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 the surface of the coverslip with poly-D-lysine, 0.1% w/v, in water. Rock gently to ensure an even coating of the coverslip surface.</li>
			<li>After culturing overnight (~12 h) at 37 &#176;C, remove the poly-D-lysine solution and rinse the surface 3x with 1 mL of sterile water.</li>
			<li>Dry the cells at least 30 min before seeding them.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Murine neural stem cell C17.2 (C17.2-NSC) culture and predifferentiation
	<ol>
		<li>Incubate C17.2-NSCs in a 25 cm2 flask in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin at 37 &#176;C and 5% CO2 for ~2 - 3 d.</li>
		<li>When the cells reach 85% confluence, remove the medium, wash the cells with 1 mL of PBS and add 1 mL of fresh trypsin (0.25%) into the flask. Leave it for 1 min; then, add 1 mL of culture medium to the flask and pipette up and down several times to ensure a single cell suspension.</li>
		<li>Count the density of the cells in the suspension using a hemocytometer. Calculate the required volume of cell suspension to give a final cell concentration of 2 x 104 cells/mL.</li>
		<li>Seed the C17.2-NSCs on the coated coverslips in the 12-well plate with the density of ~2 x 104 cells per well. Check the cell density under a microscope. 
		NOTE: For a better result, the optimal cell density must be determined before the immunofluorescence experiment.</li>
		<li>Incubate the cells at 37 &#176;C in a cell incubator for 12 h to allow for attachment.</li>
		<li>After attachment, wash the cells 2x with 1 mL of PBS and cultivate the cells with differentiation medium consisting of DMEM/F12 with 1% N2 supplement for 48 h before the plasma treatment.</li>
	</ol>
	</li>
	<li>Primary rat neural stem cell culture and predifferentiation
	<ol>
		<li>Culture the primary rat NSC suspension in rat NSC growth medium in uncoated T25 flasks at a density of 5 x 105 cells. 
		NOTE: The primary rat NSCs were isolated following the protocol of Xie et al.9.</li>
		<li>Check the formation of neurospheres under an inverted microscope. Make sure the morphology of the neurospheres exhibits spherical and transparent multicellular complexes.</li>
		<li>When the neurospheres reach a diameter of 3 mm or larger, transfer the neurosphere suspension to a 15 mL sterile centrifuge tube and let the neurospheres settle by gravity.</li>
		<li>Remove the supernatant carefully to leave the neurospheres in a minimal volume of medium.</li>
		<li>Rinse the neurospheres 1x with 5 mL of PBS and let the neurospheres settle by gravity. Remove the supernatant to leave a minimal volume of PBS.</li>
		<li>Add a suitable volume of differentiation medium to adjust the cell density to ~12 - 15 neurospheres per milliliter. The differentiation medium for primary rat NSCs consists of DMEM/F12, 2% B27, and 1% FBS.</li>
		<li>Seed 1 mL of neurosphere suspension onto each coated coverslip in the 12-well plate. 
		NOTE: Shake the plate gently to ensure that the neurospheres are evenly distributed. This step is critical for a better immunofluorescence result.</li>
		<li>Place the plate into the incubator and allow it to predifferentiate for 24 h before the plasma treatment.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Plasma Jets
<ol>
	<li>Choose a half-open quartz tube with an internal diameter of 2 mm and an external diameter of 4 mm. Insert a high-voltage wire with a diameter of 2 mm into the tube.</li>
	<li>Insert the quartz tube with the high-voltage wire into a 5 mL syringe and use a holder to mount it inside the center of the syringe. Set the distance between the sealed end of the quartz tube and the syringe tip at 1 cm.</li>
	<li>Connect a 1 m silicone rubber pipe (with an inner diameter of 12 mm) to the open end of the syringe and, then, connect it to the flowmeter and gas valve in sequence.</li>
</ol>
3. Acquisition of the Jets
<ol>
	<li>Connect the circuit as shown in Figure 1. Connect the output wire of the power supply to the plasma jet device and, then, connect the tip of the high-voltage probe with the output wire to detect the voltage. Connect the other end of the high-voltage probe to the oscilloscope to record the information of the output voltage. Check the whole circuit and make sure the power supply, the oscilloscope, and the high-voltage probe are all grounded.</li>
	<li>Check the gas line. Make sure the gas tube has been connected to the plasma jet device; then, open the gas valve of the helium (volume fraction, 99.999%) and oxygen (volume fraction, 99.999%) and set the gas flow to 1 L:0.01 L/min (He:O2). 
	NOTE: Let the gas flow for several minutes before turning on the power supply for the first time.</li>
	<li>Set the pulse amplitude, frequency, and pulse width as 8 kV, 8 kHz, and 1600 ns, respectively. Check the circuit again and, then, turn on the output button to create a plasma jet. 
	CAUTION: Do not touch the high-voltage wire at any time.</li>
</ol>
4. Plasma Treatment of Neural Stem Cell
<ol>
	<li>Set the distance between the nozzle of the syringe and the cell well hole to 15 mm. 
	NOTE: The distance is measured from the bottom of the platform where the 12-well plate is placed.</li>
	<li>Take the 12-well plate out of the incubator and change the medium of the predifferentiated cells to 800 &#181;L of fresh differentiation medium.</li>
	<li>Divide the cells into three groups: an untreated control group, a 60 s He-and-O2 (1%) gas flow treatment group, and a 60 s plasma treatment group. 
