Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Summary

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

Abstract

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

Introduction

The prairie vole (Microtus ochrogaster), a member of the Cricetidae family, is a small mammal whose life strategy develops as a socially monogamous and highly sociable species. Both males and females establish an enduring pair bond after mating or long periods of cohabitation characterized by sharing the nest, defending their territory, and displaying biparental care for their progeny^1,2,3,4. Thus, the prairie vole is a valuable model for understanding the neurobiological basis of socio-sexual behavior and impairments in social cognition⁵.

Adult neurogenesis is one of the most paramount processes of neural plasticity that leads to behavioral changes. For example, our research group reported in male voles that social cohabitation with mating increased cell proliferation in the subventricular zone (VZ) and subgranular zone in the dentate gyrus (DG) of the hippocampus, suggesting that adult neurogenesis can play a role in the formation of pair bonding induced by mating in prairie voles (unpublished data). On the other hand, although the brain regions where new neurons are generated and integrated are well known, the molecular and cellular mechanisms involved in these processes remain undetermined due to technical drawbacks in the whole brain model⁶. For instance, the signaling pathways controlling gene expression and other cellular activities have a relatively short activation period (detection of phosphoproteome)⁷. One alternative model is isolated and cultured adult neural stem cells or progenitor cells to elucidate molecular components involved in adult neurogenesis.

The first approach to maintain in vitro neural precursors from adult mammal (mouse) brain was the assay of neurospheres, which are cellular aggregates growing under non-adherent conditions which preserve their multipotent potential to generate neurons, as well as astrocytes^8,9,10. During their development, there is a selection process where only the precursors will respond to mitogens such as the Epidermal Growth Factor (EGF) and Fibroblast Growth Factor 2 (FGF2) to proliferate and generate neurospheres^8,9,10.

To our knowledge, no protocol is reported in the literature to obtain adult neural progenitors from prairie voles. Here, we established the culture conditions to isolate neuronal progenitors from neurogenic niches and their in vitro maintenance through the neurosphere formation assay. Thus, experiments can be designed to identify the molecular and cellular mechanisms involved in proliferation, migration, differentiation and survival of the neural stem cells and progenitors, processes that are still unknown in the prairie vole. Moreover, elucidating in vitro differences in the properties of the cells derived from the VZ and DG could provide information about the role of neurogenic niches in neural plasticity associated with changes in socio-sexual behavior and cognitive behaviors, and deficits in social interactions (autism spectrum disorder and schizophrenia), which could also be sex-dependent.

Protocol

The study was approved by the Research Ethics Committee of the Instituto de Neurobiología, Universidad Nacional Autónoma de México, Mexico and Instituto Nacional de Perinatologia (2018-1-163). The reproduction, care and humane endpoints of the animals were established following the Official Mexican Standard (NOM-062-Z00-1999) based on the “Ley General de Salud en Materia de Investigación para la Salud” (General Health Law for Health Research) of the Mexican Secretaria of Health.

1. Solutions and stocks preparation

1. Prepare an N2 culture medium with 485 mL of Dulbecco's Modified Eagle Medium-F12 (DMEM-F12), 5 mL of N2 supplement (100x), 5 mL of glutamine supplement (100x) and 5 mL of antibiotic-antimycotic (100x).
2. Prepare a B27 culture medium with 480 mL of Neurobasal medium, 10 mL of B27 supplement (50x), 5 mL of glutamine supplement, and 5 mL of antibiotic-antimycotic (100x).
3. Reconstitute collagenase powder in 1x PBS (Phosphate-buffered saline) to obtain aliquots with an activity of 100 units/µL (1000x) and store at -20 °C. Notice, collagenase activity depends on the lot number of the companies.
4. Prepare dispase stock aliquots by dissolving 5 mg of dispase powder in 1x PBS (50 mg/mL). Store at -20 °C.
5. Prepare an enzymatic solution with 100 mL of DMEM-F12 medium, 50 µL of stock collagenase (100 units/µL) to have a final concentration of 50 U/mL and 333 µL of stock dispase (50 mg/mL) to have a final concentration of 0.33 mg/mL.
6. To prepare a washing solution, to 1,000 mL of 1x PBS, add 0.4766 g of HEPES (final concentration 2 mM), 3.6 g of D-glucose (final concentration 20 mM) and 2.1 g of NaHCO₃ (final concentration 25 mM).
7. Prepare poly-L-ornithine stock aliquots (1 mg/mL) using sterile water and store at -20 °C.
8. Prepare a working solution of poly-L-ornithine. Dilute a stock aliquot (1mg/mL) in 49 mL of sterile water for a final concentration of 20 µg/mL.
9. Prepare a working solution of laminin. Dilute 25 µL of laminin (1 mg/mL original stock) in 5 mL of sterile water for a final concentration of 5 µg/mL.
   NOTE: After preparation, filter the culture media, working and stock solutions to avoid contamination. Use a syringe or bottle-top vacuum filters (polyethersulfone membrane with a 0.2 µm pore size). The culture media and work solutions can be stored for up to 30 days at 4 °C, while the stocks can be stored for up to four months at -20 °C.

