The overall goal of this protocol is to deliver neural stem progenitor cells to the CNS via intravenous injection in mice affected by experimental autoimmune encephalomyelitis, a disease model that mimics multiple sclerosis. This is accomplished by first deriving neural stem progenitor cells, or NPCs from the subventricular zone of the brain from adult mice. The second step is to genetically tag NPCs with viral vector technology for in vivo cell identification and tracking.
Next proliferating neurospheres are dissociated into a single cell suspension, resuspended at the desired density and injected into the tail vein of EAE mice at the peak of disease. Ultimately, postmortem organ and tissue pathology and immunohistochemistry are performed to identify and characterize the survival distribution and integration of injected stem cells. The main advantage of this technique is that it provides a minimally invasive, safe, and effective method to transplant stem and progenitor cells into the inflamm nervous system.
This method helps answer three key questions related to the safety and efficacy of systemically injected stem cells in models of inflammatory diseases of the nervous system. The visual demonstration of this method is useful especially for young scientists as it helps in the understanding of critical experimental steps from brain stem cell isolation to preparation for systemic injection in a mouse model for multiple sclerosis, The first step is to gather together all of the solutions needed for dissection. Using proper sterile technique.
Place a freshly isolated brain on a brain slicer matrix. Use two razor blades to produce coronal sections, two millimeters from the anterior pole of the brain, excluding the optic tracks and three millimeters posterior to the previous cut. Place the brain slices on a clean Petri dish and work under a dissecting microscope to isolate the subventricular zone tissue.
After mincing the tissue, add it to 30 milliliters of previously prepared and filtered digestion solution. Shake gently and incubate for 30 minutes shaking every 15 minutes. After centrifugation, remove the supernatant and resuspend the pellet in 200 microliters of complete growth.
Medium or CGM by pipetting. First with a P 1000, and then with a P 200 pipette. Once a single cell suspension is reached, bring it up to five milliliters with CGM and transfer to a new flask.
Place the cells in an incubator and allow neurospheres to form for five to seven days. When ready, collect the suspension into a 15 milliliter tube and centrifuge. Remove the supernatant and resuspend the palate in 200 microliters of CGM.
Dissociate the neurospheres mechanically by passing them through a P 200 pipette tip 100 to 150 times. Bring the suspension up to one milliliter with CGM. Next, accurately dilute the suspension.
Mix the appropriate volume with a vital stain and fill a hemo cytometer to determine the cell count. Resuspend the cells in the correct concentration and transfer to a new flask. Repeat these steps every four to five days to help the identification of transplanted cells in vivo.
NPCs are transduced with viral vectors coding for reporter genes 72 hours after NPC transduction. The expression of fluorescent reporter genes is verifiable by either immunofluorescence or flow cytometry. When ready, harvest the GFP expressing neurospheres by centrifugation and remove the supernatant.
Next, add 200 microliters of cell aggregate dissociation solution and incubate for 10 minutes. Retrieve the cells and gently resuspend them to obtain a single cell suspension. Taking care to avoid air bubbles.
After counting and diluting the cells, keep the cell suspension on ice until ready to use. Once the neurospheres are ready, retrieve mice that were previously prepared to serve as a mouse model for CNS. Damage treatment should occur at the peak of disease expression.
Begin by placing the mice into a warming box for about 10 minutes to allow the tail vein to dilate. When ready, transfer one mouse from the warming box to a mouse restrainer. Close the restrainer to restrict movement and isolate the tail.
Sterilize the injection area on the tail with an appropriate topical solution. Fill a one milliliter insulin syringe with 150 microliters of cell suspension and make sure to remove all the bubbles. Localize the vein on the dorsal surface of the tail and position the needle parallel to the vein at the mid lower part of the tail.
Here the vein is closer to the surface of the skin. Thus facilitating the procedure. Gently insert the needle into the vein assuring its correct position by withdrawing a few microliters of blood.
Then slowly inject the solution into the tail vein After the injection, remove the needle and apply pressure for a few seconds to slow the bleeding. Place the mouse in a fresh cage and monitor until recovered. Repeat the same procedure for all the mice at predetermined experimental time points.
Samples are collected from treated and control animals and analyzed to determine infiltration and integration of NPCs into the CNS and other tissues when proliferating as neurospheres NPCs express markers of mitotic activity such as phospho hisone H three shown in green and of undifferentiated neural cells like nest in shown in red when plated under appropriate differentiation conditions, NPCs express markers typical of the three neural lineages such as the astroglia marker, GFAP, the neuronal marker, micro tubular associated protein two, and the oligo dyl glial markers O four and myelin basic protein nuclei are counter stained with dpi undifferentiated MPCs that are to be therapeutically efficacious after transplantation must express cell surface adhesion molecules that include CD 44 and the alpha four subunit of the very late antigen four. These examples show that transduced NPCs express GFP, both as proliferating neurospheres as well as when differentiating in vitro upon growth factor withdrawal. Syngeneic NPCs injected systemically in EAE mice are found almost exclusively in perivascular areas of CNS damage indicated by the arrows both in the brain and spinal cord up to 45 days post transplantation.
A key step of this procedure is cell dissociation. In fact, the preparation of single cell suspension is fundamental to avoid clamps or aggregates that may lead to vessel occlusion After its development. This technique paved the way for researchers in regenerative medicine that were exploring minimally invasive protocols for efficient delivery of non periodic stem cells into the brain of small laboratory animals, and in close perspective humans affected by inflammatory neurological diseases.
After watching this video, you will hopefully have a clear understanding of the troubleshoots related to the isolation, preparation and systemic injection of tissue specific stances in laboratory animals with brain diseases.
