No abstract

Traditional approaches to characterize stem cell differentiation are time-consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) - both considered as non-invasive techniques - are applied to detect the biochemical and biophysical properties of trophoblast derived stem-like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed. Monitoring trophoblast cells differentiation.

The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx platform. The average depth of sequencing coverage was 14,611 for IVV and 17,068 for PA. Quantitative analysis of the methylation profiles of both input samples for each genomic locus showed distinct differences in methylation profiles between IVV and PA samples for six of the target loci, and subtle differences in four loci. It was concluded that high throughput sequencing technologies can be effectively applied to provide a powerful, cost-effective approach to targeted DNA methylation analysis of embryonic and other reproductive tissues.

Porcine trophoblast-derived stem-like cells grown into serum medium start to differentiate and become senescent within 30 days. However, trophoblast-derived cells, cultured in vitro in a defined and non-serum medium, have the regenerative properties, such as indefinite passage and foreign DNA receptivity, similar to stem cells. To evaluate the biochemical, biophysical, and genetic changes of the terminal differentiation of trophoblast derived cells, Raman microspectroscopy, atomic force microscopy, and qPCR were applied. It was found that Raman spectral intensities of characteristic peaks, cell morphology, and Young's modulus can be used to distinguish differentiated and undifferentiated trophoblast cells. In addition, 17 cytoskeleton and extracellular matrix-related genes were significantly impacted by medium type (non-serum versus serum). Our findings suggest that Raman microspectroscopy and atomic force microscopy-both considered as label-free, non-invasive techniques-can be applied to distinguish differentiated trophoblast cells, and cellular biochemical information and biophysical properties can be indicative of cellular differences during cell differentiation. In addition, most of cytoskeleton-related genes exhibit similar pattern to that of Young's modulus during trophoblast cell differentiation, indicating the potential connection between cytoskeleton-related genes and cellular stiffness.

The goal of this study was to evaluate global levels of a variety of histone modifications at different lysine (K) residues on Histone 3 (H3) within the chromatin of porcine germinal vesicle (GV)-stage oocytes that were aspirated from follicles of different sizes. We hypothesised that we would see evidence of a transition from open, transcriptionally active chromatin (in oocytes from smaller, growing follicles) to more closed, transcriptionally silent chromatin associated with fully grown oocytes (aspirated from large, preovulatory follicles). Cumulus-enclosed oocytes were aspirated from small (<3 mm) or large (>7 mm) follicles from abattoir-derived pig ovaries. Oocytes were denuded immediately after aspiration and then immunoprobed with antibodies specific for trimethylated (me3) H3K4, H3K9me3, and H3K27me3. Background-corrected nuclear fluorescence levels for each histone mark were collected from multiple oocytes from each of at least three experimental replicates (aspiration days). Data were subjected to one-way ANOVA with a Bonferroni multiple testing correction to determine whether there were differences in fluorescence intensities in the nuclei (germinal vesicles) of oocytes from small v. large follicles. Oocytes from large follicles displayed more intense nuclear staining for all 3 histone marks: average nuclear H3K4me3 intensity was 31.4% higher (P = 0.0004), H3K9me3 was 70.3% higher (P = 0.0218), and H3K27me3 was 32.0% higher (P = 0.0231) in oocytes from large follicles. An ancillary analysis of the data revealed no effect (P > 0.1) of pubertal status (i.e. whether small and large follicles were aspirated from pre- v. post-pubertal ovaries) on the intensity of nuclear fluorescence for any of the marks evaluated. In continuation, 3 oocytes from both follicle types were collected on each of 6 aspiration days (i.e. 18 individual oocytes from each follicle type), and the mRNA from these were used for an RT-qPCR experiment to detect the relative abundance of transcripts from 21 different genes coding for histone methyltransferase or demethylase enzymes in oocytes from large v. small follicles. Of the 21 genes tested, 5 genes (KDM4C, KDM4D, KDM5B, KDM5C, and SETD7) were not detectable in our individual oocyte samples, but transcripts from 6 of the 16 remaining genes (KDM6A, KMT2B, MLL3, SETD1B, SETDB1, and SUV39H2) were shown to be significantly more abundant in oocytes from large follicles (at least 2-fold greater abundance and P < 0.05). Although our expectation-that histone marks (and related transcripts) would consistently reflect a globally “repressive” chromatin configuration in oocytes from large follicles and a more “open” configuration in oocytes from small follicles-turned out to be untrue, the evidence suggests that the epigenetic constitution of oocytes from small follicles may indeed vary from that of oocytes from large, preovulatory follicles, and this phenomenon warrants further investigation.