The overall goal of this procedure is to differentiate human cardiomyocytes from human IPS cells cultured in feeder free conditions. This is accomplished by first plating IPS cells into ultra low attachment plates to form embryo bodies. The second step of the procedure is to transfer the formed embryo bodies into gelatin coated dishes.
When contracting areas appear, they are manually dissected under the stereo microscope and plated onto fibronectin coated dishes. For further differentiation, the final step is to obtain single cardiomyocytes by enzymatic digestion of contracting clusters. Ultimately, results can show differentiation of cardiomyocyte like cells that express typical structural markers, IE cardiac troponin through immunofluorescence microscopy.
This method can help answer a key question in the cardiovascular biology field, such as, which are the mechanism driving cardio differentiation and dysfunction during cardio development and diseases at the level of the human cardiomyocyte. This is a very interesting technique because it allows the generation of cardiomyocytes in vitro from human patients and human individuals. And in the second instance, it allows the recapitulation of the disease in the test tube so that new drugs can be experimented in vitro before going in vivo.
Experimentation Begin with confluent IPS cell cultures grown on 35 millimeter matrigel coated plates. Replace the growth medium with one milliliter of PBS containing a milligram of diss. Examine the cells using polled glass pipettes.
Remove regions with differentiated cells such as crater like or cystic structures in the center of colonies. Also remove any areas where the cells have separated, flattened, and seem to be migrating outwards. Now incubate the remaining cells at 37 degrees Celsius under a culture hood until the colony borders become brighter and begin to detach.
This usually takes between five and seven minutes. At this point, remove the dispa solution and wash the cells with one milliliter of HESM. Then discard the wash solution.
In a fresh milliliter of HESM, use a cell lifter to harvest the colonies. Collect the colonies into a 15 milliliter tube. Next, rinse the cells with another milliliter of HESM and spin them down at 1000 times G for four minutes.
Then proceed with plating the cells on matrigel and change the media daily during culturing. Begin by harvesting undifferentiated IPS colonies as described in the previous section. Once the undifferentiated populations have been isolated, gently resus suspend them in one milliliter of EBM 20.
Using a two milliliter pipette, don't break up the cell clumps too much. Limit the pipetting to two or three strokes. Check the size of the colonies under the stereoscope between strokes.
When the colonies are all about the same size, they need no further mixing. Now plate the cells on ultra low attachment plates filling one six well plate per plate of cells harvested. Don't forget to rinse the tube with one milliliter of EBM 20 and distribute the rinse to the plate.
After the third day of incubation, change the media using a microscope to avoid removal of embryo bodies. Keep the pipette close to the edge of the plate. On the seventh day, move the embryo bodies to plates coated with 0.1%Gelatin warmed at 37 degrees Celsius for 30 minutes.
Add 35 to 40 embryo bodies to each 35 millimeter dish in two milliliters of medium. During the culturing. Change the medium twice a week regularly.
Check the embryo bodies for beating areas and mark out where beading areas are located. On the top of the dish cover beading contracting areas should appear after approximately 10 to 20 days. When areas with spontaneous contraction appear, switch their medium to EBM two After the contracting areas appear, keep culturing and monitoring other areas on the plate for up to a month.
In total. In EBM two Deficiency of the differentiation process is highly dependent on several factors, the most critical of which are the specific cell lines and the batch of serum used to induce cardio differentiation. To obtain highest efficiency test several lines for the cardiogenic potential and various batches of serum Begin by coating 12.
Well plates with five micrograms per square centimeter of fibronectin in PBS 300 microliters per well should completely cover each well's surface. Allow the plates to dry uncovered in a cell culture hood. To remove the beading area of the cell culture, use a pulled glass pipette and scalpel.
Then using a one milliliter pipette transfer four to five clamps to each well of a fibronectin coated plate. Once all the collections have been made, add one milliliter of EBM two to the loaded wells on the collection plate mark areas where cells were removed. Then change the medium and return the plate to the culture hood.
If smaller beating clumps are required under a stereo microscope, pass the explanted areas through a pipette. This produces small clusters containing a few beating cells. In culture.
Change the medium twice weekly until thes are ready for analysis. First, prepare four centimeters square two chamber slides by coating them with 20 micrograms of fibronectin diluted in enough PBS to cover the surface. If a glass support is used, add an additional 20 micrograms of laminin.
Isolate the bead culture area as previously described. Using a one milliliter pipette, transfer the cells to a 15 milliliter tube containing 500 microliters of collagenase tube. Incubate the cells for 15 minutes and shake the tube once during the incubation.
After 15 minutes, move the cells through a 200 microliter pipette to remove clumps if any clumps remain. Transfer the cells to fresh collagenase two and repeat the incubation when no large clumps are left. End the digestion by adding three milliliters of EBM two without as soic acid as they are each prepared.
Store the tubes in a heated rack under the hood, then centrifuge them at 1000 times G for four minutes at room temperature. Once centrifuged, make a suspension of the cell fractions from the same initial sample in a milliliter of 0.25%trypsin EDTA in PBS. Then incubate the suspension for five minutes in the heated block, inactivate the trypsin with four milliliters of E BM two without as soic acid.
Next, pass the cells through an 18 gauge needle on a 2.5 milliliter syringe up to seven times until no big clumps are visible under the microscope. Then centrifuge the cells at 1000 times G for four minutes. Now resuspend the cell pellet in E BM two and plate them on two well chamber slides.
The cells will be ready for analyses after two to three days of incubation. CMS were generated from several IPS cell lines. These lines were initially generated on a meth feeder layer and immediately placed on matrigel using a defined medium.
The cells showed a human ESC like morphology with phase bright edges and prominent nuclei. Immunofluorescence analysis showed the IPS cells correctly expressed the transcription factor T four and expressed the TRA one 60 surface antigen. Cyto fluoro metric analysis confirmed the expression of typical surface pluripotency antigens, SS EEA four and TRA one 60.
The gating strategy is shown in the left scatter. A karyotype analysis of the IP FS cells grown on matrix gel was also performed. Induction into the cardiac lineage was triggered by the aggregation of the IPS cells into ebs, along with culturing in the presence of a specific type of serum.
And as sorbic acid contracting areas represented 20 to 35%of the total culture depending on the cell line used. Contracting CMS were isolated from these regions by enzymatic digestion and analyzed for protein expression. R-T-P-C-R found expression of cardiac specific ion channels expressed in the IPS derived beating cells, but not in non beating cells.
These channels included an alpha one, a subunit of a voltage dependent calcium channel, an alpha subunit of a type five voltage gated sodium channel, a potassium voltage gated channel of the KQT, like subfamily, and both forms of myosin. Heavy chain immunofluorescence analysis revealed that single IPS derived cardiomyocytes expressed the structural proteins, alpha sarcomeric, actin, cardiac troponin I, and the cardiac specific junction, conexion 43. After all, this technique really paved the way for the modeling of myocardial diseases in vitro and allows the experimentation of drugs in the human setting without injecting the drug in the human bodies.
After watching this video, you should have a good understanding of how to maintain IPS cells in free free conditions and to differentiate them into cardiomyocyte through a method based on embryo bodies aggregation.
