mouse blastocysts were measured using quantitative RT-PCR, immunoblotting and immunofluorescence followed by confocal microscopy. Changes to oxidative phosphorylation metabolism were measured by total ATP content and superoxide production. Statistical analyses were performed on a minimum of three experimental replicates using Students\u27 t-test or one-way ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE: P66Shc is basally expressed during in vivo mouse preimplantation development. Within in vivo blastocysts, p66Shc is primarily localized to the cell periphery of the trophectoderm. Blastocysts cultured under atmospheric oxygen levels have significantly increased p66Shc mRNA transcript and protein abundances compared to in vivo controls (P \u3c 0.05). However, the ratio of phosphorylated serine 36 (S36) p66Shc to total p66Shc decreased in culture regardless of O2 atmosphere used, supporting a shift in the mitochondrial fraction of p66Shc. Total p66Shc localized to the cell periphery of the blastocyst trophectoderm and phosphorylated S36 p66Shc displayed nuclear and cytoplasmic immunoreactivity, suggesting distinct compartmentalization of phosphorylated S36 p66Shc and the remaining p66Shc fraction. Glucose concentration in the culture medium did not significantly change p66Shc mRNA or protein abundance or its localization. Blastocysts cultured under low or high oxygen conditions exhibited significantly decreased cellular ATP and increased superoxide production compared to in vivo derived embryos (P \u3c 0.05). LIMITATIONS/REASONS FOR CAUTION: This study associates embryonic p66Shc expression levels with metabolic abnormalities but does not directly implicate p66Shc in metabolic changes. Additionally, we used one formulation of embryo culture medium that differs from that used in other mouse model studies and from clinical media used to support human blastocyst development. Our findings may, therefore, be limited to this media, or may be a species-specific phenomenon. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to show distinct immunolocalization of p66Shc to the trophectoderm of mouse blastocysts and that its levels are abnormally increased in embryos exposed to culture conditions. Changes in p66Shc expression and/or localization could possibly serve as a molecular marker of embryo viability for clinical applications. The outcomes provide insight into the potential metabolic role of p66Shc. Metabolic anomalies are induced even under the current optimal culture conditions, which could negatively impact trophectoderm and placental development. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Canadian Institutes of Health Research (CIHR) operating funds, Ontario Graduate Scholarship (OGS). There are no competing interests

Knockdown of p66Shc Alters Lineage-Associated Transcription Factor Expression in Mouse Blastocysts

The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is upregulated at the blastocyst stage. Our objective was to determine the biological function of p66Shc during mouse blastocyst development. In this study, we demonstrate that a reduced p66Shc transcript abundance following its short interfering RNA (siRNA)-mediated knockdown alters the spatiotemporal expression of cell lineage-associated transcription factors in the inner cell mass (ICM) of the mouse blastocyst. P66Shc knockdown blastocysts restrict OCT3/4 earlier to the inner cells of the early blastocyst and have ICMs containing significantly higher OCT3/4 levels, more GATA4-positive cells, and fewer NANOG-positive cells. P66Shc knockdown blastocysts also show a significantly reduced ability to form ICM-derived outgrowths when explanted in vitro. The increase in cells expressing primitive endoderm markers may be due to increased ERK1/2 activity, as it is reversed by ERK1/2 inhibition. These results suggest that p66Shc may regulate the relative abundance and timing of lineage-associated transcription factor expression in the blastocyst ICM