Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Summary

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Abstract

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as "niches" that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Introduction

The central dogma of neurobiology, laid down by the fundamental observations of the brain cytoarchitecture made by Ramón Y. Cajal over a century ago, held that neurogenesis was unlikely after adolescence given the complexity of the neural networks found in the CNS¹. Despite the work of Altman in the 1960s, and later Kaplan, demonstrating that ³H-thymidine could be found in mature neurons indicating that in fact neurons were being generated in distinct areas of the adult brain, the dogma continued to hold^2,3. Evidence continued to mount with Nottebohm’s research describing the seasonal changes in the number of neurons present in songbird brains⁴. It wasn’t until 1999, when Gould et al. published work on the generation of neurons in the hippocampus increases with the performance of associative learning tasks in rat, as well as the observations of Kornack and Rakic demonstrating continued neurogenesis in the adult macaque that the concept of a less rigid, more plastic brain, was recognized^5,6.

The search for the cellular source for these de novo generated neurons lead to the discovery of a discrete population of stem cells (SCs) that reside in areas of the brain referred to as niches⁷. The subventricular zone and sub granular zone of the hippocampus are considered to be the two principal neurogenic regions^8,9. Cells isolated from these locations display the classic characteristic of embryonic or fetal derived SCs, self-renewal and differentiation potential. In the case of neural stem cells (NSCs), they can be differentiated into neurons, astrocytes, and oligodendrocytes. In addition, these SCs  stain positive for fetal NSC markers such as the intermediate filament protein nestin¹⁰. More recent work highlights that SCs might not be limited to these two areas, and are in fact localized throughout the brain as a largely quiescent population of cells tightly associated with the vasculature¹¹.

The observations that SCs are mobilized in response to injury suggests the possibility of being able to utilize these cells for regenerative purposes to aid in the recovery from neurodegenerative disorder and stroke^12,13. This is not unlike the role that mesenchymal stem cells (MSCs)  play in the healing of connective tissues, which are found as perivascular cells that have the potential to become osteoblasts, chondrocytes, and adipose cells¹⁴. However, NSCs cannot be harvested in the same manner as MSCs from bone marrow by routine aspiration and density gradient centrifugation techniques and subsequently utilized in autologous cell based therapies. As a consequence, other sources of cells, such as the use of fetal NSCs or neuronal precursors derived from embryonic stem cells have been extensively explored in animal disease and injury models with varying degrees of success¹⁵. Induced pluripotent stem cell technologies utilizing somatic cell sources offer another potential avenue for producing therapeutically useful cell-based therapies for a wide range of applications, overcoming the limited availability and ethical concerns regarding the use of embryonic cells and fetal tissues¹⁶. However, clinical translation of these findings has proven to be a difficult task, as demonstrated in the struggles of treating various neurological conditions with SC-based therapeutic approaches^17,18, as well as a tortuous path to regulatory clearance. As an alternative approach, introduction of specific pharmacological treatments can modulate NSC numbers and facilitate recovery in models of Parkinson’s disease and stroke¹⁹. Whatever the strategy might be, understanding how to effectively manipulate these cells requires an accessible in vitro system.

Cultures of NSCs can be performed either as aggregate cultures, also known as neurospheres, or as a monolayer^8,20. Both techniques have been widely used, allowing for the establishment of defined culture conditions, i.e. use of Epidermal Growth Factor (EGF) or basic Fibroblast Growth Factor (bFGF) as a mitogen source, that provide for the expansion of multipotent precursors. While neurosphere cultures may be better suited for studying clonal propagation capability of an isolated cell type, the system has been shown to produce a mixed population of cells during expansion²¹. In addition, the closed structure of neurospheres makes pharmacological manipulation of the cells impractical, and the interpretation of the influence these factors may have could be confounded due to the microenvironment established within the neurosphere itself. Monolayer cultures, on the other hand, can be employed in high throughput screens of small molecule libraries, providing a powerful tool to explore the signal transduction mechanisms that regulate SC growth and differentiation and opens the opportunity to discover novel compounds that specifically target this cell population.

As a consequence, the ability to reproducibly generate cultures of adult NSCs from different regions of interest in the brain can be used in a broad spectrum of research applications, ranging from developmental studies of the central nervous system (CNS) to exploring novel regenerative medicine approaches. The protocol presented here demonstrates how to dissect and assess the differentiation potential of CNS SCs isolated from the adult rodent brain.

Protocol

The work adheres to the Declaration of Helsinski and the ARVO Animal Statement. Animals were used for tissue collection and all relevant methods were followed according to the instructions of the animal facility at the University of Dresden. Animals were handled and housed according to the German Federal guidelines for the use and care of laboratory animals, and the study was approved by the Landesdirektion Dresden. Please consult with your institution’s veterinary policy (IACUC or other board) regarding appropriate euthanasia methodology.

Specific stock and working concentrations of reagents can be found in the Reagent Tables accompanying this protocol.

