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09:46 min
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January 20th, 2023
DOI :
January 20th, 2023
•0:04
Introduction
0:52
Day 0: Generation of Pluripotent Aggregates from Single-Cell Suspension
2:34
Day 1: Mesoderm Induction of the Aggregates
3:41
Day 4: Vascular Induction and Priming of the Aggregates
4:20
Day 6: Aggregate Embedding and Vessel Sprout Induction
6:02
Day 11: Isolation and Maturation of the Blood Vessel Organoids (BVOs)
7:25
Results: Stepwise Progression of Human Blood Vessel Organoid Generation from hPSCs
9:13
Conclusion
필기록
This protocol outlines our generation of human blood vessel organoids from pluripotent stem cells. This technology can be used to study aspects of vasculogenesis, angiogenesis, vascular disease, as well as vascular pathologies. The reproducibility and high throughput nature, as well as its consistency across many different stem cell lines, are strong advantages of this technique.
In terms of their application, some of our previous research outlines the use of blood vessel organoids to study the morphological changes for diabetic patient vasculature and explores drug treatment avenues for diabetic vasculopathy. Properly maintaining the initial stem cell population, preventing the aggregates from clumping, and ensuring near-homogenous aggregate diameter, these are essential factors for generating good blood vessel organoids. Begin generation of aggregates using cultured human pluripotent stem cells, or hPSCs, having 70%confluency.
Using a pipette or a vacuum system, aspirate the culture medium and replace it with one milliliter of cell dissociation reagent before incubating the cells for five minutes at 37 degrees Celsius. Meanwhile, prepare the necessary volume of aggregation medium in a 15-milliliter conical tube, following the formulation mentioned in the text manuscript. After incubating the cells, aspirate the cell dissociation reagent before suspending the cells in one milliliter of aggregation medium.
Gently pipette the content up and down to create a single-cell suspension. Count the cells using an automated cell counting device or under a microscope, and calculate the total number of cells required for aggregate formation. The cell viability readout shows single cell suspension with low to no clusters.
Aspirate the supernatant from the tube and add the appropriate volume of the cell suspension to the aggregation medium in the 15-milliliter high-clarity polypropylene conical tube and gently pipette the diluted cell suspension up and down to ensure a homogenous cell distribution. Pipette three milliliters of the diluted cell suspension into each desired well of the six-well ultra-low attachment culture plate. Place the plate in the incubator and minimize any disturbance to maintain the size and shape of aggregates.
Prepare the mesoderm media as described in the text manuscript. 24 hours post-seeding of the cells, take the culture plate out of the incubator. Swirl the plate in a circular motion to accumulate the aggregates in the center of each well.
Using a one-milliliter pipette, gently transfer the aggregates with the medium from each well into the corresponding conical tube. Allow the aggregates to sediment in the conical tubes at room temperature for one hour. Once sedimented, aspirate the supernatant with a pipette or a high-sensitivity aspirating pump cautiously, without disturbing the settled aggregates.
Resuspend the aggregates in each tube by adding two milliliters of mesoderm induction medium. Next, transfer the suspension from each tube back into the respective well of the ultra-low attachment six-well culture plate. Place the plate in the incubator at 37 degrees Celsius and leave it until day four.
On day four, take the culture plate out of the incubator, then shake the plate in a circular motion to collect the aggregates in the center of each well. Using a one-milliliter pipette, gently transfer the aggregates with the surrounding medium from each well into the corresponding conical tube. Set a timer for 30 minutes to allow the aggregates to sediment in the tubes.
Once the aggregates sediment, prepare the culture plates as previously demonstrated and place them in the incubator at 37 degrees Celsius until day six. For aggregate embedding and vessel sprout induction, prepare the desired final volume of the extracellular matrix solution while working on ice. Pipette 500 microliters of the ECM into one well of a 12-well plate to form the first layer of the ECM sandwich.
To ensure effective polymerization of the first ECM layer, place the plate in an incubator at 37 degrees Celsius for two hours. Towards the end of the two-hour incubation, start working with the aggregates in the culture plate. Gather the aggregates in the center of each well before using a one-milliliter pipette to gently transfer the aggregates and medium from each well into a corresponding 15-milliliter conical tube.
Allow the aggregates to settle for 10 to 15 minutes before aspirating the supernatant. Following which, keep the conical tubes containing the aggregates on ice for five minutes. Working swiftly and carefully, resuspend the aggregates in 500 microliters of ECM without bubble formation.
Using a pipette, layer the ECM aggregate suspension atop the already-polymerized first ECM layer inside the well in the 12-well plate. In the meantime, prepare the sprouting medium as described in the text manuscript. After two hours of incubation at 37 degrees Celsius, add one milliliter of sprouting medium prewarmed to 37 degrees Celsius into the well to induce blood vessel differentiation.
Working under sterile conditions, use the rounded end of a sterile spatula to loosen the ECM sprouting matrix containing the vascular networks. Then, using sterile forceps and the rounded end of a sterile spatula, carefully transfer the loosened gel disc onto the lid of a 10-centimeter culture dish. Place the gel on the lid under a stereo microscope adjusted to the desired magnification and focus, and use sterile needles to cut out single blood vessel networks, trying to limit the amount of non-vascularized ECM obtained in the process.
Gently transfer the isolated organoids back into one well of an ultra-low attachment six-well plate containing three milliliters of sprouting medium. Next, using a one-milliliter pipette, transfer the single organoids to the appropriate number of wells in an ultra-low attachment 96-well plate. Once transferred, add 200 microliters of prewarmed sprouting medium into each well of the 96-well plate.
Four to six days post-isolation in the 96-well plate, ensure the organoids possess a round and healthy morphology before proceeding to fix and stain them. Images of stepwise progression of the human blood vessel organoid, or hBVO, generation from hPSCs were captured under bright-field. On day zero, aggregates ranging from 30 to 100 microns in diameter were generated from the hPSC culture.
Subtle changes in the size and shape of the aggregates were observed upon mesoderm induction on day one, which further changed on day four as the aggregates underwent vascular priming. Near-radially-symmetric early vessel sprouting can be observed on day seven, a day after embedding the aggregates in the sprouting matrix. Healthy organoid morphology and continued vessel sprouting was seen on day nine, which progressed to late-stage vessels sprouting by day 10 when the dense cell structures at the organoid center almost disappeared.
Morphology typical of mature human blood vessel organoids was clearly observed by day 15. Whole mount staining of the mature hBVOs on day 15 exhibited and extensive and connected endothelial network that was CD31-positive and surrounded by PDGFR-beta-positive parasites and SMA-positive alpha-smooth muscle actin. The PDGFR-beta-positive and SMA-positive mural cells encapsulating the endothelial vessel networks is well-observed.
A continuous collagen IV-positive basement membrane enveloping the vessel networks was also observed. Ensuring a proper polymerization of the extracellular matrix during the embedding step is critical to effective blood vessel sprouting. Researchers have used our blood vessel organoid technology to generate early vascular compartments in already established organoid models, such as brain and kidney, that were previously avascular.
This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.
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