	NOTE: There is no obvious liquid loss under the treatment condition.</li>
	<li>Place the 12-well plate under the plasma jets, make sure the syringe nozzle is fixed in the center of each hole, and give the relevant treatment to the different groups mentioned in step 4.3. 
	NOTE: The untreated controls were kept in differentiation medium at room temperature during the experimental procedure to ensure uniform treatment conditions. The He-and-O2 (1%) gas flow treatment group was treated with only a He and O2 (1%) gas flow, without plasma generation. All the treatments should be performed in triplicate.</li>
</ol>
5. Neural Stem Cell Differentiation
<ol>
	<li>After treatment, remove the original culture and add 1 mL of new differentiation medium to each well.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C and 5% CO2 for 6 d. Change the medium every other day.</li>
	<li>Check the differentiation status of the different groups daily under an inverted phase-contrast light microscope. Randomly select at least 12 fields and take photos to record the morphological changes.</li>
</ol>
6. Immunofluorescence Staining
<ol>
	<li>Rinsing
	<ol>
		<li>Take the samples out of the cell incubator and remove the medium by aspiration. Rinse the cells 1x with 1 mL of PBS. 
		NOTE: After the differentiation, the cells are easily detached. It is necessary to add the PBS from the side of the culture wells to avoid washing off the cells.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Fix the cells with 500 &#181;L of 4% paraformaldehyde for 20 min at room temperature. After the fixation, gently rinse the cells 3x with 1 mL of PBS, 5 min each, to remove the residual 4% paraformaldehyde. 
		NOTE: The optimal time point for cell fixation must be determined according to the cell types. After the fixation, add 1 mL of PBS from the side of the culture wells and leave the sample on the table for 5 min. Do not use a lab shaker, to ensure a minimal loss of cells.</li>
	</ol>
	</li>
	<li>Permeabilization
	<ol>
		<li>Permeabilize the samples with 0.2% TritonX-100 in PBS for 10 min at room temperature. After permeabilization, rinse the cells gently with 1 mL PBS for three times, 5 min each. 
		NOTE: The optimal time point for cell permeabilization must be determined according to cell types. After permeabilization, add 1 mL PBS from the side of the culture wells, leave the sample on the table for 5 min. Do not use a lab shaker to ensure the minimal loss of cells.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Add 1 mL of 10% goat serum in PBS to each sample and leave the sample on the table for 1 h to block any nonspecific interactions. 
		NOTE: Do not use a lab shaker, to prevent the cells from detaching from the slide. A rinse is not necessary in this step.</li>
	</ol>
	</li>
	<li>Incubation with primary antibody
	<ol>
		<li>Dilute the primary antibody using primary antibody dilution buffer. 
		NOTE: For optimal results, the final dilution ratio of the primary antibody must be determined by pretest. The dilution ratios of anti-Nestin (for undifferentiated stem cell), anti-&#946;-Tubulin III (for neuron), anti-O4 (for oligodendrocyte), anti-NF200 (for mature neurons), anti-ChAT (for cholinergic neurons), anti-LHX3 (for motor neurons), anti-GABA (for GABAergic neurons), anti-serotonin (for serotonergic neurons), and anti-TH (for dopaminergic neurons) are 1:80, 1:200, 1:100, 1:100, 1:100, 1:200, 1:100, 1:200, and 1:100, respectively.</li>
		<li>Apply 300 &#181;L of diluted primary antibodies to different samples.</li>
		<li>Incubate the cell samples overnight at 4 &#176;C. 
		NOTE: It is recommended to incubate primary antibodies at 4 &#176;C to reduce the background and nonspecific staining.</li>
		<li>Remove the primary antibodies and gently rinse the cells 3x with 1 mL of PBS.</li>
	</ol>
	</li>
	<li>Incubation with secondary antibody
	<ol>
		<li>Dilute the Cy3-conjugated or Alexa Fluor 488-conjugated secondary antibodies using 3% goat serum in PBS. 
		NOTE: For optimal results, the final dilution ratio of the secondary antibody must be determined by pretest.</li>
		<li>Apply 300 &#181;L of relevant secondary antibodies to detect each primary antibody. 
		NOTE: All the subsequent steps need to be performed in the dark to prevent fluorescence quenching.</li>
		<li>Incubate the cell sample in the dark for 2 h at room temperature.</li>
		<li>Remove the secondary antibodies and rinse the cells gently with 1 mL of PBS for 10 min at room temperature.</li>
	</ol>
	</li>
	<li>Nuclear staining
	<ol>
		<li>Apply 500 &#181;L of Hoechst 33258 working solution to immerse the cell sample.</li>
		<li>Incubate the cell sample in the dark for 8 min at room temperature to label the nuclei.</li>
		<li>Gently rinse the cells 3x with 1 mL of PBS, 10 min each, protected from light. 
		NOTE: For a minimal loss of cells, the optimal washing condition must be determined empirically.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Place one drop of mounting medium in the center of the microslide.</li>
		<li>Take out of the coverslips (with samples) using tweezers and carefully position the sample on top of the mounting medium. Avoid air bubbles.</li>
		<li>Remove any excess mounting medium with absorbent paper.</li>
	</ol>
	</li>
	<li>Fluorescence microscopy observation
	<ol>
		<li>Observe the samples under the fluorescence microscopy equipped with filters for Hoechst 33258, Alexa Fluor 488, and Cy3. For each sample, randomly select at least 8 - 12 fields and record images with a camera.</li>
	</ol>
	</li>
</ol>

