Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Summary

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.

Abstract

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.

Introduction

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 cells^2-4. 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 determination^5-7.

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 cells^8,9. 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.

Protocol

Zebrafish of the WT^CF 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

1. Prepare 10 ml of dissection medium, add 200 µl of 100x penicillin-streptomycin into 9.8 ml of DMEM/F12.
2. Prepare L-Cysteine solution: to 10 ml of water for tissue culture, add 120 mg of L-cysteine. Store the L-cysteine solution at 4 °C for up to 2 weeks, or in aliquots at -20 °C for up to 1 year.
3. Prepare DNase I solution: to 1 ml of water for tissue culture, add 10 mg of DNase I. Store the DNase I solution at 4 °C for up to 2 months.
4. Prepare papain solution: to prepare papain solution, add 100 µl papain (approximately 140 units), 100 µl DNase I (1%) and 200 µl L-cysteine (12 mg/ml) into 5 mL of DMEM/F12. Freshly prepare the papain solution every time and sterilize with a 0.22 µm pore size filter before use.
5. Prepare washing solution: to prepare 100 ml of washing solution, add 650 µl glucose 45%, 500 µL HEPES 1 M and 5 ml FBS into 93.85 ml DPBS 1x. Sterilize the washing solution with a 0.22 µm pore size filter before use. Store the washing solution at 4 °C for up to 2 months.
6. Prepare insulin solution: to prepare 2 ml of insulin solution, add a 25 µl drop of 10 N NaOH and 100 mg insulin into 2 ml of water for tissue culture.
7. Prepare EGF and FGF solutions: Dissolve both mitogens in DMEM/F12 at 100 µg/ml concentration. Store as 10 µl aliquots at -20 °C.
8. Prepare B-27 and N-2 media: store B-27 and N-2 supplements at -20 °C as 500 µl and 1 ml aliquots, respectively.
9. Prepare Z-differentiation condition medium: to prepare 100 ml of Z-differentiation condition medium, add 40 µl insulin (50 mg/ml), 500 µl B-27, 1 ml N-2, 650 µl glucose 45% and 1 ml of 100 x penicillin-streptomycin into 97.81 ml of DMEM/F12. Sterilize the Z-differentiation condition medium with a 0.22 µm pore size filter before use. Store the Z-differentiation condition medium at 4 °C for up to 1 week.
10. Prepare Z-condition medium: to prepare 50 ml of Z-condition media, add 10 µl of EGF and 10 µl of FGF into 50 ml of sterile Z-differentiation condition medium (20 ng/ml). Store the Z-condition medium at 4 °C for up to 1 week.
11. Prepare coating solution for differentiation culture: for 5 ml extracellular coating solution, add 100 µl of the extracellular matrix solution (e.g., Matrigel) into 4.9 ml of DMEM/F12. Thaw extracellular matrix solution at 4 °C on ice. Once defrosted, keep at 4 °C for up to 2 weeks.

2. Dissection of the Adult Zebrafish Brain

1. Prepare a dissection bed by filling a 100 mm x 15 mm Petri dish with gel ice packs. Then place the lid on the petri dish and incubate at -20 °C until the gel freezes. On top of the lid place a square of clean filter paper and wrap both the filter paper and petri dish with plastic film.
2. Clean and sterilize all the microdissection instruments by 70% ethanol or heat before each use. Place all sterilized dissection instruments near the dissecting microscope and, right before euthanasia, place the dissection bed under the microscope with optical fiber illumination.
3. Collect 2 adult zebrafish for a whole brain neurosphere preparation; and 3 to 4 zebrafish to generate neurospheres from dissected brain regions.
4. Euthanize adult zebrafish (8-12 months old) using a protocol approved by the Institutional Animal Care and Use Committee. Next, immerse the fish in 75% ethanol for 5-10 sec and quickly place in the dissection bed followed by decapitation at the level of the gills using a surgical blade.
   1. To euthanize animals, administer an overdose (300 mg/L) of tricaine methanesulfonate until the animal's heart-beat gradually slows down and circulation stops, then immerse in iced water.
5. Turn the head dorsal side down, and using the scissors make a longitudinal cut from the cut side to the mouth. Using the forceps expose the base of the skull and remove all the adjacent tissue. Cut the lateral walls of the skull from the beginning of the spinal cord towards the tectum.
6. Using the scissors, cut and remove the optic nerves and then remove both sides of the most lateral part of the skull at the level of the tectum. Turn the head ventral side up. Using forceps, peel off the rest of the most apical part of the skull.
7. Transfer the brain along with the remaining part of the skull into a new dish with the dissection medium (1.1). Clean the brain tissue in the dissection medium using the micro knife's plastic handle, keeping all brain structures intact.
8. From this point, continue the protocol using the whole zebrafish brain.
   1. Alternatively adapt this protocol to specific brain regions to generate neurospheres from whole zebrafish brain or from the telencephalon, tectum/diencephalon or cerebellum dissected with a fresh scalpel. Use a neural specific fluorescent zebrafish neural transgenic line to dissect the brain region of interest according to Figure 3 and reference ¹¹.

