This work was supported by the Royan Institute.

All the surgeries and procedures on animal were approved by the animal ethics committee of the Royan Institute, Tehran, Iran.
1. Preparation of Surgical Instruments, Washing Buffer (HEPES-MEM), Cell Culture Media and Cell Culture Plates
<ol>
	<li>Sterilize the surgical tools (scissors, scalpel blade handle and forceps) by autoclaving them based on the routine sterilization guidelines.</li>
	<li>Prepare an adequate volume of HEPES-MEM buffer (around 100 mL is enough for each pregnant mice). Add high concentrations of antibiotics (10% penicillin/streptomycin) to the HEPES-MEM buffer. This concentration of antibiotics is necessary to reduce the risk of microbial contamination.
	<ol>
		<li>To prepare HEPES-MEM buffer, add 0.5958 g of HEPES powder to 100 mL of MEM (minimum essential medium), and mix well. Adjust the pH to 7.3-7.4 and filter the medium using a 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Make NSC medium containing neurobasal medium (NB), supplemented with 20 ng/mL EGF, 10 ng/mL bFGF, 1% 100x N2 supplement or 1% B27 supplement (without vitamin A), 1 U/mL Heparin, 1% L-glutamine, 1% non-essential amino acid (NEAA), 1% Penicillin/streptomycin, and 0.1 mM &#946;-mercaptoethanol.</li>
</ol>
2. Animal Surgery and Micro-Dissection
<ol>
	<li>Anesthetize a pregnant mouse on the 13th day of gestation (here, use BALB/c strain aged 4 months) by intraperitoneal injection of ketamine-xylazine, 100 mg/kg and 10 mg/kg by body weight respectively.
	<ol>
		<li>Confirm deep anesthesia by exerting a painful stimulus (e.g., pinch with forceps) on the animal&#39;s foot and when there is no pain reflex, sacrifice the animal by cervical dislocation.</li>
	</ol>
	</li>
	<li>Clean and disinfectant the skin of the abdomen by spraying 70% ethanol.</li>
	<li>Control the skin over the abdomen and then cut the skin and the underlying fascia with scissors to uncover the abdominal cavity and uterine horns.
	<ol>
		<li>Remove the uterine horns containing the embryos by scissors and transfer them into a 50 mL conical tube containing 25 mL of cold HEPES-MEM buffer.</li>
		<li>Place the conical tube under the laminar flow hood and open the cap under the hood. Bring the uterine horns out from the tube and rinse 3 times with enough volume of the fresh cold HEPES-MEM buffer to eliminate debris and blood.</li>
	</ol>
	</li>
	<li>Transfer the uterine tissue to a 10 cm Petri dish containing 7-10 mL of cold HEPES-MEM buffer. Open the uterine horns using fine scissors and transfer embryos to a new dish containing 7-10 mL of cold HEPES-MEM buffer. Now put the 10 cm dish with embryos under the dissecting microscope for the micro-dissection operation.</li>
	<li>Bend the tip of a 25-27 G syringe needle by pushing its tip on a steel scalpel blade handle. Bend the tip of the needle until the tip is bent at an angle of 70-90&#176;. Check the tip of the needle under the dissecting microscope to see the bend at the tip of the needle. 
	NOTE: Naturally, embryos have a lateral position in the dish.</li>
	<li>Without changing their position, hold the neck and body of the embryo with fine forceps and then insert the bent part of the needle tip into the frontal fontanelle of the embryo head. There would be small bleeding or a blood clot there.
	<ol>
		<li>Move the needle tip superficially. While the bent part is inside the bulge toward the forehead and then toward back of the head, make sure to cut through the skin and the skull and not to damage the underlying brain.</li>
		<li>Push the skin with the needle tip laterally to uncover the brain and insert the needle from the lateral side of the skin to the area underneath the brain. Remove the brain from the skull by pushing it to come out from the dissected skull.</li>
		<li>Hold the brain steady using the curved forceps and cut the cortex of each hemisphere using micro-scissors to expose the ganglionic eminence.</li>
		<li>Hold the brain and place the dissected ganglionic eminence into the 15 mL centrifuge tube containing neural stem cell medium. The ganglionic eminence is clearly shown with its middle and lateral parts.</li>
		<li>Repeat this procedure until all the brains have been micro-dissected.</li>
	</ol>
	</li>
	<li>Cut the dissected ganglionic eminence by placing the pipette tip against the bottom of the tube. Pipette the suspension up and down 4 to 5 times to break up the tissue for a single cell suspension.
	<ol>
		<li>Centrifuge the suspension at 110 x g for 5 min. Remove the supernatant and re-suspend the cells in 1 mL of 0.05% trypsin-EDTA.</li>
