Challenges and possible clinical applications of human embryonic stem cell research

Human embryonic stem cells (hESC) are harvested from the inner cell mass of the pre-implantation embryo and possess several unique characteristics. First, they are self-renewing, meaning they can grow indefinitely in an appropriate culture environment and secondly, they are pluripotent, which means they have the potential to become nearly every cell of the human body. Consequently, hESC offer a unique insight into basic human development in vitro, allow better understanding of the genetic and molecular controls of these processes, and are of pharmaceutical interest to test or develop new drugs. The most exciting and high-profile potential application of hESC research is the possibility that such cells can be used for regenerative medicine. Still, several obstacles have to be overcome before clinical applications can be considered: (i) xeno-free derivation and culture of hESC is necessary; (ii) hESC should be safe after transplantation and (iii) their identity and behaviour should be well-known

Bta-miR-10b secreted by bovine embryos negatively impacts preimplantation embryo quality

In a previous study, we found miR-10b to be more abundant in a conditioned culture medium of degenerate embryos compared to that of blastocysts. Here, we show that miR-10b mimics added to the culture medium can be taken up by embryos. This uptake results in an increase in embryonic cell apoptosis and aberrant expression of DNA methyltransferases (DNMTs). Using several algorithms, Homeobox Al (HOXA1) was identified as one of the potential miR-10b target genes and dual-luciferase assay confirmed HOXA1 as a direct target of miR-10b. Microinjection of si-HOXA1 into embryos also resulted in an increase in embryonic cell apoptosis and downregulation of DNMTs. Cell progression analysis using Madin-Darby bovine kidney cells (MDBKs) showed that miR-10b overexpression and HOXA1 knockdown results in suppressed cell cycle progression and decreased cell viability. Overall, this work demonstrates that miR-10b negatively influences embryo quality and might do this through targeting HOXA1 and/or influencing DNA methylation

Pan-cancer pharmacogenetics : targeted sequencing panels or exome sequencing?

Aim:This study provides clinicians and researchers with an informed choice between current commercially available targeted sequencing panels and exome sequencing panels in the context of pan-cancer pharmacogenetics.Materials & methods:Nine contemporary commercially available targeted pan-cancer panels and the xGen Exome Research Panel v2 were investigated to determine to what extent they cover the pharmacogenetic variant-drug interactions in five available cancer knowledgebases, and the driver mutations and fusion genes in The Cancer Genome Atlas.Results:xGen Exome Research Panel v2 and TrueSight Oncology 500 target 71.0 and 68.9% of the pharmacogenetic interactions in the available knowledgebases, and 93.7 and 86.0% of the driver mutations in The Cancer Genome Atlas, respectively. All other studied panels target lower percentages.Conclusion:Exome sequencing outperforms pan-cancer targeted sequencing panels in terms of covered cancer pharmacogenetic variant-drug interactions and pharmacogenetic cancer variants

Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

Background: Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced. Results: In this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up. Conclusions: Our results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments

Diagnosis and treatment of male infertility-related fertilization failure

Infertility affects approximately 15% of reproductive-aged couples worldwide, of which up to 30% of the cases are caused by male factors alone. The origin of male infertility is mostly attributed to sperm abnormalities, of which many are caused by genetic defects. The development of intracytoplasmic sperm injection (ICSI) has helped to circumvent most male infertility conditions. However, there is still a challenging group of infertile males whose sperm, although having normal sperm parameters, are unable to activate the oocyte, even after ICSI treatment. While ICSI generally allows fertilization rates of 70 to 80%, total fertilization failure (FF) still occurs in 1 to 3% of ICSI cycles. Phospholipase C zeta (PLC zeta) has been demonstrated to be a critical sperm oocyte activating factor (SOAF) and the absence, reduced, or altered forms of PLC zeta have been shown to cause male infertility-related FF. The purpose of this review is to (i) summarize the current knowledge on PLC zeta as the critical sperm factor for successful fertilization, as well as to discuss the existence of alternative sperm-induced oocyte activation mechanisms, (ii) describe the diagnostic tests available to determine the cause of FF, and (iii) summarize the beneficial effect of assisted oocyte activation (AOA) to overcome FF

Treatment of human embryos with the TGFβ inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation

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