3. Single Cell Dissociation of Adult Brain

1. Perform all subsequent work in a biological safety cabinet. Transfer the brain tissue into a 1.5 ml tube containing 800 µl of dissection medium (prepared in step 1.1). Remove the dissection medium by pipetting, without touching the brain tissue.
2. Add 500 µl of papain solution (step 1.4) and digest the brain tissue at 37 °C for 10 min. Do not incubate longer than 15 min as it could decrease cell viability.
3. After incubation, transfer the brain tissue with 500 µl of the papain solution into a 15 ml conical tube using a 1,000 µl pipet tip cut at ~0.25 inches from the bottom. Gently dissociate the brain tissue by slowly pipetting up and down 10 times with the same tip. Do not pipet up and down more than 15 times and do not generate air bubbles during this step, as it could alter cell viability.
4. Incubate the brain tissue again at 37 °C for 10 min followed by pipetting up and down 10-13 times using an uncut 1,000 µl regular tip. Do not pipet up and down more than 15 times, to avoid cell death.
5. Stop the enzymatic digestion by adding 2 ml of washing solution (step 1.5), and centrifuge the cell suspension at 800 x g for 5 min at room temperature (RT). Carefully decant the supernatant and resuspend the pellet in the remaining solution by tapping the tube vigorously with a finger and then by pipetting up and down with a 1,000 µl regular tip. Next, add 2 ml of washing solution.
6. At this stage, check under the microscope that a single cell suspension has been obtained. Centrifuge the cell suspension again at 800 x g for 5 min at RT. Remove the supernatant and re-suspend the pellet using 1 ml of fresh Z-condition medium (step 1.10).
7. Stain cells by using trypan blue¹² and count the living cells using a hemocytometer by excluding blue dead cells. Prepare a 24-well plate with 200 µl of cell suspension and 300 µl of fresh Z-condition medium in each well. Seed cells at a density of ~500 cells/µl. Maintain cultured cells in an incubator at 30 °C in 5% CO₂, since zebrafish brain-derived neurospheres do not grow well at 37 °C.

4. Generation of Primary Neurospheres

1. After 1 day in vitro (DiV1), observe the single cell suspension obtained from the adult whole brain under the microscope and note whether debris has accumulated in the center of the well. Carefully remove any debris by pipetting approximately 100 µl of medium (less if possible). Next add 100 µl of fresh Z-condition medium. Single cell suspensions should be observed at this time (Figure 2A DiV1).
2. After 2 days in vitro (DiV2), expand the cultured cells. Using a 1,000 µl pipette with the tip cut, collect and transfer 250 µl of cell suspension from each well into a new well. Add 250 µl of fresh Z-condition medium into all wells. Before expanding the cultured cells, homogenize the suspension very gently by pipetting up and down throughout the well.
3. Repeat steps 4.1 and 4.2 for the following 4 days in vitro (DiV4). A progressive increase in size of neurospheres should be visible at the center and edges of the well at DiV3 and DiV4 (Figure 2B and C).
   Note: Primary neurospheres can be processed for passage or differentiation to assess multipotency. Alternatively, they can be processed for nucleofection or lipo-transfection¹¹ to characterize the role of specific genes during the differentiation process.

