In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This protocol describes an in vitro sprouting angiogenesis assay that recapitulates many features of the formation of blood vessels and can be used for testing drugs. This method is easy to implement in a lab, robust, and scalable. It has many similarities with assays using human endothelial cells and microspheres.

This model robustly mimics the phenotype showing defective endothelial tip cell selection and orientated endothelial cell migration that leads to pathological angiogenesis. The work can provide insights into the mechanisms regulating sprouting angiogenesis, the key genes, and the signaling pathways involved, notably by performing genetic screening The main challenge is maintaining the quality or pluripotency of mouse embryonic stem cells in culture. Regularly checking the pluripotency stages and morphology of the cell lines is important.

It is also important to perform a karyotyping of the cell lines as they tend to culture to lose or gain chromosomes. Begin by coating one well of the six well cell culture plate with 500 microliters of 0.1%gelatin solution and place it in a carbon dioxide incubator for 30 minutes. Then wash the gelatin coated plates with PBS and add 500 microliters of freshly prepared CM plus minus medium.

To obtain a pure population of mouse embryonic stem cells or mESCs, trypsinize the cell culture plate with 10x TVP buffer for 30 seconds at room temperature. Then resuspend the cells in one milliliter of mesoderm differentiation medium before transferring them to the gelatin coated six well plate for 30 minutes to allow the mouse embryonic fibroblasts to attach while the mESCs stay in suspension. Transfer the cell suspension to a 15 milliliter tube before counting live cells using a Neubauer hemocytometer in Trypan blue dye.

Then centrifuge the cells at 200 times G for five minutes at room temperature. Remove the supernatant and resuspend the cell pellet in an appropriate volume of mesoderm differentiation medium. Prepare four 94 millimeter low attachment polystyrene dishes by adding 15 milliliters of sterile water to the bottom.

After transferring the cell suspension into a sterile plastic reservoir, load four positions of a multi-channel pipette with 22 microliters of cell suspension per channel. Lift and place the inverted lid of the 94 millimeter dish on the clean surface of the flow cabinet with the inner side facing upwards. Deposit 40 drops of the cell suspension on the inner surface of each lid and carefully invert the lid without disturbance to place it back on the dish, so that the drops face the water.

Incubate the dishes in a carbon dioxide incubator for four days, considering this as differentiation day zero. After four days of incubation, collect the hanging drops in a 15 milliliter conical tube using a P1000 pipette. Let EB sediment in the tube before removing the supernatant.

Resuspend the EBs in three milliliters of 2x vascular differentiation medium before transferring and uniformly distributing EB suspension in a 60 millimeter agar-coated dish. Incubate the dishes in a carbon dioxide incubator until day nine with a medium refresh every two days. Prepare a 35 millimeter culture dish by adding one milliliter of the sprouting medium to its bottom.

To induce gelation, incubate the dish at 37 degrees Celsius for five minutes. Collect nine day old EBs from one agar dish in a 15 milliliter conical tube and remove the supernatant. Resuspend the EBs in two milliliters of cold sprouting medium to transfer to the culture dish coated with the first layer of sprouting medium.

Distribute the EBs all over the plate, ensuring they are at an equal distance from each other. The first sprout formation should be evident after incubation for around 24 to 48 hours. On day 12, use a spatula to carefully transfer the collagen gel containing EBs to a glass slide.

Remove the excess liquid with a pipette and dehydrate the gel by placing a gauze sheet of nylon linen and absorbent filter cards on top of the gel. Apply pressure with a weight of 250 grams for two minutes. Then remove the nylon papers to allow the slides to air dry for 30 minutes at room temperature.

After drying, fix the EBs using zinc solution overnight at four degrees Celsius. On the next day, remove the fixative before washing the slides five times with PBS. Permeabilize the EBs and PBS containing 0.1%Triton X-100.

After 15 minutes, remove the permeabilization solution to wash the slides five times with PBS for five minutes. Incubate the EBs in the blocking buffer for one hour at room temperature and wash with PBS for five minutes. Then add the primary antibody diluted in the blocking buffer and incubate at four degrees Celsius overnight.

Then incubate the slides with Goat anti-Rat Alexa 555 secondary antibody in a blocking buffer for two hours at room temperature. After incubation, wash the slides three times with PBS for five minutes and one time with water before mounting them for microscopy. The 3D sprouting angiogenesis assay was carried out on three EBs with two drugs, DC101 and DAPT.

Both compounds progressively blocked the angiogenesis in the EB model with increasing concentrations. The various concentrations of drugs in EBs showed that high doses of DC101 inhibit the number and length of the vessel sprouts. DAPT had opposite effects at the dose of one micromole per liter.

DAPT strongly increased the number of endothelial tip cells per vessel sprouts having a misguidance phenotype even at low doses, compared to DC101. Defective vessel sprouting was observed in hereditary hemorrhagic telangiectasia EBs from ACVRL1 control and ACVRL1 heterozygous genotypes with numerous misguidance phenotypes sprouting at random angles relative to the parent vessels. Mixtures of a wild-type mESC line at a one-to-one ratio with an unlabeled mESC wild-type line, led to an equal contribution to the leading endothelial tip cells.

Microscopic analysis of this representative chimeric EB was conducted to identify the genotypic origin of the leading endothelial tip cells. To quantify mural cell coverage of the vessel sprout, EBs were fixed and stained for endothelial cell markers, PECAM, and mural cell marker, alpha smooth muscle actin. A high magnification image revealed that one individual vessel sprout was surrounded by mural cells.

The binary transformation was performed after color channel separation and the ratio of PECAM positive vessels covered by alpha SMA positive mural cells was quantified. The genetic background of the embryonic stem cell line is also a key factor the researchers must consider as some lines sprout better than others. This method can be used to perform genetic screening using RNAi approaches or a bank of mouse embryonic stem cells harboring gene mutations.