Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Note: For routine culturing of neural progenitor and oligodendrocytic lineage cells, incubation is done at 37 °C in a humidified 5% CO₂ atmosphere. Every 2 days, the medium is replaced using 50 to 100% of fresh medium if culture is 40-70% confluent. At the time of near confluency, the cultures are passaged at 2-2.5e6/T75 flask usually on a weekly schedule.

1. Preparing the Coated Flask

1. To prepare coated flasks dilute 5 mg of poly-D-lysine (PDL) in 100 ml of deionized water (DI-water), then coat T75 flasks with 50 μg/ml of PDL at room temperature (RT) in the dark. After 1½ hr, aspirate the PDL and rinse the flasks once with DI-water. Let the flasks dry before seeding the cells (Table 1).

2. Starting Differentiation of Neural Progenitor Cells into Progenitor-derived Oligodendrocytes (PDO)

The protocol to isolate human CNS progenitor cells is described in a previous publication and it is not part of this protocol¹. Human CNS progenitor cells were isolated from the telencephalon of an 8-week gestation fetal brain, obtained in accordance with NIH guidelines.

1. Grow multipotential population of neural progenitor cells on PDL coated flasks in neurobasal medium supplemented with 25 ng/ml of bFGF and 20 ng/ml epidermal growth factor (EGF) (Figure 1A). Do not use them if higher than passage 11 (p11).
2. Start to differentiate the neural progenitor cells when the culture is approximately 70% confluent.
3. Make complete differentiation oligodendrocyte medium using serum-free DMEM/HAMS F12 1:1 medium supplemented with bovine serum albumin, L-glutamine, gentamicin, N2 components, T3, Shh, NT-3, bFGF, and PDGF-AA (Table 1). This medium will be defined as oligo medium with growth factors (oligo medium + GF).
4. Remove progenitor media from the flasks, rinse cells once with phosphate-buffered saline (PBS), then add oligo medium + GF (details described in 2.3) to start oligodendrocyte differentiation. Only cells committed to the oligodendrocytic lineage will grow (Figure 1B). Culture the cells in this medium for 3 weeks.
5. Every week, when the cells are at 90-95% confluence, passage them using trypsin (0.05%)-EDTA (0.1%) (4 ml for a T75 flask). Transfer the entire medium from the cells to be passaged into a 50 ml conical tube; this conditioned medium will be used to quench the trypsin. Make sure not to dry out the cells. Then add trypsin.
6. Incubate cells in trypsin for 5 min at RT, gently tapping the flask several times to help cell detachment.
7. Quench the trypsin by adding 4-5 ml of conditioned medium to the flask with the detached cells.
8. Transfer the medium and the cells to the same 50 ml conical tube. Centrifuge the medium and the cells at 1,200 rpm (~300 x g) for 5 min at RT.
9. Aspirate the supernatant, resuspend the cell pellet, gently tipping the tube, and then add fresh oligo medium + GF . Gently pipette medium and cells up and down to make a uniform cell suspension.
10. Count the cells, and transfer 2.5e6 into each new PDL-coated T75 flask.

3. Final Step in the Differentiation of Oligodendrocytes

1. After 3 weeks of culture in oligo medium + GF, when the cells are at 70-80% confluence, start the final process of differentiation (Figure 1C).
2. Prepare medium in the same way as the oligo medium + GF but without adding the four GF: Shh, NT-3, bFGF and PDGF-AA. This medium without the four GF will be defined as oligo medium – GF.
3. Aspirate the oligo medium + GF from the flask, rinse once with PBS and then add a total volume of 16 ml of medium, of which ¾ (12 ml) is oligo medium – GF and ¼ (4 ml) is PDN conditioned medium, for 6-10 days). The use of conditioned medium from PDN is not critical to the differentiation process or to the survival of the cells. We in fact observed that only direct contact of PDO with PDN cells in co-culture experiments had an effect on the length of the PDO survival. The survival time in oligo medium – GF could be prolonged to two or three weeks if the PDO cells are co-cultured with PDN cells. The protocol to differentiate human neural progenitor cells into PDN is described in a previous publication and it is not part of this protocol¹. Growth factor withdrawal from the culture medium results in progressively differentiated culture of cells with multiple processes (Figure 1D).

4. Flow Cytometry Assay

The flow cytometry assay compares the acquisition of oligodendrocyte markers during the differentiation process in relationship with those of their parental population.