<ol><li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+neural+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Neural+stem+cell+therapy+for+neurological+diseases%3A+dreams+and+reality%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((The+emerging+role+of+reactive+oxygen+and+nitrogen+species+in+redox+biology+and+some+implications+for+plasma+applications+to+medicine+and+biology%5BTitle%5D)+AND+%22Journal+of+Physics+D%3A+Applied+Physics%22%5BJournal%5D)">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/pubmed?term=((Plasma+medicine-current+state+of+research+and+medical+application%5BTitle%5D)+AND+%22Plasma+Physics+and+Controlled+Fusion%22%5BJournal%5D)">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/pubmed?term=((Gas+Plasma%3A+Medical+Uses+and+Developments+in+Wound+Care%5BTitle%5D)+AND+%22Plasma+Processes+and+Polymers%22%5BJournal%5D)">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/pubmed?term=((Low-temperature+plasmas+at+atmospheric+pressure%3A+toward+new+pharmaceutical+treatments+in+medicine%5BTitle%5D)+AND+%22Fundamental+%26+Clinical+Pharmacology%22%5BJournal%5D)">Low-temperature plasmas at atmospheric pressure: toward new pharmaceutical treatments in medicine.</a> Fundamental & Clinical Pharmacology. 28 (2), 123-135 (2014).</li>
<li>Xiong, Z., Roe, J., Grammer, T. C., Graves, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+Treatment+of+Onychomycosis%5BTitle%5D)+AND+%22Plasma+Processes+and+Polymers%22%5BJournal%5D)">Plasma Treatment of Onychomycosis.</a> Plasma Processes and Polymers. 13 (6), 588-597 (2016).</li>
<li>Xiong, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+neuronal+differentiation+of+neural+stem+cells+induced+by+nanosecond+microplasma+agitation%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation.</a> Stem Cell Research. 12 (2), 387-399 (2014).</li>
<li>Xie, Z., Zheng, Q., Guo, X., Yi, C., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+Culture+and+Identification+of+Neural+Stem+Cells+in+New-born+Rats%5BTitle%5D)+AND+%22Journal+of+Huazhong+University+of+Science+and+Technology.+%5BMed+Sci%5D%22%5BJournal%5D)">Isolation, Culture and Identification of Neural Stem Cells in New-born Rats.</a> Journal of Huazhong University of Science and Technology. [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/pubmed?term=((Establishment+and+characterization+of+multipotent+neural+cell+lines+using+retrovirus+vector-mediated+oncogene+transfer%5BTitle%5D)+AND+%22Journal+of+Neurobiology%22%5BJournal%5D)">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/pubmed?term=((Neural+progenitor+cell+engraftment+corrects+lysosomal+storage+throughout+the+MRS+VII+mouse+brain%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Multipotent+neural+precursors+can+differentiate+toward+replacement+of+neurons+undergoing+targeted+apoptotic+degeneration+in+adult+mouse+neocortex%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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>
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</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Coverslip</td><td>NEST</td><td>801008</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Beyotime</td><td>P0128</td><td></td></tr><tr><td>DMEM medium</td><td>HyClone</td><td>SH30022.01B</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>DMEM/F12 medium</td><td>HyClone</td><td>SH30023.01B</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504044</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30084.03</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Donor Horse serum</td><td>HyClone</td><td>SH30074.03</td><td>tored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Penicillin/Streptomycin</td><td>HyClone</td><td>SV30010</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Trypsin</td><td>HyClone</td><td>25300054</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>PBS solution</td><td>HyClone</td><td>SH30256.01B</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>4% paraformaldehyde</td><td>Beyotime</td><td>P0098</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>TritonX-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Normal Goat Serum Blocking Solution</td><td>Vector Laboratories</td><td>S-1000-20</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>anti-Nestin</td><td>Beyotime</td><td>AF2215</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-&amp;beta;-Tubulin III</td><td>Sigma Aldrich</td><td>T2200</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-O4</td><td>R&amp;amp;D Systems</td><td>MAB1326</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-NF200</td><td>Sigma</td><td></td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-ChAT</td><td>Sigma</td><td></td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti- LHX3</td><td>Sigma</td><td></td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-GABA</td><td>Sigma</td><td></td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-Serotonin</td><td>Abcam, Cambridge, MA</td><td></td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>anti-TH</td><td>Abcam, Cambridge, MA</td><td></td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Immunol Staining Primary Antibody Dilution Buffer</td><td>Beyotime</td><td>P0103</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Cy3 Labeled Goat Anti-Rabbit IgG</td><td>Beyotime</td><td>A0516</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>Alexa Fluor 488- Labeled Goat</td><td>Beyotime</td><td>A0428</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>Anti-Mouse IgG&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>12-well plate</td><td>corning</td><td>3512</td><td></td></tr><tr><td>25 cm2 flask</td><td>corning</td><td>430639</td><td></td></tr><tr><td>Hoechst 33258</td><td>Beyotime</td><td>C1018</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>Mounting medium</td><td>Beyotime</td><td>P0128</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>Light microscope</td><td>Nanjing Jiangnan Novel Optics Company</td><td>XD-202</td><td></td></tr><tr><td>Fluorescence microscopy</td><td>Nikon</td><td>80i</td><td></td></tr><tr><td>High &amp;ndash; voltage Power Amplifier</td><td>Directed Energy</td><td>PVX-4110</td><td></td></tr><tr><td>DC power supply</td><td>Spellman</td><td>SL1200</td><td></td></tr><tr><td>Function Generator</td><td>Aligent&amp;nbsp;</td><td>33521A</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>DPO3034</td><td></td></tr><tr><td>High voltage probe</td><td>Tektronix</td><td>P6015A</td><td></td></tr></tbody></table>

