The overall goal of this neuronal differentiation protocol is to generate neuronal networks from the human-induced pluripotent stem cells growing on microelectrode arrays in a rapid and controlled way. This method can be used to address key questions in the neuroscience fields. It can be used to study biological mechanisms underlying neurological disorders, but it can also be used to address more fundamental neurobiological questions.

The main advantage of this technique is the rapid differentiation of induced pluripotent stem cells into neurons in a controlled manner. On day one, warm DMEM/F12, CDS, and E8 medium with 1%penicillin streptomycin to room temperature. Next, add doxycycline to E8 medium to make a final concentration of four micrograms per milliliter and then add rock inhibitor to the mixture.

Aspirate the spent medium of the RTTANGN2 positive hiPSCs and add one milliliter CDS to the cells. Subsequently, incubate the cells for three to five minutes in a humidified 37 degree Celsius incubator. Under the microscope, check whether the cells are detaching from one another.

Then, add two milliliters DMEM/F12 in the well. Gently suspend the cells with a 1, 000 microliter pipette, and transfer then to a 15 milliliter tube. Afterward, add seven milliliters of DMEM/F12 to the cell suspension, and spin the cells at 200 x g for five minutes.

After five minutes, aspirate the supernatant and add two milliliters of the prepared E8 medium. Dissociate hiPSCs by putting the tip of a 1, 000 microliter pipette against the side of the 15 milliliter tube and resuspending the cells gently. Under the microscope, check whether the cells are dissociated.

Then, determine the number of cells per milliliter using a hemocytometer. For the six well MEAs, dilute the cells to obtain a cell suspension of 7.5 times 10 to the fifth cells per milliliter. For the cover slips, dilute the cells to obtain a cell suspension of four times 10 to the fourth cells per milliliter.

Aspirate the diluted laminin from the cover slips and the six well MEAs. For the six well MEAs, plate the cells by adding 100 microliters of cell suspension to the active electrode area in each well. Then plate the cells by adding 500 microliters of cell suspension to each well of the 24 well plate.

Next, place the six well MEAs or the 24 well plate in a humidified 37 degree Celsius incubator. After two hours, carefully add 500 microliters of the prepared E8 medium to each well of the six well MEAs. Subsequently, place the six well MEAs overnight in a humidified 37 degree Celsius incubator.

On day three, warm 0.05%Trypsin-EDTA to room temperature. Warm DPBS and DMEM/F12 with 1%penicillin streptomycin to 37 degrees Celsius. Then, aspirate the spent medium of the rat astrocyte culture.

Wash the culture by adding five milliliters of DPBS and swish it around gently. Afterward, aspirate DPBS and add five milliliters of 0.05%trypsin-EDTA. Swish the trypsin-EDTA around gently.

Then incubate the cells in a humidified 37 degree Celsius incubator for five to 10 minutes. Subsequently, check under the microscope to examine whether the cells are detached. Detach the last cells by hitting the flask a few times.

Then, add five milliliters of DMEM/F12 to the flask. Try to rate the cells gently inside the flask with a 10 milliliter pipette. Afterward, collect the cell suspension in a 15 milliliter tube.

Spin the tube at 200 x g for eight minutes. After that, aspirate the supernatent, and resuspend the cells in one milliliter of DMEM/F12. Determine the number of cells per milliliter using a hemocytometer.

Next, add 7.5 times 10 to the fourth astrocytes to each well of the six well MEAs. And add two times 10 to the fourth astrocytes to each well of the 24 well plate. Incubate the cells overnight in a humidified 37 degree Celsius incubator.

Acquire the data at 1, 200 times amplification, and sample the signal at 10 kilohertz using the MCS data acquisition card. Record 20 minutes of electrophysiological activity of hiPSC-derived neurons cultured on MEAs. During the recording, maintain the temperature at 37 degrees Celsius, and prevent medium evaporation and pH changes by delivering a constant slow flow of humidified gas onto the MEAs.

After that, analyze the data using a custom software packet. This figure shows the expression changes of neuronal markers MAP2 in synapsin during the differentiation process, which indications neuronal maturation. This graph shows that synapsin expression is measured for individual cells increased during the differentiation process.

The synapsin that is expressed in the cells after three weeks of differentiation co-localized with PSD-95, indicating the presence of functional synapses. Here, whole cell patch clamp was performed at different time points during the differentiation process to measure the electrophysiological activity of the cells. The cells were able to generate action potentials at the different time points.

And the percentage of spiking cells increased over time shows that the majority of the cells are able to generate action potentials, even in the early stage of differentiation. Patch clamp was also used to measure the excitatory postsynaptic currents received by the cells. The number of inputs that the cells receive increased during the differentiation process.

Both the frequency and the amplitude of the excitatory postsynaptic currents increased during the differentiation process. The electrophysiological activity of the cells differentiated on microelectrode arrays was measured during the differentiation process. Here, the neuronal network activity increased during the differentiation process, and showed synchronous events after 23 days.

Once mastered, this technique can be used to differentiate induced pluripotent stem cells into functional neuronal networks within three to four weeks. While attempting this procedure, it's important to remember to work sterile, and treat the cells gently. Following this procedure, other methods like pharmacological testing can be used to rescue the phenotype, observed in patients'cell lines.

After its development, this technique paved the way for researchers in the neuroscience field to officially study network activity defects in neurological disorders. After watching this video, you should have a good understanding of how to differentiate, induce pluripotent stem cells into neurons in a rapid and controlled manner.