The overall goal of this procedure is to generate and quantify a diverse set of induced cardiomyocyte subtypes. This method can help answer key, fundamental questions regarding subtype-specific lineage conversion during cardiac reprogramming such as the necessary cues that regulate sarcomere assembly and subtype specification. The main advantage of this technique is that it provides the method for quantified effects on cardio subtype specification which is required to perform mechanisis studies.
To begin, isolate mouse embryonic fiberblasts from stage E 12.5 embryos. Store them in liquid nitrogen as described in the accompanying text protocol. Next, harvest plat-E cells and plate them at one million cells per well into a six well plate so that the cells reach 70 to 80%confluency in 24 hours.
Cover the cells with transfaction media and incubate them under standard culture conditions. On experiment day minus one, take a 15 milliliter conical polystyrene tube and mix 60 microliters of reduced serum media with six microliters of transfection reagent per reaction. Incubate the mixture for five minutes at room temperature.
Since the transfection reagent use your add your reagent directly to the reduced serum media to avoid any decrease in transfection efficiency. Next, add a total of two micrograms of GHMT cocktail per reaction, and gently tap the tube to mix it. Incubate the reaction for 15 minutes at room temperature and then add the mixture to the plat-E cells in the drop wise manner.
Record the date and time of transfection. Incubate the transfected plat-E cells overnight. One hour before plating the mouse embryonic fibroblast, prepare a 24 well plate for imunno-cytochemistry by first adding 12 millimeter fibronectin coated cover slips to each well.
Next, add 300 microliters of bovine collagen solution to each well and incubate the plate for one hour. Near the end of the incubation, thaw a frozen vial of HCN4GFP mouse embryonic fibroblast. Quick thaw the cells, wash them once with pre-warmed fiberblast media and count the concentration of live cells.
Then aspirate the coating solution in the 24 well plate and immediately plate 30, 000 cells per well onto the collagen and fibronectin coated cover slips. 24 hours post-transfection, filter the retro-viral medium through a 0.45 micron pore size surfectant free cellulose acetate filter, and transfer it to a 15 milliliter conical tube. Then, add polybrene to a final concentration of eight micrograms per milliliter.
Carefully replenish the plat-E cells with two milliliters of fresh transfection medium. Changing the media too rapidly may cause the cells to detatch. Properly maintained plat-E cells produce an average of ten million infection units per milliliter.
High GFP expression and intensity in more than 95%of the cells, typically correlates with a successful GHNT mediated generation of induced cardiomyocyte-like myocytes. Next, aspirate the medium of the cultured embryonic fibroblasts and add the freshly collected retroviral medium. Then return meth plate to the incubator and incubate overnight.
The next day, again transfer the media from the plat-E cells to the embryonic fibroblasts. Discard the plat-E cells after the second virus collection. 48 hours post induction, aspirate the cell condition media and wash the cells once with PBS.
Then add 500 microliters of pre-warmed induced cardio myocyte-like myocyite media per well of the 24 well plate. Replace the media every two to three days for the next 12 days. 14 days post induction, carefully aspirate the media and rinse each well with 300 microliters of ice cold PBS.
Then removed the PBS and add 250 microliters of a 4%paraformaldehyde solution per well and incubate the cells for 15 minutes at room temperature. Next, permeabalize the cells by washing the wells three times for five minutes with 300 microliters of 0.1%tritonex 100 and PBS. Aspirate the excess permiableization solution after the last wash, and then add a universal blocking buffer at 300 microliters per well.
Block for 10 minutes at room temperature. Stain one pair of slides overnight with mouse anti-alpha actenin, chicken anti-GFP and rabbit anti-NPPA antibodies to identify induced pacemaker-like myocytes in induced atrial-like myocytes. The following day, wash the wells and add the appropriate secondary antibody as described in the accompanying text protocol.
Following additional washes, add 2.4 microliters of mounting media to a glass microscope slide. Take a cover slip from the 24 well plate and remove the excess solution. Then transfer it to the glass slide with the mounting media.
For each slide, start from one edge and start scanning up and down in the red fluorescent channel to identify alpha-actinin positive sarcomere positive cells. Sarcomere striations are easiest to identify in the 555 nanometer wavelength. Once identified, switch to the green channel and keep note if the cells are positive.
Then switch to the computer to asses the far red 647 nanometer wavelength to look for NPPA or MYL2 staining. Cells that are alpha-actinin positive, sarcomere positive, Hnc4-GFP negative and Nppa positive are induced atrial-like myocytes. Nppa staining will appear paranuclear and punctate.
First, count the number of cells 24 hours after seeding as described in the accompanying text protocol. Next, visually inspect each cell on a cover slip for proper alpha-actinin and sacromere staining and record the number of cells positively stained for both markers. Then, divide by the actual total cells plated.
An average reprogramming experiment will yield approximately one percent of cells that are both alpha-actinin and sarcomere positive. By day 14, the experiment can be stopped and assessed for sarcomere organization. This is identified by the positive alpha-actinin staining shown here in red.
Only cells that exhibit sarcomere organization are capable of spontaneous beating, so this is a good first check. Shown here are induced pacemaker-like cardiomyocites from Hcn4-GFP reporter mice. Three cells from this view exhibit sarcomere organization.
Three cell clusters positive for Hcn4-GFP are present in the center of the image and can be idenfied as a pacemaker cell type. Once cell at the bottom left can be observed with alpha-actinin staining but no sarcomere organization or identifiable subtype marker. In this population of induced ventricular like cardiomyocytes, the central cell exhibits sarcomere organization, this cell also stains positive for the myocin light chain marker MYL-2 which is a marker for ventricular cardio myocytes.
Once mastered, the plating and the transfection of the plat-E cells can be performed in one hour. The induction of the mouse embryonic fibroblasts in one to two hours and the myto quantification of a full 24 well plate in about eight hours. While attempting this procedure, it's important to remember that healthy mouse embryonic fibroblasts and high titer viruses are key for a successful reprogramming experiment.
After watching this video, you should have a good understanding of how to generate and quantify individual induced cardiomyocyte subtypes.
