Name: assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress
Uploaded: 2014-05-07T13:45:00.000+00:00
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Description: 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

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><tbody><tr><td>EdU</td><td>Life Technologies</td><td>A10044</td><td>1 mg/ml</td></tr><tr><td>BrdU</td><td>Life Technologies</td><td>B5002</td><td>5 mg/ml</td></tr><tr><td>IBL 637</td><td>CIS BIO International</td><td></td><td></td></tr><tr><td>Tissu Tek VIP</td><td>Leica</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM 2125 RT</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>93443</td><td></td></tr><tr><td>Click-iT EdU Alexa 488 imaging kit</td><td>Life Technologies</td><td>C10083</td><td></td></tr><tr><td>Anti-BrdU</td><td>GE Healthcare</td><td>RPN202</td><td>1/300</td></tr><tr><td>Goat anti mouse-Alex594</td><td>Life Technologies</td><td>A11001</td><td>1/400</td></tr><tr><td>Fluoromount</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>Polysine slide</td><td>Thermo scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>20012-068</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Microscope BX51</td><td>Olympus</td><td></td><td></td></tr><tr><td>Confocal microscope SPE</td><td>Leica</td><td></td><td></td></tr><tr><td>Prism software</td><td>Graphpad</td><td></td><td>Version 5.0c</td></tr><tr><td>Photoshop software</td><td>Adobe</td><td></td><td></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...

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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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

Medicine

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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 ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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. ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

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

Summary

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Chapters in this video

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

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

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury