Name: isolation and culture of adult zebrafish brain-derived neurospheres
Uploaded: 2016-02-29T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres at JoVE.com

The authors thank Guillermina Hill-Teran and Marie-Elise Schwartz for assistance. This work was supported by grants from the National Institutes of Health (5R00HL105791 to S.N.) and from the Alzheimer (NIRP 12-259162). This work was also supported by Institut National de la Sant&#233; et de la Recherche M&#233;dicale (CFC and JLT), Agence Nationale de la Recherche (13-BSV4-0002-01 (JLT), NIH (1R01EB016629-01A1 (JLT), Connecticut Stem Cell Research Fund (13-SCA-Yale-04 (JLT).

Zebrafish of the WTCF strain were raised and maintained according to protocols approved by the Yale University Institutional Animal Care and Use Committee (IACUC protocol number 2012-11473). All experiments should first be approved by all relevant governmental and institutional ethics regulating bodies regarding the use of animals for research purposes.
1. Preparations
<ol>
	<li>Prepare 10 ml of dissection medium, add 200 &#181;l of 100x penicillin-streptomycin i...

<ol>
	<li>Rietze, R. L., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+isolation+and+characterization.">Neural stem cell isolation and characterization.</a> Methods Enzymol. 419, 3-23 (2006).</li><li>Brewer, G. J., Torricelli, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+neurons+and+neurospheres.">Isolation and culture of adult neurons and neurospheres.</a> Nat Protoc. 2, 1490-1498 (2007).</li><li>Guo, W., Patzlaff, N. E., Jobe, E. 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The main aim of this protocol is to isolate and culture adult zebrafish brain-derived neurospheres for studying the cellular and molecular features of neural stem/progenitor cells. Here, we report how to select multipotent neural cells and generate the three neural cell types, i.e., astrocytes, neurons and oligodendrocytes, from the adult zebrafish brain. The protocol is highly significant since a reproducible neurosphere-forming assay had not been established in the zebrafish so far.
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Mammalian neural stem cells (NSCs) have been characterized in vitro by their ability to grow in free-floating cultures as clusters of dividing cells termed neurospheres1. In the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF), NSCs divide either symmetrically to generate self-renewing NSCs, or asymmetrically to generate two different daughter cells, i.e., a differentiating progenitor cell and a novel NSC. Neurosphere cultures are therefore a mixture of neural stem/progenitor cells and more differentiated neural cells2-4. NSCs can, however, be distinguished from other neurosphere cell-types by two specific properties: they display long-term self-renewal in free-floating cultures and they can differentiate into all neural cell lineages (i.e., neurons, astrocytes, and oligodendrocytes) following withdrawal of growth factors and adhesion to extracellular matrix substrates. In mammals, the neurosphere culture system was the first in vitro system used to demonstrate the presence of NSCs in the adult brain and remains the most commonly used tool to analyze proliferation, self-renewal capacity and multipotency of neural stem and progenitor cells. Therefore, even though sphere-forming assays suffer from some disadvantages and limitations4, this culture system is valuable for evaluating the potential of a cell to behave as a stem cell when removed from its in vivo niche4 and has been instrumental in identifying key regulators of NSC self-renewal and cell fate determination5-7.
In contrast to mammals who have limited adult neurogenesis, zebrafish constitutively produce new neurons along the whole brain axis throughout their life. The zebrafish adult brain displays multiple neurogenic niches harboring neural stem/progenitor cells making zebrafish a powerful model organism for understanding the stem cell activity in the brain as well as the molecular programs required for central nervous system regeneration. Over the past 17 years, several research groups developed methodologies for isolating and culturing zebrafish neural cells8,9. These studies were aimed at producing embryonic neuronal and glial cells in vitro, but not at maintaining NSCs and investigating their properties. Although neurospheres have been generated in the adult Apteronotus leptorhynchus (Brown Ghost Knifefish)10, a neurosphere-forming assay in the zebrafish remained to be established.
Here we describe a neurosphere-forming assay to demonstrate the role of miR-107 during zebrafish neurogenesis11. The protocol enables: 1) the collection of adult neural stem/progenitor cells either from zebrafish whole brain or from several dissected brain regions such as the telencephalon, the tectum, and the cerebellum; 2) the generation of floating and self-renewing neurospheres from adult neural stem/progenitor cells; 3) the down- and up-regulation of the expression of coding genes or small non-coding RNAs11 in neurospheres, in order to investigate their roles in the proliferation and differentiation of neural stem/progenitor cells.

