Comparative analysis of naive, primed and ground state pluripotency in mouse embryonic stem cells originating from the same genetic background

Mouse embryonic stem cells (mESCs) exist in a naive, primed and ground state of pluripotency. While comparative analyses of these pluripotency states have been reported, the mESCs utilized originated from various genetic backgrounds and were derived in different laboratories. mESC derivation in conventional LIF + serum culture conditions is strain dependent, with different genetic backgrounds potentially affecting subsequent stem cell characteristics. In the present study, we performed a comprehensive characterization of naive, primed and ground state mESCs originating from the same genetic background within our laboratory, by comparing their transcriptional profiles. We showed unique transcriptional profiles for naive, primed and ground state mESCs. While naive and ground state mESCs have more similar but not identical profiles, primed state mESCs show a very distinct profile. We further demonstrate that the differentiation propensity of mESCs to specific germ layers is highly dependent on their respective state of pluripotency

Direct comparison of distinct naive pluripotent states in human embryonic stem cells

Stem cells & developmental biolog

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells

Real-time resampling in Fourier domain optical coherence tomography using a graphics processing unit

Fourier domain optical coherence tomography (FD-OCT) requires either a linear-in-wavenumber spectrometer or a computationally heavy software algorithm to recalibrate the acquired optical signal from wavelength to wavenumber. The first method is sensitive to the position of the prism in the spectrometer, while the second method drastically slows down the system speed when it is implemented on a serially oriented central processing unit. We implement the full resampling process on a commercial graphics processing unit (GPU), distributing the necessary calculations to many stream processors that operate in parallel. A comparison between several recalibration methods is made in terms of performance and image quality. The GPU is also used to accelerate the fast Fourier transform (FFT) and to remove the background noise, thereby achieving full GPU-based signal processing without the need for extra resampling hardware. A display rate of 25 frames/sec is achieved for processed images (1024x1024 pixels) using a line-scan charge-coupled device (CCD) camera operating at 25.6 kHz

Adaptive optics assisted Fourier domain OCT with balanced detection

Two factors are of importance to optical coherence tomography (OCT), resolution and sensitivity. Adaptive optics improves the resolution of a system by correcting for aberrations causing distortions in the wave-front. Balanced detection has been used in time domain OCT systems by removing excess photon noise, however it has not been used in Fourier domain systems, as the cameras used in the spectrometers saturated before excess photon noise becomes a problem. Advances in camera technology mean that this is no longer the case and balanced detection can now be used to improve the signal to noise ratio in a Fourier domain (FD) OCT system. An FD-OCT system, enhanced with adaptive optics, is presented and is used to show the improvement that balanced detection can provide. The signal to noise ratios of single camera detection and balanced detection are assessed and in-vivo retinal images are acquired to demonstrate better image quality when using balance detection

Real-time correction of geometric distortion artefacts in large-volume optical coherence tomography

Large-volume optical coherence tomography (OCT) setups employ scanning mirrors and suffer from geometric distortion artefacts in which the degree of distortion is determined by the maximum angles over which the mirrors rotate. In this note, we describe a straightforward coordinate transformation scheme to correct for these artefacts in three dimensions, creating an alternative to previously reported ray-tracing schemes. We demonstrate that this recalibration procedure can be applied in real time by implementing the proposed algorithm on the graphics card of a standard computer, making it useful for topography applications. The accuracy of the proposed calibration procedure is validated over an imaging volume of 12.35x10.13x2.36 mm3 using optical moire measurements of a highly curved object. © 2013 IOP Publishing Ltd

Full-field thickness distribution of human tympanic membrane obtained with optical coherence tomography

The full-field thickness distribution, three-dimensional surface model and general morphological data of six human tympanic membranes are presented. Cross-sectional images were taken perpendicular through the membranes using a high-resolution optical coherence tomography setup. Five normal membranes and one membrane containing a pathological site are included in this study. The thickness varies strongly across each membrane, and a great deal of inter-specimen variability can be seen in the measurement results, though all membranes show similar features in their respective relative thickness distributions. Mean thickness values across the pars tensa ranged between 79 and 97 μm; all membranes were thinnest in the central region between umbo and annular ring (50-70 μm), and thickness increased steeply over a small distance to approximately 100-120 μm when moving from the central region either towards the peripheral rim of the pars tensa or towards the manubrium. Furthermore, a local thickening was noticed in the antero-inferior quadrant of the membranes, and a strong linear correlation was observed between inferior-posterior length and mean thickness of the membrane. These features were combined into a single three-dimensional model to form an averaged representation of the human tympanic membrane. 3D reconstruction of the pathological tympanic membrane shows a structural atrophy with retraction pocket in the inferior portion of the pars tensa. The change of form at the pathological site of the membrane corresponds well with the decreased thickness values that can be measured there. © 2013 Association for Research in Otolaryngology