The overall goal of the procedure we detail here is to dissect involvement of primary cilium during neocortical development. Primary cilia have been shown crucial for many steps of neocortical development, including progenitor cell expansion, fate determination, as well as neuronal migration and maturation. To further dissect cell involvement during neocortical development, we set up relevant tools by taking advantage of emerging 2D and 3D iPS cell-based models of neocortical development.
In other words, neural rosettes and dorsal forebrain organoids. 3D iPS cell-based models begin with embryoid body formation. Adhesion of said embryoid bodies, allow generation of 2D neural rosette structures, while free-floating culture of such EBs allow generation of dorsal forebrain organoids.
The protocol we set up to generate cerebral organoids is based on previous published protocols using the gagging method by adding pre-patterning factors to specifically generate dorsal forebrain organoids by using inhibitors of the activin nodal and BMP pathway, also known as dual SMAD inhibition. To take advantage of the 3D organization of the organoids, we set up a simple and fast method, allowing in total immunolabeling, clearing, and lighted acquisition of the entire organoid with high resolution. And to focus on primary cilium biogenesis and functions, we adapted immunohistochemistry on free-floating sections cut with a vibratome.
After immunostaining and clearing of the sections, we acquired the images using resonant scanning confocal microscopy. Start with iPS cell cultures harboring large regular colonies, exhibiting less than 10%differentiation and are no more than 80%compliant. Manually dissect each iPS cell colony using a needle, allowing to catch precisely each colony in horizontal and vertical directions to create a checkerboard pattern, dividing each colony into equal clusters.
Detach the colony using a cell scrapper and transfer them onto an ultra low attachment culture plate. Let them float overnight in the incubator so that they can form embryoid bodies. The next day, turn the medium, taking care not to aspirate the embryoid bodies, and transfer them into Poly-L-ornithine Laminine-coated dishes until the formation of neural rosettes.
That takes approximately 12 to 14 days. Daily refresh induction, medium, and check under microscope for formation of neural rosette structures. From this step, neural rosettes can be expanded, differentiated, and processed for immunostaining analysis or disassociated to get isolated neural stem and progenital cell cultures.
First of all, start with iPS cultures harboring large regular colonies exhibiting less than 10%differentiation and which have been passaged almost once on the monolayer. On day zero, allow iPS cell to form embryoid bodies in EB medium containing low concentration of fJ2 and a high concentration of ROCK inhibitor required for hiiPS cell survival to avoid disturbing EB formation incubator plates for three days without medium change. On day three, EBs should measure approximately 400 micrometers and show regular borders.
If past criteria rich for most EBs, change the medium with induction medium one containing dual SMAD inhibitors certainly to brightening up the contra DBs by best six to seven, indicating neural tomaly concession. And they turn other DBs into gross-factor reduced basement membrane metrics. From this step always use sterile scissors to get the opening of the peptides to avoid damaging of the organoids.
First transfer about 15 organoids to a clinical tube. Let the EB settle and remove the medium at 50 microliters of induction medium two and transfer the organoids to a microsensitive tube containing 100 microliters of matrix and homogenized by pipetting up and down. Spread the mixture onto the center of a well of a neutral attachment plate.
And finally, space EBs so that they do not touch each other to avoid them to fuse. Let the matter solidify in the incubator for 45 minutes, add induction medium two to each well and incubate in the incubator. Refresh induction medium two every other day.
On day 17, disassociate the basement membrane matrix manually by pipetting up and down 10 times with five ml pipettes. Incubate the organoid plate in the translation medium one under agitation to improve nutritional absorption. Importantly, use a different incubator for stationary culture and culture on orbital shaker to avoid any vibrations detrimental for adherent iPS cell growth.
After fixation, permeabilization, blocking and incubation with primary and secondary antibodies takes 10 days, including washing steps. The clearing method we favored relies on TDE, a glycol derivative. For optimal clearing, intubate the organoid in TDE solution with gradually-increased concentration for 24 hours each.
For organoid embedding, use custom main molding system made from the one ml syringe, which tip is cut off using a scalpel. Low melting point Agarose is recommended, as its melting temperature is only approximately 60 degrees Celsius. Prepare 4%low melting Agarose in 60%TDE solution and let it to cool just about 37 degrees Celsius in a water base pre-organoid embedding.
Put in the syringe 600 microliters of the gel solution using the plunger, then position the sample and fill in the syringe with 400 microliters of the gel solution. Let the gel polymerize, and finally, the sample is mounted ideally within the gel and can be easily pushed out within the lighted chamber to position the organoid in front of the objective. Lighted fluorescence microscopy is ideal for first imaging of such clear organoid.
We've reduced photo imaging and weighed sub-cellular resolution. Here, we used a 20x objective immerged in 80%TDE solution added in the sample chamber to allow to accurately adjust to the refractive index of our clearing method. The largest sample order has been designed to accommodate a one-milliliter syringe that can be inserted from the top with the plunger that can be operated once the sample order is mounted to position the organoid in front of the objective.
The acquisition of an entire organoid takes approximately 5 to 10 minutes. For immuno staining on free-floating sections, collect and fix your organoids in four-person part overnight at four degrees Celsius. Embed the organoids into 4%low-melting Agarose and section by using a vibratome to obtain 150 micrometer of six sections transferred in PBS solution, using a paintbrush to avoid damaging them.
