神経毒性試験のための、ヒト誘導多能性幹細胞のニューロンとグリアの混合培養物への分化のためのプロトコール

The overall goal of this procedure is to differentiate neural stem cells derived from human induced pluripotent stem cell colonies into mixed cultures of neuronal and glial cells. This model is suitable for drug and chemical toxicity screening. This in vitro model can be applied for assessing chemical induced perturbations of signaling pathways that are activated during the process of neuronal and glial differentiation.

The main advantage of this system is that use a human model derived from induced pluripotent stem cells that represents alternative to traditionally used cancer cells and animal primary cultures and allowing to study neurotoxicity in mixed population of neuronal and glial cells. The procedure will be demonstrated by Francesca Pistollato, our post-doc, and Carolina Nunes, a post graduate student from our lab. Begin by passaging the hiPSC colonies.

Working under a stereoscopic microscope at four X magnification in a laminar flow cabinet, use a one milliliter syringe with a 30 gauge needle. Cut the stem cell colonies in squares of about 200 microns by 200 microns. Cutting out the colonies requires good manual skills to obtain fragments of equal size.

The use of commercially available cutting tools can help. Then use a 200 microliter pipette to detach the colony fragments from the dish surface by gently pipetting the medium underneath to lift the pieces. Transfer 100 or so colony fragments to a qualified matrix DMEM F12-coated 60 millimeter dish filled with four milliliters of complete hiPSC medium.

Then incubate the new dish at 37 degrees Celsius and 5%carbon dioxide. Perform a total medium change every day and inspect the morphology of the colonies using a phase contrast microscope at four X and 10X magnifications. On day zero of the differentiation procedure, refresh the hiPSC medium by adding three milliliters of medium per 60 millimeter petri dish.

Then cut and detach the 200 micron fragments as before. Pipette the detached fragments and medium into a 15 milliliter tube. Rinse the dish with two milliliters of complete hiPSC medium and recover all fragments.

Then centrifuge at 112 times G for one minute. Following centrifugation, aspirate the supernatant and gently resuspend the fragments in five milliliters of complete hiPSC EB medium. Plate the full volume in a 60 millimeter ultra low attachment tissue culture dish.

Incubate the dish overnight at 37 degrees Celsius and 5%carbon dioxide. The next day check the cells with the inverted microscope. The day one embryoid bodies should appear rounded and distinct from one another as seen here.

Then pipette the embryoid bodies and medium from the dish and transfer to a 15 milliliter tube. Centrifuge the embryoid bodies at 112 times G for one minute. On day two aspirate standard matrix coating solution from a previously prepared 60 millimeter dish and add five milliliters of complete neuroepithelial induction medium, or NRI, to the dish.

Working under the stereoscopic microscope at four X magnification and using a 200 microliter pipette, collect around 50 floating embryoid bodies and transfer to one coated dish. Then incubate the dish at 37 degrees Celsius and 5%carbon dioxide. The next day, day three of the differentiation procedure, check the dish under the microscope at 10X magnification to ensure that the embryoid bodies are all attached.

Then gently perform a total medium change with complete NRI medium. Change the NRI medium every other day up to day seven when rosette-like neuroepithelial aggregates should be visible. On day seven, coat a 96-well plate by pipetting 100 microliters of standard matrix diluted in culture medium into each well.

On day eight, cut the rosette-like aggregates into fragments under a stereoscopic microscope at 10X magnification in sterile conditions. Note that the rosettes tend to easily detach from the dish when touched with the needle. In this case, partially disaggregate the detached rosette with the needle tip.

If the rosettes will remain partially attached, use a 200 microliter pipette to complete the detachment of the rosette fragments. Rosette cuttings require good manual skills and precision in order to avoid cutting no neurexidermal derivatives. Next collect the rosette fragments and their medium into a 15 milliliter conical tube.

Rinse the dish with two milliliters of NRI medium to recover all fragments. Then spin down the rosette fragments at 112 times G for one or two minutes. After aspirating the supernatant, gently resuspend the pellet in one milliliter of prewarmed one X DPBS without calcium and magnesium.

Gently pipette the rosette fragments up and down to partially dissociate them. Then add four milliliters of complete NRI medium and count the cells using trypan blue and an automated cell counter. Next aspirate the standard matrix coating solution from the 96-well plate and deposit the cells into the wells at a density of around about 15, 000 cells per square centimeter in NRI medium.

Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide. On day 10, perform a total medium change using complete neuronal differentiation medium. This representative image shows rosettes after 12 days in vitro stained for nestin in green and beta three tubulin in red.

Here cells that have been grown for 28 days in vitro have been stained for beta three tubulin in red and NF200 in green. This representative image shows neuronal cells differentiated from NSCs after 21 days in vitro stained for NF200 in red and tow in green. Here glial cells from the same culture are stained for GFAP in red.

This histogram compares quantification of nestin, MAP two, GFAP, gamma-amino butyric acid, vesicular glutamate transporter one and tyrosine hydroxylase positive cells in IMR90 hiPSC derivatives and cells differentiated from IMR90 hiPSC derived NSCs. These phase contrast images taken using a 10X objective show neuronal stem cells undergoing differentiation for 21 days. By following these procedures, other readouts such as quantitative real-time PCR and multilateral reanalysis can be performed in order to assess the physiology and the phenotype of neuronal and glial cells and assess the effect of chemicals.

Don't forget that the working with chemical compounds can be extremely hazardous. That's why your relevant precautions should be always taken using goggles, gloves or mask under the flow hood especially when performing chemical treatments. After watching this video, you should have a good understanding how to obtain mixed differentiated population of neurons and glial cells starting from human induced pluripotent stem cells which represents suitable model, in vitro model, for development on neurotoxicity testing.