2. Preparation before starting the microdissection

1. Sterilize surgical instruments by autoclaving or with a hot glass bead dry sterilizer.
2. Clean the microdissection surface area under strict aseptic and antiseptic conditions (e.g., with ozonized water).
   NOTE: The timing of microdissection of both neurogenic niches from each vole brain is approximately 30 min. Working with 1-4 animals for the entire procedure is recommended.

3. Extraction of the whole brain

1. Anesthetize the adult vole (12-16 weeks) with an overdose of pentobarbital (6.3 mg/animal) through intraperitoneal injection. Verify the depth of anesthesia by the absence of pedal reflex in response to a firm toe pinch.
2. Once the vole is entirely anesthetized, induce euthanasia by decapitation and recover the head.
3. Dissect the skin from the skull with scissors, making a caudal-rostral incision (15 mm long) to expose the skull.
4. Cut the occipital and interparietal bones and trace an incision into the skull along the sagittal and parietal sutures.
5. Make a hole in the skull at the junction of frontal and parietal bones using scissors, being very careful not to damage the brain tissue.
6. To expose the brain, remove the remaining cranium fragments that cover both brain hemispheres with sharp-pointed tweezers.
7. Use a stainless-steel spatula to lift the entire brain from the cranial base.
8. Collect the brain into a centrifuge tube (50 mL) with 20 mL of cold wash solution.
9. Wash the brain twice with the cold wash solution.

4. Microdissection of the neural tissue

1. Place a Petri dish on a surface surrounded by ice.
2. Deposit the brain on the dish and add 20 mL of cold wash solution.
3. With a scalpel, in the coronal plane, divide the brain into two blocks of tissue (rostral and caudal). As a neuroanatomical reference, perform the coronal cut at Bregma level in the anterior-posterior axis¹¹ (Figure 1A, solid line).
4. From the rostral block, extract the VZ tissue (Figure 1B), while from the caudal block remove the DG (Figure 1C).
5. Dissect the VZ under a stereo microscope.
   1. With a Dumont forceps, hold one of the hemispheres; then, insert, at the height of the ventricle, the fine tips of a second Dumont forceps under the tissue that lines the caudate-putamen (Figure 2A).
   2. Open the forceps along the dorsoventral axis to separate the tissue.
   3. Collect the VZ tissue per individual in a centrifuge tube with 2 mL of cold wash solution. Do not pool the tissue of more than two animals.
   4. Repeat the microdissection in the other hemisphere.
   5. Store the tube containing the bilateral VZ tissue on ice and continue dissecting the DG.
6. Dissect the DG from the caudal block under a stereo microscope.
   1. With a scalpel, make a coronal cut into the block to obtain two slices, in which the hippocampal formation is observed. As a landmark, the cut is made at -2 mm Bregma coordinates in the anterior-posterior axis according to the mouse brain atlas¹¹ (Figure 1A, dotted line and Figure 1C).
   2. With a Dumont forceps, hold one of the slices, and with fine-point Dumont forceps make a horizontal cut between DG and CA1 and then perform a vertical incision between the DG and CA3 to separate the DG (Figure 2B).
   3. Repeat the dissection in the first slice of the other hemisphere.
   4. Repeat the dissection in both hemispheres in the second slice.
   5. Collect the four DG pieces of each vole in a centrifuge tube. Do not pool the DG tissue of more than two animals.
      NOTE: If dissection of more than one animal is required, store the centrifuge tubes with the VZ or DG tissue on ice while continuing to dissect the rest of the brains. Remove all blood vessels that cover the brain tissue while dissecting. If the vessels are not discarded, the culture could be mixed with an excess of erythrocytes and disturb neurosphere formation.

