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 ventricle into a 1.5 mL tube, add 500 &#181;L of 0.05% trypsin-EDTA in PBS, and incubate the tube for 15 min at 37 &#176;C in a water bath.</li>
	<li>After 15 min, add 500 &#181;L of stopping medium and gently pipet 20 - 30 times with a 1 mL tip. Avoid forming air bubbles during pipetting. 
	NOTE: This step is critical for cell survival.</li>
	<li>Spin down the cells at 500 x g for 8 min. Discard the supernatant, add 1 mL of PBS, and resuspend the cells by gently pipetting 5x with a 1 mL tip.</li>
	<li>Spin down at 500 x g for 8 min. Discard the supernatant using a 1 mL tip and add 1 mL of basal medium.</li>
	<li>(Optional) If cellular debris are observed, pass the cells through a 70 &#181;m cell-strainer.</li>
	<li>Count the number of cells with a hemocytometer; in general, about 30,000 - 60,000 cells/SVZ are obtained.</li>
	<li>Plate the cells from one SVZ into a 10 cm dish with 10 mL of NSC medium and culture at 37 &#176;C with 5% CO2.</li>
	<li>(Optional) To avoid fusion between spheres49, put 1,000 cells in a single well of an ultra-low-binding 6-well plate that is prefilled with 1.5 mL of NSC medium and culture at 37 &#176;C with 5% CO2. 
	NOTE: After 5-7 days, neurospheres can be observed (Figure 2A). The culturing period may differ with the age of mouse or the genetic background.</li>
	<li>Add 2 mL of NSC medium every 3-4 days to maintain the culture (do not remove the existing medium).</li>
</ol>
2. Analysis of the Differentiation Capacity of Neurospheres and Ciliogenesis Tests
<ol>
	<li>To analyze the differentiation capacity, analyze the neurospheres under adherent conditions in differentiation medium.</li>
	<li>Sterilize 12 mm round coverslips by autoclaving or with UV exposure prior to use. For an adherent cell culture, put a sterilized 12 mm round cover glass into a well of a 24-well plate under aseptic conditions.</li>
	<li>Coat the cover glass for 10 s with 500 &#181;L of 0.002% poly-L-Lysine (PLL). Aspirate the solution and dry it for 10-15 min.</li>
	<li>Add 500 &#181;L of laminin solution (5 &#181;g/&#181;L). Incubate the cover glass for 1 h at 37 &#176;C.</li>
	<li>Aspirate the laminin and add 500 &#181;L of differentiation medium or NSC medium (undifferentiated control).</li>
	<li>For the differentiation assay, pick up a 100 - 200 &#181;m sphere with a 200 &#181;L tip under the microscope. Add 5-10 neurospheres to each well of a 24-well plate and culture for 7-10 days in differentiation medium.</li>
	<li>To analyze undifferentiated neurospheres, add 5-10 neurospheres to each well of a 24-well plate and culture for 1-2 days in NSC medium. Attached neurospheres spread and grow as a monolayer (Figure 2B).</li>
	<li>After carefully removing the medium, fix the cells with 4% paraformaldehyde (PFA) in PBS for 15 min at RT, and then wash with PBS twice for 5 min at RT. The plate can be stored at 4 &#176;C for 1-2 months. 
	NOTE: To visualize neural stem/progenitor cells in NSC medium and differentiated cells in differentiation medium, perform immunostaining against Nestin (neural stem/progenitor cell marker), &#946;&#8211;tubulin III (TUJ1 monoclonal, neuronal marker), GFAP (Glial Fibrillary Acidic Protein, astrocyte marker), and O4 (oligodendrocyte marker) (Figure 2B-E). To analyze the cilia, perform immunostaining against Arl13b (primary cilia marker) and Gpr161 (ciliary GPCR) (Figure 3).</li>
	<li>Mount the cover glass with mounting solution onto a slide glass. Tilt the slide glass to remove excess solution.</li>
</ol>
3. Analysis of Ciliogenesis in Neurospheres
<ol>
	<li>To analyze cells in intact neurospheres by immunostaining, transfer 1 mL of culturing medium containing multiple neurospheres to a 1.5 mL tube and spin down at 500 x g for 8 min. Fix the spheres with 4% (PFA) after removing the medium for 15 min and wash with PBS.&#160;Spin down spheres at 500 x g for 8 min, remove supernatant, and incubate the spheres O/N with 30% sucrose at 4&#176;C.&#160;</li>
	<li>Discard the supernatant using a 1 mL tip and add 500 &#181;L of OCT solution using a cut 1 mL tip. Cut the edge of the tip to widen the opening, as OCT is viscous.</li>
	<li>Transfer the OCT solution containing the neurospheres into a disposable plastic freezing mold (10 mm x 10 mm x 5 mm).</li>
	<li>Freeze the mold on dry ice for at least 15 min.</li>
	<li>(Optional) Stop the experiment and store the mold in a -80 &#176;C freezer for up to 1 year.</li>
