An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

The overall goal of the following experiment is to establish a co-culture system for evaluating the ability of human-induced pluripotent stem cell derived neurons to form functional connections using an genic approach based on photo stimulation of cultured neurons via channel RUSIN two or CR two. This is achieved by first isolating primary mouse neural progenitor cells or NPCs, and allowing them to differentiate into astrocytes. Next primary rat cortical neurons are transfected with CH R two and plated onto the astrocyte layer, which serves as the existing neuronal population.

Then human NPCs derived from induced pluripotent stem cell or IPSC cultures are transduced with synapsin one 2D tomato virus, then seated onto the astrocyte neuron culture and induced to differentiate subsequently forming synaptic connections. The results show that the IPSC derived neurons form synaptic connections with rat cortical neurons based on electrophysiological recordings of postsynaptic currents in the IPSC derived neurons upon genic photo stimulation of the CHR two expressing cortical neurons. The main advantage of this matter over the existing ones like those co culturing of only astrocytes with human IPSC derived neurons is that the maturation time of this human neurons is shorter.

This is because the presence of primary neurons can accelerate maturation of this human neurons demonstrating the procedure will be Uni Chin, a graduate student from my laboratory and suing Y from George Augustine's laboratory After harvesting embryonic rat brains and isolating the cortical tissue according to the text protocol, transfer the cortical tissues to a dish containing the prewarm cortical tissue digestion solution. Mince the cortical tissues as fine as possible before incubating at 37 degrees Celsius for 30 minutes. After the incubation, transfer the cortical tissues to a 50 milliliter centrifuge tube, dissociate the tissues by using a serological pipette until a homogenous solution with no tissue pieces is produced.

Pass the tissue solution through a 70 micron cell strainer to filter out any remaining tissue clumps. Aliquot three milliliters of solution into 15 milliliters. Centrifuge tubes.

Add 800 microliters of 7.5%BSA in one XPBS to the bottom of each tube, forming a layer underneath the tissue layer centrifuge at 200 Gs for five minutes. Then remove the supernatant by aspirating from the interface of the two layers. Avoid disturbing the selp pellet at the bottom of the tube.

After using one milliliter of primary neuron medium to resuspend each cell palette, combine them all in a 15 milliliter centrifuge tube. Then use a hemo cytometer to determine the cell density centrifuge. 6 million rat cortical neurons at 200 Gs for five minutes, and remove the supernatant.

At least 6 million cortical neurons are required for electroporation. To ensure the formation of a neuronal network by surviving neurons, add 100 microliters of electroporation solution to the cell pellet and gently resuspend. Then combine 100 microliters of the cell suspension with six micrograms of CR two plasmid and transfer it into a certified vet.

Select and apply the appropriate program for electroporation according to the manufacturer's instructions. Following electroporation, add 500 microliters of MEM to the vet. Transfer the cells into 11.5 milliliters of primary neuron, medium, and gently resus suspend.

Then add 500 microliters into each well of a 24 well plate containing mouse astrocytes. Incubate the plate at 37 degrees Celsius and 5%CO2. After generating induced pluripotent stem cells or IPCs, induce and maintain the cells as a human neural progenitor cell or NPC culture according to the text protocol.

In this example, the NPCs are labeled using the lentiviral vector synapse in one TD tomato the day after adding primary neurons to an astrocyte culture. Use one XPBS to wash human NPCs once, then add cell detachment solution and incubate for five minutes at 30 degrees Celsius. Ensure that all the cells have detached.

Then transfer them into a 15 milliliter tube containing twice the amount of D-M-E-M-F 12 and centrifuge at 200 G for five minutes. Remove the supernatant and use neuronal differentiation medium to gently resuspend the pellet into single cells. Use a hemo cytometer to count the cells.

Next, carefully aspirate the medium from the astrocyte cortical neuron cultures plate. The human NPCs at 5, 000 cells per square centimeter per well in neuronal differentiation.Medium. Incubate at 37 degrees Celsius and 5%CO2, and replace the medium every two or three days for at least 28 days.

To confirm whether human IPCs behave like mature neurons, use a confocal microscope equipped with a 60 x water immersion lens to find cells differentiated from human IPCs. By visualizing TD tomato. Pull a recording pipette to a resistance of four to six ohm perfuse.

The cells at room temperature in an external solution bubbled constantly with 95%oxygen, 5%carbon dioxide. Use an internal solution to fill the recording micro pipette patch a TD tomato positive cell in whole cell mode and record action potential firing in response to current injection in the current clamp. To confirm that the action potential of cortical cells expressing CHR two is reliably evoked by light stimulation.

First, find cortical cells expressing CR two by visualization of GFP through light illumination with a mercury arc lamp, and using a wavelength of excitation filter of 480 over 40 nanometers. Check that an action potential is reliably evoked via current clamp recording in the whole cell mode. To perform genic stimulation, patch a TD tomato positive cell in whole cell mode and set the voltage clamp at negative 70 millivolts with the whole field mercury lamp and a 480 over 40 excitation filter.

Stimulate the whole field for 30 seconds. Record postsynaptic currents or PSCs of a patched cell induced by photo stimulation of CHR two expressing presynaptic cortical neurons. Set the threshold for amplitude and currents area at five Pico Amp here and PICO respectively manually inspect these events to discard any non PSC traces.

As demonstrated in this video, several steps are required to generate the co-culture of neurons derived from IPCs primary cortical neurons and astrocytes. This image shows that at day two of differentiation of mouse NPCs into astrocytes, glial fibrillary acidic protein or GFAP positive astrocytes can be readily observed. Mouse NPCs were allowed to differentiate for at least a week before plating cortical neurons, expressing CR two onto the astrocyte monolayer, and both are identified in this co-culture through immunofluorescent staining of GFAP and CHR two.

After three to four weeks of human NPC differentiation, TD tomato positive neurons were detected in the cultures as shown here. When stimulated by light action potentials were generated in cortical neurons expressing CHR two IPSC. Derived neurons are also excitable, showing increased action.

Potential firing as the amplitude of depolarizing current pulses was increased. In the absence of light IPSC derived neurons received spontaneous synaptic inputs. These inward post-synaptic currents were predominantly mediated by AMPA receptors.

Photo stimulation of the cortical neurons was sustained throughout the 32nd long light flash while the frequency of PSCs were elevated. When CHR two expressing cortical neurons were photo stimulated, their amplitude was unaffected. Following this procedure, we can use other co-culture systems with different patient specific cell types in order to answer additional questions such as the effect of candidate drug compounds on specific diseases of interest.