Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

The overall goal of this procedure is to generate highly pure sinus nodal cells. This is accomplished by first inducing cell differentiation by modifying the medium and making hanging drops that result in the formation of embryonic bodies of a defined size. Next, the embryonic bodies are seated on a gelatin coated dish so that they form a cell layer.

Then the antibiotic selection is initiated and only cardiomyocytes survive. Finally, the cell layer is dissociated to obtain fast beating single sinus nodal cells, or small sinus nodal bodies. Ultimately, live cell imaging is used to show high beating frequencies of sinus nodal cells.

The main advantage of this method is that you can produce highly pure sinus nodal cells, for example, for drug testing. The method can help us answer key questions in sinus node disease because the cells will give us insights into the underlying mechanisms. The technology will be presented in the lab by Julia Yung, our PhD student who develop the method, Beginning with an ES cell line and pluripotent stem cells co cultivated with irradiated feeder cells as outlined in the text protocol two days before the differentiation procedure removed the PSCs from the feeder cells by first aspirating the medium.

Use 10 milliliters of PBS to wash the cells. Then add seven milliliters of collagenase four solution and incubate the cells for 10 minutes at 37 degrees Celsius. Next place a sterile 40 micrometer filter in a 50 milliliter tube.

Then carefully rinse the PSC colonies by pipetting the collagenase solution up and down five times. Transfer the cell suspension to the filter and use eight milliliters of PBS to rinse the filter three times. Then turn the filter over and place it upside down in a Petri dish and pipette 10 milliliters of PBS on the bottom of the filter.

To remove the PSC colonies, now transfer the cell suspension to a 15 milliliter tube and centrifuge for five minutes at 300 times. G.Remove the PBS, suspend the cells in one milliliter of Accutane and incubate at 37 degrees Celsius for five minutes. After the incubation, add 10 milliliters of PBS.

Mix the cell solution by pipetting up and down five times and centrifuge for five minutes at 300 times G.Once the PBS is removed, use 10 milliliters of cell cultivation medium to resuspend the cells and determine the cell number seed. 15, 000 cells per square centimeter in a 75 square centimeter filter flask and cultivate them for two days at 37 degrees Celsius and 5%carbon dioxide when the flask should be 50 to 70%confluent. After harvesting the cells and Resus suspending in differentiation medium according to the text protocol, pipette 20 milliliters of water and five milliliters of hanging drop or HD solution into a quadratic petri dish to avoid drying out the hds.

Next, pipette up to 50 milliliters of cell suspension in a tray. Then turn over the lid of the Petri dish and using a 12 channel pipette place 180 HDS each containing 20 microliters of cell suspension onto the lid. Carefully turn over the lid and place it on the Petri dish.

After incubating for two days to let the cells form ebs carefully turn over the lid of the quadratic Petri dish and transfer the EBS derived from two Petri dishes into a 50 milliliter tube. Wait 10 minutes to let the EBS settle to the bottom of the tube, then aspirate as much medium as possible. Use 10 milliliters of differentiation medium to suspend the EBS and transfer the suspension to a 10 centimeter Petri dish.

Once the cells have been cultured in suspension for four days, according to the text protocol, transfer the EBS to one gelatin coated 10 square centimeter cell culture dish and incubate over the next two to six days under a microscope, check the cells for beating foci and the formation of a cell layer three days after the cells have started beating, aspirate the medium and add 10 milliliters of fresh differentiation. Medium containing 400 micrograms per milliliter of G four 18 to the cells four days later. Carefully remove the medium without aspirating the cell layer.

Then add 10 milliliters of PBS to wash the cells. After carefully removing the PBS, add 10 milliliters of collagenase four solution and transfer the cells to a 50 milliliter tube. Next, after incubating at 37 degrees Celsius for five minutes vigorously mix the suspension by pipetting up and down 10 times and incubate the cells at 37 degrees Celsius for an additional five minutes.

A suspension of small clusters should be present. Then add 20 milliliters of PBS and centrifuge for 10 minutes at 300 times. G, carefully aspirate the PBS and if generating induced sino atrial bodies or sabs, use 12 milliliters of differentiation medium containing 400 micrograms per milliliter of G four 18 to Resus, suspend the cells and see two milliliters each onto six wells of a 24 well gelatin coated plate to generate single nodal cells rather than adding differentiation medium.

After removing the PBS, use five milliliters of Accutane to suspend the cells and incubate at 37 degrees Celsius for five minutes. Pipette up and down to vigorously. Mix the suspension after incubating at 37 degrees Celsius for five minutes.

Add 20 milliliters of PBS and centrifuge for five minutes at 300 times G with 12 milliliters of differentiation. Medium containing G four 18. Suspend the cells and see two milliliters each onto six wells of a 24 well gelatin coated plate culture and perform analysis of cells according to the text protocol.

Using the protocol demonstrated in this video, isab S can be generated from pluripotent stem cells with a beating frequency of about 450 beats per minute as seen here after dissociation of isab S.The observed single cells show the typical shapes of cells of the sinus node that includes spindle and spider cells. These cells show high expression levels of proteins essential for the function of the sinus node like hyperpolarization activated cyclic nucleotide gated cation. Channel four HCN four, conexion four five conexion 30.2 and MYH six.

When treated with the channel blocker ZD 7 2 88, or the muscarinic receptor agonist caracol, the isab derived cells exhibit a significantly reduced beating frequency. The beta adrenal receptor agonist isoprene, on the other hand, leads to an elevated beating frequency. Overall, our technique will pave the way to better understand cardiac pacemaker functionality and diseases.

After watching This video, you should have a good understanding how to generate fully functional sinus nodal cells from PLU potent stem cells.