This approach allows for investigating cellular identity, integration, and functionality of transplanted interneurons over extended time in developing healthy and diseased brains. The protocol describes a fast and highly efficient generation of human stem cell-derived GABAergic interneuron like precursors that survive and mature into the hippocampus of naive and transgenic mice. This approach help us evaluate the therapeutic potential of using cell therapy in developmental disorders where interneurons are compromised.
To begin, remove the cell culture medium from the human-derived interneuron precursors and carefully rinse with DPBS without calcium and magnesium. Add 400 microliters of the cell detachment solution to each well of the six-well plate. Incubate for two to three minutes at 37 degrees Celsius in the incubator until the border of the cells starts to look shiny.
Then, add 600 microliters of fresh N2 medium to each well of the six-well plate to stop the cell detachment solution and mechanically detach the cells using a pipette to obtain a single-cell suspension. Transfer the cell suspension to a plastic tube and centrifuge at 180 G for four minutes at room temperature. Discard the supernatant using a vacuum system and resuspend the pellet in the transplantation medium.
Count the total cells in the suspension using a counting chamber and a tally counter, and adjust the volume to a final concentration of 100, 000 cells per microliter. Keep the cell suspension in a closed tube on ice until transplantation for a maximum of four hours. Immediately after anesthesia, place the pup onto a wet tissue on the surface of wet ice for three minutes until the upper limbs become whitish.
Resuspend the cells carefully with the pipette and load the syringe with the cell suspension. Position the pup by placing it on the stereotaxic frame and using the ear bars in the opposite direction. Next, clean the surface of the skin using a soft tissue soaked in ethanol.
Identify lambda and set the coordinates to zero on the digital display console of the stereotaxic instrument. After relocating the Hamilton syringe to the desired coordinates, use a 90-degree bent insulin needle to penetrate the skull, creating a tiny hole. Then, bring the Hamilton syringe down until the needle crosses the skull and zero the dorsoventral coordinate.
Lower the needle until the desired dorsoventral coordinates are achieved. Retract the needle slowly once the stem cells have been injected. End the procedure by warming up the pup with the hands until it starts moving before giving it back to the mother.
No immune reaction or local inflammation against the transplanted cells was found either at postnatal day 14 or two months post transplantation, as assessed by the absence of reactive microglia identified using Iba1, CD68 and galectin-3. The extent of astrogliosis is determined by the glial fibrillary acidic protein and inflammatory cytokines, such as interleukin-1, and the absence of cytotoxic lymphocytes. In the Cntnap2 knock-out mice, the human stem cell-derived interneuron-like cells survived up to nine months post transplantation and were localized at the injection site.
Although, they were also dispersed across the ipsilateral and even the contralateral hippocampus, as observed in the wild-type mice. The protocol requires practice to ensure the minimal disruption and compression of the brain, as well as keeping a good balance between the speed of the procedure and the precision of the coordinates. This protocol is compatible with a range of techniques, such as connectivity studies or functional readouts like electrophysiology or behavioral studies, depending on your research, question of interest.
