The overall goal of this article, is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells, CSCs from the Adult Mouse Heart. This method can help answering key questions, in the field of cardiovascular disease and regional therapy such as, cardiac stem cell therapy and heart failure. The main advantage of this technique is that it is cost effective, with high aid in small period of time.
Start this experiment by preparing all the necessary materials, instruments, and isolation buffers in a sterilized condition as described in the protocol. After removing the skin from the abdomen of a euthanized mouse, cut the ribcage inside the sterile hood to open the thoracic cavity. Expose the heart and then remove the blood near the heart using a one milliliter syringe.
To remove any blood near the heart, wash the surrounding area with ice cold PBS. Using surgical scissors and tweezers to dissect the heart, place it in a 100 millimeter Petri dish containing 10 milliliters ice cold PBS. Use curved shank forceps to palpitate the heart, and remove the residual blood inside the heart.
Transfer four to five isolated hearts to a 100 milliliter Petri dish containing 10 milliliters of ice cold Ham's Balanced Salt Solution. Palpate the hearts again to wash them and remove any residual blood inside the heart. To ensure the complete removal of the blood, change the HBSS solution after each wash.
Hold each heart with a pair of forceps and use a surgical blade to cut each heart into two to four millimeter pieces in a 100 millimeter Petri dish, containing five to seven milliliters of HBSS. Frequently change the solution to ensure complete blood removal. Finally, mince the hearts in the HBSS solution.
Centrifuge the minced hearts placed in a 50 milliliter conical tube at 500 times G, at four degrees Celsius for five minutes, and then discard the supernatant. Add five to six milliliters, 0.2%Collagenase II solution to the pellet to digest the tissue. Re-suspend the pellet thoroughly by rocking the tube on a shaker at 75 times G, at 37 degrees Celsius for 45 to 60 minutes.
Every 20 to 30 minutes, shake the tube vigorously by hand to enhance the lysis. Triturate the lysed tissue mix using five milliliters serological pipette, and a one milliliter pipette tip to dissociate the tissue lump into the single cell suspension. To stop the enzymatic lysis of the tissue, add two to three times as much commercially available CSC maintenance medium as the volume of the lysed tissue.
Pass the digested cell suspension through a 100 micrometer cell strainer placed on a 50 milliliter conical tube to remove any undigested tissue pieces. To separate the cells to density gradient centrifugation, add 37 degrees Celsius, pre-warmed, polysucrose and sodium diatrizoate solution to a 50 milliliter conical tube. Then gently and slowly add an equal volume of filtrate over the solution, avoiding any mixing of the two solutions.
It is critical to add filtrate over polysucrose and sodium diatrizoate solution slowly without mixing by tilting the tube at 45 degrees and against the wall of the tube. Do not add polysucrose and sodium diatrizoate solution over filtrate. Place the tube very slowly in a swing bucket centrifuge making sure not to disturb the solutions.
Centrifuge at 500 times G for 20 minutes at room temperature set at a lower acceleration and deceleration speed to avoid mixing the two solutions, and for proper separation of the cardiac stem cells. It is critical to put the gradient solution very carefully without disturbance in the centrifuge. Put the acceleration and deceleration of the centrifuge must be at lowest speed, such as one or zero.
Use a one millimeter pipette to transfer the buffy coat containing the CSCs along with extra solution from the upper layer to a 15 milliliter sterilized tube. Add an equal volume of CSC maintenance medium, and mix it properly to neutralize the residual polysucrose and sodium diatrizoate solution. Centrifuge this suspension at 500 times G for five minutes at four degrees Celsius.
Discard the supernatant. Repeat the washing of CSCs with incomplete DMEM Solution at least for two times. Centrifuge at 500 times G for five minutes at four degrees Celsius to get rid of any residual polysucrose and sodium diatrizoate solution.
After removing the supernatant, resuspend the pellet containing purified CSCs in one milliliter CSC maintenance medium, and then count the cells. Seed the CSCs in a six-well culture plate coated with a 0.02%gelatin solution containing 0.5%fibronectin, and add two milliliters of complete maintenance medium. Grow the cells at 37 degrees Celsius in 5%CO2.
When the CSC culture reaches confluency, transfer the cells to a new gelatin and fibronectin coated plate as described in the text protocol. Culture these transferred P0 CSCs in complete CSC maintenance medium at 37 degrees Celsius in a 5%CO2 incubator until confluent. Repeat the cell transfer to a new plate to make P1 CSCs which can be used for further experiments.
To maintain the CSC culture at its initial stage, seed the cells further as described in the text protocol. To characterize the cultured cells, place the CSC containing plate on the objective of a fluorescent microscope. Use ten times magnification to focus the cells.
To observe the cells, use phase-contrast aperture, and increase the magnification to 20 times by changing the objective lens, and then image the CSCs. After performing the immunostaining, place the culture plate under the fluorescence microscope. Focus the cells at different magnifications, and observe the cells under phase contrast and different excitation filters, and save the images.
For cell differentiation, grow the CSCs in a six-well plate with two milliliters of 37 degrees Celsius, pre-warmed CSC maintenance medium. When the cells reach 80 to 95%confluency, replace the maintenance medium with two milliliters, 37 degrees Celsius, pre-warmed cardiomyocytes differentiation medium. Incubate at 37 degrees Celsius, in a 5%CO2 for up to two to three weeks.
Change the differentiation medium every two to three days. During the medium change, observe the cell morphology under a microscope to ensure the good culture conditions. After 12 days, image the cells for differentiation markers following a standard immunostaining protocol.
Two to three days cultured CSCs show a spindle-shaped morphology when observed under a phase-contrast microscope. After seven days of culture, they show a change in morphology becoming elongated. After immunostaining for markers of pluripotency, CSCs show expressions of OCT4, SOX2, and Nanog.
Furthermore, CSCs proliferate in culture medium, and express the proliferation marker Ki67. To characterize the cardiac origin of CSCs, expression of cardiac markers was examined, and the cells showed to be positive for cardiac markers Sca-1, NKX2.5, and GATA4. After culturing in cardiomyocyte differentiation medium for 12 days, differentiated CSCs show expression of cardiomyocyte markers ACTININ, and TROPONIN-I.
While attempting this procedure, it is important to remember to not allow mixing of the gradient solution and the filtrate. It is also important to set the acceleration and deceleration at lowest speed. Whoever contaminates them, it is recommended to perform all cell isolation process inside a biosafety cabinet.
Following this procedure, other methods like magnetic-bead based separation or flow cytometic based cell sorting methods can be performed in order to answer additional questions such as, how clear is the homogeneous population of cardiac stem cells? Crack radius in a cardiac stem cell is big markers, and there are differences toward cardiomyocytes. After it's development, this technique will pave the way for the researchers in the field of cardiovascular disease who often high yield of cardiac stem cells from mouse heart which could be important for regenerative therapy in ischemic heart failure.
Do not forget that working with surgical blade, bleach and DMSO can be hazardous, and precautions such as using Personnel Protective Equipments, PPE, should always be taken while performing this procedure.
