Culture scheme of neural stem cells
Neural stem cells are a class of stem cells found in the central nervous system and are pluripotent stem cells. Neural stem cells play an important role in the embryonic development of organisms such as humans and mice, and are also present in the adult bodies of these organisms. These cells have the ability to differentiate into neurons, astrocytes and oligodendrocytes. During embryonic development, neural stem cells are called "radial glial cells".
Figure 1. Neurogenic areas in the mammalian brain
Methods
Isolation of neural stem cells from neural tissue
2. The brain was removed and transferred to a 50 mL test tube into a 10 cm Petri dish (C7071) containing 20 mL of cold solution A (1×HBSS (H377731) containing 30 mM glucose (G116304), 2 mM Hepes (H109408), 26 mM NaHCO3 (S112331)). A small piece of filter paper was placed on the tissue chopper, the filter paper was slightly moistened with a wet sterile cotton swab and the brain was then placed on the wet filter paper using curved-tip forceps. The brain was cut into 400 μM coronal sections and sections containing SVZ (~6 sections) and hippocampus (~5 sections) were collected using a wet sterile swab into a 6 cm Petri dish (B5222) filled with 5 mL of solution A.
Key steps: Place the Petri dish containing the brain section and solution A on ice during cutting and dissection.
3. Dissect out the SVZ and dentate gyrus under a dissecting microscope (brain map) and save the dissected tissue in separate 15 mL centrifuge tubes (C1506), each containing 10 mL of cold solution A.
Key steps: This and subsequent steps are optimised for the isolation of NSC from SVZ and DG preparations from one mouse. If you intend to isolate cells from the pooled tissue of more than one mouse, we recommend that you add a Percoll (P331004) purification step to remove excess myelin and other cell types.
4. After dissection of the brain sections, the tissue blocks were spun down using a low-speed centrifuge at 200g for 1 minute at room temperature (20-25°C).
5. Remove the supernatant in a laminar flow hood and add 1 mL of 0.05% trypsin-EDTA (T301862) to each 15 mL conical tube containing tissue blocks. Spin the conical tubes for 10-20 minutes at room temperature.
Caution: Do not incubate for more than 30 minutes as this will reduce the viability of the cells.
6. Add 1mL of Trypsin Inhibitor Solution (T113170) to each tube and rotate for a further 10-20 minutes.
Caution: Do not incubate for more than 30 minutes as this will reduce the viability of the cells.
7. Pre-wet a fire-ground glass pipette and triturate the tissue (M108433) by pipetting up and down 10-20 times until there are no tissue clumps, then add 8 mL of N2 medium, N2 supplement, and L-glutamine (G105425) to each tube. Cells were pellet at 200g for 5 minutes at room temperature.
Caution: Avoid creating air bubbles when titrating the tissue as this will reduce the viability of the cells.
Key steps: It is important to dissociate SVZ and DG into single cells, as any remaining aggregates will result in reduced yield.
8. Wash the cells twice with 10 mL of N2 medium, each time spinning at 200 g for 5 minutes at room temperature.
9. If it is desired to isolate cells from pooled tissue from greater than 2 mice, resuspend each cell pellet with 5.5 mL of N2 medium, add 5.5 mL of Percoll/PBS solution and mix by inverting the test tube. The cells were allowed to settle at 400g for 15 minutes at room temperature. Cells were then washed 3 times with 10 mL of N2 medium and collected by spinning at 200g for 5 minutes each time at room temperature.
Key steps: Gently remove the supernatant as the cell pellet may not be firmly attached to the bottom of the tube.
Key steps: It is critical to remove Percoll by thorough washing as residual Percoll can affect cell viability.
10. Wash once more with 8 mL of Initial Proliferation Medium (IPM) (Neural Stem Cell Basal Medium, L-Glutamine, 1×Pen-Strep, 20 ng/mL FGF-2 and 20 ng/mL EGF.
11. Resuspend each cell pellet with 1 mL IPM (DG), 2 mL IPM (SVZ), and load the cells into one well of a 24-well tissue culture plate for DG and 2 wells for SVZ cells. Cells were incubated in a CO2 incubator for 48 hours.
