Name: nerve stem cell differentiation by a one-step cold atmospheric plasma treatment in vitro
Uploaded: 2019-01-11T14:00:00
Description: Watch this Scientific Journal Video about Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro at JoVE.com

This work was supported by the Huazhong Scholar Program, the Independent Innovation Fund of the Huazhong University of Science and Technology (No. 2018KFYYXJJ071), and the National Natural Science Foundation of China (Nos. 31501099 and 51707012).

1. Cell Cultures and Predifferentiation
<ol>
	<li>Neural stem cell culture and predifferentiation
	<ol>
		<li>Prepare poly-D-lysine-coated coverslips. Put a sterile coverslip (20 mm in diameter) into a 12-well plate. Coat the cover glass with poly-D-lysine, 0.1% w/v, in water (Table of Materials) for better cell adhesion on the coverslips by following the next steps. 
		NOTE: Optimal conditions must be determined for each cell line and application.
		<ol>
			<li>Aseptically coat t...

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414 (6859), 112-117 (2001).</li><li>Rossi, F., Cattaneo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+therapy+for+neurological+diseases:+dreams+and+reality.">Neural stem cell therapy for neurological diseases: dreams and reality.</a> Nature Reviews Neuroscience. 3 (5), 401-409 (2002).</li><li>Graves, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+reactive+oxygen+and+nitrogen+species+in+redox+biology+and+some+implications+for+plasma+applications+to+medicine+and+biology.">The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology.</a> Journal of Physics D: Applied Physics. 45 (26), 263001 (2012).</li><li>Weltmann, K. D., von Woedtke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+medicine-current+state+of+research+and+medical+application.">Plasma medicine-current state of research and medical application.</a> Plasma Physics and Controlled Fusion. 59 (1), 014031 (2017).</li><li>Lloyd, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+Plasma:+Medical+Uses+and+Developments+in+Wound+Care.">Gas Plasma: Medical Uses and Developments in Wound Care.</a> Plasma Processes and Polymers. 7 (3-4), 194-211 (2010).</li><li>Yousfi, M., Merbahi, N., Pathak, A., Eichwald, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+plasmas+at+atmospheric+pressure:+toward+new+pharmaceutical+treatments+in+medicine.">Low-temperature plasmas at atmospheric pressure: toward new pharmaceutical treatments in medicine.</a> Fundamental &amp; Clinical Pharmacology. 28 (2), 123-135 (2014).</li><li>Xiong, Z., Roe, J., Grammer, T. 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[Med Sci]. 23 (2), 75-78 (2003).</li><li>Ryder, E. F., Snyder, E. Y., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+multipotent+neural+cell+lines+using+retrovirus+vector-mediated+oncogene+transfer.">Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer.</a> Journal of Neurobiology. 21 (2), 356-375 (1990).</li><li>Snyder, E. Y., Taylor, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cell+engraftment+corrects+lysosomal+storage+throughout+the+MRS+VII+mouse+brain.">Neural progenitor cell engraftment corrects lysosomal storage throughout the MRS VII mouse brain.</a> Nature. 374 (6520), 367-370 (1995).</li><li>Snyder, E. Y., Yoon, C., Flax, J. D., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+neural+precursors+can+differentiate+toward+replacement+of+neurons+undergoing+targeted+apoptotic+degeneration+in+adult+mouse+neocortex.">Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (21), 11663-11668 (1997).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Fixatives+for+Immunostaining.">Cell Fixatives for Immunostaining.</a> Methods in Molecular Biology. 588, 55-61 (2010).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+Cell+Membranes.">Permeabilization of Cell Membranes.</a> Methods in Molecular Biology. 588, 63-66 (2010).</li></ol>

C17.2-NSCs is a kind of immortalized neural stem cell line from neonatal mouse cerebellar granular layer cells, developed by Snyder and others10,11. C17.2-NSCs can differentiate into neurons, astrocytes, and oligodendrocytes and are widely used in neuroscience12. In our previous study, CAPs could enhance the differentiation of C17.2-NSCs into neurons. A proof-of-principle study was also carried out using primary rat NSCs, and the effect of...

