First, centrifuge a single-cell human pluripotent stem cell, or HPSC suspension, at 200 times g for five minutes. Following the centrifugation, aspirate the wash medium, and re-suspend the cells in aggregation medium, at a density of 1.2 times 10 to the sixth cells per milliliter. Next, seed one milliliter of the cell suspension to each well on the prepared microwell plate, and evenly distribute the cells.
To seed a large number of wells, periodically vortex the cell suspension to prevent settling. After seeding, centrifuge the plate at 200 times g for five minutes. After the spin, observe the plate under the microscope to confirm cells have spun to the bottom of each microwell.
Then, incubate the plate for 24 hours at 37 degrees Celsius in a 5%C-O two, 5%O two hypoxic incubator. One day following aggregation, inspect the aggregates under the microscope. Compared to the immediate post-centrifuge aggregation, they should appear intact with smooth edges.
Remove the supernatant by holding the microwell plate horizontally, and placing the tip of a P-1000 micropipettor at the surface of the culture medium, and against the edge of the well. Then, slowly remove the medium, being careful not to disturb the aggregates at the bottom of the microwells. After reducing the medium level to about one to two millimeters from the textured microwell surface, slowly tilt the plate, and slowly aspirate the remaining medium from the well.
Add one millileter of freshly-prepared prewarmed stage one induction medium by holding the pipette tip against the inside edge of the well, and very slowly dispensing the medium against the inside wall of the well. Return the plate to the incubator under hypoxic conditions for three days. On day four, use a five-milliliter serological pipette to harvest the aggregates from each well of the microwell plate.
Collect up to 10 wells of aggregate suspension in a 15-milliliter conical tube. Allow the aggregates to settle for 15 minutes in a hypoxic incubator. After the aggregates have settled, carefully aspirate the supernatant, and re-suspend the aggregates in ten milliliters of prewarmed wash medium, to remove residual inductive cytokines.
Centrifuge the aggregates at 50 times g for two minutes. Following the centrifugation, aspirate the supernatant, and re-suspend the pelleted aggregates in prewarmed stage two induction medium. Transfer the aggregate suspension to a 24-well ultra-low attachment plate at one milliliter per well.
Observe the aggregates under the microscope as uniform tight-cell clusters. Incubate under hypoxic conditions until day six. On day six, pool up to 10 milliliters of aggregates per 15-milliliter conical tube as before.
After the aggregates have settled, aspirate the supernatant, and re-suspend the aggregates in the prewarmed stage three induction medium. Using a five-milliliter serological pipette, redistribute the aggregates into a 24-well ultra-low attachment plate, at one milliliter per well. Perform a complete medium change on day 10 of culture.
On day 12, transfer the cells to normoxic culture conditions of 20%oxygen and 5%carbon dioxide for the remainder of the culture period. After 12 days of differentiation, which corresponds to six days in cardiac induction stage three medium, forceful aggregate-wide contractions were observed. At day 17, 74.8%of the cells are positive for cardiac troponin T, by flow cytometry, as indicated by the filled portion of the histogram.
Also shown are cells stained with the secondary antibody alone, shown by the unfilled section of the histogram. While attempting this procedure, it's important to wash the aggregates well on day four, to remove trace levels of cytokines, specifically activin-A, which promotes endoderm differentiation, at the expense of cardiac induction. Following this procedure, other methods, such as cool culture of inductive cell types within the aggregate, can be performed to answer other questions, such as how paracrine signaling from neighboring embryonic tissues influences human pluripotent stem cell differentiation into the cardiac lineage.