5. Isolation of neural cells

1. Place the centrifuge tubes inside the biosafety cabinet and wait about 10 min for the tissue fragments to precipitate by gravity.
2. Remove the wash solution and add 1 mL of the warm enzymatic solution to each tube.
3. Incubate the tubes at 37 °C for 10 min.
4. Disintegrate the tissue fragments; pipette up and down with a 1 mL tip. Do not pipette more than 30x.
5. Carry out a second incubation of 10 min at 37 °C.
6. At the end of the second incubation, pipette to break up the tissues. Do not pipette more than 30x.
   NOTE: After pipetting, the tissue fragments should be completely disintegrated; if they are not disintegrated, incubate for another 10 min at 37 °C and re-pipette. The digestion period should not exceed 30 min.
7. Add 9 mL of N2 medium per tube to dilute the enzymatic treatment.
8. Centrifuge the tubes at 200 x g for 4 min at room temperature.
9. Discard the supernatant and wash with 10 mL of N2 medium.
10. Centrifuge under the same conditions as step 5.8.
11. Remove the supernatant from each tube and resuspend the cell pellets of the VZ and DG in 2 mL and 1 mL of the B27 medium, respectively.
12. To remove any non-disintegrated tissue, filter each cellular suspension using a cell strainer (size 40 µm).

6. Neurospheres formation

1. Culture the cells passed through the strainer into an ultra-low attachment, 24-well plate. Use two wells for the VZ and one well for the DG (1 mL of B27 medium/well).
2. Add 20 ng/mL of FGF2 and 20 ng/mL of EGF to each well (final concentration 1x).
3. Incubate at 37 °C, 5% CO₂ and high humidity (90-95%). Do not disturb for 48 h (day 1 and day 2 of culture, D1-D2).
4. On the third day (D3), remove half of the culture medium and replace it with fresh B27 medium (500 µL per well) supplemented with double concentration (2x) of growth factors.
5. Repeat every third day, change the culture medium (half of it) and replace it with a fresh B27 medium supplemented with double concentration (2x) of growth factors.
6. On days when it is not necessary to change the culture medium, add growth factors to a final concentration of 1x.
7. Ensure that the neurospheres are formed around D8-D10.
8. At the D10, change the complete culture medium to remove all debris.
   1. Collect the medium and neurospheres individually of each well in centrifuge tubes.
   2. Incubate for 10 min at room temperature. This procedure allows neurospheres precipitation by gravity.
   3. Remove the supernatant and resuspend in 1 mL of fresh B27 medium supplemented with growth factors.
   4. Place the neurospheres back into the same ultra-low attachment plate and incubate at 37 °C, 5% CO₂.
9. From D10 to D15, continue changing half of the medium and adding growth factors.

7. Passage of the neurospheres

1. At D15 of the primary culture, collect the neurospheres into centrifuge tubes using 1 mL pipette. Cut the pipette tip to increase the size of the opening to avoid damage to the neurospheres.
2. Incubate for 10 min at room temperature. Neurospheres precipitate by gravity.
3. Remove the medium and add 1 mL of the cell detachment medium per tube.
4. Incubate the tubes for 7 min at 37 °C.
5. Pipette up and down with a 1 mL tip to dismantle the neurospheres.
6. Dilute the cell detachment medium with 3 mL of B27 medium per tube.
7. Centrifuge the cell suspension for 5 min at 200 x g.
8. Discard the supernatant and resuspend each cell pellet with a fresh B27 medium supplemented with growth factors.
   1. Resuspend the VZ-derived cells in 4 mL of medium and the DG-derived cells in 2 mL of medium.
9. Culture the cells (passage 1) in a new ultra-low attachment plate by doubling the number of wells that were used in the primary culture (4 and 2 wells for VZ and DG, respectively).
10. Change half of the medium every third day and add growth factors daily.
11. After 10 days (D10) in passage 1, change to adherent conditions in the next passage.

8. The passage in adherent conditions

1. Before carrying out the passage 2, prepare coated plates with poly-L-ornithine and laminin.
   1. In 24-well plates, add 500 µL of 1x poly-L-ornithine (20 µg/mL) per well. Incubate at 37 °C overnight.
   2. Remove the poly-L-ornithine and wash 4x with 1x PBS (500 µL/well).
   3. Add 200 µL (minimum volume to cover the surface of a single well) of 1x laminin (5 µg/mL) per well and incubate for 2-3 h at 37 °C before cultivating the cells.
2. Collect the neurospheres with 1 mL pipette with cut tips into a centrifuge tube.
3. Incubate for 10 min at room temperature to precipitate the neurospheres by gravity.
4. Discard the supernatant and resuspend the neurospheres in fresh B27 medium without growth factors.
5. Aspirate the laminin from the coated plate and deposit the neurospheres into the wells using 1 mL pipettes with cut tips.
   NOTE: Prevent coated wells from drying out between laminin removal and plating neurospheres.
6. Divide the culture into two conditions:
   1. Maintain differentiated neurospheres for 6 days (D6). Change the medium every third day and add growth factors daily.
   2. Observe differentiation of the neurosphere-derived cells by 12 days (D12). Change the medium every third day without growth factors.
      NOTE: At the end of D6 for undifferentiated or D12 for differentiation conditions, the cells can be used for conventional immunohistochemistry, cell sorting analysis, 5-Ethynyl-2´-deoxyuridine (EdU) staining, RNA extraction, among others.