	<li>Cut the sections with a cryostat; the thickness of sections should be 15-30 &#181;m.</li>
	<li>To visualize the primary cilia in a neurosphere, perform immunostaining against Arl13b (Figure 4).</li>
</ol>
4. Culture and Passage of Neurospheres and Adherent NSCs
<ol>
	<li>Passage the neurospheres while the sphere size is between 100-200 &#181;m; when the neurospheres are too big (300 &#181;m or above), they are not ideal for experiments.</li>
	<li>Transfer the neurospheres into a 50 mL tube using a 1 mL tip and spin down at 500 x g for 8 min.</li>
	<li>Discard the supernatant using an aspirator, add 500 &#181;L of 0.05% trypsin-EDTA in PBS, and incubate for 5 min at 37 &#176;C. The amount of trypsin varies depending on the number of spheres.</li>
	<li>Add 500 &#181;L of serum medium and gently pipet 20 times with a 1 mL tip.</li>
	<li>Spin down at 500 x g for 8 min. Discard the supernatant using a 1 mL tip and add 1 mL of basal medium.</li>
	<li>(Optional) If cellular debris or undissociated neurospheres are seen, pass the cells through a 70 &#181;m cell-strainer.</li>
	<li>Passage the cells in a 10 cm dish at a density of 10,000 cells/cm2 in NSC medium; the cells will be ready for the next passage after a week.</li>
	<li>(Optional) To freeze the cells, add freezing medium to generate 500,000 to 1,000,000 cells/mL&#160;suspension. Freeze the cells using cryo-freezing containers; undissociated neurospheres can also be frozen.</li>
	<li>For the adherent culture, dillute the cells to 50,000 cells/cm2&#160;onto PLL- and Laminin-coated coverslips with NSC medium, and culture for 1-2 days.</li>
</ol>
5. Starvation and Analysis of Cilia
<ol>
	<li>Prepare starvation medium (Table 1).</li>
	<li>1-2 days after the initial plating of adherent cells after dissociation, change to NSC medium in one well (control) and starvation medium in another well (experiment).</li>
	<li>Culture the adherent cells for 24 h.</li>
	<li>Fix the cells with 4% PFA in PBS for 15 min at RT and wash twice with PBS&#160;for 5 min each at RT.</li>
	<li>(Optional) Stop the experiment and store the 24-well plates with coverslips in PBS at 4 &#176;C for 1-2 months.</li>
	<li>Perform an immunofluorescence protocol for staining7,50.</li>
	<li>Mount the coverslips using mounting solution. Dry the slide glass O/N&#160;at RT in the dark.</li>
	<li>Acquire images on a compound microscope at the necessary magnification. Use a microscope, a camera, and objectives (40X/1.3 oil and 63X/1.4 oil), controlled using the accompanied software. Take sufficient z-sections at 0.5-0.8 &#181;m intervals (Figure 5).</li>
	<li>For the quantitative analysis of ciliary localization in adherent cells, acquire stacks of images from 3-8 consecutive fields with confluent cells by looking into the DAPI channel. Quantify the number of primary cilia using ImageJ/Fiji. Typically, use the &#34;Cell counter&#34; tool in the ImageJ Plugins&#62;Analyze dialog box to count the cells with GPCR-positive cilia; maximal projections from stacks of images can also be exported from ImageJ/Fiji.Use similar image intensity and contrast parameters for all images from the same experiment for counting and exporting purposes.</li>
</ol>
6. Transfection of Neurospheres
<ol>
	<li>Adhere dissociated cells to coverslips for 24 h. Use a cell density typically between 75,000 and 150,000 cells in 500 &#181;L of NSC medium in a single well of a 24-well plate.</li>
	<li>Mix 25 &#181;L of reduced serum medium and 1.5 &#181;L of transfection reagent in a 0.5 mL microtube by vortexing for 5 s.</li>
	<li>Add 2.5 &#181;g of endotoxin-free plasmid DNA to a separate 0.5 mL microtube containing 25 &#181;L of reduced serum media and mix by vortexing for 5 s.</li>
	<li>Add 1 &#181;L of transfection reagent to the second microtube containing DNA and mix by vortexing for 5 s.</li>
	<li>Add the mixture from the DNA-containing tube to the first microtube and mix by pipetting.</li>
	<li>Incubate the mixture for 10-15 min at RT. After incubation, gently add the transfection mix dropwise to the wells on top of the NSC medium (500 &#181;L/well).</li>
	<li>Change the medium 24 h post-transfection to control medium (NSC medium) or starvation medium (500 &#181;L/well).</li>
	<li>Fix the cells by changing the medium to 4% PFA&#160;after 24 h and perform immunostaining; typically, up to a 10% transfection efficiency is obtained using this protocol.</li>
</ol>