12. Replace half of the IPM to avoid removing any cells. Continue to culture the cells for 7-14 days, replacing half of the IPM every other day and monitoring the cells for neurosphere formation. Neurospheres should form in both cultures within1-2weeks.
Differentiation of human iPSCs to NSC
Dual SMAD inhibition is a well-established method for obtaining neural progenitor cells from human ES/iPS cells in monolayer culture. The protocol uses two SMAD inhibitors, Noggin and SB 431542, to drive rapid differentiation of ES/iPS cells into a highly enriched population of NPCs. Noggin acts as a BMP inhibitor and SB 431542 inhibits Lefty/Activin/TGFβ by blocking phosphorylation of the ALK4, ALK5 and ALK7 receptors pathway. In order to clarify and optimise the neuronal differentiation protocol, the researchers modified the original protocol to create an entirely small molecule-based approach to differentiation that relies on three small molecules to inhibit the GSK-3β, CHIR99021 (C125082), TGFβ, SB431542 and Notch, Compound E (C275008) signalling pathways, and human LIF. This novel small molecule-based neural differentiation protocol improves the kinetics of neural differentiation and allows the derivation of truly pluripotent neural stem cells that respond to regional pattern cues specifying forebrain, midbrain and hindbrain neural and glial subtypes.
Characteristics of neural stem cells
Currently, the identification of neural stem cells is usually based on the presence of molecular markers associated with stem and/or progenitor cell status and the lack of more differentiated phenotypes assessed by marker analysis. NSCs positively express the stem cell markers Nestin, Sox-2 and Musashi, while lacking more differentiated lineage markers, including βIII-tubulin for neurons, astrocytes GFAP and O1 in oligodendrocytes.
Cell Type | Sox-2 | Nestin | Musashi | b-Tubulin | GFAP | O1 |
Pluripotent Stem Cells | + | - | - | - | - | - |
Neural Stem Cells | + | + | + | - | - | - |
Neurons | - | - | - | + | - | - |
Astrocytes | - | - | - | - | + | - |
Oligodendrocytes | - | - | - | - | - | + |
Table 1. Neural stem cell markers
Neural stem cell expansion
Culturing NSCs in 3D neurospheres
13. After 7-12 days of neurosphere culture (steps1-12above), collect all primary spheroids without disturbing the attached cells; spin at 200g for 5 minutes at room temperature.
14. Carefully remove the medium and add 1mL of 0.05% trypsin/EDTA (T301862) to each tube. Digest the spheres by pipetting up and down 20 times with a 1mL blue tip over 2 minutes to dissociate the spheres. Add 1mL of trypsin inhibitor solution (T113170) to each tube and pipette up and down 10 more times. Add 5mL of IPM to each tube, invert the tube several times to mix and settle the cells at 200g for 5 minutes at room temperature.
15. Resuspend DG cell pellets in 1 mL IPM and SVZ cell pellets in 2 mL N2 medium. A 10 μL aliquot of each sample was diluted in 10 μL of 0.5% Trypan blue (T293438) and viable cells were counted using a haemocytometer.
16. Incubate the cells by loading DG cells into one well of a 24-well plate and SVZ cells into one well of a 6-well plate.
17.Replace half of the IPM medium for DG cells and fresh N2 medium for SVZ cells every other day for 1 week. Avoid removing any cells while replacing half of the medium. After the first phase, maintain hematopoietic stem cells in N2 medium for DG and SVZ and seed hematopoietic stem cells at a rate of 3×104-1×105 cells/mL every 2-3 days.
Cultivation of NSCs in two-dimensional monolayer cultures
To enable better growth and adhesion of NSCs, we recommend applying poly-L-ornithine and laminin to the tissue culture plastic or glassware.
18. Dilute the stock concentration of poly-L-ornithine (0.1mg/mL) with water to obtain: a. 20 μg/mL for polystyrene plates b. 50 μg/mL for glass plates.
19. Add enough poly-L-ornithine solution to cover the entire surface of the tissue culture vessels. 2 mL volume for 3.5 cm plates, 5 mL volume for 6 cm plates and 10 mL volume for 10 cm plates and T75 flasks. Incubate in a humidified 37°C incubator for at least one hour.
