In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Our method aims to generate heart field-specific cardiac progenitor cells from mouse embryonic stem cells. This heart field reporter stem cell line allows for the high-throughput study of the mechanisms underlying congenital heart disease, providing a system that is amenable to genetic and pharmacological manipulation. There are several chamber-specific congenital heart diseases.

By recapitulating cardiogenesis in vitro, this protocol allows the study of disease mechanism and the development of future regenerative therapies. This protocol can provide insight into how cardiac progenitor cells of different chambers are specified during heart development. Begin by growing transgenic mouse embryonic stem cells in 0.1%gelatin-coated T25 flasks in 2i medium.

When the cells reach 70 to 80%confluence, rinse the cultures with PBS and add one milliliter of trypsin per flask to dissociate the culture into single cells at 37 degrees Celsius for three minutes. When the cells have detached, neutralize the reaction with four milliliters 10 FBS in DMEM and count the cells by an appropriate method. Dilute the cells to an approximately three times 10 to the five cells per fresh medium concentration, and collect the cells by centrifugation.

Then resuspend the pellet in five milliliters of fresh 2i medium for replating onto new 0.1%gelatin-coated T25 flasks. For CPC generation from cardiac spheroids, collect 2.5 times 10 to the six of the detached transgenic mouse embryonic stem cell sample by centrifugation, and resuspend the cells in 25 milliliters of SFD medium. Plate the cell suspension into one 150-by-25-millimeter sterile plate for incubation at 37 degrees Celsius and 5%carbon dioxide for 48 hours.

At the end of the incubation, collect the cardiac spheroids into a conical tube. Sediment the spheroids by centrifugation to facilitate the selective isolation of the spheroids and to avoid single cells. Resuspend the spheroids in 25 milliliters of fresh SFD medium supplemented with one nanogram per milliliter of activin A and 1.5 nanograms per milliliter of bone morphogenetic protein four.

Replate the spheroids back onto the same culture plate for a 24-hour incubation in the cell culture incubator. The next day, recollect all of the cardiac spheroids by centrifugation. Resuspend the differentiated embryoid bodies in 25 milliliters of fresh SFD medium for plating in an ultra-low attachment, 75-centimeter squared flask in the cell culture incubator for 48 hours.

For isolation of heart field-specific CPC using fluorescent reporters, collect the embryoid bodies by centrifugation and dissociate the 3D cultures with one milliliter of trypsin at 37 degrees Celsius for three minutes. At the end of the incubation, mix well by pipetting to dissociate the cells and neutralize the reaction with four milliliters of 10%FBS in DMEM. Pass the mixture over a 70-micrometer strainer to remove any non-dissociated embryoid bodies, and sediment the filtered cells by centrifugation.

To sort the CPCs by their fluorescent reporter expression, resuspend the pellet in 500 microliters FACS solution and filter the cells through a 40-micrometer cell strainer into a five-milliliter polystyrene round-bottom tube on ice. Then sort the cells to isolate the RFP-and GFP-expressing cells by FACS, collecting the cells in one milliliter of FBS. To isolate first versus second heart field CPCs based on their surface protein Cxcr4 expression, resuspend single mouse embryonic stem cell RFP-expressing cardiac progenitor cells in 300 microliters of 10%FBS in PBS supplemented with fluorescence-conjugated anti-Cxcr4 antibody.

After five minutes at room temperature, wash the cells three times in one to two milliliters of fresh, cold PBS per wash. After the last wash, resuspend the cells in 500 microliters of FACS solution and filter the cells through a 40-micrometer strainer into a five-milliliter, round-bottom tube. Then sort the cells by their Cxcr4 expression, collecting both the Cxcr4-positive and negative populations into individual tubes containing one milliliter of FPS per tube on ice.

To re-culture the FACS-isolated, heart field-specific cardiac progenitor cells, collect the sorted cells by centrifugation and resuspend the pellets in SFD medium. Then seed approximately three times 10 to the four cells per well in a 0.1%gelatin-coated, 384-well plate. If increased cell death is noted after sorting, add 10-micromolar ROCK inhibitor to each well.

After two days of culture, spontaneous beating should be observed. To analyze the ability of plated CPCs to differentiate to cardiomyocytes, collect the cells at day 12 of differentiation with trypsin, as demonstrated, to isolate the single cardiomyocytes and resuspend the cells in 4%paraformaldehyde. After 30 minutes at room temperature, collect the fixed cells by centrifugation and wash the pellet in PBS to remove the excess fixative.

Next, resuspend the cells in 10%FBS in PBS, and incubate half of the cell sample with mouse anti-troponin T antibody and use the other half of the sample as a negative control. After 30 minutes at room temperature, wash the cells two times in fresh PBS and resuspend the samples in 10%FBS plus PBS supplemented with an appropriate secondary antibody. After another 30-minute incubation at room temperature, wash the cells two times in fresh PBS per wash and resuspend the cells in 200 microliters of PBS per tube for analysis on a flow cytometer.

After approximately 132 hours of differentiation, Tbx1-RFP and Hcn4-GFP cardiac progenitor cells can be detected by fluorescence microscopy. Generally, both GFP and RFP cells appear approximately around the same time, and the two populations of progenitor cells continue to expand in close proximity and typically in a complementary pattern. Adjusting the concentrations of activin A and bone morphogenetic protein four will alter the percentages of first versus second heart field cardiac progenitor cells.

Similarly, using an RFP reporter mouse embryonic stem cell line, after 132 hours of differentiation, RFP-positive cardiac progenitor cells appear. After immunostaining for Cxcr4, RFP-positive, Cxcr4-positive, and RFP-positive, Cxcr4-negative cells can be isolated. Immunostaining for cardiac troponin T at day 12 of differentiation confirms that first heart field cells differentiate mainly into myocytes.

Similarly, cells derived from RFP-positive, Cxcr4-negative cardiac progenitor cells give rise to cardiomyocytes at much higher percentages compared to Cxcr4 single positive cardiac progenitor cells. Occasionally, mouse embryonic stem cells fail to differentiate efficiently and form very low numbers of heart field-specific cardiac progenitor cells. The timing, concentration, and consistency of the addition of the cytokines and the number of the cells are critical for a successful generation of chamber-specific cardiac progenitor cells.

Following this procedure, investigators can analyze the physiological properties of the different populations of cardiac progenitor cells to obtain further insight into their specification and function.