Results

Neurospheres were formed from neural stem cells isolated from the VZ and DG of both female and male adult prairie voles. About 8-10 days after starting the culture, cells should have formed the neurospheres. Note that the plate may contain debris in the primary culture (Figure 3A). However, in passage 1 the culture should only consist of neurospheres (Figure 3B).

A higher number of neurospheres were obtained from the female VZ as comp...

Discussion

A stage to obtain a neural stem cell culture is the digestion period with the enzymatic solution, which should not exceed more than 30 min because it might decrease cell viability. The neurospheres should emerge at 8-10 days after initial culture; if they do not emerge by day 12, discard the culture and repeat the experiment, reducing the digestion period. Another issue is the blood vessels that cover the brain tissue. They should be completely removed during the dissection because the excess of erythrocytes can interfer...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies			Antibody ID
Anti-Nestin	GeneTex	GTX30671	RRID:AB_625325
Anti-Doublecortin	MERCK	AB2253	RRID:AB_1586992
Anti-Ki67	Abcam	ab66155	RRID:AB_1140752
Anti-MAP2	GeneTex	GTX50810	RRID:AB_11170769
Anti-GFAP	SIGMA	G3893	RRID:AB_477010
Goat Anti-Mouse Alexa Fluor 488	Thermo Fisher Scientific	A-11029	RRID:AB_2534088
Goat Anti-Rabbit Alexa Fluor 568	Thermo Fisher Scientific	A-11036	RRID:AB_10563566
Goat Anti-Guinea Pig Alexa Fluor 488	Thermo Fisher Scientific	A-11073	RRID:AB_2534117
Culture reagents
Antibiotic-Antimycotic	Thermo Fisher Scientific/Gibco	15240062	100X
B-27 supplement	Thermo Fisher Scientific/Gibco	17504044	50X
Collagenase, Type IV	Thermo Fisher Scientific/Gibco	17104019	Powder
Dispase	Thermo Fisher Scientific/Gibco	17105041	Powder
DMEM/F12, HEPES	Thermo Fisher Scientific/Gibco	11330032
Glucose	any brand		Powder, Cell Culture Grade
GlutaMAX	Thermo Fisher Scientific/Gibco	35050061	100X
HEPES	any brand		Powder, Cell Culture Grade
Mouse Laminin	Corning	354232	1 mg/mL
N-2 supplement	Thermo Fisher Scientific/Gibco	17502048	100X
NAHCO3	any brand		Powder, Suitable for Cell Culture
Neurobasal	Thermo Fisher Scientific/Gibco	21103049
Phosphate-Buffered Saline (PBS)	Thermo Fisher Scientific/Gibco	10010023	1X
Poly-L-ornithine hydrobromide	Sigma-Aldrich	P3655	Powder
Recombinant Human EGF	Peprotech	AF-100-15
Recombinant Human FGF-basic	Peprotech	AF-100-18B
StemPro Accutase Cell Dissociation Reagent	Thermo Fisher Scientific/Gibco	A1110501	100 mL
Disposable material
24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates	Corning/Costar	3473
24-well Clear TC-treated Multiple Well Plates	Corning/Costar	3526
40 µm Cell Strainer	Corning/Falcon	352340	Blue
Bottle Top Vacuum Filter, 0.22 µm pore	Corning	431118	PES membrane, 45 mm diameter neck
Non-Pyrogenic Sterile Centrifuge Tube	any brand		with conical bottom
Non-Pyrogenic sterile tips of 1,000 µl, 200 µl and 10 µl.	any brand
Sterile cotton gauzes
Sterile microcentrifuge tubes of 1.5 mL	any brand
Sterile serological pipettes of 5, 10 and 25 mL	any brand
Sterile surgical gloves	any brand
Syringe Filters, 0.22 µm pore	Merk Millipore	SLGPR33RB	Polyethersulfone (PES) membrane, 33 mm diameter
Equipment and surgical instruments
Biological safety cabinet
Dissecting Scissors
Dumont Forceps
Motorized Pipet Filler/Dispenser
Micropipettes
Petri Dishes
Scalpel Blades
Stainless-steel Spatula