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, ...

nerve-stem-cell-differentiation-one-step-cold-atmospheric-plasma

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JoVE Journal

Engineering

7.3K Views.  Huazhong University of Science and Technology. Deze methode heeft het potentieel om de differentiatie van neurale stamcellen in vitro te verbeteren en bij te dragen aan de behandeling van neurologische aandoeningen zoals de ziekte van Parkinson. De belangrijkste voordelen van deze techniek is dat de koude atmosferische plasma's in één stap kunnen worden toegepast voor de veilige gerichte differentiatie van neurale stamcellen voor weefseltransplantatie. Koud atmosferisch plasma kan de differentiatie van neurale stamcellen binnen een kort...

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Developmental Biology

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neuroscience

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required.
We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

Here, we present a method for reproducible generation of ventral midbrain patterned astrocytes from hiPSCs and protocols for their characterization to assess phenotype and function.

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells

Source: Becker-Koji&#263;, Z. A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61634">GM-Free Generation of Blood-Derived Neuronal Cells.</a> J. Vis. Exp. (2021).
This video demonstrates a technique to generate neuronal cells from blood-derived pluripotent stem cells (BD-PSCs). The BD-PSCs are obtained via reprogramming blood progenitor cells. The undifferentiated BD-PSCs are cultured on cell culture substrate-coated coverslips using a neural induction medium and a neural differentiation medium, triggering differentiation into cells with a neuronal phenotype.

Ethic approvals were obtained when performing the experiments.
1. Isolation of human peripheral blood mononuclear cells (PBMNCs)

	Ensure that all donors ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

Co-culture and Interaction Studies

Stimulating Nerve Stem Cell Differentiation Using Cold Atmospheric Plasma Treatment

Source: Xiong, Z., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58663">Nerve stem cell differentiation by a one-step cold atmospheric plasma treatment in vitro.</a> J. Vis. Exp. (2019).
This video presents a method for triggering differentiation in neural stem cells by utilizing cold atmospheric plasma. This generates UV radiation and reactive molecules to stimulate nerve cells to produce projections, indicating neuronal cell differentiation.

1. Cell Cultures and Predifferentiation

	Neural stem cell culture and predifferentiation
	
		Prepare poly-D-lysine-coated coverslips. Put a sterile ...

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

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