Name: using primary neurosphere cultures to study primary cilia
Uploaded: 2017-04-14T12:30:00
Description: Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Work in S.M.'s laboratory is funded by recruitment grants from CPRIT (R1220) and NIH (1R01GM113023-01).

1. Isolation of Neurospheres from the Adult Mouse Brain
<ol>
	<li>Euthanize an adult mouse (around 2 months old) by an overdose of isoflurane. Double-check that the mouse has stopped breathing and dissect immediately after death.</li>
	<li>Using scissors, make a midline incision to open the skull. Remove the brain.</li>
	<li>Place the brain in cold PBS in a 10 cm dish on ice. Follow the whole-mount dissection method to obtain the SVZ from the lateral ventricle48.</li>
	<li>Place the lateral vent...

Here, we describe a method to generate and maintain neurosphere cultures from adult mouse SVZ. A few pertinent points regarding the cultures are as follows. First, the sizes of the spheres are typically between 50 - 200 &#181;m. In our experience, when a neurosphere gets larger than 300 &#181;m in diameter, the optimal time for passaging has been missed. These larger spheres contain dead cells in the core. Second, as neurospheres are commonly used to study neural stem/progenitor cells, it is important to use EGF and FGF-...

The primary cilium is a microtubule-based dynamic subcellular compartment that functions as a sensory antenna in coordinating cellular signaling pathways, including the Sonic Hedgehog (Shh) pathway during embryonic neuronal development1,2, and compartmentalized subcellular signaling in adult neuronal function3,4. Signaling components of these pathways, such as the Shh receptor Patched5; the pathway activator Smoothened (Smo)6; and Gpr1617, an orphan G Protein-coupled Receptor (GPCR) that negatively regulates the Shh pathway, localize to cilia in a dynamic fashion. Multiple GPCRs have been reported to localize to the cilia in neurons in the brain7,8,9,10,11,12,13,14,15,16. Defects in cilia and cilia-generated signaling pathways affect multiple tissues and are collectively known as ciliopathies17,18,19. The ciliopathy disease spectrum frequently includes neurodevelopmental defects, such as craniofacial abnormalities20,21,22. In addition, primary cilia in hypothalamic neurons regulate central satiety pathways, and defects result in central obesity23, mirroring obesity in syndromic ciliopathies such as Bardet Biedel syndrome24. In addition, neuropeptide receptor signaling in cilia regulates central satiety pathways11,14. Ciliary localization of Adenylyl Cyclase III (ACIII) and GPCRs such as somatostatin receptor 3 in hippocampal neurons result in novel object recognition defects and memory deficits25,26 and parallels a lack of ciliary integrity27. The developmental aspects of cilia-generated signaling are closely tied to tissue homeostasis; in particular, cilia are important to the progression of Shh-subtype medulloblastomas arising from granule progenitors in the cerebellum28,29. Thus, primary cilia play important roles during embryonic, postnatal, and adult neuronal development and function.
Neural Stem Cells (NSCs) reside in the subventricular zone (SVZ) of the lateral ventricle, the subgranular zone of the dentate gyrus of the hippocampus, and the ventricular zone of the third ventricle in the hypothalamus in mammals30,31,32. NSCs are multipotent, possess the capacity for self-renewal, and are important for brain development and regenerative medicine30. Most NSCs in the SVZ are quiescent and possess a solitary primary cilium that, in many cases, extends out to the lateral ventricle33. The primary cilium signals via the localization of various receptors, inducing downstream cellular responses, particularly in relation to Shh, TGF&#946;, and receptor tyrosine kinase pathways2,34,35,36. Since primary cilia extend into the lateral ventricle, it is hypothesized that primary cilia detect cytokines in the cerebrospinal fluid (CSF) to activate NSCs37. Recent studies suggest that the Shh signaling pathway and primary cilia are critical for the activation of stem cells in the repair and regeneration of multiple tissues, including the olfactory epithelium, lung, and kidney38,39,40,41. However, the mechanisms by which CSF communicates with NSCs and whether primary cilia are involved are not known. Adherent NSCs in culture are ciliated; localize Shh pathway components, such as Smo and Gpr161 in cilia; and are Shh responsive42. Thus, NSCs can serve as an important model system to study the Shh pathway, ciliary trafficking, and neuronal differentiation pathways. In addition, neurons differentiated from NSCs can also be used for ciliary trafficking assays.
Neurospheres are constituted of clusters of free-floating cells arising from the proliferation of neural stem/progenitor cells that grow in the presence of specific growth factors and nonadhesive surfaces43,44. Neurospheres serve as important in vitro culture models to study neural stem/progenitor cells in normal development and disease31,45,46,47. Here, we describe a neurosphere-based assay for culturing neural stem/progenitor cells and for differentiation into neurons/glia. We particularly emphasize the trafficking of signaling components to cilia of neural stem/progenitor cells and differentiated neurons (Figure 1). As opposed to culturing primary neurons, primary neurospheres are relatively easy to culture, are amenable to multiple passages and freeze-thaw cycles, and can undergo differentiation into neurons/glia. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Neurosphere-based culturing methods can serve as an ideal model system for studying ciliogenesis and ciliary trafficking in NSCs and differentiated neurons.