20. Remove the poly-L-ornithine solution and rinse once with sterile water. Rinse and aspirate.
21. For the culture, propagation and differentiation of NSCs, laminin is diluted to a final concentration of 5 μg/ml. coated plates and flasks can be stored in laminin solution at 2-8°C for 3 weeks or at -20°C for 6-8 months. Before use, bring the coated plates or flasks to room temperature and aspirate the laminin solution, rinsing the plates once with 1 x PBS (P301982) before use.
22. Vials of neural progenitor cells were removed from the liquid nitrogen and incubated in a 37°C water bath and monitored closely until the cells were completely thawed.
Caution: Do not vortex the cells.
23. Immediately after complete thawing of the cells, sterilize the exterior of the vial with 70% ethanol. In a laminar flow hood, transfer the cells to a 15mL sterile conical tube using a 1 or 2mL pipette. Be careful not to introduce any air bubbles during the transfer.
24. Using a 10mL pipette, slowly add 9mL of Neural Extension Medium (pre-warmed to 37°C) dropwise to the 15mL conical tube.
Caution: Do not add the entire volume of medium to the cells at once. This may result in a decrease in cell viability due to osmotic shock.
25. Gently mix the cell suspension by slowly pipetting up and down twice, without introducing any air bubbles during the operation.
Caution:Do not vortex the cells.
26. Centrifuge the cells at 200g for 3-5 minutes at room temperature to pellet the cells.
27. Decant as much of the supernatant as possible.
28. Cells were resuspended in a total volume of 2 mL of neural extension medium containing FGF-2, 20 ng/mL (pre-warmed to 37°C).
29. Load the cell mixture into 3.5 cm tissue culture plates coated with poly-L-ornithine and laminin.
Caution:For optimal growth, thawing cells on tissue culture plates larger than 3.5cm is not recommended.
30. Cells are incubated at 37°C in a humidified 5% CO2 incubator.
31. The next day, the medium was exchanged with fresh neural extension medium (pre-warmed to 37°C) containing FGF-2 (20 ng/mL). Thereafter, the medium was exchanged with fresh medium every other day.
32. When the cells are approximately 90-100% confluent, they can be isolated with Accutase™ and passaged or frozen for later use. Cells should always be at a high cell density, so a passaging ratio of 1:2 to 1:6 is recommended.
Figure 2. culture characteristics of neural stem cells. NSCs can be grown as floating three-dimensional neurospheres (A) or as attached two-dimensional monolayers (B) on poly-L-ornithine/laminin-coated plates. Pluripotent neural stem cells express Nestin (C) and Sox-2 (D).
Figure 3. Differentiation of neural stem cells. Pluripotent NSCs expressing Nestin/Sox-2 can differentiate into BIII-Tubulin-positive neurons (B) or GFAP-positive astrocytes (C) under appropriate culture conditions.
Frequently Asked Questions
1. What is the doubling time of NSCs? How often do they divide the culture?
Human haematopoietic stem cells double every 48-72 hours. Murine NSCs are doubled every 24 hours. Cells should be isolated at a ratio of 1:2 to 1:6 after 80% confluence, approximately every 3-5 days.
2. What could be the cause of the lower than normal cell growth rate and how can it be resolved?
Change the medium daily to ensure rapid proliferation. Use fresh medium and freshly supplemented bFGF. wait until the cells are 80-90% confluent and then isolate at a ratio of 1:2 to 1:4. Check to ensure that the cells are not differentiated.
3. My cells do not adhere to the P/L-coated plates, what should I do to establish a monolayer NSC culture?
Plates coated with Poly-L-Ornithine/Laminin are problematic. Re-coat the new plates with fresh Poly-L-Ornithine/Laminin solution.
4. There are a large number of dead/floating cells in my culture. Is this normal?
This is a loosely adherent cell culture and it is common for cells and cell debris to float in the medium. As long as the adherent cells on the plate are growing well in the monolayer, there is no need to worry about floating cells, they will be washed away when the medium is changed.
5. How do you differentiate the cells into a specific neural phenotype?
The differentiation protocols/media provided are intended as a base system. For specific cell types, differentiation protocols that include additional neurotrophic factors can be developed according to the interest of the end user.