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			<td>DPBS 1X</td>
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			<td>11330-032</td>
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			<td>L-Cysteine hydrochloride monohydrate&#160;</td>
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			<td>C6852-25g</td>
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			<td>Life Technologies</td>
			<td>17504-044</td>
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			<td>N-2 supplement (100x) liquid&#160;</td>
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			<td>HEPES&#160;</td>
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			<td>D-(+)-Glucose 45%&#160;</td>
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			<td>Penicillin-streptomycin&#160;</td>
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			<td>AF-100-15</td>
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			<td>Insulin&#160;</td>
			<td>Sigma</td>
			<td>I5500-50 mg</td>
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			<td>DNAse</td>
			<td>Sigma</td>
			<td>DN25-10mg</td>
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			<td>Papain&#160;</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003126</td>
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			<td>Matrigel&#160;</td>
			<td>Becton Dickinson</td>
			<td>356234</td>
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			<td>PFA&#160;</td>
			<td>TCI</td>
			<td>P0018</td>
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			<td>PBS</td>
			<td>AmericanBio</td>
			<td>AB11072-04000</td>
			<td></td>
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		<tr>
			<td>Tricaine MS-222</td>
			<td>Sigma</td>
			<td>A5040</td>
			<td>stock solution of 4 mg/ml.&#160;</td>
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		<tr>
			<td>Trycold gel&#160;</td>
			<td>Sigma</td>
			<td>TGP8</td>
			<td>gel pack</td>
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			<td>Amaxa Basic Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPI-1004</td>
			<td></td>
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			<td>Trypan blue stain&#160;</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
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isolation and culture of adult zebrafish brain-derived neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

General Scheme of the Adult Zebrafish Neurosphere Culture
Here we describe all the steps of the protocol of a neurosphere-forming assay performed from the adult zebrafish brain. First, adult neural stem/progenitor cells have been collected either from zebrafish whole brain or from several dissected brain regions such as the telencephalon, the tectum and the cerebellum (Figures 1A-C). Single cell suspension of ad...

Watch this Scientific Journal Video about Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres at JoVE.com

