Session 1F: Modeling CHARGE Syndrome in Human Fibroblast Cells via CRISPR/Cas9

Mutations in chromodomain helicase DNA binding protein 7 (CHD7) gene located in human chromosome 8 have been linked to “CHARGE” syndrome, a cluster of disease seen in a number children, for which there is no pharmacotherapy. As there is no cellular or animal model system to study the function of CHD7, the goal of this investigation is to mimic CHARGE disease in a dish by the use of CRISPR/Cas9 to edit the CHD7 gene in human fibroblast cells. Accordingly, I have generated lentivirus particles encoding Cas9 and CHD7 guide(g)RNA in all-in-one vector, and transduced fibroblast cells. CHD7 mutation will be confirmed by qRT-PCR and DNA sequencing; microscopy, Western blot analyses, and the function of mutant protein will be analyzed. Thereafter, the fibroblast cells that harbor a specific mutation in CHD7 gene will be used to screen drugs using a chemical compound library. Through this assay, a molecule that acts on mutant CHD7 protein as an agonist or antagonist could be found, thereby one could fantasize a therapeutic approach to alleviate CHARGE syndrome. There are limitations to cell-based assays, therefore, animal experiments will be needed to address the toxicity and efficacy of any chemical that might be discovered

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

Rationale The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation. Methods and results Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2\u27-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network- like structures in the presence of VEGF. Conclusion Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tubeforming cells in a 2D matrix in VEGF-dependent manner

Dinoflagellate fossils: Geological and biological applications

International audienc

Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca2+]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca2+]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca2+]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

<div><p>Rationale</p><p>The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.</p><p>Methods and results</p><p>Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the <i>VEGFR-2/FLK1</i> promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.</p><p>Conclusion</p><p>Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.</p></div

Chromatin modification mediate expression of mesodermal and endothelial cells marker proteins.

<p>Total protein extracts prepared from: a) d2 control cells; b) d3 cells treated once with Aza + TSA; c) d4 cells treated twice with Aza + TSA; d) d5, treated with a third dose of Aza + TSA and TDG were resolved by denaturing SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and analyzed by Western blotting (WB) with antibodies to: <b>A</b>) β-catenin, <b>B</b>) OCT4, and <b>C</b>) VEGFR-2/FLK1; <b>D</b>) Brachyury; <b>E</b>) N-cadherin; <b>F</b>) TIE-2; <b>G</b>) CD31; <b>H</b>) LPP3; and (<b>I</b>) β-tubulin. β-tubulin antibody was used to judge equal loading of proteins across the lanes. (<b>J</b>) Representative image of ponceau-S stained nitrocellulose membrane. Molecular weights are given in kiloDaltons (kDa). The numbers at the lowermost rows of each image indicate signal intensities that were quantified by NIH ImageJ. Experiments were carried out three times.</p

Chromatin modification strategy, timeline of experiments, and gene expression analyses.

<p><b>A</b>) The strategy and timeline used for all the experiments described in this study. <b>B</b>) Descriptions of the four experimental groups: (<b>a</b>) day-2 (d2) dish is control untreated cells; (<b>b</b>) day-3 (d3) dish received one dose of Aza + TSA for 24 hours before the cells were used; (<b>c</b>) day-4 (d4) received a second dose of Aza + TSA for 24 hours before use; (d) day-5 (d5) received a third dose of Aza + TSA and TDG 24 hours before use. <b>C</b>) Total mRNAs prepared from these cells were subjected to q-RT-PCR using gene-specific primers for <i>18S RNA</i> (control), <i>VEGFR-2/FLK1</i>, <i>OCT4</i>, and <i>LPP3</i>. Experiments were carried out three times, with triplicate mRNAs. * p<0.05 or ** p<0.01 vs. control <i>18S RNA</i> from same group.</p

Microscopic analysis of β-catenin, OCT4, N-cadherin and vWF in epigenetically modified cells.

<p>hADFCs were plated on coverslips, left untreated or treated with epigenetic modifiers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176496#pone.0176496.g001" target="_blank">Fig 1A</a>, and stained with anti-β-catenin (A-D, green) or anti-OCT4 (E-P, green) or anti-vWF (Q-T, red). Representative microscopic images of: <b>A&E</b>) d2 control untreated cells; <b>B&F</b>) d3 cells treated once with Aza + TSA; <b>C&G</b>) d4 cells received two doses of Aza + TSA; <b>D&H</b>) d5, treated with a third dose of Aza + TSA and TDG. White arrowheads indicate β-catenin; magenta arrowheads show nuclear accumulation of OCT4. <b>I-L</b>) d5 cells treated with a third dose of Aza + TSA and TDG were stained with DAPI (blue), TRITC-phalloidin (red), and OCT4 (green). Magenta arrows (<b>L</b>) indicate nuclear localization. <b>M-P</b>) d5 cells treated with a third dose of Aza + TSA and TDG were stained with DAPI (blue), OCT4 (green), and N-cadherin (red). <b>Q-T</b>) d2, d3, d4 and d5 hADFC were stained with DAPI (blue) and vWF (red). Magnification is as shown.</p

Human gene specific primers used for q-RT-PCR data shown in S1 Fig.

<p>Human gene specific primers used for q-RT-PCR data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176496#pone.0176496.s001" target="_blank">S1 Fig</a>.</p

A model of the conversion process of fibroblast cells into tube-forming cells.

<p>hADFCs are mature differentiated cells with highly condensed chromatin containing histones tightly bound to DNAs. However, upon treatment with Aza and TSA for two or three cycles, histone acetylation increases, DNA methylation decreases, the chromatin becomes relaxed, so that the transcription factors could activate the expression of mesodermal-endothelial genes. These events mediate the formation of tube-like structures in 2D Matrigel in a VEGF-dependent manner.</p