Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

The overall goal of this protocol is to produce uniform aggregates of mouse embryonic stem cells that undergo self-organization and axial elongation and suspension culture. This method can help answer key questions in the field of developmental biology, such as the role of tissue interactions in access specification and self-organization in processes like gas relation. The main advantage of this technique is that it allows developmental events to be studied from a novel deconstructive perspective outside of the embryo Prior to aggregation.

Maintain mouse embryonic cells or M MECs in ES.Lift medium on gelatin coated 25 square centimeter tissue culture treated flasks for aggregate generation. Start with the tissue culture flask containing cells at 40 to 60%Confluence aspirate the medium and rinse the flask twice. With PBS, add one milliliter of point 25%trypsin EDTA and incubate at 37 degrees Celsius for approximately five minutes to detach the cells from the flask.

Next, add five milliliters of Es.Lift medium to the detached cell suspension and mix thoroughly. Transfer the cells to a 50 milliliter centrifuge tube and take out a one milliliter aliquot to count the cells. Add five times 10 to the fourth cells to five milliliters of warm PBS and centrifuge the cells at 170 times G for five minutes.

Aspirate the solution without disturbing the pellet. Then add five milliliters of PBS gently and repeat the centrifugation. Aspirate out the maximum volume without disturbing the cell pellet.

To prevent carryover of PBS. Re suspend the cell pellet in one milliliter of warm N two B 27 medium. Mixing well with a P 1000 pipette to generate a homogenous cell suspension.

Then dilute it with an additional four milliliters of N two B 27 medium. Transfer the cell suspension to a sterile reservoir using a multi-channel pipette. Mix the cells thoroughly and transfer 40 microliters of the cell suspension from the reservoir to each well in a ubo non tissue culture treated.

96 well plate. A non tissue culture plate is specifically used to minimize adherence of the cells to the plate. Incubate the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 48 hours to allow cell aggregation after 48 hours.

Remove the plate from the incubator and observe the cells under an inverted microscope and confirm the presence of aggregates. Add a volume of 10 millimolar CHI 9 9 0 2 1 stock solution to warm N two B 27 to make a final concentration of three micromolar. This is a secondary medium for cell stimulation.

Using a multi-channel pipette dispense 150 microliters of the secondary medium to each well. With enough force to dislodge any adherent aggregates. Return the plate to the cell culture incubator for 24 hours after the incubation.

Carefully remove the medium without aspirating the cell aggregate. Add 150 microliters afresh N two B 27 to each well with enough force to dislodge any adherent aggregates. Repeat this every 24 hours for a total incubation of approximately five days.

To prepare aggregates for fixation, first gently aspirate the incubating solution from each well holding the pipette at a 30 degree angle. To prevent aspirating the cell aggregate, add 150 microliters of PBS to each. Well wait for a few minutes for the aggregate to settle at the bottom and then aspirate the PBS.

Repeat this washing step twice more using sterile scissors. Cut a P 1000 pipette tip at approximately three millimeters above its dispensing. End with this tip, pipette up and release a small volume of liquid into a well.

To agitate the aggregate and loosen it from the bottom of the well. Then draw up the aggregate into the tip and transfer the aggregate into a glass drosophila dissection. Well using the same procedure, collect all aggregates that will undergo identical immunostaining procedures into a single dissection.

Well place the dissection well under the microscope and swirl it to move the aggregates towards the center. Then carefully aspirate the PPS from the edge of the well. Taking care not to remove any aggregates.

To fix the aggregates add one milliliter of freshly prepared 4%formaldehyde solution and incubate the dishes in an orbital shaker for two hours at four degrees Celsius. Set at a low speed. Aspirate the formaldehyde solution with care to prevent suction of any aggregates, and then add one milliliter of PBS to the, well.

Return the plate to the orbital shaker for 10 minutes. Remove the PBS from the well and repeat this washing step two more times. Next, add one milliliter of P-B-S-F-T solution and place the plate on the orbital shaker for 10 minutes.

Aspirate the solution and repeat the wash two more times. Add one milliliter of P-B-S-F-T per well to block the aggregates from nonspecific immunoreactivity. Place the plate on the orbital shaker at low speed for one hour at four degrees Celsius.

Following incubation, aspirate the blocking solution carefully from the wells and add 500 microliters of the primary antibody diluted in P-B-S-F-T. Seal the plate with paraform to prevent evaporation and incubate overnight at four degrees Celsius on an orbital shaker at low speed. Next, replace the primary antibody solution with pre chilled P-B-S-F-T.

Wash the aggregates by placing the plate on the orbital shaker. Set at low speed at four degrees Celsius for five minutes. Repeat the wash one more time.

Perform two additional wash cycles with P-B-S-F-T at four degrees Celsius at low speed on the orbital shaker. As indicated in the text protocol, remove the wash solution and incubate with 500 microliters of secondary antibody diluted in P-B-S-F-T. Add one microgram per milliliter of herst in order to visualize the nuclei incubate overnight at four degrees Celsius on a gyratory rotor protected from light.

Perform another wash cycle with PBS FT at four degrees Celsius, protected from light under a foil wrapped box. Perform additional washes with PBS containing 0.2%FBS and 0.2%Triton X 100 or PBT buffer at room temperature. After carefully aspirating the solution, incubate the aggregates with one milliliter of one-to-one glycerol to PBT for 30 minutes in the dark at room temperature.

Then incubate them with one milliliter of seven to three glycerol to PBT for 30 minutes. To prepare the samples for confocal imaging at aspirate the glycerol to PBT solution and replace it with one milliliter of mounting medium. Then mount the aggregates in 17 microliter droplets on a glass slide.

Using a pipette tip, prepare a spacer by folding a piece of double-sided tape four times upon itself. Then place one spacer on either side of the aggregates on the slide, invert the cover, slip on the spacers, and then image the aggregates using confocal microscopy. 48 hours after plating aggregates of around 150 micrometer diameter are formed displaying smooth boundaries.

On the other hand, poor medium quality, non-optimal cell number plating or cell adherence could result in failure to aggregate properly. 120 hours of continuous treatment with CHI 9 9 0 2 1 a glycogen synthase kinase three inhibitor causes the spherical aggregate to become elongated. Also, there is distinct localization of SOX GFP positive cells at one end of the aggregate after treatment, different treatments yield different morphologies changes in the morphology of a single aggregate and in fluorescent marker.

Localization within the aggregate can be tracked over time. Once mastered, the researcher will be able to generate aggregates within about 45 minutes if it is performed properly. Once the aggregates have been formed, other methods like Q-R-T-P-C-R can be performed instead of the immuno staining to investigate changes in gene expression within the aggregates.

After watching this video, you should have a good understanding of how to form aggregates from MECs, apply specific stimuli and prepare aggregates to immuno staining and confocal microscopy analysis.