The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n&#176; 323267, from Electricit&#233; de France (EDF) and from l&#8217;Agence Nationale de la Recherche &#8211; Sant&#233;-Environnement et Sant&#233;-Travail (ANR-SEST, Neurorad).

1. Animal Procedures
This protocol has been designed in compliance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and has been approved by our institutional committee on animal welfare (CETEA-CEA DSV IdF).
<ol>
	<li>Injections with EdU/BrdU and irradiation of E14.5 pregnant mice (Figure 2A).
	<ol>
		<li>Prepare a solution of EdU at 1 mg/ml&#160;and BrdU at 5 mg/ml&#160;in PBS. Perform an intra-peritoneal injection (IP) of 100 &#181;l of EdU solution using a 25 G needle into pregnant mice. 1.5 hr later, irradiate the mice (total body irradiation) at 0 Gy or 2 Gy (0.6 Gy/min). Immediately after, inject (IP) 200 &#181;l of BrdU solution using a 25 G&#160;needle into the pregnant mice.</li>
	</ol>
	</li>
	<li>Preparation of coronal slices of embryonic brains.
	<ol>
		<li>At 1 hr or 4 hr after irradiation, euthanize the pregnant mice by carbon dioxide.</li>
		<li>Open the abdominal cavity over three centimeters using scissors. Separate the two uterine horns from the rest of the uterus by sectioning both extremities of the horns. Transfer the uterine horns into a Petri dish containing PBS 0.6% glucose. Open the uterine horns with the forceps in order to isolate the embryos. Remove the amniotic sac of each embryo using forceps.</li>
		<li>Dissect embryonic heads with forceps and fix them by immersion in 4% paraformaldehyde (PFA, pH=7.4) O/N at 4 &#176;C.</li>
		<li>Rinse embryonic heads in PBS for at least one night at 4 &#176;C. Heads can be kept in PBS for several days.</li>
		<li>Embed heads in paraffin using a vacuum infiltration processor as indicated in Table 1. Heads embedding can also be performed under a chemical hood for ethanol and xylen baths, and then in a 65 &#176;C oven for the paraffin baths.</li>
		<li>Prepare 5-&#181;m thick coronal sections with a microtome.</li>
		<li>Mount onto polylysine-coated microscope slides.</li>
		<li>Slides can be kept at room temperature (RT) for several months.</li>
	</ol>
	</li>
</ol>
2. EdU/BrdU Staining
<ol>
	<li>Deparaffinization and antigen unmasking
	<ol>
		<li>Deparaffinize the paraffin-embedded brain sections by immersion of slides in 3 baths of toluene for 5 min.</li>
		<li>Rehydrate for 3 min in 2 baths of each solutions of decreasing ethanol concentration as following: ethanol 100%, ethanol 95%, ethanol 70%, and 5 min in H2O. Slides can be kept in deionized water for several hr.</li>
		<li>Boil the slices for 10 min in citrate solution (10 mM, pH 6), and then incubate in deionized water for 5 min.</li>
	</ol>
	</li>
	<li>EdU staining
	<ol>
		<li>Perform EdU detection according to the manufacturer&#8217;s protocol. Permeabilize cells with 0.5% triton X-100 in PBS for 10min.</li>
		<li>Prepare 1X EdU buffer additive by diluting the 10X solution in deionized water. This solution should be freshly made and used on the same day.</li>
		<li>Prepare EdU reaction cocktail, including EdU Alexa Fluor 488 azide. It is important to add the ingredients following the order listed in Table 2, otherwise, the reaction will not proceed optimally. Use the EdU reaction cocktail within 15 min of preparation.</li>
		<li>Remove the permeabilization buffer (step 2.1.1), then wash twice with PBS.</li>
		<li>Add 150 &#181;l&#160;of EdU reaction cocktail, put a coverslip, and incubate for 30 min in the dark at RT.</li>
		<li>Remove the EdU reaction cocktail, then wash once with PBS.</li>
	</ol>
	</li>
	<li>BrdU staining
	<ol>
		<li>Prepare saturation buffer with 7.5% goat serum and 7.5% fetal bovine serum in PBS. Add 150 &#181;l&#160;of saturation buffer on brain slices, put a coverslip, and incubate for 1 hr at RT to block non-specific antibody binding.</li>
		<li>Remove the coverslip. Add 150 &#181;l&#160;of mouse anti-BrdU primary antibody diluted at 1/300 in saturation buffer, put a coverslip and incubate O/N at 4 &#176;C.</li>
		<li>Remove the coverslip, then wash 3 times in PBS.</li>
		<li>Add 150 &#181;l&#160;of goat anti-mouse-Alexa594 secondary antibody diluted at 1/400 in saturation buffer, put a coverslip, and incubate for 1 hr at RT.</li>
		<li>Remove the coverslip, then wash once in PBS.</li>
		<li>Nuclear staining: add 150 &#181;l of 4&#8217;,6-diamidino-2-phenylindole (DAPI) at 1 &#181;g/ml in PBS, put a coverslip and incubate for 2 min at RT.</li>
		<li>Mount slides in mounting medium.</li>
	</ol>
	</li>
</ol>
3. Analysis
<ol>
	<li>Examine brain sections under a fluorescence microscope with a 20X objective or a confocal microscope and acquire images in three channels (488 nm, 594 nm and UV) as separates files.</li>
	<li>Stack images with Photoshop software.</li>
	<li>Analyze brain sections in a standard sector of the dorsomedial cerebral wall. This sector is 100 &#181;m in its medial-lateral dimension and is divided into 18 bins (or more) of 10 &#181;m height in its radial dimension using a grid superposed on images (Figure 1) as previously described31.
	<ol>
		<li>Align the grid such as the first bin is at the ventricular surface, with its long axis parallel to the ventricular border. Then number the labeled EdU, BrdU and/or pyknotic nuclei (corresponding to apoptotic nuclei) in each bin. Number a nucleus located on the boundary of two bins in the bin which contains its larger part, or in the lower bin, when the nucleus occupies equal areas within the two bins.</li>
	</ol>
	</li>
	<li>Statistical analysis.</li>
</ol>
Analyze at least two cortical slices per embryo. Repeat experiments on at least 3 embryos from different litters. Results should be given as mean &#177;&#160;standard error of the mean (SEM). Statistical analyses are conducted with Graphpad Prism using two-way ANOVA and Bonferroni multiple comparison posthoc tests.

