Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts

In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53

Improved Membrane Proteomics Coverage of Human Embryonic Stem Cells by Peptide IPG-IEF

Protein biomarkers are fundamental tools for the characterization of stem cells and for tracking their differentiation and maturation down developmental lineages. Technology development allowing increased coverage of difficult cellular proteomes should allow for the discovery of new and novel membrane protein biomarkers for use by the stem cell research community. The amphipathic and highly hydrophobic nature and relative low abundance of many membrane proteins present significant analytical challenges. These difficulties are amplified when the source material (tissue or cells) is only available in limited quantities (e.g., embryonic stem cells). Recent advances in enrichment for purer membrane fractions, the enzymatic and chemical digestion of membrane proteins in the presence of solvents or chaotropes, and the use of "shotgun" proteomics methodologies have gradually resulted in increased membrane proteome coverage with numbers of predicted integral membrane proteins now in excess of 1000 being routinely reported. We have recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients (peptide IPG-IEF) followed by reversed phase chromatography and tandem MS to increase membrane proteome coverage. This study looked at achieving a similar level of membrane proteome coverage using modifications to reported methodologies while restricting the number of characterized human embryonic stem cells to 107 cells. Two-thousand two-hundred and ninety-two (2292) nonredundant proteins were identified with two or more high accuracy peptide matches from 260 μg of a human embryonic stem cell membrane enriched fraction with a false discovery rate of 0.32%. Gene Ontology (GO) mapping predicted 1279 (44.9%) of this list to be membrane proteins of which 395 proteins were predicted to be derived from the plasma membrane compartment. The TMHMM algorithm predicted 904 integral membrane proteins with up to 16 transmembrane helices. Collectively, we assert that the substantial membrane proteome coverage achieved using these procedures will enable rapid advances in the identification and quantitation of novel membrane proteins as markers of differentiation status and/or genetic mutation from relatively low numbers of cultured embryonic stem cells.8 page(s

Derivation of three new human embryonic stem cell lines

Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines