The overall goal of this procedure is to generate high quality cultures of cardiomyocytes from pluripotent stem cells and to appropriately characterize the end product using flow cytometry. To accomplish this high quality monolayer cultures of undifferentiated pluripotent stem cells are generated. Cardio myogenesis is then induced by modulating the wind pathway using small molecules.
Next, the cardiomyocytes are fixed, permeated and stained using conditions that are specifically optimized for each antibody. Finally, the cells are analyzed by flow cytometry. Ultimately, results can be obtained that show cultures that are highly positive for cardiac markers, TNNI three and IRX four and demonstrate ventricular like action potentials.
The main advantage of this technique over existing methods like those in employee and braid bodies, are that it is relatively simple to implement and is cost-effective. Moreover, the technique generates cultures of cells that are highly positive per cardiac troponin I.Without the inner underlying variation and efficiency observed by other protocols, visual demonstration of the method is critical as the pre differentiation cell quality will significantly affect differentiation efficiency. Also improper fixation and permeable prior to antibody staining will lead to suboptimal flow cytometry results demonstrating the procedure will be shabo bachar, a graduate student from my laboratory.
The most challenging aspect of the following procedure is maintaining high quality undifferentiated pluripotent stem cells prior to differentiation. The use of cells with suboptimal growth characteristics will significantly affect the efficiency of differentiation. Begin this procedure with well-established HPCs grown in six well HESC qualified matrix coated dishes.
Check the cells under the microscope to ensure that they exhibit a homogenous morphology. If the cells exhibit neural epithelial or fibroblast like morphology as shown here, they should not be used. Ensure that the cells proliferate robustly when seated at 0.75 million per well passage them approximately every three days at 75%Confluence passage the cells with dissociation to the single cell level at least five times prior to the start of differentiation To passage.
The cells first aspirate the E eight growth medium and wash the cells twice with four milliliters of PBS prewarm to room temperature. Next, add one milliliter of room temperature cell detachment solution to each well and leave the well undisturbed until the cell boundaries begin to round up. This typically takes three to seven minutes and can be observed macroscopically and confirmed using a microscope.
Next, using a cotton plugged glass pipette with a bulb dislodged the cells and transfer them into a 15 milliliter conical tube containing one milliliter of DM EMF 12 medium. This will inactivate the cell detachment solution. Remove a 10 microliter aliquot of the cell solution and mix it with 10 microliters of trian blue in a micro centrifuge tube.
Count the cells and then to collect them centrifuge, the 15 milliliter conical tube at 130 GS for five minutes. At room temperature during the centrifugation count the cells using an automated hemo cytometer. Expect 70 to 80%viability going forward.
Base all cell numbers on total cell counts as opposed to viable cell counts. After the spin aspirate the medium and resuspend the cell pellet in D-M-E-M-F 12 to a final concentration of 0.75 times 10 to the six cells per 500 microliters, add 0.5 milliliters of the cell suspension to each well of a six well plate in which each well contains two milliliters of E eight medium. With rock inhibitor, gently rock the plate front to back and side to side to uniformly disperse the cells across the well.
Return the cells to the incubator at 5%carbon dioxide and 37 degrees Celsius. 24 hours after packaging. Begin replacing the medium daily using two milliliters per well of E eight with without rock inhibitor.
Optimize the seeding density to achieve 100%confluence prior to the start of differentiation. On day zero, cells are 100%confluent with compact morphology and minimal cell debris. Begin the differentiation process by replacing the E eight medium with two milliliters of differentiation.Medium.
Number one, add 1.2 microliters of CHIR to each. Well on days one and two, it is common to observe significant 40 to 50%cell death. However, the attached cells will retain compact morphology.
During this time, the medium becomes orange and turbid. If pink medium, which indicates excessive cell death is observed, confirm cell viability with triam blue and discontinue if cell death exceeds 70%Otherwise, on day one, repeat the process of replacing the media with two milliliters of fresh differentiation.Medium. Number one, add 1.2 microliters of CHIR to each well and on days two to three, replace with fresh differentiation medium.
Number one, during days three through four, the cells will recover and the density will increase on day four. Replace the medium in each well with fresh differentiation. Medium number one and one microliter of IWR one.
During days five through six, minimal cell death occurs and dense patches may begin to appear on day five, replace the medium with fresh differentiation. Medium number one, and add one microliter of IWR one to each well on day six through seven, replace the medium with fresh differentiation. Medium number one.
By day seven through eight, a confluent monolayer is achieved with compact morphology interspersed with dense patches. By day eight, the cells will begin to spontaneously contract and the first contractions will often be visible in the dense patches. On day eight, replace the medium with fresh differentiation.Medium.
Number two, continue to replace with differentiation medium number two, every other day for the duration of the culture. By day 10 more robust contraction is observed and will continue to spread throughout the culture in subsequent days. This video shows the cells at day 30 and here the cells are shown at day 55.
To collect cells for flow cytometry, perform the cell dissociation collection and counting as performed earlier. Note that the cells do not need to be kept sterile at this point. While gently vortexing the cell pellet, add 100 microliters of fixation solution to the cell pellet dropwise.
Then incubate the tube on ice for 15 minutes. Add three milliliters of cell wash solution, collect cells by centrifugation after the spin, aspirate the solution while vortexing. Add 100 microliters of permeable solution to the cell pellet dropwise as before.
Then incubate the tube on ice for 30 minutes. Add three milliliters of cell wash solution. Collect the cells by centrifugation and aspirate the solution.
Repeat for a total of two washes after permeation. Perform antibody staining as described in the accompanying document using a P 1000 pipette at 400 microliters of cell maintenance solution to the cells and disaggregate them by pipetting up and down. Next, pre-wet a cell strainer cap on a round bottom tube with 50 microliters of cell maintenance solution.
Set the tube on ice. The cell strainer cap prevents cell aggregates from clogging the flow cytometer. Next, transfer the cell solution to the cell strainer cap and allow the cell suspension to drain by gravity.
Tap the bottom of the tube gently on the bench top so that cells are collected into a tube. Place the tube back on ice as quickly as possible. Rinse the strainer with 250 microliters of cell maintenance solution to ensure maximal recovery of cells.
Maintain the cells on ice and protect them from light until they are analyzed by flow cytometry to determine the quality of the cardiomyocytes generated. Using this protocol, cells were differentiated for 10 days and stained with antibodies against TNNI three a, pan cardiomyocyte marker, TNNT two a striated muscle marker, and IRX four A ventricular cardiomyocyte marker. Note that fixation and permeable conditions were optimized for each antibody to achieve the maximum percentage of positive cells.
Histograms show that 99%of gated cells were positive for TNNI three and 96%were positive for IRX four. Additionally, the cells were highly positive for other markers of cardiac differentiation, including TNNT two, MLC two V and MLC two. A note that the assertion regarding cardiomyocyte purity and cell identity is based on TNNI three and IRX four.
Staining MC two V and MC two A are not chamber restricted during development and TNNT two is also found in skeletal muscle. The action potential shown here was recorded from a single spontaneously contracting cardiomyocyte on day 46 of differentiation using whole cell patch clamping. The action potential profile indicates that ventricular like cardiomyocytes were obtained.
A small percentage of nodal like profiles was also observed. After watching this video, you should have a good understanding of how to generate high quality cultures of cardiomyocytes from pluripotent stem cells and how to characterize them by flow cytometry. Generally, individuals new to this method will struggle because of the common challenges encountered in maintaining the stem cells as high quality monolayers prior to the start of differentiation.
Do not forget that working with a fixative can be hazardous and personal. Protective equipment such as a lab coat or gloves should always be worn while performing this procedure.