The authors declare no competing financial interests.

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.

<table><tbody><tr><td>12 mm round cover glass</td><td>Fisherbrand</td><td>12-545-80&amp;nbsp;</td><td></td></tr><tr><td>24-well plate</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>4% paraformaldehyde (PFA)</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 &amp;micro;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></tr><tr><td>B-27 Supplement (50X), serum free</td><td>ThermoFisher Scientific</td><td>17504001</td><td>B27</td></tr><tr><td>Centrifuge</td><td>Thermo scientific</td><td>ST 40R</td><td></td></tr><tr><td>Cryogenic vial</td><td>Corning</td><td>430488</td><td></td></tr><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></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D8418-100ML</td><td>DMSO</td></tr><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&#039;s Phosphate-buffered Saline 10X, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma</td><td>D1408-500ML</td><td>DPBS</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine science tools</td><td>11254-20</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F9026-500ML</td><td></td></tr><tr><td>Fluoromount-G solution</td><td>Southern Biotech</td><td>0100-01</td><td>mounting solution</td></tr><tr><td>GFAP</td><td>DAKO</td><td>Z0334</td><td></td></tr><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 (200 mM)</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&amp;nbsp;</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 (PLL)</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-100</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>&amp;beta;-tubulin III</td><td>Covance</td><td>MMS-435P</td><td>TUJ1</td></tr></tbody></table>

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, ...

using-primary-neurosphere-cultures-to-study-primary-cilia

Research

JoVE Journal

Developmental Biology

9.3K Views.  University of Texas Southwestern Medical Center.  De primaire cilium fundamenteel belang neurale proliferatie, neuronale differentiatie en volwassen neuronale functie. Hier beschrijven we een werkwijze voor ciliogenese en het transport van signalerende eiwitten trilharen neurale stam / progenitorcellen en gedifferentieerde neuronen in het primaire neurosfeerculturen bestuderen. 

Video: Using Primary Neurosphere Cultures to Study Primary Cilia

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

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

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

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

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

Neuroscience

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Biology

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...

Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.

We present a three-dimensional (3D) in vitro differentiation protocol generating neurospheres of reproducible size to produce cranial neural crest cells from mouse embryonic stem cells. We show that this methodology reduces variability compared to previous protocols and how it can be used for multiplexed assay to study cranial neural crest cell development.

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest ...

Volumetric Imaging and Analysis of Primary Cilia in Musculoskeletal Tissue using the ARL13B-CENTRIN-2 Mouse Model

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton and in coordinating cell responses to biochemical and mechanical cues from their surrounding environment. Due to its micrometer-scale size, the primary cilium is challenging to image in situ to characterize in tissues, in a high throughput manner and without optical biases. An understanding of its organization in its 3D environment in vivo will be key to understanding ciliary roles in physiology and disease. The ARL13B-CENTRIN-2 mouse line presents an mCherry tag on the ciliary axoneme protein ARL13B and a green fluorescent protein (GFP) tag on the centriole protein CENTRIN-2, localized to the base of the cilium. This mouse line allows for imaging of the primary cilium without the need for staining, which reduces the number of steps in image collection and circumnavigates issues with signal-to-noise. This publication describes a step-by-step protocol to image primary cilia in musculoskeletal tissues whilst preserving the fluorescent signal. It also describes a universally applicable image analysis pipeline, enabling unbiased quantification of cilia features, such as length and 3D orientation.

This method describes the exploitation of the endogenous signal in an ARL13B-CENTRIN-2 transgenic mouse model to image primary cilia in situ, in three dimensions across large areas of mineralized tissue, from tissue collection to analysis of cilia organization. This protocol may also be applied to other types of tissues.

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton ...

Using Primary Neurosphere Cultures to Study Primary Cilia

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