1. Culture Dish Preparation

1. Coat dishes with a sufficient volume of 75 μg/ml poly-L-ornithine (i.e. 2 ml/well of a 6-well plate) overnight in the cell culture incubator 2 days prior to the scheduled dissection date.
2. Aspirate the poly-L-ornithine the following day and wash each well 2x with 3 ml of PBS for 5 min.
3. Remove the final wash, then add fibronectin (diluted 1:250, 4 μg/ml in PBS) to the plate and place the dishes in the incubator overnight.
4. Aspirate the fibronectin on the day of dissection and wash the dishes 2x with PBS. Return the plates still containing the final PBS wash to the incubator until the dissociated tissue is ready for culturing. At that time, remove the PBS prior to plating the tissue.
5. Store plates coated with poly-L-ornithinein the incubator for up to 3 weeks. Plates coated with fibronectin can be stored for up to 1 week. Fibronectin coating can be done in as little as 2 hr if urgent. Fibronectin is susceptible to denaturation; do not agitate the solution or let dishes dry.

2. Dissection/Plating

1. This protocol applies to the use of 3 adult rats (3-6 months of age).
2. Chill the brain sectioning block on ice prior to euthanizing the rat.
3. Place a 15 ml Falcon tube that contains 5 ml of N2 medium with added bFGF on ice.
4. Euthanize the rats according to your institution’s veterinary policy.
5. Remove the brain from the skull by making an incision down the midline of the head. Use forceps to peel away the bone allowing for the brain to be carefully extracted.
6. Place the brain into a 10 cm Petri dish containing ice cold PBS. This will remove any residual hair and blood. (Figure 1A)
7. Place the brain into the chilled sectioning block. (Figure 1B)
8. Insert one new clean razor blade into the block as pictured in Figure 1C.
9. Insert a second clean razor blade 3 mm caudal to the first as illustrated in Figure 1D.
10. Carefully lift the blades from the block, taking with them the section of interest.
11. Float the section off the blade by immersing it in PBS. (Figure 1E)
12. Harvest tissue from the area of interest (in this example we used the anterior commissure, (anterior) ACA, Figure 1F) using #5 forceps and collect into a 15 ml conical tube containing 1 ml N2 medium containing bFGF.
13. Move the tube into a laminar flow cell culture hood, continuing the remainder of the protocol under sterile conditions.
14. Mechanically dissociate the tissue using a 1 ml pipette tip attached to a P1000 pipettor. Pipette up and down several times (approximately 20x at a rate of 1 pipetting cycle per second) while pressing the opening of the pipette tip against the bottom of the conical tube (Figures 1G 1-3). Stop trituration when the solution becomes homogeneous.
15. Allow the solution to sit for 2 min to allow any larger nondissociated tissue to settle to the bottom. (Figure 1G 4)
16. Collect the homogeneous supernatant and place into a conical tube containing 8.5 ml of N2 media plus 20 ng/ml bFGF, 500 ng/ml Dll4, 500 ng/ml Ang2, and 200 nMJAK Inhibitor.
17. Plate them into 3 wells of a 6-well plate using 3 ml of diluted cell solution/well.
18. Place the plate into a humidified cell incubator at 37 °C, 5% CO₂, 5% O₂.

3. Daily Care

1. Replace the medium on the culture dishes with N2 Medium containing bFGF, Ang2, Dll4, and JAK inhibitor 24 hr after plating.
2. Add a bolus of bFGF, Dll4, Ang2, and JAK inhibitor on the next day.
3. Continue this alternating process of media changes and factor additions for a total of 10-14 days.

4. Induction of Differentiation

1. Withdraw bFGF, Dll4, Ang2, and JAK inhibitor containing medium and replace it with N2 medium that does not contain any additional factors.
2. Change the medium every second day.
3. Fix the cells after 10 days and perform immunocytochemistry.

5. Immunocytochemical Detection

1. Aspirate the medium and fix cell culture plates with 2 ml of 4% paraformaldehyde for 20 min.
2. Wash 2x with 3 ml of PBS for 5 min each.
3. Aspirate PBS, then permeabilize and block the cells by adding 2 ml of PBS containing 5% normal donkey serum (NDS) and 0.1% Triton X-100 for 20 min.
4. Prepare the primary antibody mix in PBS containing 5% NDS. Anti-nestin antibody can be used for identifying SCs, while TuJ1 (class III β-tubulin), GFAP, and CNPase antibodies can be used together for a triple staining of differentiation markers.
5. Aspirate the blocking solution and add 1.5 ml of the primary antibody mixture to each well. Incubate at room temperature for 90 min.
6. Remove the primary antibodies and wash 2x with 3 ml of PBS, and 1x with 3 ml of PBS containing 5% NDS for 5 min each.
7. Prepare the secondary antibodies in PBS containing 5% NDS.
8. Aspirate the final wash and add 1.5 ml of secondary antibody solution to each well. Incubate in the dark for 40 min at room temperature.
9. Remove the secondary antibody solution and add 2 ml of a freshly prepared DAPI stain (500 ng/ml in PBS). Incubate for 3 min.
10. Aspirate the DAPI solution then wash 3x with 3 ml of PBS for 5 min each. The cells can now be visualized with a fluorescence microscope.

