Astrocytes are an interesting autogenous population to target for direct neuronal programming. This protocol provides a reliable technique to isolating culture astrocytes with high purity from different region or the central nervous system. This protocol is designed and optimized to investigate the ability of astrocytes to be reprogrammed into functional neurons without confounding factors such as differences in astrocytes purity of different startup population.
Demonstrating the procedure will be done by Bob Hersbach, Postdoc in the lab, and Tatiana Simon a technical assistance in our laboratory. To begin, place the torso of a euthanized mouse in a 35 millimeter Petri dish and keep it on ice. Open the skin with scissors and remove the vertebra with small scissors.
Then extract the spinal cord and place it in a dissection buffer on ice. Under a stereotactic dissection microscope with two times magnification, remove the meninges from the isolated spinal cords using forceps and transferred dissected and cleaned spinal cord tissue to a C tube. Add 50 microliters of enzyme P from the neural tissue dissociation kit and 30 microliters of previously prepared enzyme mix to each C tube.
Invert the C tubes and place them on a heated dissociated ensuring that all tissue is collected in the tube's lid. Elude cells by removing the column from the separator adding 800 microliters of astrocyte culture medium and pushing cells out of the column with the included plunger. Plate cells in the previously prepared culture flasks by adding 4.2 milliliters of astrocyte culture medium supplemented with 10 nanograms per milliliters of EGF and bFGF.
It is not necessary to coat the culture flasks for astrocytes isolated from other brain regions but it provides a better substrate for astrocytes isolated from the spinal cord. Culture the cells at 37 degrees Celsius and 5%carbon dioxide. Perform astrocyte seating under a biological safety cabinet with a safety level one and prepare 24 well plates with Poly-D-Lysine coated glass cover slips as described in the text manuscript.
For plating, isolate six spinal cords from P2 mice yields around 1 million cells. Aspirate media from the T-12.5 culture flasks containing the cultured astrocytes and wash once with 1x PBS. Detach the astrocytes from the culture flask by adding 0.5 milliliters of 0.05%trypsin EDTA and incubate at 37 degrees Celsius for five minutes.
Gently tap the side of the flask to release cells from the culture flask surface and check detachment under a brightfield microscope using a 10 times magnification. Stop trypsinazation with 2.5 milliliters of astrocyte culture medium and collect cell suspension in 15 milliliter tubes. Centrifuge at 300 RCF for five minutes at four degrees Celsius.
Aspirate supernatant and resend cells in one milliliter of astrocyte culture medium. Calculate cell concentration using a hemocytometer or an automated cell counting system. Based on the number of cells, dilute the cell suspension with fresh astrocyte medium supplemented with EGF and bFGF at 10 nanograms per milliliter per factor.
Add 500 microliters of the cell suspension to each well of the previously prepared 24 well plates and culture cells at 37 degrees Celsius and 5%carbon dioxide. Prepare neuronal differentiation medium by adding one milliliter B27 supplement to 49 milliliters of basic culture medium. After 24 to 48 hours, depending on whether cells have been transduced or transfected, replace astrocyte medium with one milliliter neuronal differentiation medium per well.
Culture the cells at 37 degrees Celsius with 9%carbon dioxide. To increase the reprogramming efficiency, supplement the neuronal differentiation medium with forskolin and dorsomorphin to a final concentration of 30 micromolar and one micromolar respectively when replacing the astrocyte medium with the differentiation medium. When opting to treat cells with forskolin and dorsomorphin provide a second dose of dorsomorphin two days after the initial treatment.
Add this second treatment directly to the culture medium. Primary cultures of astrocytes reached 80 to 90%confluency between seven to 10 days after magnetic assisted cell sorting and plating. The day after plating, cells typically covered 50 to 60%of the cover slip surface.
Cultures majorly consisted of astrocytes at this stage, while other cell types such as neuro blasts were virtually absent. At seven days post transduction or transfection, induced neuronal cells were clearly distinguishable from astrocytes. The neuronal cell soma was smaller than unreprogrammed Astrocytes had long processes and was positive for neuronal marker, beta three tubulin, and negative for astrocyte marker GFAP.
At 21 days post transduction or transfection, many induced neurons were capable of firing action potentials and were positive for the mature neuronal marker NeuN and the pan synaptic protein Synaptophysin. It is essential to properly dissect the region of interest. Avoiding contamination from surrounding tissue.
Care must be taken to remove the meninges and the dorso root ganglia from the isolated spinal cord. This method can be used to isolate astrocytes from various regions of the central nervous system including the cerebral cortex, dorsal midbrain, and cerebellum. In the future, this technique will allow us to expand our knowledge on regional astrocytes and their potential to be reprogrammed into functional neurons.