1. Use neural progenitors as control. Seed PDO cells at 2.5×10⁶ cells in two T75 flasks coated with poly-D-lysine in oligo medium + GF.
2. A week later, replace medium with fresh oligo medium + GF in flasks as a control culture for cell viability, while other flasks had oligo medium – GF.
3. To dissociate PDO cells in both media use 20 U/ml of papain and not trypsin. This is because papain has a gentler effect on the cells during the dissociation preserving the cellular morphology.
4. Add 5 ml of Earle’s Balanced Salt Solution (EBSS) to a papain vial. Place the vial at 37 °C for 10 min or until the papain is completely dissolved and the solution appears clear. This is the papain solution used to dissociate the cells.
5. Add 0.5 ml of EBSS to a Deoxyribonuclease I (DNase) vial (Table 1). Mix gently. Add 0.25 ml of this solution to the vial of dissolved papain. This preparation contains a final concentration of approximately 20 U/ml papain and 0.005% DNase.
6. Place papain solution (as described above) on the cells and incubate at 37 °C for 15-30 min; monitor the detachment of the cells using a microscope. Stop the reaction with 5 ml of medium with or without GF and centrifuge at 1,200 rpm for 7 min.
7. Transfer cell pellets from both flasks into two different 5 ml polypropylene tubes and wash them with normal physiological medium (NPM: 145 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 10 mM Hepes and 10 mM glucose) supplemented with 1 mg/ml bovine serum albumin (NPM+BSA).
8. Perform surface immunostaining at 4 °C for 20 min using specific and appropriately titrated primary antibodies for A2B5, O4, GalC (Table 2).
9. Wash cells with NPM+BSA at 4 °C for 5 min and incubate them with specific secondary antibodies at 4 °C for 20 min (Table 2).
10. To stain intracellular antigens including nestin, glial fibrillary acidic protein (GFAP), class III β-tubulin, and myelin basic protein (MBP) (Table 2), fix cells in 2% paraformaldehyde (PFA) for 20 min at RT and permeabilize with cold 70% ethanol for 15 min at -20 °C.
11. Wash cells with 1x PBS and incubate with specific secondary antibodies (Table 2).
12. To discriminate between intact cells and subcellular debris during flow cytometry analysis, stain cells with 4,6-diamidino-2-phenyllindole, dihydrochloride (DAPI) (Life Technologies, Grand Island, NY).
13. In optimizing the above protocol, we performed all the appropriate control experiments to confirm the specificity of each immunoreagent. Briefly, for directly labeled antibodies, the appropriate negative control was a directly conjugated antibody of the same immunoglobulin isotype class (or subclass) and fluorochrome conjugate as the primary antibody (i.e., the isotype control). For indirect immunostaining of primary antibodies, the appropriate negative control was the secondary antibody conjugated to the fluorochrome of interest that specifically targeted the immunoglobulin class (or subclass) of the host in which the primary antibody was generated. When primary antibodies from multiple hosts (mouse, rabbit, chicken) were used together, we used the secondary antibody targeting the primary antibody of each host and each secondary antibody used was typically cross-adsorbed against the immunoglobulins from the other hosts to minimize cross-host immunoreactivity. The positive controls included direct or indirect immunostaining of cell preparations known to express the antigens of interest using either direct or indirect immunoreactions with the primary antibodies specific for these antigens. The above optimization methods were carried out once for each type of immunostaining experiments. Control immunoreactions revealed no significant cross-epitope immunoreactivity among primary and secondary antibodies. Flow cytometry was performed on uniformly suspended fluorescently labeled cells using a FACVantage SE cell sorter (BD Biosciences, San Jose, CA) equipped with three lasers, which provide excitation wavelengths tuned to 488 nm, 647 nm, and a broad UV (351-364 nm) (Figure 2).

5. Immunofluorescence Assay

Note: For immunofluorescence experiments, PDO are plated in oligo medium + GF or oligo medium – GF at 2.5e5 in 6 well plates coated with PDL. The cells are fixed in 2% PFA at various time points after withdrawal of growth factors . The antibody stain is performed on plastic 6 well plates instead of glass coverslips.

1. Remove media and add the 2% PFA. Incubate for 10 min at RT. Discard PFA. Wash cells 3 times with PBS, 5 min each time. Be careful not to dry out the cells.
2. Permeabilize cells for intracellular markers using 0.25% triton solution in PBS for 10 min at RT.
3. To prevent non-specific binding, block cells for 10 min with HHG (1 mM HEPES buffer, 2% horse serum, and 10% goat serum in Hanks’ balanced salt solution). Dilute specific primary antibodies (Table 2) to a pre-determined concentration in HHG. Place diluted antibodies onto cells. Incubate 1 hr at RT on the shaker or at 4 °C overnight.
4. Wash cells 3 times with PBS, for 5 min each time. Dilute appropriate secondary antibodies (Table 2) coupled with a fluorochrome to a pre-determined concentration in HHG. Place the mixture of diluted secondary antibodies onto cells. Incubate 1 hr at RT on a shaker in the dark.
5. Wash cells 3 times with PBS, for 5 min each time.
6. To avoid photobleaching add 10 μl of Prolong Gold with DAPI to the cells and place a glass coverslip on top of them. Visualize labeled cells using an Axiovert 200M fluorescence microscope (Zeiss, Thornwood, NY) (Figure 3).