		<li>Incubate the cell suspension in a 37 &#176;C for 10 min, and then add an equivalent volume of soybean trypsin inhibitor (25 &#181;g/mL) to stop the trypsin activity. Pipette the cell suspension up and down to make sure that the trypsin has been totally inactivated.</li>
		<li>Centrifuge the cell suspension at 110 x g for 5 min. Remove the supernatant and re-suspend the cells in 1 mL of complete NSC medium and count the number of cells.</li>
	</ol>
	</li>
	<li>Plate the cells (2x105 cells/mL) at NSC medium into the suitable size tissue culture flask. Transfer 5 mL to T25, 20 mL to T75 and 40 mL to T175 flasks. 
	NOTE: Neurospheres will appear after 3-days culture at 37 &#176;C in a humidified incubator with 5% CO2. After 6-7 days, the spheres should measure between 150-200 &#181;m in diameter and will be ready for subculture.By cultivating one million primary NSCs after seven days the number of cells will increase to 3-4 million.</li>
</ol>
3. Trypsinization and Passaging of Neurospheres
<ol>
	<li>To subculture the neurospheres, transfer the medium with suspended neurospheres to the centrifuge tube, centrifuge at 110 x g for 5 min and then discard the supernatant. Add 1 mL of 0.05% trypsin-EDTA and incubate at 37&#176;C for 10 min.
	<ol>
		<li>Inactivate the trypsin using soybean trypsin inhibitor and pipette the neurospheres gently up and down to make them single cells.</li>
	</ol>
	</li>
	<li>Centrifuge the cell suspension at 110 x g for 5 min, discard the supernatant and re-suspend the cells in 1 mL of NSC medium. These cells are ready for next subculture or culture on Laminin and Poly-L-Ornithine coated surfaces for advanced experiments.</li>
	<li>For the next subculture, transfer NSCs into the new flask (follow step 2.8) and culture them in the NSC medium. For induction of neuronal differentiation culture NSCs in the NSC medium in the absence of bFGF and EGF on Laminin and Poly-L-Ornithine coated plates (Figure 1B).</li>
	<li>For the coating procedure, fully coat the cell culture surface by adding enough volume of diluted poly-L-ornithine solution (25 &#181;g/mL) to the cell culture plate and incubate at 37 &#176;C for 2 h.
	<ol>
		<li>Aspirate the poly-L-ornithine and rinse the well 2x with PBS. Add enough volume of 5 &#181;g/mL laminin to completely cover the culture surface area and incubate at 37 &#176;C for 2 h. Aspirate the laminin before plating cells or store plate at 4 &#176;C until needed.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry Assay
NOTE: The expression of specific markers of NSCs, neurons and glial cells can be measured by flow cytometry assay13. The protocol is based on Menon et al.,13 with minor modifications.
<ol>
	<li>Dissociate neurospheres or detach cultured NSCs from coated plates by trypsinization to make them single cells. Add 500 &#181;L of fixation buffer (2% paraformaldehyde (PFA) in PBS) to 100 &#181;L of the cell suspension (around 1 million cells are enough for staining with each antibody), and then incubate the tubes at RT for 15 min.</li>
	<li>Wash cells by adding 1 mL of PBS to the tube and centrifuge at 110 x g for 5 min. Discard the supernatant and resuspend the cell pellet, leaving approximately 100 &#181;L in the tube.
	<ol>
		<li>Permeabilize cells with Tween-20 (0.7% Tween-20 in PBS) by adding 500 &#181;L of Tween-20 buffer to 100 &#181;L of cell suspension. Incubate the tubes at RT for 15 min on an orbital shaker (100 rpm).</li>
	</ol>
	</li>
	<li>Repeat the centrifugation and the PBS wash. Remove the supernatant from the tubes completely, leaving only the pellet behind. Add 100 &#181;L of diluted primary antibodies (rabbit anti mouse &#946;tub-III, GFAP and Nestin antibodies at 1:200 concentration) in dilution buffer containing 1% bovine serum albumin, 10% serum (goat serum) and 0.5% Tween-20 in PBS and gently triturate to mix. Incubate the tubes at RT for 30 min on an orbital shaker.</li>
	<li>Wash the cells once with PBS and remove the supernatant from the tubes completely, leaving only the pellet behind.
	<ol>
		<li>Add 100 &#181;L of diluted secondary antibody (goat anti rabbit FITC conjugated at 1:500 concentration) in PBS to the cell pellet and gently triturate to mix. Incubate the tubes at RT for 30 min on a shaker in the dark.</li>
		<li>Wash the samples 2x with PBS and then wash once with flow buffer. Centrifuge and re-suspend the cells in approximately 150 &#181;L of flow buffer for flow cytometer analysis.</li>
	</ol>
	</li>
</ol>

