Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

This is the first report demonstrating that echocardiography-guided, percutaneous intramyocardial injection of cardiomyocytes significantly enhance the reparative property of human iPS-derived cardiomyocytes. This method is feasible, satisfactory, repeatable less invasive and effective procedure for delivering human iPS-derived cardiomyocytes therapy. We believe that percutaneous intramyocardial delivery is a safe and efficient method for local drug delivery for diseases including ischemic heart disease, myocardial infarction, and heart failure.

This project is supervised by Professor Jinfu Yang and Professor Jun Peng. demonstrating the procedure will be Xun Wu, Kele Qin, and Kun Xiang, MD PhDs from my laboratory and Di Wang, a PhD from Hunan Provincial Key Laboratory of Cardiovascular Research. To begin, vacuum aspirate the culture supernatant from a 60-day culture of hiPSC-CMs.

Wash the cells with PBS thrice. Then dissociate the hiPSC-CMs into individual cells using the cell detachment enzyme mixture at 37 degrees Celsius for three to five minutes. Collect the cells in a 15 milliliter tube containing five milliliters of RPMI plus B27 medium and mix.

Count the cells and calculate the total number of cells. Centrifuge the cell suspension at 300G for two minutes at room temperature. Discard the supernatant and resuspend the pellet in cardiomyocyte support medium to a concentration of 6 x 10 of the 4th cells per microliter.

Place the tube at 37 degrees Celsius til the injection step. After the mouse is sufficiently anesthetized, fix the limbs and tail with tape. Then, fix the mouse incisors with a 2/0 silk thread and tape.

Sterilize the median neck and left chest with three alternating rounds of betaine followed by alcohol. Make a median neck incision using fine anatomical scissors and fine dissecting forceps. Cut off a few anterior cervical muscles to stabilize and fully expose the trachea.

Insert a 20-gauge catheter puncture needle as a tracheal tube through the mouth. Connect the tracheal tube to the ventilator and check for thoracic movement to ensure both lungs are well-ventilated. Adjust the breathing parameters.

Next, refix the left hind limb to the lower-right side and loosen the tape around the left upper limb. With fine dissecting scissors and forceps, perform a thoracotomy at the three to four intercostal space of the left thorax. Using forceps, peel off the pericardium and locate the left anterior descending coronary artery.

Use a 6/0 silk thread to ligate the proximal end of the artery. To ensure that the acute myocardial infarction model is established, wait till the distal side of the artery at the ligation site changes from red to white. Close the chest layer by layer and suture the skin with 4/0 thread.

For the sham group of animals, perform all the surgical procedures except for ligation. In the first two weeks following myocardial infarction, place the mice in an anesthesia box connected with 5%isofluorane and perform tracheal intubation. Three days before administering hiPSC-CMs, inject cyclosporine A into the intraperitoneal cavity at a dose of 10 milligrams per kilogram per day.

Remove the hair from the chest and upper abdomen of the mouse with depilatory cream. Supply inhalation isofluorane til the heart rate is maintained at 400 to 500 beats per minute. Fix the mouse limbs and tail with tape on the detection console and set the platform temperature at 37 degrees Celsius.

Apply the ultrasound jelly on the upper abdomen. Using a high resolution veterinary ultrasound probe, obtain mouse liver images. Using a five microliter micro syringe, draw approximately 3 x 10 to the 5th hiPSC-CMs.

Hold the syringe and insert a three millimeter needle at the left paraxiphoid process. Under ultrasound guidance, follow the upper edge of the diaphragm. Observe the respiratory rhythm and range.

Enter the pericardium at the end of the expiratory circle, avoiding damage to the liver and lung. Then acquire the parasternal long axis image of the mouse heart by B-mode echocardiography. Under ultrasonic guidance, inject 1 x 10 to the 5th hiPSC-CMs cells into each of the three marginal areas of the infarct site.

Remove the ultrasound probe and rinse off the jelly. Wean the mouse off the anesthesia and return it to its cage. Inject cyclosporine A continuously for one month after transplantation.

Echocardiogram of the left ventricle showed that the injuries due to myocardial infarction were reversed in the multiple dose group. Transplantation of hiPSC-CMs also resulted in increased ejection fraction and fraction shortening. Measurement of cardiac parameters using Sirius Red and Fast Green staining showed that the infarct size and collagen volume fraction decreased, whereas the left ventricular anterior wall thickness increased after transplantation.

Labeling for non-specific alpha actin in green, DAPI in blue, and human-specific troponin T in red showed the presence of transplanted cells in infarct and marginal infarct area. The number and percentage of transplanted cells were higher in the multiple dose group compared to single dose. 28 days after myocardial infarction, the multiple dose group showed an increase in the number of cells expressing the endothelial cell marker, I-B4.

Wheat germ agglutinin and sarcomeric alpha actinin staining on cardiomyocytes showed a reduction in the minimum fiber diameter. Lower number of tunnel positive cardiomyocyte nuclei in the multiple dose group indicated reduced apoptosis. PCR analysis detected the human mitochondrial DNA only in the heart, showing specific transplantation.

When inserting the needle, make sure it enters the pericardial cavity along with the upper margin of diaphragm and at the end of the expiratory circle to avoid the injury to the liver or the lung.