The overall goal of this experimental procedure is to induce and observe the cardiomyogenic induction of mesenchymal stem cells. This method can help answer key questions in the mesenchymal stem cell field such as, what remedies can be induce by environmental signals to generate functional cardiomyocytes. The main advantage of this technique, is that it applies aggregate formation and cardiac fetal layers, instead of pharmacological agents or genetic material.
The implications of this technique extend towards therapy of cardiovascular disease because it elaborates on the cardiomyogenic potential of mesenchymal stem cells. Demonstrating this procedure with me will be, Miss Farwah Iqbal, a PhD student of our laboratory, Max Librach and Nadav Gasner, both trainees of our laboratory. After isolating rat pup parts according to the text protocol, cut the ventriculi in half and transfer them to a 10 centimeter dish with 10 milliliters of PBS and pinstripe on ice, to let the blood wash out.
Then using curved scissors, cut the ventricular walls into small pieces with a diameter of two to three millimeters. Using a serological pipette, transfer the heart pieces from 10 to 12 animals to a 50 milliliter tube and let them settle. Remove as much PBS P/S as possible, without removing any heart pieces.
Then add 10 millimeters of fresh PBS P/S. To isolate the cardiomyocytes, after the heart pieces settle, use 0.15%trypsin in PBS, to replace the PBS P/S and incubate the tissue at 37 degree celsius with shaking, for 10 minutes. Discard the supernatant.
Then, repeat the trypsin digestion three more times, except, decanter supernatants into 50 milliliter collection tubes containing 10 milliliters of 100%FBS. Next, centrifuge the tubes of cells and aspirate the supernatant. Then use DMEM-F12 with 10%FBS and 1%P/S to resuspend the cells and seed them onto a six well plate.
Once the cells have attached, replace the medium with two milliliters of fresh medium. To prepare pre-stained MSCs, remove the medium from MSC cultures that have reached 70%to 80%confluency in 10 centimeter dishes and add three milliliters of cell dissociation solution. Incubate the dishes at 37 degree celsius and 5%carbon dioxide for five minutes.
Transfer the dissociated cells to 15 milliliter tube and centrifuge at 400 x G for five minutes. Aspirate the supernatant without disrupting the cell pellet and re-suspend the cells. Then use an automated cell counter to count the cells.
Dilute the cells to a concentration of 1 x 10 to the sixth MSCs per milliliter, in DMEM-F12 containing 10%FBS and 1%P/S. Then, to the cells in 1.5 milliliter centrifuge tubes, add viable, non-transferrable fluorescent dye and incubate the MSCs for half an hour. After centrifuging the tubes at 400 x G for five minutes, aspirate the supernatant and re-suspend the pellet at a concentration of 1 x 10 to the sixth MSCs per milliliter.
Then transfer the MSCs onto cardiomyocytes at a concentration of 10 x 10 to the fourth cells per well of the six well plate. To make aggregate co-cultures, prepare a single cell suspension of MSCs in alpha MEM, supplemented with a 10%FBS and 1%P/S. Initiate aggregate formation, by placing 25 microliter drops of cell suspension on the inner surface of the lids of 10 centimeter tissue culture dishes.
Place the lids on their bottom counterparts containing PBS P/S. Incubate the dishes at 37 degree celsius and 5%carbon dioxide. Under a stereo microscope, observe aggregate formation in the drops after three days.
If over 80%of the drops contained formed aggregates, use a one milliliter micropipette to collect the drops from the lids and transfer the aggregates directly onto primary rat cardiomyocyte monolayers. Incubate the aggregate co-cultures for up to two weeks. Changing the full volume of medium every 72 hours.
On a daily basis, use bright field microscopy to observe aggregates attaching to fetal cell layers. Record contracting aggregates when observed. The active aggregates contract in a synchronized manner, we suggest a conductance between them throughout the fetal layer, which has no physical activity.
Aspirate the medium and add two milliliters of PBS per well of the six well dish. Then remove the PBS and add two milliliters of dissociation solution per well. Incubate the dishes at 37 degree celsius and 5%carbon dioxide for three minutes.
As shown here, HMSCs of three different types displayed similar abundance and appearance in direct co-cultures. Two to three days after transferring hanging drops onto cardiac cells in aggregate co-cultures, FTMs and term huck PBCs appeared in larger aggregates compared to those seen from BMSCs. In this experiment, flow cytometric analysis of CD49F expression in undifferentiated MSCs showed that 96%of FTM huck PBCs, termed 89%of huck PBCs and 53%of BMSCs, were positive for cell surface CD49F.
Flow cytometry on TRA won 85 cells, showed the upregulation of the cardiomyocyte associated marker, SIRPA in FTM huck PBCs and term huck PBCs, but not in BMSCs from direct cardiomyocyte co-cultures. As seen here, cx43 upregulation was significantly higher in FTM huck PBCs compared to term huck PBCs or BMSCs in direct co-cultures. Fluorescent microscopy analysis revealed that differentiating FTM and term huck PBCs, both upregulated cx43.
With term huck PBCs at a higher portion than FTMs. However, fluorescence microscopy revealed that cx43 positive puncta were observed in the cell membrane of FTM huck PBCs and they were predominantly cytoplasmic in term cells. Finally, as seen here, high magnification fluorescence microscopy was performed on sorted human cells, after co-culturing and the high abundance of cx43 in the plasma membrane was confirmed.
Once mastered, the key steps of this procedure can be done in one hour each, if they are performed properly. When attempting this procedure, it is important to maintain aseptic precautions. Primary animal tissues involved which may introduce contamination, if not handled properly.
Following this procedure, other methods like further culturing of contracting aggregates can be performed to answer additional questions such as, longevity of these structures. After its development, this technique enables researchers in the field of MSCs to explore the differentiation potential of younger sources of mesenchymal stem cells such as, extra embryonic tissues. After watching this video, you should have a good understanding on how to prepare MSC aggregates and combine them with direct primary tissue fetal layers, in a manner that provides a proper induction for cardiomyogenic differentiation.
Don't forget, that working with BRDU can be extremely hazardous and precautions such as, proper handling of cytotoxic waste, should always be taken while performing this procedure.
