The overall goal of the following experiment is to identify the crucial temporal regulation required for cardiomyocyte differentiation in mouse embryonic stem cells. This is achieved by first forming embryonic bodies in a 96 well round bottom plate to standardize EB formation as a second step. Protein or chemical modulators are added to EBS over a predetermined time course, which will allow for temporal control of the pathways under examination.
Next, beating EBS are quantified manually to determine the induction efficiency of various signal modulation schemes. Results are obtained that show temporal requirements of essential signaling pathways for cardiomyocyte differentiation based on the percentage of beating EBS formed and confirmation using the hanging drop method. The main advantage of this technique over existing methods like hanging drops, is that it standardizes and simplifies a labor intensive procedure and allows for a straightforward readout for quantification.
This method can help answer key questions in the stem cell biology field, such as identifying essential signaling coagulation that directs differentiation towards the desired cell type To begin this procedure. Gro mouse embryonic stem cells in 10 centimeter cell culture plates with mouse ES cell medium supplemented with leukemia inhibitory factor. When the ES cells are at 50 to 70%confluence, they're ready for use.
Remove the ES media and rinse the cells once with five milliliters of sterile PBS. Add two milliliters of 0.05%tripsin EDTA to each plate, and incubate the plates at 37 degrees Celsius for three to five minutes. Next, quench the trips in EDTA with three milliliters of ES media per plate and transfer the cells to a 50 milliliter centrifuge tube.
Spin at 1000 rotations per minute for three minutes. After centrifugation, remove the supinate and resuspend the cell pellet with the desired amount of EB media. Count the cell number and then dilute the cells to five times 10 to the three cells per milliliter.
In EB media using a multi-channel pipette, add 100 microliters of EB media containing cells into each well of a 96 round bottom well microtiter plate. The number of 96 well plates required will depend on the number of conditions and time points of the experiment. Place the 96 round bottom well microtiter plates into a humidified 37 degrees Celsius 5%carbon dioxide incubator to identify critical time windows of key signaling pathways for cardio genesis.
Modulators of specific signaling pathways are added to the ES cells. In the 96 round bottom well plates add the signaling modulator, which is diluted in media into each well at various time points. Half of the wells in each 96 well plate are used for each starting time point of treatment.
A vehicle control is added to the other 48 wells for each time point At different stopping points, we're gonna wash out the signaling modulators from each well by changing the media and great care must be taken when doing this to prevent disrupting the embryonic bodies At different stopping time points. Wash out the modulators by changing the EB media for the wells. Tilt the 96 well plate by about a 10 degree angle and gently remove the medium from the well with a multichannel pipette.
Then add back EB media without modulator to each well for any treatment longer than 48 hours. Change the EB media supplemented with fresh modulators every two days until the desired stopping time. Points seven days after EBS were induced by transferring ES cells to 96 well plates examine EB contraction under a microscope.
Score the wells with contracting EBS as positive ones to get percentages of contracting EBS for each different treatment time course. The time windows of signaling for cardio genesis identified by the 96 well plate experiments can be verified in EBS made from hanging droplets to make hanging EB droplets. First prepare Petri dishes by adding three to four milliliters of PBS to preventive aberration of the droplets later.
Next, an ES cell suspension prepared at a final density of 2.5 times 10 to the four cells per milliliter is poured into a bacterial dish for easy access. Using a multichannel pipette add 20 microliter drops of ES cells onto the inverted lids of the Petri dishes. Do not allow the individual droplets to touch one another.
Generally, one lid can fit up to 80 drops. Then flip the lid back over the Petri dish containing PBS incubate for 48 hours in a 37 degrees Celsius 5%carbon dioxide incubator to allow EB formation. After 48 hours, wash down the EBS formed from hanging droplets from each lid with three milliliters of EB media.
Pull two lids of EBS into a new Petri dish. Incubate the Petri dish for four days in a 37 degrees Celsius 5%carbon dioxide incubator at day four, transfer EBS in suspension onto 0.2%Gelatin coated six well plates in general. 30 EBS are transferred to each well incubate signaling modulators at the time period identified from the 96 well plate experiments seven days after the hanging droplets were made.
Observe the EBS under the microscope for EB contraction ES cells grown in the round bottom wells of a 96 well plate will form EBS as shown in this representative image of an EB formed at day four. The critical time points for ES cardio genesis are the time windows that contain the highest percentage of contracting EBS treated by signaling modulators when compared to the vehicle control. Shown here is a spontaneously contracting focus of cardiomyocytes formed from dorsal morphine treated ES cells at day 10 of differentiation on a 96 well microtiter plate.
After watching this video, you should have a good understanding of how to efficiently identify critical temporal regulation, windows of key signaling pathways in ES cardio genesis.