<ol><li>Navarro Quiroz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+Signaling+in+Neuronal+Stem+Cells%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Cell Signaling in Neuronal Stem Cells.</a> Cells. 7 (7), (2018).</li>
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<li>Aligholi, H., Hassanzadeh, G., Gorji, A., Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Biopsy+Method+for+Isolating+Neural+Stem+Cells+from+the+Subventricular+Zone+of+the+Adult+Rat+Brain+for+Autologous+Transplantation+in+CNS+Injuries%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">A Novel Biopsy Method for Isolating Neural Stem Cells from the Subventricular Zone of the Adult Rat Brain for Autologous Transplantation in CNS Injuries.</a> Methods in Molecular Biology. 1462, 711-731 (2016).</li>
<li>Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishing+embryonic+mouse+neural+stem+cell+culture+using+the+neurosphere+assay%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Establishing embryonic mouse neural stem cell culture using the neurosphere assay.</a> Journal of Visualized Experiments. (47), (2011).</li>
<li>Ferrari, D., Binda, E., De Filippis, L., Vescovi, A. L. Isolation of neural stem cells from neural tissues using the neurosphere technique. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFerrari%2C+D+%22Current+Protocols+in+Stem+Cell+Biology%22">Current Protocols in Stem Cell Biology</a>. , Chapter 2 Unit2D.6 (2010).</li>
<li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nature Protocols. 7 (2012), (2005).</li>
<li>Liu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passage+Number+is+a+Major+Contributor+to+Genomic+Structural+Variations+in+Mouse+iPSCs%5BTitle%5D)+AND+%22Stem+Cells+and+Development%22%5BJournal%5D)">Passage Number is a Major Contributor to Genomic Structural Variations in Mouse iPSCs.</a> Stem Cells and Development. 32 (10), 2657-2667 (2014).</li>
<li>Diaferia, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+Chromosomal+Analysis+of+Cultured+Mouse+Neural+Stem+Cell+Lines%5BTitle%5D)+AND+%22Stem+Cells+and+Development%22%5BJournal%5D)">Systematic Chromosomal Analysis of Cultured Mouse Neural Stem Cell Lines.</a> Stem Cells and Development. 20 (8), 1411-1423 (2011).</li>
<li>Hemmer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+Neural+Stem+Cells+Achieve+Long-Term+Survival+and+Functional+Integration+in+the+Adult+Mouse+Brain%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Induced Neural Stem Cells Achieve Long-Term Survival and Functional Integration in the Adult Mouse Brain.</a> Stem Cell Reports. 3 (3), 423-431 (2014).</li>
<li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+Cytometry+Protocols+for+Surface+and+Intracellular+Antigen+Analyses+of+Neural+Cell+Types%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types.</a> Journal of Visualized Experiments. (94), 52241(2014).</li>
<li>Zhang, Z., Wang, Z., Rui, W., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnesium+lithospermate+B+promotes+proliferation+and+differentiation+of+neural+stem+cells+in+vitro+and+enhances+neurogenesis+in+vivo%5BTitle%5D)+AND+%22Tissue+and+Cell%22%5BJournal%5D)">Magnesium lithospermate B promotes proliferation and differentiation of neural stem cells in vitro and enhances neurogenesis in vivo.</a> Tissue and Cell. 53, 8-14 (2018).</li>
<li>Zilkha-Falb, R., Gurevich, M., Hanael, E., Achiron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prickle1+as+positive+regulator+of+oligodendrocyte+differentiation%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Prickle1 as positive regulator of oligodendrocyte differentiation.</a> Neuroscience. 364, 107-121 (2017).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligodendrocyte+differentiation+from+human+neural+stem+cells%3A+A+novel+role+for+c-Src%5BTitle%5D)+AND+%22Neurochemistry+International%22%5BJournal%5D)">Oligodendrocyte differentiation from human neural stem cells: A novel role for c-Src.</a> Neurochemistry International. 120, 21-32 (2018).</li>
<li>Palanisamy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxytocin+alters+cell+fate+selection+of+rat+neural+progenitor+cells+in+vitro%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Oxytocin alters cell fate selection of rat neural progenitor cells in vitro.</a> PLoS ONE. 13 (1), e0191160(2018).</li>
<li>Llewellyn-Smith, I. J., Basbaum, A. I., Braz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term%2C+dynamic+synaptic+reorganization+after+GABAergic+precursor+cell+transplantation+into+adult+mouse+spinal+cord%5BTitle%5D)+AND+%22Journal+of+Comparative+Neurology%22%5BJournal%5D)">Long-term, dynamic synaptic reorganization after GABAergic precursor cell transplantation into adult mouse spinal cord.</a> Journal of Comparative Neurology. 526 (3), 480-495 (2018).</li>
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No conflict of interest declared.

