The overall goal of this procedure is to induce migration in typically non-migratory cells, to identify specific compounds that activate or block migration. This method can help to answer key questions in the field of cell migration and invasion, and may be of particular interest to scientists working with neural stem cells. The main advantage of this technique is that non-migratory and non-invasive cells can be analyzed during the process of becoming migratory and invasive.
The implications of this technique extend toward the identification of novel compounds that block epithelial to mesenchymal transition, or EMT, which occurs in human high-grade gliomas believed to derive from neural stem cells. Though this method can provide insight into EMT and neural stem cells, it can also be used to study other epithelial cancers, such as breast, lung, or colon cancer. On embryonic day 14.5, confirm the correct age of the rat embryos by the presence of beginning digit formation in a rat forelimb.
Next, transfer the embryos into individual, uncoated petri dishes filled with ice-cold PBS, which are placed under a stereo microscope. Use a pair of fine-tipped forceps to remove the embryonic hulls. Keep embryos in expansion medium on ice.
Using two autoclave-sterilized fine forceps, and starting at the intersection of the developing telencephalon and diencephalon, pull simultaneously anteriorly and posteriorly, to remove the head skin and skull. Then, identify the midbrain-hindbrain boundary separating the mesencephalon from the rhombencephalon, and cut the rhombencephalon just at or below the midbrain-hindbrain boundary. Holding the block of the mesencephalon with a pair of forceps, sever the connection between the telencephalon and diencephalon at the skull base with the facial skeleton, and immerse the telencephalon, diencephalon, mesencephalon block in an uncoated petri dish of expansion medium on ice.
When all of the neural blocks have been harvested in the same manner, transfer one telencephalon, diencephalon, mesencephalon block into fresh, ice-cold PBS under the microscope. To separate the mesencephalon from the diencephalon, first sever the tissue between the diencephalon and the telencephalon. Next, split the two telencephalic hemispheres, and identify the medial and lateral ganglionic eminences, which are visible through the future foramen interventriculare.
Dissect along a straight line at the intersection between the medial and lateral ganglionic eminences and the anterior cortex, followed by a straight line through the cortex, hippocampus, and choroid plexus. Cut a second straight line through the cortex, hippocampus, and choroid plexus at the intersection of the caudal ganglionic eminence and the posterior cortex. Then, flatten out the telencephalon, identify the cortical hem containing the hippocampus and the choroid plexus.
Separate the cortical hem from the cortex, leaving the size of the cortex identical to the size of the ganglionic eminences. Flip the cortex-ganglionic eminence block so that the ventricular side is touching the dish surface, and the meninges are facing up. Pinning the ganglionic eminences to the dish surface with one hand, use forceps in the other hand to peel off the meninges.
Then, cut each cortex along the lateral ganglionic eminence, about half of the distance of the diameter of the lateral ganglionic eminence. To prepare the explant cultures, cut the cortices into explants of less than 400 microns in diameter, using a 400 micron grid below the dissection dish as a reference if necessary. Then, transfer all of the explants into a fresh petri dish with ice cold expansion medium.
Next, remove the excess fibronectin from a previously-coated 35 millimeter tissue culture dish and allow the dish to dry. Then, place the fibronectin-coated dish over a 10 by 10 millimeter grid and add one milliliter of cold expansion medium to the center of the dish, allowing the medium to form a spherical drop. Place up to eight explants into the center of the drop with at least three millimeters of space between each piece of tissue.
Then, carefully place the dish in a cell culture incubator without shaking to allow the explants to attach to the fibronectin-coated surface. After one hour, fill the dish to a final volume of two milliliters of expansion medium plus the growth factors or test substances of interest, and return the plate to the incubator. In this experiment, explants were seeded at the center of 500 micron-grid culture dishes and cultured in different growth factors.
In the control and TGF Beta 1 alone treated explants, a strong proliferations, coupled with almost no migration was observed. BMP4 however, induced an intermediate cell migration and proliferation response. With the combination of BMP4 and TGF Beta 1 inducing the strongest migration and proliferation of all the groups.
Once mastered, the neural stem cell can be isolated from 10 to 15 embryos in approximately two hours. While attempting this procedure, it is important to maintain all of the embryos and cell explants in ice-cold medium. This cost-efficient and highly-reproducible system provides the flexibility of using either single cells or explant cultures to study cell migration and invasion in vitro.
Culturing the cells in large multi-well plates allows analysis of the effects of a variety of compounds on migration and invasion in parallel. Further, the system can be easily adapted for high throughput screening of novel compounds. After watching this video, you should have a good understanding of how to isolate neural stem cells with the potential to undergo EMT from the appropriate region of the developing cortex.