The directed differentiation of NSCs into a certain lineage for tissue transportation is considered one of the most promising therapies for neurodegenerative and neurotraumatic diseases1. For example, catecholaminergic dopaminergic neurons are especially desired in Parkinson&#39;s disease (PD) treatment. However, traditional methods to prepare the desired cells for transportation have many drawbacks, such as chemical toxicity, scar formation, or others, which largely hampers the applications of NSCs in regenerative medicine2. Therefore, it is very necessary to find a novel and safe way for NSC differentiation.
Plasma is the fourth state of matters, following solid, liquid, and gas, and it constitutes more than 95% of matters in the whole universe. Plasma is electrically neutral with unbound positive/negative and neutral particles and is usually generated by a high-voltage discharge in the lab. In the last two decades, the application of plasma in biomedicine has attracted huge attention worldwide as the development of cold atmospheric pressure plasma technology. Thanks to this technic, stable low-temperature plasma can be generated in the surrounding air at atmosphere without arc formation and consists of various reactive species, such as reactive nitrogen species (RNS), reactive oxygen species (ROS), ultraviolet (UV) radiation, electrons, ions, and electrical field3. CAPs have unique advantages for micro-organism inactivation, cancer therapy, wound healing, treatment of skin diseases, cell proliferation, and cell differentiation4,5,6,7. In previous work, we demonstrated that cold atmospheric plasma jet can enhance the differentiation of NSCs in both murine neural stem cell C17.2 (C17.2-NSCs) and primary rat neural stem cells, exhibiting a great potential to become a powerful tool for the directed differentiation of NSCs8. Although the mechanism of CAP enhancement of NSC differentiation is not fully understood yet, NO generated by CAPs has been proved to be a key factor in the process. In this work, we aim to provide a detailed experimental protocol of using an atmospheric pressure helium/oxygen plasma jet for the treatment of NSCs in vitro, cell preparation and pretreatment, morphology observation by inverted microscope, and fluorescence microscopy observation of immunofluorescence staining.

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			<td height="40" style="height:40px;width:216px;">Coverslip</td>
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			<td>Gibco</td>
			<td>17502048</td>
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			<td>Gibco</td>
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			<td>stored at -20 &#176;C and protect from light</td>
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			<td height="40" style="height:40px;">Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td>HyClone</td>
			<td>SH30074.03</td>
			<td>tored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td>HyClone</td>
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			<td>stored at 4 &#176;C</td>
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			<td>HyClone</td>
			<td>25300054</td>
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			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
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			<td height="40" style="height:40px;">4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
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			<td>T8787</td>
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			<td>AF2215</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="40" style="height:40px;">anti-&#946;-Tubulin III</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="40" style="height:40px;">anti-O4</td>
			<td>R&#38;D Systems</td>
			<td>MAB1326</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="40" style="height:40px;width:216px;">anti-NF200</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">anti-ChAT</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="40" style="height:40px;width:216px;">anti- LHX3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="63" style="height:63px;width:216px;">anti-GABA</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="63" style="height:63px;width:216px;">anti-Serotonin</td>
			<td style="width:123px;">Abcam, Cambridge, MA</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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			<td height="63" style="height:63px;width:216px;">anti-TH</td>
			<td style="width:123px;">Abcam, Cambridge, MA</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Immunol Staining Primary Antibody Dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
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			<td height="21" style="height:21px;">Cy3 Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488- Labeled Goat</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
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			<td height="21" style="height:21px;">Anti-Mouse IgG&#160;</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Hoechst 33258</td>
			<td>Beyotime</td>
			<td>C1018</td>
			<td>stored at -20 &#176;C and protect from light</td>
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			<td height="21" style="height:21px;">Mounting medium</td>
			<td>Beyotime</td>
			<td>P0128</td>
			<td>stored at -20 &#176;C and protect from light</td>
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			<td>Nanjing Jiangnan Novel Optics Company</td>
			<td>XD-202</td>
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			<td>Nikon</td>
			<td>80i</td>
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			<td height="21" style="height:21px;">High &#8211; voltage Power Amplifier</td>
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			<td height="21" style="height:21px;">DC power supply</td>
			<td>Spellman</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Function Generator</td>
			<td>Aligent&#160;</td>
			<td>33521A</td>
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			<td height="21" style="height:21px;width:216px;">Oscilloscope</td>
			<td style="width:123px;">Tektronix</td>
			<td style="width:344px;">DPO3034</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High voltage probe</td>
			<td style="width:123px;">Tektronix</td>
			<td style="width:344px;">P6015A</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
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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.

Cell morphology was observed under the inverted microscope every day after the CAP treatment. Figure 2 shows the ordinary inverted phase-contrast light microscope images of the cell differentiation in both cell lines. The plasma-treated group exhibits an accelerated differentiation rate and a high differentiation ratio compared to the control and gas flow group.
The immunofluorescen...

Watch this Scientific Journal Video about Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro at JoVE.com

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.

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, ...