Using a suitable source of neural stem cells is very important for neuroscientists. Neural stem cells could be harvested from different areas of the embryo brain and they can generate specific types of neurons and glial cells. There are several methods for induction of differentiation of neural stem cells to induce them to generate mature neurons and glial cells. There are numerous reports indicating that under specific culture conditions and trophic factor regiments, they could generate neurons14...

Neural stem cells (NSCs) reside in different regions of the adult and embryo brain and they have a tendency to generate different types of neurons and glial cell1. The subventricular zones in the adult mammalian brain2 and ganglionic eminence in the embryo brain are NSC rich regions3. In the developing brain, the ganglionic eminence delivers most of the cortical interneurons and particularly GABAergic interneurons3. There are also less invasive methods for neural stem cell generation from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that reduce the demand for animal use. Despite the fact that generating NSCs from ESCs or iPSCs is possible4,5, it has some advantages and disadvantages compared to the isolation of neural stem cells from the adult or embryo brain6,7,8. The protocols for inducing the differentiation of ESCs and iPSCs toward neuronal phenotypes are always time and cost consuming and the rate of success (70-80% Nestin positive cells)5 is lower compared with direct isolation of NSCs from the animal brain (more than 99% Nestin positive cells)9. Moreover, stem cells lose their genetic stability and differentiation tendency after several passages10,11. Even though there are other new reports about direct conversion of somatic cells into NSCs, these cells are genetically engineered and are not easily accessible in every lab12. Therefore, there is still a big demand for isolation of neural stem cells from the animal brain; it is possible to reduce the quantity of animal usage by improving the surgical techniques. By reducing the surgical time and improving the techniques, it is possible to keep cells away from damage and obtain the highest rate of NSCs from each animal.
Here, we introduce a simplified and reproducible technique for isolation of neural stem cells from mouse E13 embryo brain.

