Protocol for Differentiation of Human Embryonic Stem Cells into Neural-Related Cells

Directed differentiation of human embryonic stem cells (hESCs) to a functional cell type, including neurons, is the foundation for the application of hESCs. We describe here a reproducible, chemically-defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons.

Make ESC aggregates

• 10 min before start, warm up DMEM/F12, dispase and ES cell growth medium in 37°C water bath. Aspirate medium from each well of the 6-well plate containing the ESCs.
• Wash the cells with DMEM/F12 once to remove serum replacement. Aspirate DMEM/F12 and add to each well 1 ml dispase.
• When the edges of ESC colonies begin to curl, aspirate the dispase-containing medium from the 6-well plate and wash the cells gently with 2 ml DMEM/F12.
• Resuspend the ESCs in the ESC growth medium and transfer them to flasks (for one entire 6-well plate resuspend the cells in 50 ml and culture them in a T75 flask; for 1-3 wells of cells use 15 ml ESC growth medium and transfer to a T25 flask).

Neuroepithelial differentiation

• After 4 days, aspirate the ESC growth medium completely after the ESC aggregates are settled down to the corner of the flask. Rinse the aggregates with the neural induction medium once and resuspend the aggregates in the neural induction medium. Change half of the medium 2 days later.
• On the following day, the anaphase suspension is transferred to a centrifuge tube and left for 10 to 15 min to allow the colonies to settle to the bottom of the tube, and the medium is removed.
• Cells are resuspended with NIM culture medium containing 20 μg/mL Laminin, and hES colonies from 1 T75 vial are inoculated into 2 6-well plates. The Laminin-containing NIM culture medium is aspirated on the second day, and 2 mL of fresh NIM is added to each well of the 6-well plate.
• The cells in the clones will form a wreath-like structure after 2 to 3 days of apposition, a period we call primitive neuroepithelium. After further incubation with NIM for 4-5 days, the tightness of the wreaths will increase and each clone may contain multiple neural tube-like inner cavities of the wreaths, a period that can be referred to as the neuroepithelium.

Differentiation of neurons from neural progenitors

• Neuroepithelial colonies can be cultured in suspension for weeks to months.
• Circular coverslips are placed in 24-well plates and incubated overnight with 0.1 mg/mL polyornithine before use, after which NDM containing 20 μg/mL laminin is added and left at 37°C for more than 3 h.
• Accutase enzyme, DMEM/F12 and NDM medium are warmed in a 37°C water bath.
• Collect neuroepithelial cell colonies in 15 mL centrifuge tubes and allow the colonies to settle naturally at the bottom of the tube. Aspirate off the culture medium.
• Add 500 µL of Accutase enzyme and incubate at 37°C for 2 to 3 min. Add an equal amount of NIM medium to dilute the enzyme digestion and centrifuge at 1000 r/min for 3 min at room temperature.
• Aspirate the supernatant and wash once with DMEM/F12.
• Add 1 mL of NDM medium to the tube and use the tip of a 200 μL pipette to pipette back and forth at the bottom of the tube to break up the cells into small clusters or individual cells.
• Cells are inoculated on coverslips and Petri dishes that had been previously treated with polyornithine and Lamin. 50 to 60 colonies can be inoculated per well in a 6-well plate, and 8-10 colonies can be inoculated on one coverslip.
• Observe whether the cells adhere to the bottom of the Petri dish 2 to 3 h after cell inoculation, and if they have adhered to the wall, add 2 mL of NDM to each well of a 6-well plate and 0.5 mL of NDM to each well of a 24-well plate.
• The cells are changed every two days with the same medium, and neuronal synapses can be observed after 2-3 days.

Differentiate human ESCs from midbrain dopaminergic neurons

• Differentiation from hES cells into primitive neuroepithelial cells was performed as previously described.
• Aspirate off old medium and add 3 ml neural induction medium supplemented with FGF8b (50 ng/ml) and SHH (100 ng/ml). Continue feeding the cells every other day with the same medium with FGF8b and SHH for 7 days until the formation of neural tube-like rosettes.
• When differentiated to the stage of enriched and expanded neuroepithelial cells, FGF8b (50 ng/mL), SHH (100 ng/mL), B27 (1×), and Vc (200 µM) are added to the NIM medium and the solution is changed every other day.
• After 7 days of suspension culture, neural precursor cell colonies were digested with accutase to form small clusters and inoculated into culture dishes with surfaces pre-lined with PDL and laminin.
• After cell apposition, the medium was changed to NDM medium containing FGF8b, SHH, Vc, cAMP, TGFβ3, BDNF, and GDNF every other day for three weeks.
• After 44 days of differentiation, the medium containing FGF8b, and SHH was withdrawn and the cells continued to be maintained in an NDM medium containing Vc, cAMP, TGFβ3, BDNF, and GDNF.
• After 35 days of differentiation, differentiated dopaminergic neurons can be identified by immunofluorescence staining, such as staining for the dopaminergic neuron-specific marker TH.

Creative Bioarray Relevant Recommendations

• Creative Bioarray provides embryonic stem cells from both humans and mice. Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo.
• We also offer Neural Precursor Cell Medium and Neural Precursor Cell Differentiation Medium, which is a complete medium designed for optimal growth of and differentiation of normal human neural precursor cells in vitro.

• The undifferentiated state of ESCs is very important for efficient neural induction. If there are differentiated cells in the ESC culture, floating ESC aggregates are easy to attach to the flask and neurons will be easily found in the culture dish when these aggregates are plated down.
• Usually, the size of the clusters is around twice the size of ESCs for passaging/splitting. Cells will die and the yield of ESC aggregates will be quite low if the size of the clusters is too small. The differentiation will be unsynchronized if the size is too large.

Embryonic Stem Cells

For research use only. Not for any other purpose.