My group is interested in the mechanisms that regulate gene expression in neural progenitor cells of the developing neocortex. And particularly, we focus on epigenetic mechanisms. These are important for the potential of neural progenitor cells to proliferate and differentiate, and therefore for proper brain development and function.
Cortical organoids are exciting new models that allow us to study the development of the human brain. They also allow us to model the development of other primate species. In this protocol, we describe the electroporation of human cortical organoids to study gene function.
The organoids are sliced with a vibratome, which makes them particularly accessible for injection and electroporation. The electroporation of human cortical organoids represents a powerful technique to study gene function. We've used the method to study the primate express growth factor EPIREGULIN, which is implicated in brain development and evolution.
We've also used the method to study the activity of enhancer regions from different primate species. To begin, embed the human cortical organoids derived from human-induced pluripotent stem cells in 3%low melting point agarose. Slice the organoids on a vibratome into 500-micrometer thick sections.
After slicing, continue culturing organoids in a six-well ultra-low attachment plate containing four brain medium three on an orbital shaker at 120 rpm. Next, set the microcapillary puller to the specified settings to pull the borosilicate glass capillaries for injections. After pulling, store the capillaries in a large Petri dish and secure them with tape.
On the day of electroporation, pre-warm Tyrode's solution to 37 degrees Celsius and prepare the CRISPR Cas9 RNP at a final concentration of 24 micromolar. Add pCAG-GFP plasmid and 0.1%Fast Green solution in water to the final injection mix. Select the human cortical organoids with well-developed ventricle-like structures and discard the organoids that lack these structures.
Transfer up to 10 organoids to a 6 centimeter Petri dish containing Tyrode's solution. Using a microloader pipette tip, fill a glass microcapillary with 10 microliters of the injection solution. Open the capillary by pinching off the thin end with tweezers.
Check if the capillary is open by releasing some of the injection mix into Tyrode's solution. Insert the capillary into the center of the ventricle-like structures, and press the foot switch of a microinjector to inject 0.2-0.5 microliters of the mix. Use fine forceps to push the organoid to restrict its movement without grabbing it.
After injection, using a wide bore pipette tip, transfer one to two injected organoids into an electroporation chamber containing Tyrode's solution. Orient the injected organoids toward the electrodes, ensuring that the apical radial glia and ventricle-like regions face outward. Attach the cables to the electroporation chamber with the injected side of the organoid facing the positive pole.
Press the Pulse button on the electroporator to deliver five pulses of 38 volts for 50 milliseconds each at 1 second intervals. After electroporation, return the human cortical organoids to the four brain medium three, and culture them without shaking for 24 hours. After fixation and cryosectioning of electroporated human cortical organoids, perform antigen retrieval in citrate buffer for one hour at 70 degrees Celsius in a water bath for immunohistochemical analysis.
Wash the sections once in PBS for five minutes. Then dry the slides briefly and use a wax pen to circle the sections. Quench the sections using 0.1 molar glycine in PBS for 30 minutes at room temperature.
Then wash the sections twice with PBS for five minutes each. Add blocking buffer onto the sections and incubate for 30 minutes at room temperature. Then incubate the section in primary antibodies overnight at 4 degrees Celsius.
The next day, wash the sections three times with PBS for five minutes each. Incubate the sections with secondary antibodies and DAPI in a blocking buffer for one hour at room temperature. After washing the section with PBS, mount the slide with mounting medium and image the sections using a fluorescent microscope.
Immunohistochemistry of electroporated organoids revealed multiple targeted ventricles within an electroporated cortical organoid with optimal electroporations targeting ventricle-like structures located in the outer regions of the organoid. Troubleshooting is necessary if excessive cell death is observed. This may be caused by high plasmid concentrations or suboptimal electroporation settings.
Electroporations facing the interior of the organoid are often more difficult to interpret due to cortical plate-like areas merging with adjacent ventricles. Disruption of the apical surface may occur when excessive injection volume or strong electrical pulses are applied, leading to ventricle ruptures and cell delamination. Knockout of the Polycomb repressive complex 1 protein PCGF4 was confirmed by immunohistochemistry, showing reduced signal intensity in GFP positive cells.
Signal intensity analysis of GFP positive cells revealed a significant reduction in PCGF4 protein levels in knockout samples compared to controls.