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The authors have no conflict of interest.

The experimental design described here based on incorporation of EdU 1.5 hr before irradiation and incorporation of BrdU immediately after irradiation allowed the demonstration that NSPCs are able to activate S and G2/M checkpoints but not the G1/S checkpoint during the 1st hr after a genotoxic stress in the fetal mouse brain. We performed other experiments in which EdU has been injected at different times after irradiation and BrdU, just 1 hr before mice sacrifices, allowing us to specifically appreciate the ...

During embryonic brain development, projection neurons of the cerebral cortex are generated in the ventricular zone, a pseudostratified epithelium composed of different types of neural stem and progenitor cells (NSPC) that lines the lateral ventricles. Among NSPC, the radial glial cells (RGC), which serve as neural stem cells, undergo interkinetic nuclear migration (INM): they perform mitosis at the surface of the ventricle and S-phase at the basal limit of the ventricular zone (VZ)1,2,3. They can divide either symmetrically to generate two RGC or asymmetrically to generate one RGC and one neuron or an intermediate progenitor cell (IPC)4,5. IPC migrate to an overlying proliferating layer called the subventricular zone (SVZ), where after one last symmetric division they generate two immature projection neurons6-8. Contrary to RGC, IPC do not undergo INM (reviewed in 9). Newly generated neurons migrate radially along the radial fibers through the intermediate zone (IZ) to reach their final destination in the cortical plate (CP)10,8. The perfect timing of all these events is essential for a correct cortical development. For instance the switch from proliferation to differentiation of neural progenitor populations is controlled by the G1 phase duration11,12. The lengthening of the G1 phase correlates thus with cell differentiation.
Genotoxic stress, such as ionizing radiation, severely impair brain development (reviewed in 13). We and others have shown that NSPC are highly prone to radiation-induced apoptosis14,15,16. Ionizing radiations induce DNA double strand breaks that are the most severe damage to proliferating cells. One essential component of DNA Damage Response (DDR) in cycling cells is the activation of cell cycle checkpoints at the G1/S or G2/M transitions or during S phase (intra-S checkpoint)17-21. They block the cell cycle progression to provide time for DNA damage repair or elimination of too damaged cells. Consequently, cell death as well as delayed cell cycle progression may alter brain development in response to ionizing radiation exposure22-24. It was thus interesting to develop a method to assess the activation of cell cycle checkpoints in NSPC in the irradiated mouse embryonic brain.
The progression of the cell cycle is routinely followed using the incorporation of a thymidine analog, 5-Bromo-2&#8217;-deoxyUridine (BrdU). BrdU is incorporated during the S phase of the cell cycle, when DNA is replicating. The use of an antibody against BrdU allows thereafter the detection of cells that were in S phase during the pulse of BrdU.
A novel thymidine analog, 5-Ethynyl-2&#8217;-deoxyUridine (EdU) is detected by a fluorescent azide. The different ways to detect EdU and BrdU do not cross-react25, enabling the simultaneous detection of both thymidine analogs, which is useful for the study of cell cycle progression. Usually cells are first pulsed with EdU and then pulsed with BrdU, where the time between both incorporations lasts couple of hours25,26. Addition of BrdU in culture media containing EdU results in preferentially incorporation of BrdU into the DNA with the exclusion of EdU, while simultaneous addition of EdU25 with equimolar or half equimolar BrdU to the media results in only BrdU incorporation27. This simplifies the dual labeling protocol by eliminating the wash steps that are normally required to remove the first label from the culture media prior to addition of the second label. This also is of a particular interest for in vivo study, where the washing steps are not possible, to determine the precise timing of S phase entry or exit of cell population.