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			<td>12 mm round cover glass</td>
			<td>Fisherbrand</td>
			<td>12-545-80&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>4 % paraformaldehyde</td>
			<td>Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>95 mm X 15 mm petri dish, slippable lid</td>
			<td>Fisherbrand</td>
			<td>FB0875714G</td>
			<td>10 cm dish</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Affinipure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>711-545-152</td>
			<td>Donkey anti Rabbit, Alexa 488 secondary antibody</td>
		</tr>
		<tr>
			<td>Arl13B, Clone N295B/66</td>
			<td>Neuromab</td>
			<td>AB_11000053</td>
			<td></td>
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		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>ThermoFisher Scientific</td>
			<td>17504001</td>
			<td>B27</td>
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		<tr>
			<td>Centrifuge</td>
			<td>Thermo scientific</td>
			<td>ST 40R</td>
			<td></td>
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		<tr>
			<td>Cryogenic vial</td>
			<td>Corning</td>
			<td>430488</td>
			<td></td>
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		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancrease</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>Dnase I</td>
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			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D8418-100ML</td>
			<td>DMSO</td>
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		<tr>
			<td>Disposable Vinyl Specimen Molds</td>
			<td>Sakura Tissue-Tek Cryomold</td>
			<td>4565</td>
			<td>10 mm X 10 mm X 5 mm</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline 10&#215;, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td>PBS</td>
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		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine science tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Sigma</td>
			<td>F9026-500ML</td>
			<td></td>
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		<tr>
			<td>Fluoromount-G solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>mounting solution</td>
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		<tr>
			<td>GFAP</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td></td>
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		<tr>
			<td>Goat anti Mouse IgG1 Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21127</td>
			<td>Goat anti Mouse IgG1, Alexa 555 secondary antibody</td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG2a Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21137</td>
			<td>Goat anti Mouse IgG2a, Alexa 555 secondary antibody</td>
		</tr>
		<tr>
			<td>Gpr161</td>
			<td>home made</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>human bFGF</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td>FGF</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>0.100 mm deep</td>
			<td>improved neubauer</td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Henry Schein</td>
			<td>NDC 11695-0500-2</td>
			<td>Isofluorane</td>
		</tr>
		<tr>
			<td>Laminin from Engelbreth-Holm-Swarm Sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Laminin</td>
		</tr>
		<tr>
			<td>L-Glutamine (200mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Nalgene&#160;</td>
			<td>5100-0036</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502001</td>
			<td>N2</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>Sakura Tissue-Tek</td>
			<td>4583</td>
			<td>OCT</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human EGF protein, CF</td>
			<td>R and D systems</td>
			<td>236-EG-200</td>
			<td>EGF</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Fine science tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slide</td>
			<td>Fisher scientific</td>
			<td>12-550-15</td>
			<td>slides</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>Bio-Rad</td>
			<td>161-0407</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution (10X)</td>
			<td>Sigma</td>
			<td>T4174-100</td>
			<td>Trypsin</td>
		</tr>
		<tr>
			<td>COSTAR 6 Well Plate, With Lid Flat Bottom Ultra-Low Attachment Surface Polystyrene, Sterile</td>
			<td>Corning</td>
			<td>3471</td>
			<td>ultra-low binding 6 well plate</td>
		</tr>
		<tr>
			<td>&#946;-tubulin III</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>TUJ1</td>
		</tr>
	</tbody>
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using primary neurosphere cultures to study primary cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

After plating the cells from the SVZ in NSC medium for a week, floating neurospheres were observed (Figure 2A). The sizes of the spheres varied between 50 and 200 &#181;m. To examine if the spheres were derived from neural stem/progenitor cells, the neurospheres were plated onto PLL- and Laminin-coated coverslips in NSC medium for 2 days. They were then immunostained against the neural stem/progenitor cell marker, Nestin. Two days were needed to allow the spheres to attac...

Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

The overall goal of this procedure is to utilize neurosphere based culturing methods for primary neural stem and progenitor cells to study primary cilium biogenesis and trafficking. This method can help answer key questions in the primary cilia field such as cilia biogenesis and trafficking of cilia molecules to the primary cilia. In primary neural stem cells, neural progenitors and neurons.The main advantage of this technique is that we can use the primary cell criteria of the neural stem cells, neural progenitors and neurons to analyze biogenesis and ciliary pathways in the context of the primary cilium. Generally, individuals new to this method will struggle because culturing neural stem cells is technically challenging for beginners. We'll show a demonstration of this method is critical, as dissection of the lateral ventricle and steps such as culturing neurospheres and quantification of primary cilia are easily learned by observing the critical steps.Begin this procedure with a subventricular zone of the lateral ventrical dissected from an adult mouse using the whole mount dissection method. Place the tissue containing the SVZ into a 1.5 milliliter tube. Add 500 microliters of 0.05%Trypsin-EDTA in PBS and incubate 15 minutes at 37 degrees Celsius in a water bath.Following the incubation, add 500 microliters of stopping medium and gently pipette 20 to 30 times using a 1 milliliter tip. Avoid forming air bubbles during pipetting. After this, spin down the cells at 500 times G for eight minutes.Discard the supernatant then add 1 milliliter of PBS and resuspend the cells by gently pipetting five times using a 1 milliliter tip. Centrifuge at 500 times G for eight minutes. Then aspirate the medium using a 1 milliliter tip and add 1 milliliter of basal medium.After counting the cells, plate the cells from one SVZ into a 10 centimeter dish with 10 milliliters of NSC medium and culture at 37 degrees Celsius with 5%carbon dioxide. After five to seven days, neurospheres can be observed. To analyze cells in attached neurospheres by immunostaining, transfer 1 milliliter of medium containing multiple neurospheres to a 1.5 milliliter tube and spin down at 500 times G for eight minutes.After removing the medium, fix the spheres with 4%paraformaldehyde for 15 minutes. After removing paraformaldehyde incubate the spheres with 30%sucrose, overnight. And then centrifuge again.Wash with PBS. Then, after centrifuging as before, remove and discard the supernatant. Then, fit a pipette with a cut 1 milliliter tip and pipette 500 microliters of OCT solution onto the neurospheres.Transfer the OCT solution containing the neurospheres into a disposable plastic freezing mold. Then, freeze the mold on dry ice for at least 15 minutes. Cut sections with a cryostat.To visualize the stem cells in the neurosphere perform immunostaining against the stem cell marker Nestin. To visualize the primary cilia in a neurosphere, perform immunostaining against ARL13B. Begin this experiment one to two days after seeding adherent dissociated neural stem cells onto Poly-L-Lysine and Laminin coated cover slips.Change the medium in the experimental wells to starvation medium, and to fresh NSC medium in the control wells. Incubate for a further 24 hours. The next day, fix the cells with 4%paraformaldehyde in PBS for 15 minutes at room temperature.After fixation, wash with PBS twice for five minutes each, at room temperature. And then perform immunoflourescence using standard protocols. Mount the cover slips with mounting solution.Then, dry the slide glass overnight at room temperature in the dark. The next day, acquire images on a compound microscope at the necessary magnification. The microscope camera and objectives should be controlled using the accompanying software.Take sufficient z-sections at 0.5 to 0.8 micron intervals. For the quantitative analysis of ciliary localization in adherent cells, select three to eight consecutive fields with confluent cells by viewing the DAPI channel. Acquire stacks of images from each field.Quantify the number of primary cilia using ImageJ. Open the Plugins Analyze dialog box and select Cell Counter tool to count the cells with GPCR positive cilia. Use similar image intensity and contrast parameters for all images from the same experiment for counting and exporting purposes.24 hours after culturing dissociated cells in NSC medium or starvation medium cells were fixed and immunostained against GPR161 shown in green, ARL13B which appears red, and DNA shown as blue. GPR161 positive cilia are observed in control cells on the left, and starved cells on the right. As seen in these enlarged views.The white arrows and arrowheads indicate GPR161 positive and negative cilia, respectively. This image shows quantification of ARL13B positive ciliated cells. The quantification is performed from two fields of view each from two technical replicates of a single experiment.The percentage of ARL13B positive ciliated cells increases upon starvation. This image shows quantification of GPR161 localizing in ARL13B positive cilia in control and starved cells. The percentage of GPR161 localizing in ARL13B positive cilia, also increases upon starvation.Once mastered this technique can be done in less than two hours if it's performed properly. Following this procedure, another method of culturing primary tumor derived cells can be performed in order to answer additional questions like the role of cilia in tumor genesis. After its development, this technique paved the way for researchers in the field of primary cilia to explore ciliary biogenesis and trafficking in primary neurosphere based systems.After watching this video, you should have a good understanding of how to analyze primary cilia in neural stem cells, neural progenitors and neurons that are derived from neurosphere based cultures.

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