In addition to the protocol for mouse embryonic stem cell differentiation into neuron cells, we also show a set of experiments that can comprehensively analyze the differentiation process. These techniques are pertinent to early ectodermal embryonic development, providing researchers with options to analyze neuron cell differentiation. When attempting this procedure, starting with stem cells in optimal condition is essential.
Hence it is important that the EBs, MPCs and neurons are cultured in fresh and optimum media. Visual demonstration of this protocol is important, because it contains minute detail that can affect its success and effectiveness, especially the differentiation part. Begin by setting up a hanging drop cell culture.
For a 10-centimeter cell culture plate, adjust the concentration to 500 cells per 20-microliters of differentiation medium. Prepare enough cell suspension for 50 20-microliter droplets. Use a micro-pipette or a repeater pipette to place 20-microliter droplets of cell suspension onto the lid of a tissue culture plate, making sure that the droplets aren't too close together, to prevent them from merging.
Fill the plate with five to 10-milliliters of PBS and carefully put the lid back on the plate. Incubate the culture in the 37 degrees Celsius incubator. After two days, collect the droplets from the lid, and place them in a 10-centimeter cell culture suspension plate filled with 10-milliliters of differentiation medium, then place the plate on an orbital shaker set to low speed in the incubator.
After another two days, centrifuge the cells at 200 times G for three minutes, and remove the supernatant. To induce embryoid body differentiation into neural progenitor cells, re-suspend the cells in differentiation medium with five-micromolar retinoic acid. Incubate the plate for two days, then replace the least half of the medium with fresh medium, by tilting the plate and pipetting it out.
Collect the cells two days later by centrifuging them at 200 times G for three minutes, and removing the supernatant. Fix the embryoid bodies and neural progenitor cells with 4%PFA in PBS for 30 minutes at room temperature. Then remove the PFA and wash the cells in PBS for five minutes.
Incubate the cells serially in PBS 10, 20 and 30%sucrose solutions, at 25 to 28 degrees Celsius, with each incubation lasting 30 minutes or longer until the cells settle at the bottom of the tube. And use it to transfer the sample to the center of a Cryomold, pipetting out the excess liquid. Carefully add optimum cutting temperature or OCT solution to the mold, without re-suspending the sample, then remove excessive bubbles with a pipette.
Placed the mold with the sample on a laboratory mixer at low speed for 15 minutes, which will settle the embryoid bodies and neural progenitor cells to the bottom of the OCT solution. Then quickly freeze the sample by placing the mold in liquid nitrogen. Block OCT sections or culture chambers containing neurons in 10%normal donkey serum, and 0.1%Triton X-100 in PBS for one hour at room temperature.
Then incubate the samples in primary antibody, diluted in 5%normal donkey serum, and 0.05%Triton X-100 in PBS, at four degrees Celsius overnight. On the next day, wash samples in PBS with 0.1%Triton X-100 three times for five minutes per wash. Incubate them with secondary antibody, diluted in 5%normal donkey serum, and 0.05%Triton X-100 in PBS, for one hour at room temperature.
Repeat the washes in Triton X-100 and PBS, then incubate the sample in one microgram-per-milliliter DAPI. Mount the sample with a cover slip and some mounting medium, allow it to dry, and image it with a fluorescence microscope. To perform neuron differentiation, E14 cells were cultured on gamma-irradiated mouse embryonic fibroblasts until the fibroblast population diluted out.
The pluripotency of the E14 cells was confirmed with alkaline phosphatase staining. After differentiation, embryonic bodies had a round shape that continuously increased in size. By day 10, neural progenitor cells differentiating into neurons, had an elongated cell shape.
Immunofluorescence experiments were performed on neural progenitor cells at day eight, and on neurons at day 12. Positive staining was observed for nesting in neural progenitor cells, and for neurofilament in neurons. A mouse embryonic stem cell line expressing a Sox1 promoter-driven GFP reporter was differentiated, and flow cytometry was performed on the embryonic stem cells and neural progenitor cells.
It was found that 58.7%of total cells at the NPC stage are GFP positive, while 0%are GFP positive at the ESC stage. RNA sequence experiments were performed on E14 embryonic stem cells, embryoid bodies, and neural progenitor cells. The genes in the RNA sequence heat map was sorted based on their expression levels.
Gene ontology analysis of the four gene clusters showed that they correspond to distinct cellular functions or pathways. When performing this protocol, EBS should appear as spherical, blackened masses of cells. To ensure that EBS grow with such, change the medium on day one, if the medium starts to become yellowish.
The same approach is important for MPC culture, as both EBs and MPCs are in a critical stage of early embryonic development. Other functional genomic assays, such as ChIPseek and chromatin confirmation capture assays from the different stages of neuron differentiation, can be performed to understand the underlying biological processes. Integrating by chemical and functional genomics assays in the neuron differentiation process, allows for researchers to better delineate the molecular mechanisms of early embryonic development.