After permeabilization and blocking, incubate the free-floating sections with primary antibody overnight at four degrees Celsius and under agitation. Wash the sections carefully before incubation with secondary antibody for two hours at room temperature and under agitation. Finally, for optimal clearing, incubate the section in TDE solution with gradually increased concentration for one hour and 30 in 60%TDE and then in 80%TDE overnight at room temperature.
Store the section in 80%TDE and TLAC solution at four degrees Celsius. Mount free-floating section in a cell chamber, endeavoring to maintain the sample in 80%TDE solution and designed to adapt on a motorized stage of clinical microscopes. First, put one round cover slip in the chamber and transfer carefully the clear protection using a paint brush.
Fill the chamber with 80%TDE solution and then add two standard cover slips plus one second round cover slip and a Silicon seal. Finally, screw on the screwing ring of the chamber to perfectly seal the system. Nightsheet, as well as resonance acquisitions are processed thanks to a software enabling 3D utilization and an analysis of the entire immunostain sample.
Such software allows you to rapidly open huge data we generate to make easily snapshots and animations. It allowed to move the sample in different orientation and we can generate 2D views of the image thanks to a 2D slicer tool in different orientation, X, Y and Y, Zed, for example. In addition, such software allows automatic detection of both centrosome and primary cilia, enabling to quantify the number in pathological versus control conditions.
It also allows 3D reconstitution of primary cilia for precise measurement of the sites. The protocol we described for 2D iPS cell-based modeling of new cortical development allows generation of neural rosette structures that contain new cortical progenitors and neurons similar to those seen in the developing neocortex. Detailed validation can be performed by conventional immunostaining analysis.
Applicable progenitors should be double-stained with us to impact sensitivities while intermediate progenitors are revealed by TBI2 staining and early bone neocortical neurons by CTIP2 positive staining. Such neural rosette-like structures model interkinetic nuclear migration of radical progenitors that can be visualized by immunostaining with antibodies raised against phospho vimentin that stain mototic nuclei and TBX2 that sustains the mitotic spindle. Finally, ciliagenesis can be analyzed by immunostaining with antibodies raised against pericentric, that stains the back body of primary cilia and are of certain means that stains the axiom.
On such rosette structure, primary cilia extracted from the apical poly Fabri-Kal progenitors and densely above the central lumen of each ventriculite region while they are also protruding from CTIP2-positive neurons. Dissociation of adenoneural rosettes allows to generate isolated neural stem and progenitor cell cultures that are highly useful to analyze primary cilium biogenesis and function. In particular, we detail the procedure to test the induction of the sonic drug pathway after treatment with recommended sonic drug or osmosin agonists.
Subsequent RTPCR SA tests the induction of sonica drug target genes, such as GLI1 and PTCH1, and immunofluorescence analysis tests the dynamics of key sonic drug components along the primary cilium that get be quantified, thanks to open source tools, ilastik and CiliaQ. Different ciliary parameters, including length and orientation, are investigated but also intensity of primary cilium components are shown here for GLI2 and GPR161 that, respectively, accumuulate or exit from the primary cilium in response to recommended sonica drug treatment. The protocol we detail for out 3D iPS cell-based modeling of neocortical development allows generation of homogeneous cerebral organoid with dorsal forebrain identity that can be characterized either by conventional immunostaining analysis or by woman staining, as shown here, with optimal penetration of the antibodies raised, again, N-cadherin, PAX6, and CTIP2 that stain respectively neuronal cadherin, in pink, apical progenitors in the ventricular zone like region, in green, and all newborn neocortical neurons, located in the cortical plate like region.
Here is a second movie of the whole immunostain organoid with antibodies raised again, CTIP2, in red, possibly maintain in pink, and TPX2, in green, allowing to test several characteristics of neocortical progenitors, such as, interkinetic nuclear migration of Fabri-Kal progenitors. Interestingly, phospho vimentin positive progenitors are also observed in the supraventricular zone like region and harbor a unique positive process extending to the positive surface, reminiscent of outer radial glial progenitors and illustrating the high resolution of this method. Here is a movie of an immunostain, free-floating section, acquired thanks to a resonance-scanning microscope showing primary cilia detected with pericentrin antibody that's stained the back of the body, in green, and ARLT3B antibodies that stain the axiom, in pink.
Primary cilia extend from all neuronal stem and progenitor cells, in particular, at the epical surface of the epical progenitors but also in neocortical neurons of the pre-plate like region. Finally 2D neuron rosettes and isolated stem and progenitor cells recapitulate several aspects of cerebral cortical development and represent complementary, useful tools to test ciliary biogenesis and function. The protocol we detect here allowed us to successfully generate dorsal forebrain organoids from this distinct iPS cell lines that give rise to homogeneous results, ensuring the robustness of this procedure.
The protocols we set up for immunostaining, clearing, and imaging of entire organoids or free-floating sections are simple, fast, and cost-effective. Combining such cell-based models and 3D imaging analysis on iPS cells, generated either by reprogramming ciliary perturbation cells or by using CRISPR 9 technology to specifically edit ciliary genes, should allow significant progress in the understanding of the contribution of the primary cilium during normal and pathological development of the cerebral cortex.