Recently, a method of dual-pulse labeling in embryonic mouse brain using EdU and BrdU23,24,22 has been developed to analyze cell cycle progression and INM of NSPC after in utero irradiation. Moreover it has been demonstrated that, during S phase, mouse cells replicate first the euchromatin regions and then the pericentric heterochromatin28,29,30. Interestingly, pericentric heterochromatin of different chromosomes clustered in interphasic nuclei to form heterochromatic foci also known as chromocenters and easily detectable by DAPI staining as bright foci. Therefore, the differential EdU and BrdU stainings of euchromatin and chromocenters helped us to analyze more precisely S phase progression of NSPC.
This method allowed the demonstration of the apparent lack of G1/S checkpoint in NSCP22-24, which is quite surprising since this checkpoint is supposed to be critical for genome stability.&#160; Several experimental designs based on various combinations of EdU and BrdU pulses have been used to analyze cell cycle progression in the ventral and dorsal telencephalon. Here, we give an example of protocols allowing the study of the acute DNA damage of NSPC within the first 4 hr&#160;following in utero irradiation of E14.5 mouse embryos.

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:228px;">EdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A10044</td>
			<td style="width:163px;">1 mg/ml</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">BrdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">B5002</td>
			<td style="width:163px;">5 mg/ml</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:228px;">IBL 637</td>
			<td style="width:212px;">CIS BIO International</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Tissu Tek VIP</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Microtome</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;">RM 2125 RT</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Triton X-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td align="right" style="width:125px;">93443</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:228px;">Click-iT EdU Alexa 488 imaging kit</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">C10083</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:228px;">Anti-BrdU</td>
			<td style="width:212px;">GE Healthcare</td>
			<td style="width:125px;">RPN202</td>
			<td style="width:163px;">1/300</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:228px;">Goat anti mouse-Alex594</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A11001</td>
			<td style="width:163px;">1/400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Fluoromount</td>
			<td style="width:212px;">SouthernBiotech</td>
			<td style="width:125px;">0100-01</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Polysine slide</td>
			<td style="width:212px;">Thermo scientific</td>
			<td style="width:125px;">J2800AMNZ</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Paraformaldehyde</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">P6148</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:228px;">PBS</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">20012-068</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:228px;">DAPI</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">D9542</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Microscope BX51</td>
			<td style="width:212px;">Olympus</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Confocal microscope SPE</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Prism software</td>
			<td style="width:212px;">Graphpad</td>
			<td style="width:125px;"></td>
			<td style="width:163px;">Version 5.0c</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Photoshop software</td>
			<td style="width:212px;">Adobe</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

In the experiment described in Figure 2, EdU was administered 1.5 hr&#160;before irradiation and BrdU just after irradiation. Four types of cells were then distinguished in the cortical slices prepared at 1 or 4 hr post-irradiation, according to the incorporation of either EdU or BrdU, both or none (Figures 2A and 2B). Importantly, neither EdU nor BrdU incorporation changed the level of radiation-induced apoptosis (data not shown). Moreover, the staining methods allow on...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress | Neuroscience | Video

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

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15.2K Views.  CEA DSV iRCM SCSR. Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Video: Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

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.

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