Reestablishment of the inactive X chromosome to the ground state through cell fusion-induced reprogramming

The restricted gene expression pattern of a differentiated cell can be reversed by fusion of the somatic cell with a more developmentally potent cell type, such as an embryonic stem (ES) cell. During this reprogramming process, somatic cells obtain most of the characteristics of pluripotent cells. Reactivation of an inactive X chromosome (Xi) is an important epigenetic marker confirming the pluripotent reprogramming of somatic cells. Female somatic cells contain one active X chromosome (Xa) and one Xi, and following the fusion of these cells with male ES cells, the Xi becomes activated, resulting in XaXaXaY fusion hybrid cells. To monitor Xi reactivation, transgenic female neural stem cells (fNSCs) carrying a green fluorescent protein (GFP) reporter gene expressed on the Xa (X-GFP), but not on the Xi, were used for reprogramming. XaXi^GFP NSCs, whose GFP reporter was silenced, were fused with HM1 ES cells (XY) to induce pluripotent reprogramming. The Xi^GFP of NSCs were found to be activated on day 4 post-fusion, indicating reactivation of the Xi. Hybrid cells showed pluripotent cell-specific characteristics cells including inactivation of the NSC marker Nestin, DNA demethylation of Oct4, DNA methylation of Nestin, and reactivation of the Xi. Following differentiation of the (GFP-positive) hybrid cells through embryoid body formation, the proportion of GFP-negative cells was found to be approximately 26?%, indicating that there was random inactivation of one of the three Xas. Here, we showed that the Xi of somatic cells is reprogrammed to the Xa state and that cellular differentiation occurs randomly, i.e., regardless of the Xa or Xi state, indicating that the memory of the Xi of somatic cells has been erased and reset to the ground state (i.e., inner cell mass-like state), indicating that random X-chromosome inactivation occurs upon differentiation.     

Blastomere biopsy influences epigenetic reprogramming during early embryo development,which impacts neural development and function in resulting mice

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.     

Distribution of sister chromatid exchanges in different types of chromatin in the X chromosome ofMicrotus cabrerae

We used the X chromosomes ofMicrotus cabrerae as a model to analyze the distribution of sister chromatid exchanges (SCEs) on different types of chromatin, because of the marked heterogeneity of the heterochromatin in the entire short arm and a portion of the long arm of this chromosome. Computer-simulated distributions, according to an algorithm that makes it possible to modify the distribution on the basis of any possible hypothesis, were compared with real distributions by log-linear models. We found that the frequency of SCEs in different types of heterochromatin was higher than that expected for a random distribution, and located an SCE hot-spot at the junction between euchromatin and heterochromatin. The possible relationship between the distribution of SCEs and base composition or chromatin accessibility are discussed.     

La FAM fatale: USP9X in development and disease

Deubiquitylating enzymes (DUBs), act downstream of ubiquitylation. As such, these post-post-translational modifiers function as the final arbitrators of a protein substrate’s ubiquitylation status, thus regulating its fate. In most instances, DUBs moderate the absolute level of a substrate, its locality or activity, rather than being an “all-or-none” phenomenon. Yet, disruption of this quantitative regulation can produce dramatic qualitative differences. The ubiquitin-specific protease 9X (USP9X/FAM) is a substrate-specific DUB, which displays an extraordinarily high level of sequence conservation from Drosophila to mammals. It is primarily the recent revelations of USP9X’s pivotal role in human cancers, both as oncogene or tumour suppressor, in developmental disorders including intellectual disability, epilepsy, autism and developmental delay that has led to a subsequent re-examination of its molecular and cellular functions. Results from experimental animal models have implicated USP9X in neurodegeneration, including Parkinson’s and Alzheimer’s disease, as well as autoimmune diseases. In this review, we describe the current and accumulated knowledge on the molecular, cellular and developmental aspects of USP9X function within the context of the biological consequences during normal development and disease.     

Effect of cell proliferation on the condensation of the X chromosome in culture medium of the individual karyotype 49 XXXXX]

The influence of cell proliferation on the condensation of the X chromosome was observed in vitro in human fibroblasts with 49 XXXXX karyotype. The frequency of cells with four Barr-bodies is low during the logarithmic growth phase and increases to 80% when the cells are becoming confluent or, independently of cell contact, when cell growth is arrested in a medium with low serum content. The condensation of th X chromosomes is reversible when the cells start growing again in medium with a higher serum content.     

iPSCs-based generation of vascular cells: reprogramming approaches and applications

Recent advances in the field of induced pluripotent stem cells (iPSCs) research have opened a new avenue for stem cell-based generation of vascular cells. Based on their growth and differentiation potential, human iPSCs constitute a well-characterized, generally unlimited cell source for the mass generation of lineage- and patient-specific vascular cells without any ethical concerns. Human iPSCs-derived vascular cells are perfectly suited for vascular disease modeling studies because patient-derived iPSCs possess the disease-causing mutation, which might be decisive for full expression of the disease phenotype. The application of vascular cells for autologous cell replacement therapy or vascular engineering derived from immune-compatible iPSCs possesses huge clinical potential, but the large-scale production of vascular-specific lineages for regenerative cell therapies depends on well-defined, highly reproducible culture and differentiation conditions. This review will focus on the different strategies to derive vascular cells from human iPSCs and their applications in regenerative therapy, disease modeling and drug discovery approaches.     

Active and inactive renin in dog plasma before and after bilateral nephrectomy

Summary No significant amounts of inactive renin could be demonstrated by in vitro treatment (acidification or cryotreatment) of dog plasma obtained before and after bilateral nephrectomy. After bilateral nephrectomy, total and active renin were cleared from the plasma following similar disappearance curves, and dropped to half of their initial value within 30 min.The authors gratefully acknowledge the assistance of M. Cober-Stinissen, K. Van Horenbeeck-Bijttebier, Y. Vanhulst-Toremans, L. Cocx and J. Huysecom. The work of this laboratory is supported in part by the Belgian Research Institute IWONL.     

Reactivation of juvenile hormone synthesis in adult drones of the honey bee,Apis mellifera carnica

Juvenile hormone synthesis was measured by the corpora allata radiochemical assay in vitro. No hormone was produced during pupal stages, but soon after adult eclosion the corpora allata were reactived. The rate of juvenile hormone III synthesis increased until day 10 after emergence. Possible functions of juvenile hormone in adult drones are discussed.