In mammals, X chromosome inactivation (XCI) is a process in which one of the two X chromosomes is silenced, following XIST expression. Mouse female pluripotent stem cells do not express Xist, and harbor two active X chromosomes. However, analysis of XCI in human embryonic stem cells (hESCs), mainly based on XIST expression, was not conclusive. Here, we studied XCI in hESCs by meta-analysis of the expression of the entire set of genes on the X chromosome in 21 female hESC lines. Thus, we could divide the ES cell lines into three categories: lines with no XCI, lines with full XCI, and lines with partial XCI. The partial inactivation of the X chromosome always involved the middle of the chromosome, surrounding the XIST transcription site. The status of XCI in some of the cell lines was validated by either allelic-specific expression or DNA methylation analysis. Interestingly, analysis of 10 female human-induced pluripotent stem cell (hiPSC) lines demonstrated similar heterogeneity in the inactivation of X chromosome and could also be classified into the same three categories detected in hESCs. Thus, we could show that in some hiPSC lines, the X chromosome was activated on reprogramming. Based on our analysis, we propose a model of the dynamics of XCI in pluripotent stem cells.

Female human pluripotent stem cells show vast heterogeneity regarding the status of X chromosome inactivation. By comparing the gene expression profile of cells with two active X chromosomes (XaXa cells) to that of cells with only one active X chromosome (XaXi cells), a set of autosomal genes was shown to be overexpressed in the XaXa cells. Among these genes, we found significant enrichment for genes regulated by the X-linked transcription factor ELK-1. Comparison of the phenotype of XaXa and XaXi cells demonstrated differences in programmed cell death and differentiation, implying some growth disadvantage of the XaXa cells. Interestingly, ELK-1-overexpressing cells mimicked the phenotype of XaXa cells, whereas knockdown of ELK-1 with small hairpin RNA mimicked the phenotype of XaXi cells. When cultured at low oxygen levels, these cellular differences were considerably weakened. Our analysis implies a role of ELK-1 in the differences between pluripotent stem cells with distinct X chromosome inactivation statuses.

Background: In Alzheimer’s disease (AD), brain butyrylcholinesterase (BChE) co-localizes with β-amyloid (Aβ) fibrils. Aims: In vitro testing of the significance of this phenomenon to AD progress. Methods: A thioflavine T (ThT) fluorogenic assay, photo-induced cross-linking and quantifiable electron microscopy served to compare the effect on Aβ fibril formation induced by highly purified recombinant human BChE (rBChE) produced in the milk of transgenic goats with that of serum-derived human BChE. Results: Both proteins at 1:50 and 1:25 ratios to Aβ dose-dependently prolonged the ThT lag time and reduced the apparent rate of Aβ fibril formation compared to Aβ alone. Photo-induced cross-linking tests showed that rBChE prolonged the persistence of amyloid dimers, trimers and tetramers in solution, whereas Aβ alone facilitated precipitation of such multimers from solution. Transmission electron microscopy showed that rBChE at 1:100 to Aβ prevented the formation of larger, over 150-nm-long, Aβ fibrils and reduced fibril branching compared to Aβ alone as quantified by macro programming of Image Pro® Plus software. Conclusion: Our findings demonstrate that rBChE interacts with Aβ fibrils and can attenuate their formation, extension and branching, suggesting further tests of rBChE, with unlimited supply and no associated health risks, as a therapeutic agent for delaying the formation of amyloid toxic oligomers in AD patients.

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The metabolic syndrome (MetS) is a risk factor for type 2 diabetes mellitus (T2DM). However, the mechanisms underlying the transition from MetS to T2DM are unknown. Our goal was to study the potential contribution of butyrylcholinesterase (BChE) to this process. We first determined the hydrolytic activity of BChE in serum from MetS, T2DM and healthy individuals. The ‘Kalow’ variant of BChE (BChE-K), which has been proposed to be a risk factor for T2DM, was genotyped in the last two groups. Our results show that in MetS patients serum BChE activity is elevated compared to T2DM patients and healthy controls (P < 0.001). The BChE-K genotype showed similar prevalence in T2DM and healthy individuals, excluding this genotype as a risk factor for T2DM. However, the activity differences remained unexplained. Previous results from our laboratory have shown BChE to attenuate the formation of β-amyloid fibrils, and protect cultured neurons from their cytotoxicity. Therefore, we next studied the in vitro interactions between recombinant human butyrylcholinesterase and amylin by surface plasmon resonance, Thioflavine T fluorescence assay and cross-linking, and used cultured pancreatic β cells to test protection by BChE from amylin cytotoxicity. We demonstrate that BChE interacts with amylin through its core domain and efficiently attenuates both amylin fibril and oligomer formation. Furthermore, application of BChE to cultured β cells protects them from amylin cytotoxicity. Taken together, our results suggest that MetS-associated BChE increases could protect pancreatic β-cells in vivo by decreasing the formation of toxic amylin oligomers.