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

The overall goal of this procedure is to examine adult neurogenesis using a neurosphere assay derived from the adult zebrafish brain, and to manipulate gene expression in zebrafish neurospheres. So, this method can help to answer a key question in neurogenesis, and, in particular, the key mechanism that drive neural stem cell self-renewal and the determination of this cell. The main advantage of this technique is that it's fairly simple, it's reliable, and it will help to obtain a neural stem progenitor cell from the zebrafish brain, and to understand their property in vitro.So we first add the idea for this method, when we were investigating epigenetic regulation in the zebrafish developing brain, and using our previous experts, we adapted mouse neurosphere protocol to zebrafish. To begin, prepare a dissection bed by filling a Petri dish with gel packs. Then, cover the dish with its corresponding lid, and incubate it at 20 degrees Celsius.Once the gel is frozen, remove the dish from the freezer, and place a clean square of filter paper on top of its lid. Proceed to wrap both the paper and dish with plastic film. Next, treat all micro-dissection instruments with 70%ethanol.Place the sterilized tools next to a dissecting microscope, and position the completed dissection bed under the microscope with optical fiber illumination. Immediately place a previously-prepared head specimen on top of the dissection bed and orient it so that its dorsal side is facing down. Then, use scissors to make a longitudinal cut through the soft tissue from the back of the head to the mouth.Afterwards, expose the base of the skull with forceps. And remove all of the adjacent tissue. Next, cut one of the lateral walls of the skull, starting at the back of the head, and moving towards the tectum region of the brain.Repeat this process for the contralateral side. Proceed to cut the optic nerve. And then remove the two lateral-most sides of the skull at the level of the tectum.Finally, turn the head ventral side up, and with forceps, peel off the most apical part of the skull to expose the brain. Afterwards, transfer the brain and any remaining parts of the skull to a dish containing dissection medium, which is composed of DMEM/F12 supplemented with penicillin streptomycin. With the plastic handle of a micro-knife, under the microscope, clean the brain tissue, being careful not to damage any neural structures.Use up to two zebrafish brains to generate whole brain-derived neurospheres. Move this dish containing the tissue into a bio-safety cabinet, then transfer the brain to a sterile 1.5 milliliter tube containing 800 microliters of dissection medium. Remove the media from the tube by pipetting, being careful not to touch the tissue.Proceed to digest the brain by adding 500 microliters of previously prepared papain solution. And incubating it at 37 degrees Celsius for 10 minutes. Following incubation, transfer the papain solution and digested brain into a 15 milliliter conical tube using a cut 1, 000 microliter pipette tip.Using the same tip, gently pipette up and down 10 times to dissociate the tissue, avoiding the formation of air bubbles. Incubate the conical again at 37 degrees Celsius for 10 minutes, and afterwards, dissociate the brain tissue further by pipetting up and down approximately 10 times, using an uncut 1, 000 microliter tip. To stop the enzymatic reaction, add two milliliters of previously prepared washing solution to the tube, and centrifuge it at 800 x g for five minutes at room temperature.Carefully decant the cell suspension, pour off the supernatant, and then vigorously tap the conical. To finish cell resuspension, pipette up and down very carefully the cells in the remaining solution with a regular 1, 000 milliliter tip. Then, add two milliliters of washing solution, and check the cells under a microscope.If a single cell suspension has been attained, proceed to centrifuge the tube using the conditions previously described. Remove the supernatant after centrifugation, and resuspend the pellet in one milliliter in fresh Z-condition medium. Proceed to stain the cells with Trypan blue, and then count them using a hemocytometer.To prepare for neurosphere generation, fill each well of a 24-well plate with 300 microliters of fresh Z-condition medium. Then, add 200 microliters of the cell suspension to each well, seeding them at a density of approximately 500 cells per microliter. Afterwards, incubate the plate at 30 degrees Celsius and 5%carbon dioxide.After one day of culture, view the cells in the 24-well plate under a microscope. Expect to observe single cell suspensions at this stage. If any debris is seen to have collected in the center of a well, remove it by pipetting out approximately 100 microliters of medium, and replace this liquid by adding 100 microliters of fresh Z-condition medium.Once all debris has been removed, incubate the plate under the conditions described previously. After an additional day of culture, transfer 250 microliters of cell suspension from a single well into a new, empty well. Repeat this step for all 24 wells.Then, add 250 microliters of fresh Z-condition medium to each well, and homogenize the suspensions by gently pipetting up and down. Incubate the plates again under the same conditions. And repeat this expansion procedure after an additional day of culture.Expect to observe a progressive increase in the size of neurospheres on the third, and fourth days of this period. To begin passaging, after four days, remove 250 microliters of medium, but no neurospheres, from each well. Then, use a one milliliter pipette to mechanically dissociate the neurospheres in the remaining medium volume.Proceed to pool the cell suspension from each dissociated well. Then, count the cells with a hemocytometer, and distribute 250 microliters of primary culture supernatant, containing 800 cells per microliter, into each well of a new 24-well plate. Continue by adding 250 microliters of Z-condition medium to each well.and then incubating the plate as previously described. Zebrafish primary neurospheres are capable of self-renewal, as demonstrated by the formation of secondary neurospheres at passage one, and tertiary structures at passage two, which suggests a pool of stem, or progenitor, cells. These stem or progenitor cells can also be differentiated, as supported by the observation that neurospheres cultured for four days under differentiation conditions give rise to glia and cells with axonal-like projections, indicated by arrows here.Differentiation experiments can also be performed on transfected neurospheres. Here, neurospheres were treated with either a scrambled control, or an antagonist of micro R-107, a small non-coding RNA, and differentiated as before. Antagonist transfected neurospheres demonstrate cells with abnormal axonal processes, which were significantly thicker than processes in controls.RT-PCR analysis reveals that inhibition of micro R-107 leads to a significant increase in the expression of certain neuroblast or axon-specific molecules compared to controls, but does not significantly affect glial markers. In contrast, neurospheres that were treated with the micro R-107 mimic, resulting in the gain of micro R-107 expression, and subsequently differentiated, demonstrate significantly decreased levels of neuroblasts or axon markers compared to controls. These results suggest that micro R-107 acts as a regulator of neural differentiation in zebrafish.Once mastered, this technique is fairly fast, it can be performed in about two hours, and it's a technology that will help scientists to advance other methods, such as neural regeneration in adults and zebrafish brain, in particular to understand the property of neural stem cell activation. So after its development, this technique paves way for researchers in the field of development and neuroscience to investigate neurogenesis in zebrafish. After watching this video, you should have a good understanding on how to perform a neurosphere assay using the adult zebrafish brain, and apply it to investigate biological questions in neurogenesis research as our stem and progenitor cells differentiate.

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