This protocol allows us to create a complex 3D structure that closely resembles the developing human heart in a simple cost effective way, that is reproducible across cell lines. It consists of a single protocol with three steps of differentiation, which gives rise to all the cell types of the heart, including chambers and a vascular network. This technique allows us to model a wide range of human cardiovascular diseases and to screen effective pharmacological treatments.
Thanks to the high throughput nature of its design. This method is useful for investigating the developing human heart as well as diseases commonly associated with it. One of them being congenital heart defects.
This protocol requires basic cell culture skills. We recommend attentive and delicate pipetting as well as gentle handling of the organoids. Begin by creating embryoid bodies or EBs two days before differentiation.
Wash sub-confluent HPSCs with DBPS for at least 10 seconds. Then aspirate the DPBS. Add one milliliter of room temperature ACUTA and gently tap the plate approximately five times every minute for three to five minutes to induce detachment, while checking under the microscope.
Then add one milliliter of PSC medium and Thias Avin or Thias to stop the reaction. Pipette the media up and down in the well, two to three times to generate a single cell suspension and collect the cells. Then transfer the single cell suspension to a centrifuge tube and spin for five minutes at 300 times G.Aspirate the supernatant and re-spend the cells in one milliliter of PSC medium and Thias.
Dilute the cells in DPBS. Count the cells using a cell counter or hemo cystometer and dilute the cells in PSC medium with Thias to a concentration of 100, 000 cells per milliliter. Add 100 microliters of diluted cells suspension to each well of a round bottom ultra low attachment 96 well plate.
Centrifuge the plate at 100 times G for three minutes and incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. After 24 hours carefully remove 50 microliters of medium from each well and add 200 core litters of fresh PSC medium, warm to 37 degrees Celsius for a volume of 250 microliters per well. Incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
Remove 166 microliters or two thirds of medium from one side of each well, and add 166 microliters of RPMI 1640 containing insulin free B27 supplement, six micromolar cheer 990211, 0.875 nanograms per milliliter of bone morphogenetic protein four and 1.5 nanograms per milliliter of Activin A.Incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. After 24 hours remove 166 microliters of medium from one side of each well and add 166 microliters of fresh RPMI 1640 with insulin free B27 supplement. Incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
To induce cardiac mesoderm specification on day two, remove 166 microliters of medium from each well and add 166 microliters of RPMI 1640, containing insulin free B27 supplement and three micromolar Wnt-C59 and continue incubation for 48 hours. On day four, remove 166 microliters of medium from each well and add 166 microliters of fresh RPMI 1640 with insulin free B27 supplement, then incubate another 48 hours. On day six, remove 166 microliters of medium from each well and add 166 microliters of RPMI 1640 with B27 supplement.
For this and all subsequent media changes, use B27 supplement with insulin, incubate for another 24 hours. On day seven, the cell culture is ready for pro epicardial differentiation. Remove 166 microliters of medium from each well and add 166 microliters of fresh RPMI 1640 containing B27 supplement and three micromolar cheer 99021.
Then in incubate for one hour, after one hour remove the plate from the incubator, remove 166 microliters of medium from one side of each well and add 166 microliters of fresh RPMI 1640 containing B27 supplement continue incubation for another 48 hours. Repeat this process us every 48 hours until collection. Organoids are ready for analyses and experimentation on day 15 To transfer the whole organoids, cut the tip of a P200 pipette, five to 10 millimeters from the opening with a diameter of approximately two to three millimeters.
Press the pipette plunger and insert the tip vertically into the round bottom well with the organoid. Take up approximately 100 to 200 microliters of medium enough to collect the organoids and transfer them to the target destination. Before differentiation EBS appear as blacks spherical masses the differentiation process starts on day zero with a Wnt pathway activation and growth factor addition for exactly 24 hours.
Further the edit of Wnt C59 on day two results in enlargement of the organoid from approximately 200 micrometers to a maximum of one millimeter. The human heart organoids began beating as early as day six and 100%of the organoids showed visible beating by day 10. High magnification images of day seven organoid revealed that most hand one expressing cells were cardiomyocyte in origin and hand two were non myosite cells from second Heartfield, progenitor cells.
RNA transcripts for both hand one and hand two were expressed from day three onwards in RNA sequencing data. The FHF markers were highly expressed during days three and 11 while the SF marker was more highly expressed after day 13. Immunofluorescence stain revealed the presence of various human heart cell type lineages that make up the human heart.
Such as myocardial and epicardial tissue, endocardial cells, endothelial cells, and cardiac fibroblast. The markers expressing the above cell lineages were also observed in the RNA SAC gene expression profiles. Live calcium imaging of individual cells in whole organoids was used to measure the electrophysiological function of the organoids.
Fluo-4 fluorescence intensity varied over time due to calcium entry exit from the cell revealing regular action potentials. Heat maps showing calcium intensities over a high magnification region of the organoid, show increased intensity due to calcium transients and individual cells. When attempting this protocol, keep in mind that the EBs and subsequent organoids are not attached to the bottom of the plate, and we'll move around with the removal in addition of media requiring attentive media changes.
Organoids can be culture for longer periods of time introduced to maturation techniques, or even exposed to external stimuli, such as mechanical or electrical conditioning, and even stressors such as toxins. This technique allow as access to stages of human fetal heart development that are otherwise inaccessible in research, paving the way for new insights into heart development and disease etiology.
