The overall goal of this video is to introduce a technique for differentiating human pluripotent stem cells into cardiomyocytes, and to demonstrate the use of these cells for studying myocardial repair after an ischemia reperfusion injury. This method can help answer key questions about engineering heart tissue with human iPS cell derived from cardiac cells for myocardial repair. The main advantage of this procedure is that it allows scientists to collect purified cardiomyocytes, smooth muscle cells, and endothelial cells, and generate heart tissue with these progenitor cells.
Ling Gao is a post-doc in Doctor Zhang's own lab in UAB and Greg Walcott is a cardiologist in UAB hospital. Trina is a MRI specialist in UAB hospital. To induce pluripotent stem cells into cardiomyocytes, first, coat the wells of a six-well plate with growth-factor reduced gelatinous protein mixture cooled to 4 degrees Celsius.
Then, refrigerate the plate overnight at 4 degrees Celsius. The next day, aspirate the gelatinous protein mixture and seed the plates with 100 million stem cells per well. Never keep the stem cells out of the incubator for more than 15 minutes.
Also, always use fresh media. In this case, use mTeSR1 medium, supplemented with 10 micromolar of ROCK inhibitor. Then, culture the cells with 5%carbon dioxide at 37 degrees Celsius, and refresh the medium daily by gently aspirating away the old medium without touching the cells, and using a transfer pipette to apply fresh medium.
Grow the cells until they are 90%confluent. Three days into the differentiation process, start checking for clusters of contracting cells. As soon as contractions are noted, which can take a week or so, collect the cells.
Count the contracting cells using a hemocytometer, and then culture the cells on 10 centimeter plates at a concentration of one million cells per milliliter. It is also possible to make endothelial cells from stem cells, as well as smooth muscle cells. The processes are different, but are carefully detailed in the text protocol.
First, surgically induce a myocardial infarction in a swine heart. Briefly perform a fourth intercostal thoracotomy to expose the left anterior descending coronary artery. Then, occlude the coronary artery using a ligature for 60 minutes.
Defibrillate the heart as necessary. Suspend five milligrams of microspheres in one milliliter of fibrinogen solution, and load this into a syringe. Third, fill another syringe with one milliliter of thrombin solution.
After an hour has passed, remove the ligature used to create the myocardial infarction. Then, position a plastic ring on the epicardium of the infarcted region, and simultaneously inject the microspheres and fibrinogen and the thrombin solutions into the ring. Allow the mixture of solutions to solidify, which takes about 30 seconds.
This makes the patch. Then, insert the needle of the cell-containing syringe through the patch into the infarcted myocardium and inject the cells. Now, close the muscle layers, subcutaneous tissue, and the chest wall with 30 or 20 Monocryl suture, depending on the animal's size.
One week and four weeks after the procedure, evaluate the animal's cardiac function using a cardiac MRI. As described by the first presented protocol, human pluripotent stem cells were differentiated into cardiomyocytes. To assess their purity, flow cytometry for cardiac troponin T showed it was present in excess of 90%of cells.
Other markers were also observed. Nearly all of the cells expressed slow myocin heavy chain, and nearly all the cells expressed alpha sarcomeric actin. About one quarter of the cells expressed 2v isoform of myosin light chain, a marker for ventricular cardiomyocytes.
In comparing stem cell lines, it was found that the efficiency of the differentiation was substantially higher with cells from the GRiPS line than from the PCBC16iPS line. Using the patch-mediated cell transplantation protocol, a mixture of cells, made up of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells was injected into the injured tissue. Four weeks later, more cells were retained with the patch than without it.
Within one week, treatment using the cell injection with the patch resulted in the most improvement of the infarct size. Also, there was no appreciable difference in injecting with just cardiomyocytes compared to injecting with three cell types. After watching this video, you should have a good understanding on how to differentiate human iPS cells into three different cardiac ligature cells, including cardiomyocytes, smooth muscle cells, and endothelial cells.
You should also have a good understanding of how to generate human heart tissue with the progenitor cells for myocardial repair. Since the development, this technique has advanced investigations on myocardial repair and stem cell tissue engineering. It should be noted that when attempting this procedure, it is very important to use high quantity of iPS cells and their cardiac derivatives.
