This protocol allows a non-invasive transplantation of iPSMG into immunocompetent mouse brain by combining pharmacological on/off of a CSF1 receptor antagonist PLX5622 with transnasal transplantation. The main advantage of this technique is that iPSMG can be transplanted without inducing brain damage even in immunocompetent mice. Microglial transplantation may serve as a potential therapy to exchange dysfunctional microglia with functional ones in various neurodegenerative diseases in the future.
Demonstrating the procedure will be Bijay Parajuli, a researcher from my laboratory. Begin by quickly thawing the frozen induced pluripotent stem cell-derived human microglia cells in a 37-degree Celsius water bath. Swirl the samples until all visible ice has melted.
Add the thawed iPSMG to the culture media, warmed to 37 degrees Celsius. Add one milliliter of cells to 10 milliliters of culture media. Centrifuge the cells at 300 times g for five minutes to obtain a cell pellet.
Remove the supernatant without disturbing the cell pellet. Add the transplantation medium to obtain a cell concentration of 10 to the fifth cells per microliter. Place the iPSMG on ice, and immediately proceed to transplantation.
To prepare mice for transnasal transplantation, feed the male mice with a diet containing PLX for seven days. At the end of the seventh day, cease the PLX diet and feed the mice with a normal diet. One hour before transnasal transplantation of iPSMG, administer 2.5 microliters of hyaluronidase in PBS to each nostril twice using a 10-microliter pipette tip to increase the permeability to the nasal mucosa.
Place the mice in the supine position after the hyaluronidase application. Administer 2.5 microliters of hyaluronidase ten minutes before the transnasal transplantation of iPSMG. Apply 2.5 microliters of cell suspension into one nostril of the mouse using a 10-microliter pipette tip.
Place the mouse in the supine position for five minutes before administration of cell suspension to the other nostril. Administer the cell suspension four times, applying a total volume of 20 microliters per animal. 48 hours after the cessation of PLX feeding, repeat the administration of hyaluronidase and cell suspension on the same mice.
For administration of cytokines, apply 2.5 microliters of the transplantation medium into one nostril of the mouse using a 10-microliter pipette tip. After two months of transnasal transplantation, the number of transplanted cells can be determined by counting cells that are positive for both human-specific and pan-microglial markers. Endogenous mouse microglia are positive for pan-microglial marker only.
In control mice, only mouse microglia were detected in both cortex and hippocampus. In the cortex of iPSMG transplanted mice, only mouse microglia were detected, whereas in the hippocampus iPSMG were detected. When attempting this protocol, complete removal of supernatant is essential to prevent the dilution of cytokines.
It is crucial to apply cytokine every 12 hours for the viability of transplanted cells. Depletion of endogenous mouse microglia is required for the transplantation of iPSMG. This procedure allows the transplantation of iPSMG into a normal or diseased mouse brain.
Thus, characteristic of iPSMG as well as the response to disease can be determined. This technique is suitable to determine the in vivo characteristic of iPSMG in the mouse brain. Thus, transplanting iPSMG into disease model mice will help us understand the response and may pave the way for a new therapeutic target.
