eoe sub articles

EoE Sub Articles

1. Manipulation of O9-1 cells
<ol>
	<li>Performing siRNA knockdown in O9-1 cells
	<ol>
		<li>Thaw and coat a 24-well plate with a basement membrane matrix before starting the experiment. Recover and seed O9-1 cells, allowing them to grow to 60% to 80% confluency.</li>
		<li>Dilute liposomes in serum-free media according to the appropriate well volume. Dilute siRNA in serum-free media to the final. Add diluted siRNA to diluted liposomes in a ratio recommended by the manufacturer. Mix well by pipetting and then incubate. 
		NOTE: Follow the manufacturer&#39;s guide on the volume used, time, and temperature of incubation.</li>
		<li>Add an appropriate volume of siRNA-lipid complex to cells according to the manufacturer&#39;s recommendations.</li>
		<li>Incubate cells for 24 h in a standard cell culture incubator (37 &#176;C, 5% CO2). Perform downstream steps as appropriate (extract RNA, extract proteins, staining, etc.) 
		NOTE: siRNA knockdown times and concentrations can vary depending on the individual experiment.</li>
	</ol>
	</li>
</ol>
2. O9-1 cell differentiation
<ol>
	<li>Differentiation of O9-1 cells into osteoblasts
	<ol>
		<li>To prepare osteogenic differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 0.1 &#956;M dexamethasone, 100 ng/mL bone morphogenetic protein 2 (BMP2), 50 &#181;g/mL ascorbic acid, 10 mM b-glycerophosphate, 10% FBS, 100 U/mL penicillin, and 100 &#956;g/mL streptomycin.</li>
		<li>Detect the osteoblast marker, osteocalcin, in differentiated osteoblasts by immunostaining, as shown in Figure 1. 
		&#8203;NOTE: Osteogenic differentiation can also be evaluated by using other markers of osteoblasts or with Alizarin red staining or alkaline phosphatase staining.</li>
	</ol>
	</li>
	<li>Differentiation of O9-1 cells into smooth muscle cells
	<ol>
		<li>To prepare smooth muscle cell differentiation media, dilute the following in DMEM (final concentrations are indicated): 100 U/mL penicillin, 100 &#181;g/mL streptomycin, and 10% FBS.</li>
		<li>Asses smooth muscle cell differentiation with immunofluorescence staining by using antibodies against markers of smooth muscle cells, such as smooth muscle actin (SMA), shown as an example in Figure 2.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>O9-1 mouse cranial neural crest cell line</td>
			<td>Millipore Sigma</td>
			<td>SCC049</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, no glutamine</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (fetal bovine serum)</td>
			<td>Millipore Sigma</td>
			<td>ES-009-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin - streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200mM (100X)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% in HBSS</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#39;s phosphate buffered saline) without calcium or magnesium</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids (MEM NEAA) 100X</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M-7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human fibroblast growth factor-basic (rhFGF-basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Roche</td>
			<td>10107409001</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (dimethylsulfoxide)</td>
			<td>Millipore Sigma</td>
			<td>MX1458-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I (1X)</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium, alpha 1X with Earle&#39;s salts, ribonucleosides, deoxyribonucleosides, &#38; L-glutamine</td>
			<td>Corning</td>
			<td>10-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>ON-TARGETplus Wwtr1 siRNA</td>
			<td>Dharmacon</td>
			<td>L-041057</td>
			<td></td>
		</tr>
		<tr>
			<td>ON-TARGETplus Non-targeting Pool</td>
			<td>Dharmacon</td>
			<td>D-001810</td>
			<td></td>
		</tr>
		<tr>
			<td>ON-TARGETplus Yap1 siRNA</td>
			<td>Dharmacon</td>
			<td>L-046247</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS (fetal calf serum)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ITS (insulin-transferrin-selenium )</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-b3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMP2 (bone morphogenetic protein 2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

sirna-mediated gene silencing in neural stem cells

Source: Nguyen, B. H., et al. <a href="https://app.jove.com/v/58346">Culturing and Manipulation of O9-1 Neural Crest Cells.</a> J. Vis. Exp (2018)
This video demonstrates the use of small interfering RNA (siRNA) to silence specific genes of interest in neural stem cells and differentiate them into desired cell types using appropriate differentiation medium. This procedure enables the study of the critical role of specific genes in the differentiation process of neural stem cells.

<img alt="Figure 1" src="/files/ftp_upload/22404/22404_Figure1.jpg" /> 
Figure 1: Immunofluorescence staining of osteoblast marker osteocalcin (Ocn) indicating that O9-1 cells gave rise to osteoblast cells under osteogenic differentiation conditions. Cells were stained with osteoblast marker Ocn antibody (green), and nuclei were stained with DAPI (blue). Arrows indicate Ocn-positive cells. Osteocalcin (Ocn, an osteoblast marker); DAPI (&#8242;,6-diam...

siRNA-Mediated Gene Silencing in Neural Stem Cells

1. Manipulation of O9-1 cells

	Performing siRNA knockdown in O9-1 cells
	
		Thaw and coat a 24-well plate with a basement membrane matrix before starting ...

Take small interfering RNA, or siRNA, a type of RNA involved in gene silencing.
Add spherical lipid vesicles called liposomes.
The liposome encapsulates the siRNA, protecting it from degradation.
Add siRNA-liposome complexes to a culture well containing neural stem cells. Keep one well as a control, and incubate.
The liposome fuses with the cell membrane, releasing the siRNA.
The siRNA binds to the RNA-induced silencing complex, or RISC, where one strand is removed.
The remaining strand targets the mRNA responsible for cell differentiation, leading to its degradation.
Add a suitable differentiation medium to the wells.
In the control well, the growth factors and other nutrients in the medium promote the differentiation of neural stem cells into osteoblasts.
Using suitable staining techniques, stain osteoblast-specific markers on the cells and stain the nucleus with a blue fluorescent dye.&#160;&#160;
The absence of osteoblast-specific markers in the gene-silenced cells indicates their inability to differentiate.

sirna-mediated-gene-silencing-in-neural-stem-cells

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

55 Views. Source: Nguyen, B. H., et al. Culturing and Manipulation of O9-1 Neural Crest Cells. J. Vis. Exp (2018) This video demonstrates the use of small interfering RNA (siRNA) to silence specific genes of interest in neural stem cells and differentiate them into desired cell types using appropriate differentiation medium. This procedure enables the study of the critical role of specific genes in the differentiation process of neural stem cells. 

Video: siRNA-Mediated Gene Silencing in Neural Stem Cells - Concept

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...

JoVE Journal

Engineering

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Developmental Biology

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

siRNA-Mediated Gene Silencing in Neural Stem Cells

Transcript

siRNA-Mediated Gene Silencing in Neural Stem Cells

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration