Developmental Biology
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Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions
Chapters
Summary January 7th, 2019
This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.
Transcript
Differentiation of cardiac progenitor cells, or CPCs, upon amplification requires a phase switch from a proliferating to a committed stage, and this protocol uses efficient endothelial lineage in CPCs. A major advantage of the technique is that it is applicable to CPCs isolated based on several techniques and from different species. This protocol can be used to enhance the therapeutic potentials of CPCs for cell therapy.
It can also be used to investigate the potentials and mechanisms of differentiation and how these are affected by cardiac disease. CPCs are an inhomogeneous population of cells that may differ in shape, size, and potency. Therefore, the protocol has to be adjusted according to the specific subpopulation of CPCs used.
After anesthetizing the mouse, wipe down its chest with 70%ethanol. Using scissors, cut the skin and the thoracic wall to expose the thoracic cavity. Use forceps to lift the heart, and then use scissors to cut it at the base.
Transfer the heart to a P60 culture dish containing five milliliters of cold PBS, and then use forceps to pump the heart and eject the blood out of the cavities. Place the heart in a P60 dish containing five milliliters of cold PBS and wash it. Use small scissors to remove the atria, cut the heart into two longitudinal pieces, and wash them in five milliliters of ice cold PBS.
Using small scissors, cut the heart pieces into smaller sections. Add a drop of a Hank's balanced salt solution containing collagenase B at a concentration of one milligram per milliliter, and use a sterile razor blade to mince the small heart pieces thoroughly. Next, add 2.5 milliliters of the collagenase B solution to the dish.
Place the dish at a 30 degree angle in an incubator at 37 degrees Celsius. Let the minced heart pieces incubate for a maximum of 30 minutes while repeatedly passing them through a Pasteur pipette every 10 minutes during the incubation. First, add five milliliters of cold HBSS supplemented with 2%FBS to the minced and homogenized heart pieces.
Using a 100 micrometer filter, filter the heart pieces to remove any undigested tissue. Centrifuge at 470 times G and at room temperature for five minutes. Discard the supernatant and re-suspend the pellet in five milliliters of red blood cell lysis buffer.
Incubate on ice for five minutes with occasional shaking. After this, add 10 milliliters of PBS and filter the sample through a 40 micrometer filter to exclude larger cells including residual cardiomyocytes. Centrifuge at 470 times G and at room temperature for five minutes without the break.
Then, re-suspend the cells in DMEM supplemented with 10%FBS, aiming for a final cell concentration of one million cells per milliliter. Distribute the cells into two tubes for staining and sorting, adding 1.5 milliliters of the cell solution to one tube and 3.5 milliliters to the second tube. Add Hoechst solution and incubate at 37 degrees Celsius for 90 minutes with occasional flipping to ensure equal distribute of the dye and prevent clotting of the cells.
The side population appears at the Hoechst low lower left corner aside of the main population. In the FACS plot it can be identified based on the negative control in which verapamil prompts the side population to disappear. Warm up medium one to 37 degrees Celsius and cool down the centrifuge to four degrees Celsius.
Centrifuge the sorting tubes at 470 times G and at four degrees Celsius for six minutes. Then, re-suspend the cells in medium one. Transfer the cells to a gas-permeable P60 dish containing four milliliters of medium one.
Change the medium every three days until the cells have reached a confluence between 70 and 80%Culture SP-CPCs for two to three days in T75 flasks using eight milliliters of medium one in each flask. Next, carefully aspirate the medium and gently rinse the flask with five milliliters of warm HBSS. Treat the cells with five milliliters of trypsin EDTA for five minutes at 37 degrees Celsius with 5%carbon dioxide.
Then, add five milliliters of medium one to stop the trypsin activity, and transfer the cell suspension to a 15 milliliter tube. Centrifuge at 470 times G for five minutes at room temperature. Next, re-suspend the cells in either medium one or medium two.
Plate the cells on P60 dishes, plating 250, 000 SP-CPCs per dish with three milliliters of medium containing different serum concentrations. After two days of culturing, collect the medium from each dish into a 15 milliliter tube to collect the dead cells. Trypsinize the adherent cells as previously described and collect the cell suspension into the 15 milliliter tube that contains the collected medium.
Centrifuge at 840 times G for five minutes at room temperature, then aspirate the supernatant and add one milliliter of either medium one or medium two for cell counting. Stain the SP-CPCs with trypan blue and count the viable and dead cells. First, aspirate the medium from the prepared cells, and rinse them gently with five milliliters of warm HBSS.
Treat the cells with five milliliters of trypsin EDTA for five minutes in the cell incubator. Then, add five milliliters of medium one to stop the trypsin activity. Transfer the cell suspension to a 15 milliliter tube and centrifuge at 470 times G for five minutes at room temperature.
Aspirate the supernatant and add medium two for cell counting. Next, seed 80, 000 cells into each well of a pre-coated six-well culture plate with three milliliters of medium two. Keep the plate at 37 degrees Celsius for 20 to 24 hours, and then change the medium in each well to three milliliters of medium three.
Culture the plate for 21 days, changing the medium every three days. After this, stain the cells with an endothelial marker and perform fluorescence microscopy to verify the endothelial nature of the differentiated cells. First, aspirate the medium from the prepared cells and rinse them gently with five milliliters of warm HBSS.
Treat the cells with two milliliters of trypsin EDTA for five minutes in the cell incubator, then add three milliliters of medium three to stop the trypsin activity. Transfer the cell suspension to a 15 milliliter tube and centrifuge at 470 times G for five minutes at room temperature. Aspirate the supernatant and add one milliliter of medium three, pipetting gently to mix.
Count the cells and seed between 2, 000 and 4, 000 cells in 100 microliters of medium three to each matrix coated well of a 96-well plate. Incubate the plate at 37 degrees Celsius for 16 hours. After this, use a bright-field microscope at two times magnification to take a picture of the cells.
In this study, suitable conditions are explored for the facilitation of endothelial lineage commitment of cardiac progenitor cells. When the cell confluency is at or below 60%no significant difference is seen in the samples treated with 3.5%FBS. When cells at this confluency are treated with 0.1%FBS, they show a decrease in cell proliferation on laminin when compared to fibronectin, but show no increase in cell death.
In contrast, cells at a high confluency show no significant difference in either cell proliferation or cell death between the two conditions. Changes in cell shape appear to be an indicator of suitable culture conditions in which endothelial differentiation is going to be successful. Within seven to 14 days in the endothelial differentiation medium, successful cultures are seen to contain larger cells with different morphology.
Interestingly, these cells disappeared towards the end of the differentiation phase and tended to appear in lower numbers and at later time points in high-density cultures. Tube formation is consistently successful in cells plated at low density and differentiated on laminin, while it is seen to mostly fail in cells plated at high density. While tube formation is mostly unsuccessful in cells cultured on fibronectin irrespective of cell density, those differentiated at a low density sometimes form rudimentary tubes.
The serum concentration and cell density to induce endothelial lineage are crucial, and they have to be adjusted to guarantee slowing of proliferation while maintaining viability. Upon lineage induction, CPCs from humans or preclinical disease models may be used for cell transplantation studies to assess their in vivo differentiation potential. This protocol could help to study or establish novel cardiac regeneration tools, and it was previously used to study molecular mechanism of early lineage commitment.
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