Results

Identifying the region of interest from which to harvest neural stem cells is the critical first step and will define the amount of time it takes to get a confluent plate of cells. For example, the SVZ is a classic neurogenic area and therefore has a higher relative proportion of neural stem cells. However, the technique presented here can be used with other regions not often recognized as having a robust neurogenic potential during adulthood. For the purposes of this protocol, we used the anterior commissure (anterior p...

Discussion

Many of the steps critical to successful isolation, expansion, and differentiation of neural stem cells from the adult brain are shared in common with standard tissue culture techniques. The objective to bear in mind is that NSCs isolated from the adult brain should maintain as many of their in vivo characteristics as possible (Figure 2J). Often a balance between necessary caution and appropriate speed needs to be struck. Care with the isolation of the brain from the skull will make the ide...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well tissue culture dishes	BD Biosciences	353934
Poly-L-ornithine	Sigma-Aldrich	P-3655	5 mg/ml stock solution prepared in double distilled water (Stable for several months at -20 °C). Working concentration 0.5 mg/ml in water (Stable for 1 month at 4 °C).
Fibronectin	R&D Systems	1030-Fn	Do not agitate stock solution
Filtration Apparatus	Corning Life Sciences	430769
DMEM/F-12	Mediatech	10-090-CV	See note below for complete N2 media preparation
Apo-transferrin	Sigma-Aldrich	T-2036
Insulin	Sigma-Aldrich	I-0516
Putrescine	Sigma-Aldrich	P-5780	1 M stock solution in ddH2O (Stable at 20 °C for 6 months)
Sodium Selenite	Sigma-Aldrich	S-5261	500 µM stock solution in ddH2O (Stable at -20 °C for 6 months)
Progesterone	Sigma-Aldrich	P-8783	100 µM stock solution in ethanol (Stable at -20°C for 6 months)
Penicillin/Streptomycin	Invitrogen	15140-122
Phosphate Buffered Saline	Mediatech	21-040-CV
Basic Fibroblast Growth Factor (bFGF)	R&D Systems	233-FB	Working concentration 20 ng/ml
Delta4 (Dll4)	R&D Systems	1389-D4	Working concentration  500 ng/ml
Angiopoetin 2 (Ang2)	R&D Systems	623-AN	Working concentration  500 ng/ml
JAK Inhibitor	Calbiochem	420099	Working concentration 200 nM
Bovine Serum Albumin	Sigma-Aldrich	A-2058
15- and 50-ml Conical tubes	Corning Life Sciences	430053, 430829
Other necessary items include: General dissection instruments, including razor blade and forceps, Adult rat (3-6 months old; Sprague-Dawley or Long Evans), CO2 intoxication chamber, Laminar flow hood for cell culture and incubator Incubator (humidified, 37 °C, 5% CO2, 5% O2). Note: For complete N2 media preparation, to one bottle of DMEM/F-12 (500 ml) add 0.05 g of apotransferin, 0.0125 g of insulin (freshly predissolved in 1 ml of 10 mM NaOH), 50 μl of putrescine, 30 μl of sodium selenite, 100 μl of progesterone stocks, and 5 ml of penicillin/streptomycin solution.  Adjust pH to 7.2, if needed. Filter-sterilize and store at 4 °C for up to 3 weeks and protect from light.
Immunofluorescence Reagents Table
Paraformaldehyde	Electron Microscopy Sciences	15719
Normal Donkey Serum (NDS)	Sigma-Aldrich	D-9663
Triton X-100	Sigma-Aldrich	T-8787
4,6-Diamidino-2-phenylindole (DAPI)	Sigma-Aldrich	D-8417	5 mg/ml stock solution in methanol
Primary Antibody Table
Nestin	Chemicon	MAB353	Dilution Factor: 1:400 Species: Mouse IgG1
Hes3	Santa Cruz	sc-25393	Dilution Factor: 1:100 Species: Rabbit IgG
Sox2	R&D Systems	MAB2018	Dilution Factor: 1:100 Species: Mouse IgG2a
CNPase	Chemicon	MAB326	Dilution Factor: 1:200 Species: Mouse IgG1
β-tubulin III (TUJ1)	R&D Systems	MAB1195	Dilution Factor: 1:500 Species: Mouse IgG2a
Glial Fibrillary Acidic Protein (GFAP)	Dako North America	Z0334	Dilution Factor 1:500 Species: Rabbit
Secondary Antibody Table
Alexa   568	Invitrogen	A-21124	Dilution Factor: 1:200 Species: Goat anti Mouse IgG1
Alexa 488	Invitrogen	A-21131	Dilution Factor: 1:200 Species: Goat anti Mouse IgG2a
Cy5	Jackson ImmunoResearch	59883	Dilution Factor: 1:200 Species: Goat anti Rabbit