3D Stem Cell Model Explained

This blog post intends to provide an extra layer of scientific detail to explain how Dr. Irene Pedersen and her team use stem cells to build human blood vessels in 3D. Please read the PANDAS Network funding announcement of Dr. Pedersen’s work for the introduction to this article.

Using Inducible Stem Cells to Model the Neurovascular Unit

Below is a step-by-step guide explaining how Dr. Pedersen and her team ‘build’ three-dimensional human brain blood vessels on a special tissue culture plate.

Step #1 – Force stem cells to become endothelial cells, pericytes, and astrocytes.

The first step in creating a synthetic neurovascular unit requires growing stem cells in a dish with a different cocktail of molecules in the “liquid food” known as cell culture media. The varying molecule cocktail encourages the stem cells to become endothelial cells, pericytes, or astrocytes.

Now that the cells have differentiated they are ready for step #2.

Step #2 – Add the differentiated cells to a specialized tissue culture plate

Next, Dr. Pedersen and her team add the newly differentiated cells into the wells of a specialized tissue culture chamber, the Mimetas platform.

After several hours the endothelial cells (ECs, cartoon in red) form a tube structure resembling a human blood vessel found in the brain. Pericytes migrate from the orange channel and surround the endothelial cell tube (purple cells below).

The image above in panel B is a microscopic examination of what the 3D stem cell model looks like when stained with fluorescently tagged antibodies.

  • Red = astrocyte marker
  • Purple = pericyte marker
  • Green = blood endothelial cell marker
  • Yellow = (merge of endothelial cells and astrocytes – green and red)

Although the pericytes are placed in well #3 image above, they migrate north to wrap around the blood vessel tube, mimicking the behavior of astrocytes in our brains.

The blood vessels resemble what is found in the brain, but do behave like a vessel in the brain should?

Does the 3D Stem Cell Model Work?

If these blood vessels behave like vessels in the brain they should form a tight barrier to prevent the entry of substances past the vessel wall.

To test this, Dr. Pedersen’s team pipettes a green dye inside the blood vessel channel (red channel) and measured how much green dye crosses to the “brain side” (orange channel).

Below is an image measuring how much green dye crosses the channel when no blood vessel is present (no cells, left) and when a blood vessel is present (cells, right).

Are vessels incubated with PANDAS serum more leaky?

To determine if there is some factor circulating in the blood of a PANDAS patient that increases the permeability of brain blood vessels, Dr. Pedersen’s team added green dye to blood vessel chambers that were incubated in PANDAS patient sera for 24 hours.

They observed more green dye leaking past the blood vessel compared to chambers that were incubated with sera from children without PANDAS (control sera, grey bar compared to black bar).