<table><tbody><tr><td>15 mL tubes</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>50 mL tubes</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Adson Forceps, 12 cm, Straight</td><td>WPI</td><td>14226</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504-44</td><td></td></tr><tr><td>bFGF</td><td>Sigma</td><td>F0291</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>dish 10cm&amp;nbsp;</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>Dressing Forceps, 12.5 cm, Straight</td><td>WPI</td><td>15908</td><td></td></tr><tr><td>Dumont Tweezers, 11 cm, Straight</td><td>WPI</td><td>500342</td><td></td></tr><tr><td>EGF</td><td>Sigma</td><td>E9644</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>100983&amp;nbsp;</td><td></td></tr><tr><td>Glutamate</td><td>Sigma&amp;nbsp;</td><td>G3291</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>goat anti rabbit FITC conjugated secondary antibody</td><td>Sigma</td><td>AP307F</td><td></td></tr><tr><td>goat serum</td><td>Sigma</td><td>G9023</td><td></td></tr><tr><td>Heparin&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>h3149</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>83264</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>90909C</td><td></td></tr><tr><td>insulinsyringe with 25-27 gauge Needle&amp;nbsp;</td><td>SUPA medical</td><td>A1SNL127</td><td></td></tr><tr><td>laminin</td><td>sigma&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>L2020</td><td></td></tr><tr><td>MEM</td><td>Sigma</td><td>M2279</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>NB medium</td><td>Gibco</td><td>21103-31</td><td></td></tr><tr><td>Non-essential amino acid (NEAA)</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>PBS without Ca and Mg</td><td>Gibco</td><td>20012050</td><td></td></tr><tr><td>Penicilin/ Streptomycin</td><td>Gibco</td><td>5140122</td><td></td></tr><tr><td>Poly-L-ornithine</td><td>Sigma</td><td>&amp;nbsp;P4957</td><td></td></tr><tr><td>rabbit anti mouse beta tubulin-III antibody</td><td>Sigma</td><td>T2200</td><td></td></tr><tr><td>rabbit anti mouse GFAP antibody</td><td>Sigma</td><td>G4546</td><td></td></tr><tr><td>rabbit anti mouse Nestin antibody</td><td>Sigma</td><td>N5413</td><td></td></tr><tr><td>Scalpel Handle #3</td><td>WPI</td><td>500236</td><td></td></tr><tr><td>Scissors curve</td><td>WPI</td><td>14396</td><td></td></tr><tr><td>Scissors sharp straight</td><td>WPI</td><td>14192</td><td></td></tr><tr><td>Soybean trypsin inhibitor</td><td>Roche</td><td>10109886001&amp;nbsp;</td><td></td></tr><tr><td>Tissue culture flasks, T25</td><td>BD</td><td>353014</td><td></td></tr><tr><td>Tissue culture flasks, T75</td><td>BD</td><td>353024</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Vannas Scissors,&amp;nbsp; 8 cm, Straight&amp;nbsp;</td><td>WPI</td><td>14003</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M6250</td><td></td></tr></tbody></table>

isolation and culture of embryonic mouse neural stem cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Micro-Dissection, Cell Isolation and Neurosphere Culture. Here, we presented a rapid and efficient method for mouse E13 brain microsurgery and isolation of neural stem cells. This article shows that it is possible to remove the whole brain from an E13 mouse embryo skull through a fine rupture in frontal fontanelle. With this method, the brain endures less damage and it is possible to isolate cells from different parts of the brain. The harvested cell...

Watch this Scientific Journal Video about Isolation and Culture of Embryonic Mouse Neural Stem Cells at JoVE.com

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

isolation-and-culture-of-embryonic-mouse-neural-stem-cells

Research

JoVE Journal

Neuroscience

17.9K Views.  ACECR. Het doel van dit videoprotocol is het vaststellen van een bron en efficiënt protocol voor embryonale muis neurale stamcelisolatie en -cultuur. Hallo, ik ben Maryam Sadeghi-Zadeh. Ik ben een master student cellulaire en moleculaire biologie in Royan Institute.Vandaag willen ik en mijn collega je laten zien hoe je neurale stamcel oogsten van 13 embryomuis ganglionic eminentie. Hallo, ik ben Bahareh Alizadeh-Shoorjestan. Ik ben een master student voor cellulaire en moleculaire biologie i...

Video: Isolation and Culture of Embryonic Mouse Neural Stem Cells

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Developmental Biology

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Source: Kim, J. Y., et al. <a href="https://app.jove.com/v/54929">Isolation and culture of adult neural stem cells from the mouse subcallosal zone.</a> J. Vis. Exp. (2016)
The video demonstrates the process of isolating and culturing neural stem cells from the mouse brain&#39;s subcallosal zone. It involves dissecting tissue, enzymatic dissociation, and culturing them with differentiation factors to develop into mature neural cells.

1. Preparation of Materials and Culture Medium

	For the dissection and dissociation of the SCZ (Subcallosal zone), wrap the brain matrix, double-edged ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

Source: Landeira, B. S., et al. <a href="https://app.jove.com/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Source: Edamakanti, C. R., et al. <a href="https://app.jove.com/v/60554">Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum.</a> J. Vis. Exp. (2020).
This video demonstrates a method for isolating stem cells from the postnatal mouse cerebellum and differentiating them into neural cells. Stem cells from the isolated cell suspension are purified using antibody-coated magnetic beads and are cultured to form neurospheres. The neurospheres are then maintained in a differentiation medium to generate neural cells.

1. Preparation of Solutions

	Prepare tissue dissociation solution made from sterile phenol red-containing Dulbecco&#39;s phosphate-buffered saline (DPBS) ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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