The technique we present today allows for the analysis of spinal cord neuron activity as a result of intrinsic neuronal factor and the influence of surrounding astrocytes. This technique reliably generates both human neurons and human astrocytes that are spinal cord specific and uses a functional output measure that can be scaled for reproducibility. The technique we present allows to investigate spinal cord neurons specific diseases such as amyotrophic lateral sclerosis, using neurons from patients with either sporadic or genetic disease.
To begin with, examine the human iPSC plates for sufficiently dense colonies such that the center of the colonies displays scattered white areas. Then to start packaging the cells, draw grid lines on the bottom outer surface of the plates with a marker to facilitate accurate coverage of the entire plate. Use a phase contrast microscope of 10x magnification in a culturehood to detach differentiated colonies by manually scraping with a 200 microliter pipette tip.
Next, rinse the plates twice with PBS. And then add five milliliters of tissue dissociation protease pre-warmed at 37 degrees Celsius. Incubate the cells at 37 degrees Celsius for four to seven minutes until the edges of the colonies start to round up, prior to lifting colonies from the plate.
Carefully aspirate the dissociation protease and gently rinse the plate twice with PBS without dislodging the colonies. Add five milliliters of pre-warmed human iPSC medium to the culture plate. Then scratch the entire plate with a tip of a five milliliter pipette using a back and forth motion from top to bottom and lift the colonies while breaking them up into smaller cell aggregates.
For differentiation of human iPSC into neural progenitor cells. Once human iPSC forms a monolayer and becomes 90%confluent, start differentiation as described in the manuscript. On the 12th day, after trypsin treatment, if the cells are not in suspension, detached cells mechanically by ejecting neural induction medium from a 1000 microliter pipette tip onto the cells with a circular motion to cover the whole surface.
If the neural progenitor cell cultures were frozen, thaw them. After medium exchange and PBS rinsing as described in the manuscript, change the medium with a motor neuron differentiation medium containing 0.02 micromolar cytosine arabinoside then incubate the cells for 48 hours at 37 degrees Celsius and 5%carbon dioxide when glial committed progenitors emerge as single proliferating flat cells under post mitotic motor neuron progenitors, aggregated in cell clusters. Later, performed medium exchange as indicated in the manuscript.
On the day of plating or before, start coating the 24 well MEA plates by first diluting PLO in water or PBS. Then add 15 to 20 microliters of PLO to each well forming a droplet on the center of the well, covering the electrode area ensuring not to damage electrodes with the pipette tip. Add water to the compartments surrounding wells, ensuring sufficient humidity.
And incubate PLO at 37 degrees Celsius for one to two hours. Then using a plastic micro pipette tip, aspirate as much PLO as possible without touching the electrodes. Next, wash the well with 250 microliters of water three times.
Using a pipette tip, remove as much water as possible and let the surface dry with the lid removed. Next, add 15 to 20 microliters of diluted laminin to cover each electrode array. After adding water to the humidity compartments and replacing the lid, incubate at 37 degrees Celsius for a minimum of two hours up to overnight.
On the day of plating, after rinsing the motor neurons and astrocyte cultures once with PBS, add 0.05%trypsin and incubate at 37 degrees Celsius for about five minutes to lift cells. Collect cells into a 15 milliliter conical tube containing trypsin inhibitor. And wash plates with medium or base to ensure that all the cells are collected.
Pellet down the cells by centrification. And then using a 1000 microliter pipette, resuspend motor neurons and astrocytes with a co-culture medium containing 20 micromolar of ROCK inhibitor to generate one milliliter of a single cell suspension. Using a hemocytometer, count the motor neurons and astrocytes.
And while counting, kept the cell suspensions and place them in a styrofoam rack at four degrees Celsius. Centrifuge one milliliter of both cell suspensions at 300 times G for five minutes. Calculate the desired volume and resuspend the pellets.
Combine the required volume of neuron and astrocyte suspensions in equal ratios, and mix gently by pipetting twice to combine thoroughly. Then remove laminin from each well of the plate and transfer 10 microliters of the final combined cell suspension to each well, forming a small droplet covering the electrode array. Incubate the plates with an undisturbed cell droplet at 37 degrees Celsius for 20 to 30 minutes to form initial attachments on the plates.
Then pipette 250 microliters of warm co-culture media containing ROCK inhibitor down on the wall of each well and pipette the same volume on the opposite wall of each well and incubate the plate at 37 degrees Celsius for 24 to 36 hours. The next day, examine the plates for debris or dead cells. And if required, exchange the medium with a fresh co-culture medium containing ROCK inhibitor.
Otherwise, perform half medium exchanges twice a week using a co-culture medium without a ROCK inhibitor starting on co-culture day two. To perform recording, start as soon as possible after co-culture day one by setting the temperature to 37 degrees Celsius and carbon dioxide at 5%Then transfer plates to the recording machine and equilibrate for at least five minutes before recording. Record baseline activity every other day or weekly over one to 15 minutes depending on the experimental design.
To investigate the transient electrophysiological effects of compounds targeting either neurons or astrocytes, remove the plate lid but keep the machine lid closed and record baseline activity for a minimum of one minute. Manually open the machine lid without stopping the electrophysiological recording and exchange 25 microliters of medium with the appropriate drug vehicle. Then close the lid manually and record for additional minutes if required.
Similarly, test the drug of interest. In this protocol, hiPSCs were maintained and passaged as non-confluent colonies. Neurogenesis was initiated through dual SMAD inhibition by inactivating BMP and TGF-beta pathways.
The resulting neuro epithelial stem cells divided and generated a multi-layered epithelium. The early exposure to neuronal growth factors and a gliogenic ciliary neurotrophic factor generated a mixed population of progenitor cells. Neurons emerged spontaneously from this mixed population.
The gliogenic switch induced astrocyte differentiation through activation of the JAK-STAT pathway. The spinal cord identity of notch inhibitor treated and differentiated motor neuron cells was supported by high choline acetyltransferase expression levels. On the 90th day post in vitro culture, human iPSC astrocytes displayed maturing phenotype as indicated by S100 beta and GFAP expression while their spinal cord regional identity was supported by expression of hawks before.
Co-cultured cells rearranged spontaneously with astrocytes creating a feeding layer at the bottom and neurons connecting networks at the top. Neurons aggregated in large cell clusters when cultured alone, while the co-culture of human iPSC motor neurons with human iPSC astrocytes resulted in evenly distributed monolayers. Human iPSC astrocytes enhanced the electrophysiological maturation of human iPSC motor neurons as shown by higher degrees of spiking and bursting activity in the co-cultures.
In our experience, the stem cell culture and early NPC differentiation are the most difficult and sensitive to technical error. At this stage, the techniques must not be performed with consistency and accuracy with little margin for troubleshooting. The co-culture techniques described here can be used in other cell type specific models as well.