5. Passaging of Primary Neurospheres

1. Remove 250 μl of tissue culture from each well and mechanically dissociate DiV4 neurospheres with a 1 ml pipette. Do not pipet up and down too rapidly as air bubble formation may increased cell death.
2. Count the cells with a hemocytometer and distribute 800 cells/µl in 250 µl of primary culture supernatant into each well of a 24-well plate.
3. Add 250 µl of Z-condition medium to each well.
4. After 2 days in vitro (DiV2), expand the cultured cells as reported in step 4.2 and 4.3.

6. Differentiation of Primary Neurospheres

1. Sterilize cover glasses by immersion in 70% ethanol for 10 min, then dry cover glasses at RT by placing them into each well of a 12-well plate.
2. Add 300 µl of extracellular coating solution (step 1.11) at the center of the cover glass in such a way that it can expand widely and cover the entire cover glass. Then, incubate at 37 °C for 1 h (using the humidified cell culture incubator).
3. Remove the extracellular coating solution after incubation, and dry the cover glass for 2 hr at 37 °C.
4. Remove 250 µl of tissue culture from each well. Mechanically dissociate all the DiV4 primary neurospheres per well with a 1 ml pipette (with wider-end tip) by pipetting up and down.
5. Seed dissociated neurosphere cell suspensions at a cell density of 4 x 10³ cells/ml on previously prepared extracellular matrix-coated coverglasses (step 6.1-6.3) and keep for at least 30 min, at 30 °C in 5% CO_2, until attached to the substrate. Then, remove the culture medium and rapidly add 1 ml of pre-warmed fresh Z-differentiation condition medium (step 1.9) before culturing the cells for 24 hr at 30 °C in 5% CO₂.
6. Every two days, replace half of the Z-differentiation condition medium during the time of cell differentation.

7. Gene Manipulation of Primary Neurospheres

1. Collect DiV3-4 primary neurospheres (step 4.3) by centrifuging for 8 min at 80 x g at 4 °C. Prepare for liposome transfection of commercial oligonucleoatides as previouly described¹¹ or nucleo-transfection of DNA plasmid vectors.
2. Resuspend neurosphere pellet at a cell density of 4 x 10³ cells/μl in 100 μl of a reaction mixture (82 μl nucleofactor solution and 18 μl of supplement).
3. Mix the neurosphere suspension with 5 μl of the DNA plasmid vectors of choice (plasmid stocks at 1 μg/μl). Transfer the neurosphere/DNA mixture into a nucleofector cuvette for nucleotransfection and select nucleofector program according to the manufacturer's instructions provided by the nucleofector kit.
4. Immediately after nucleofection, add a volume of 500 ml of pre-warmed Z-condition media (1.10) to the cells and plate the transfected neurospheres for studying their cell renewal and/or differentiation properties, as described above.

Results

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...

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
DPBS 1x	Life Technologies	14190-144
DMEM/F12 1x	Life Technologies	11330-032
L-Cysteine hydrochloride monohydrate	Sigma	C6852-25g
B-27	Life Technologies	17504-044
N-2	Life Technologies	17502-048	N-2 supplement (100x) liquid
HEPES	Life Technologies	15630	1 M
D-(+)-Glucose 45%	Sigma	G8769
Penicillin-streptomycin	Life Technologies	15140-122
Fetal Bovine Serum	Life Technologies	16000044
Human FGF-basic	Peprotech	100-18B
Human EGF	Peprotech	AF-100-15
Insulin	Sigma	I5500-50 mg
DNAse	Sigma	DN25-10mg
Papain	Worthington Biochemical Corporation	LS003126
Matrigel	Becton Dickinson	356234
PFA	TCI	P0018
PBS	AmericanBio	AB11072-04000
Tricaine MS-222	Sigma	A5040	stock solution of 4 mg/ml.
Trycold gel	Sigma	TGP8	gel pack
Amaxa Basic Nucleofector Kit	Lonza	VPI-1004
Trypan blue stain	Life Technologies	15250061