標準化され、再現性のある前脳型人間から脳オルガノイドの生成誘導多能性幹細胞

The overall goal of this procedure is to generate highly standardized and reproducible forebrain-type organoids from human pluripotent stem cells. This method is a powerful tool for modeling mechanisms of new development NTZs. In particular, it can help to address key questions associated with early human cortical genesis.

The main advantage of this technique is that it allows the standardized and time-efficient generation of human cortical tissue with little variations between individual organoids and organoid batches. Organoid technology can provide insight into the development, structure, and function of the human brain, especially for those aspects not found in lower species'brain models. To generate pluripotent stem cell aggregates, when the stem cell cultures reach 70 to 90%confluency, add 500 microliters of cell dissociation reagent to one well of the six well culture plate to detach the cells.

Induced pluripotent stem cells, which are adapted to monolayer culture conditions, are a good cell type to generate aggregates as they are less prone to stress-induced cell death during the dissociation and aggregation procedure. After five to 10 minutes at 37 degrees Celsius gently tap the plate, and use two milliliters of DMEM/F-12 to wash the cells from the bottom of the well. Transfer the resulting cell suspension into a 15 milliliter conical tube, and bring the volume of the cell solution up to 10 milliliters with more DMEM/F-12 medium.

After counting, transfer 4.5 times 10 to the third cells per pluripotent stem cell aggregate into a new 15 milliliter tube, and collect the cells by centrifugation. We suspend the pellet in the appropriate volume of pluripotent stem cell medium, supplemented with 50 micromolar rock inhibitor to achieve a 4.5 times 10 to the third cells per 150 microliters of medium concentration. Next, add 150 microliters of cells to individual wells of a 96-well low attachment U-bottom plate, and place the plate in a 37 degree Celsius and 5%carbon dioxide incubator.

For monitoring anterior neuroectoderm induction, use a tissue culture microscope to closely observe the morphologic changes of the pluripotent stem cell aggregates every day under low magnification. On day one, cell aggregates with clear borders should be observed. On day two, carefully aspirate approximately two thirds of the medium, without disturbing the cell aggregates at the bottom of each well, and replace the discarded medium with 100 microliters of fresh pluripotent stem cell medium.

Between four and six days later, when the cell aggregates reach 350 to 450 micrometers in diameter, and exhibit smooth edges, use a modified 100 microliter pipette tip to transfer up to 20 aggregates into a single six centimeter low attachment culture plate containing five milliliters of cortical induction medium. Replace the cortical induction medium with fresh medium once every three days, monitoring the morphology of the aggregates daily under the 4X objective. After four to five days in the cortical induction medium, the edges of the cell aggregates should begin to brighten at the surface, indicating neuroectodermal differentiation and the radial organization of a pseudostratified epithelium should emerge.

To embed the neuroectodermal aggregates in a matrix scaffold thaw basement membrane extract on ice for two to three hours. While the extract is thawing, use sterile scissors to cut plastic paraffin film into one four by four centimeter square per 16 organoids, and place each piece of film over an empty 100 microliter micropipette tip tray. Press the paraffin film with a gloved fingertip so that small dimples appear, and clean the film with 70%ethanol.

After UV radiation in a closed, sterile biosafety cabinet for 30 minutes, use a modified 100 microliter pipette tip with a one and a half to two millimeter opening to transfer each cell aggregate into one dimple in the film. When all of the aggregates have been transferred, use an uncut 100 microliter pipette tip to carefully aspirate the medium from each dimple, and add 40 microliters of undiluted basement membrane extract to each cell aggregate. Be careful not to damage the organoids by rough manipulation or aspiration into the pipette tip as this would damage the developing neuroepithelium.

Use the pipette tip to position the aggregates into the middle of each drop, and use sterile forceps to carefully transfer the plastic paraffin film sheet into a 10 centimeter petri dish. Place the dish in the incubator for 15 to 20 minutes. While the extract is solidifying, add five millimeters of fresh cortical induction medium to one six centimeter low attachment culture dish.

At the end of the incubation, flip the paraffin film sheet over and use sterile forceps to gently squeeze up to 16 polymerized droplets into each six centimeter dish. Then return the aggregates to the cell culture incubator. The next day, transfer the organoid culture dishes onto a rocking cell culture shaker, tilted at a five degree angle at 14 rpm within a cell culture incubator, with daily light microscopy monitoring, until the aggregates reach the differentiation stage of interest.

On day 20, use a modified one milliliter pipette tip with a three to three and a half milliliter opening to collect six organoids from the aggregate cultures. Place three of the organoids into one well of a 24-well plate containing 500 microliters of PBS, and use a five milliliter pipette to wash the organoids two times with 500 microliters of fresh PBS per wash. After the second wash, fix the organoids in cold 4%paraformaldehyde for 15 minutes, followed by three 10 minute washes in room temperature PBS, and a final overnight dehydration in 30%sucrose solution at four degrees Celsius.

The next day, pre-stain the dehydrated organoids with a one to 50 Trypan Blue dilution for 10 minutes to allow visualization of the organoids during the cryosectioning procedure, and replace the sucrose with freshly prepared embedding medium. Heat the plate on a 60 degree Celsius heating pad for 15 minutes to equilibrate the organoids in the embedding medium, and cover the bottom of one embedding mold per organoid with fresh embedding medium. Place the mold on ice to solidify the embedding medium, transfer the organoids to the embedding molds, and add fresh embedding medium until each organoid is covered.

Then shock freeze the organoids in a 100%ethanol dry ice freezing bath for at least one minute, and use a cryostat to obtain 20 micrometer thick sections of each organoid for immunocytochemical analysis. To generate forebrain-type organoids, use only IPSC cultures that present as a homogeneous monolayer of undifferentiated cells in the starting population. On day two, aggregates should have formed compact cell buds with smooth edges.

Day 10 aggregates should show smooth and optically smooth and optically translucent tissue on the outer surface, representing the induction of the neuroectoderm. If the protocol has been closely followed, highly standardized organoid batches that demonstrate greater than or equal to 90%homogeneity in polarized neuroectoderm formation within and across batches will be generated. Immunocytochemical analysis of day 20 organoids reveal stratified neuroepithelial loops that express the neural stem cell marker Sox2, the forebrain markers Pax6 and Otx2, and the dorsal cortical marker Emx1.

These cortical loops are further characterized by an apical localization of N-cadherin and ZO-1, ventricular zone radial glial cell derived microtubule, which spans from the apical to the basal side of the structures, and apical located dividing cells that stain positive for phosphorylated vimentin. To analyze the division plane of apical radial glial cells double staining for vimentin and Tpx2, a microtubule associated protein that can be used to visualize the mitotic spindle and the apical processes during interkinetic nuclear migration can be performed. After watching this video, you should have a good understanding of how to generate highly standardized and homogenous forebrain-type organoids from human pluripotent stem cells.

This technique facilitates the study of the human-specific aspects of neurodevelopmental disorders using a complex 3D cell model arranged in a neurotissue-specific manner. Once the technique has been mastered, these cortical organoids can be used for a variety of applications, such as neurodevelopmental, evolutionary, or gene function studies, including disease modeling and potentially drug testing or therapy.