Neuronal differentiation from mouse assays is excellent model for illustrating the key mechanisms involved in neurogenesis. We demonstrate an optimized method for embryoid neurogenesis using a company toro screening strategy. This technique has the advantages of high efficiency, low cost, and easy operation, and is suitable for popularization among laboratory.
It is a powerful tool for neuroscience research. To perform differentiation using protocol one, seed 20, 000 mouse embryoid stem cells in two milliliters of basal differentiation medium into each well of a 0.1%gelatin-coated six-well plate. After cell attachment, wash the cells with two milliliters of PBS and add two milliliters of basal differentiation medium to each well, then put the cells back in the incubator.
Allow the cells to differentiate for eight days, replacing the medium every two days. To perform protocol two, add 1.5 million mouse embryoid stem cells into a non-adhesive bacterial dish in 10 milliliters of basal differentiation medium and incubate the plate at 37 degrees Celsius and 5%carbon dioxide to allow for embryoid body formation. After two days transfer the cell aggregates into 15 milliliter centrifuge tubes and let them settle by gravity.
Remove the supernatant and add 10 milliliters of fresh basal differentiation medium to re-suspend the embryoid bodies. Then replant them into a new non-adhesive dish and incubate them for another two days. Collect and seed about 50 embryoid bodies in two milliliters of basal differentiation medium per well onto a gelatin-coated six well plate.
For retinoic acid induction, add two microliters of RA stock into each well to a final concentration of one micromolar. Return the plate to the incubator and differentiate the cells for another four days. To perform differentiation using protocol three, repeat protocol two but allow only two days for embryonic body formation and six days for RA induction.
Quality control of embryoid bodies formation should be carried out using microscopy. Only those with bright cores can differentiate successfully in the subsequent process. For protocol four, seed 20, 000 mouse embryonic stem cells per well into the gelatin coated plates and follow protocol one.
After allowing the cells to differentiate for four days add two microliters of all trans RA stock into each well and incubate the plate for another four days. To perform protocol five, repeat protocol four but begin RA induction after only two days of differentiation. For protocol six, plant 1.5 million stem cells into a non-adhesive bacterial dish in 10 milliliters of N2B27 medium two and incubate the plate at 37 degrees Celsius and 5%carbon dioxide to allow for embryoid body formation.
After two days, collect cell aggregates as previously described and re-suspend the embryoid bodies with 10 milliliters of fresh N2B27 medium two. Replant them into a new non-adhesive bacterial dish and allow differentiation for another two days. On the fourth day, collect and seed about 50 embryoid bodies per well onto gelatin-coated six well plates with two milliliters of N2B27 medium two.
Add two microliters of all trans RA stock into each well and induce differentiation for another four days. To perform protocol seven, seed about 20, 000 cells in two milliliters of basal differentiation medium per well onto the gelatin-coated plates. After cell attachment and two washes with PBS add two milliliters of N2B7 medium two to each well and allow the cells to differentiate for eight days at 37 degrees Celsius and 5%carbon dioxide.
After performing phase one differentiation using protocol three, seed about 500, 000 mouse embryonic stem cell derivatives in two milliliters of basal differentiation medium per well onto the 0.1%gelatin-coated plates. Randomly divide the derivatives into three groups in order to test three phase two differentiation protocols. Incubate the plate for six hours, then wash the cells twice with two milliliters of PBS and add two milliliters of basal differentiation medium N2B27 medium one or N2B27 medium two to each well, depending on the protocol.
Place the blades into the incubator and allow the cells to differentiate for another 10 days, changing the corresponding medium every two days. Seven protocols were tested to determine the optimal protocol for the differentiation of mouse embryonic stem cells in general precursor cells or NPCs. The protocols were tested on both A2lox locks and 129 cells and the differentiation status of each group was monitored using a light microscope.
Most A2lox and 129 derivatives showed well-stacked and neurite-like morphologies under protocol three indicating the formation of NPCs. However, cells differentiated using protocol two appeared apoptotic which may be due to the lack of nutrients within the embryoid bodies. To further confirm the formation of NPCs nestin-positive cells were detected using an immunofluorescence assay.
Protocol three resulted in the highest percentage of nestin-positive cells reaching up to 78 and 69%in A2lox and 129 derivatives respectively. Three protocols were tested for the differentiation of NPCs into neurons. It was determined that protocol three most effectively induces the differentiation.
Most A2lox and 129 derivatives in phase two protocol three had prolonged neuron-like structures with clear neurites and cell body extensions. Immunofluorescence assays further confirmed the generation of neurons with the percentage of beta tubulin three positive cells up to 68 and 59%in A2lox and 129 derivatives respectively using protocol three. When attempting this procedure, it is important to ensure that mouse assays are healthy and non-differentiated before differentiation.
In addition, strict quality control should be carried out after formation in bore out bodies and before phase two differentiation. This technique provides a standard model for researchers of neurobiology and developmental biology and is expected to be a powerful tool for illustrating the key mechanisms involved in neurogenesis.
