Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The overall goal of this procedure is to differentiate mouse embryonic stem cells to a neural lineage in a serum free monolayer cell culture. This is accomplished by first coating the culture vessel with gelatin. In the second step, the embryonic stem cells are associated into a single cell suspension in differentiation medium and diluted to the appropriate plating densities.

In the final step, the cells are seeded onto the gel ENC coated plate. Ultimately, immunofluorescence microscopy is used to confirm the success of the differentiation by visualization of the neural markers of interest. Generally, individuals new to this method will struggle as plating at the appropriate density is critical to achieving a good differentiation.

And there are several factors that affect the plating density both directly and indirectly. Before plating the cells warm an aliquot of 1%gelatin in a 37 degree Celsius water bath until dissolved. Then mix the gelatin with 500 milliliters of warm PBS and cover the bottom of the cell culture container with just enough of the solution to coat the surface while the gelatin is coating.

Rinse the sub confluent culture of mouse embr stem cells two times in PBS. Then add one milliliter of dissociation reagent to the culture. After two to five minutes, tap the vessel to dislodge the monolayer into a single cell suspension and to collect the culture in a total of 10 milliliters of medium and cell dissociation reagent.

Transfer the cells into a 50 milliliter conical tube and spin them down in a centrifuge. Then after careful aspiration of the supinate, resuspend the pellet in 10 milliliters of prewarm N two B 27 by pitting against the side of the tube. To avoid creating bubbles, now count the cells recording the concentration and re suspend them at the appropriate number of cells per unit of volume per well in prewarm N two B 27 according to the table.

Then aspirate the gelatin from the culture vessel and plate the cells without swirling the container. Place the cultures in a humid incubator at 37 degrees Celsius and 5%carbon dioxide. Replacing the medium every one to two days.

With fresh N two B 27, the cells will begin to differentiate and exhibit socks, one expression within four to six days to stain the cells for immunofluorescence imaging. Remove the differentiation medium from the cultures and fix the cells in 500 microliters of 4%Para formaldehyde after 15 minutes, wash and permeate the cells two times with two milliliters of PBS with tween. Then block any non-specific binding with a one hour incubation of the cells.

In one milliliter of PBS tween supplemented with serum on a plate rocker after washing the cells as just demonstrated. At 500 microliters of mouse IgG against beta three tubulin to the cultures for an overnight incubation on the plate rocker at four degrees Celsius. The next day.

After washing the cells, incubate the samples in fluorescently tagged anti-US IgG secondary antibody at room temperature protected from light with rocking after one hour. Rinse the cells again and stain them in 500 microliters of DPI for five minutes. Finally, after washing the cells one last time, store them in PBS tween for up to two weeks at four degrees Celsius.

Until imaged. In this experiment, mouse embryonic stem cells with an endogenous SOX one GFP reporter were used to track neural differentiation. On day six, the cells were fixed and stained for the neuronal marker beta three tubulin.

At the optimal density, the cells differentiated into neural progenitors and neurons, whereas placing it to higher density resulted in a decreased number of SOX one GFP and beta three tubulin positive cells. The volume of the cell culture container affects the optimal plating density as well. For example, cells grown in a 96 well plate require a cell density of about 10 times higher than that.

The cells plated in a six well culture dish to reach the optimal and two confluence cell densities. During differentiation, embryonic stem cells gradually change their morphology. For example, in these images, the cell and colony morphology from days one to six after blazing at the optimal cell density can be observed.

On day four, the cells begin to become neural progenitors as evidence by the appearance of green fluorescent signal from the SOX one GFP reporter. And by day six, most of the cells are SOX one positive. Although the existence of some non neural cells is expected After watching this video, you should have a good understanding of how to initiate the neural differentiation of mouse emry stem cells in a mono layer cell culture.