This method has the potential to enhance the differentiation of neural stem cells in vitro and to contribute to the treatment of neurological disease such as Parkinson's disease. The main advantages of this technique is that the cold atmospheric plasmas may be applied in one step for the safe directed differentiation of neural stem cells for tissue transplantation. Cold atmospheric plasma can enhance neural stem cells differentiation within a short treatment period.And with need more damage to the cells, providing the desired cells for transplantation. Begin by seeding about five times ten to the fifth murine neural stem cells in a 25 centimeter square flask. Containing five milliliters of complete DMEM for two to three days at 37 degrees celsius and five percent carbon dioxide.When the cells reach 85 percent confluency, wash the culture with one milliliter of PBS. Before treatment, with one milliliter of 25 percent trypsin for one minute. When the cells have detached, stop the reaction with one milliliter of culture medium.And pipette several times to generate a single cell suspension. After counting, dilute the cells to a two times ten to the fourth cells per milliliter concentration. Seed one milliliter of stem cells onto one poly-D-lysine coated cover slip per well in nine wells of a 12 cell culture plate.Check the cell density under a microscope. And return the cells to the incubator for 12 hours. When the cells have attached, wash the cover slips two times with one milliliter of PBS per wash, and treat the cells with differentiation medium for 48 hours.For jet acquisition, first connect the output wire of the power supply to the plasma jet device. And the tip of the high voltage probe with the output wire to detect the voltage. Then connect the other end of the high voltage probe to the oscilloscope to record the information from the output voltage.Check the whole circuit to make sure that the power supply, oscilloscope and high voltage probe are all grounded. And check the gas line to make sure that the gas tube is connected to the plasma jet device. Next, open the helium and oxygen gas valves and set the gas flow to a one liter to 01 liters per minute ratio of helium to oxygen.Check the circuit again and press the output button to create a plasma jet. To plasma treat the neural stem cells, set the distance between the syringe nozzle and the first well of cells to 15 millimeters. Replace the supernatants in the neural stem cell cultures with 800 microliters of fresh differentiation medium.And place the plate under the plasma jets. Making sure that the syringe nozzle is fixed over the center of each well. Create three wells with plasma for 60 seconds per treatment, and three wells with one percent helium and oxygen gas for 60 seconds per treatment.Leaving three wells untreated, as controls. Then replace the supernatants with one milliliter of fresh differentiation medium. And return the cells to the incubator for six days.Check the differentiation status of the different groups daily, under an inverted phase contrast light microscope. After the treatment, allow the cells to fully equilibrate in the condition medium for about one hour before replacing the supernatant with fresh differentiation medium. For immunofluorescence analysis, fix the cultures with 500 microliters of four percent paraformaldehyde per well for 20 minutes at room temperature.Followed by three gentle five minute rinses, with one milliliter of PBS per wash. Next, permeabilize the fixed samples with 2 percent Triton X-100 in PBS for 10 minutes at room temperature. Followed by gentle rinsing as demonstrated.After the last wash, block any non specific binding with one milliliter of 10 percent goat serum in PBS per sample for one hour at room temperature. At the end of the incubation, label the cells in each well with 300 microliters of the primary antibody of interest overnight at four degrees celsius. Followed by three washes in PBS as demonstrated.After the last wash, label the cells with 300 microliters of the appropriate secondary antibodies. Incubate for two hours at room temperature, protected from light. Then rinse the cells gently with one milliliter of PBS per well for ten minutes at room temperature.Image the cells on a fluorescence microscope equipped with the appropriate filters. Plasma treated neural stem cells exhibit an accelerated differentiation rate, and a higher differentiation ratio compared to untreated control, and gas flow treated stem cells. After plasma treatment, Nestin decreases.Beta-Tubulin III significantly increases. And O4 slightly increases, compared to control treated and gas flow treated stem cells. Further, after 60 seconds of plasma treatment, a robust expression of mature neuron, cholinergic neuron, and motor neuron markers is observed.With very few GABAergic and serotonergic neurons, and no dopaminergic neurons detected. While attempting these procedures, it is important to remember, treat the cells with proper plasma dosage, because a long and intense plasma treatment period will induce cell apoptosis or necrosis. Remember to also pre test the cell viability before downstream application.

Research

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We demonstrate a method for the study of the heat and mass transfer and of the freezing phenomena in a subcooled brine environment. Our experiment showed ...

Spark Plasma Sintering Apparatus Used for the Formation of Strontium Titanate Bicrystals

A spark plasma sintering apparatus was used as a novel method for diffusion bonding of two single crystals of strontium titanate to form bicrystals with ...

New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells

The atmospheric pressure plasma jet (APPJ) has attracted the attention of many researchers from multiple disciplines in recent years because its emissions ...

Atmospheric Pressure Fabrication of Large-Sized Single-Layer Rectangular SnSe Flakes

Tin selenide (SnSe) belongs to the family of layered metal chalcogenide materials with a buckled structure like phosphorene, and has shown potential for ...