2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Summary

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Abstract

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

Introduction

Primary cilia (PC) are microtubule-based organelles that sense and transduce a plethora of chemical and mechanical cues from the extracellular environment. In particular, PC is the central organelle for the transduction of the Hedgehog signaling pathway in vertebrates^1,2. While most neural cells have long been shown harboring a PC, the contribution of this organelle in shaping the central nervous system has long been undervalued. Studies on neocortical development have led to the discovery of multiple neural stem and progenitor cells (NSPCs), all harboring a PC, the location of which has been proposed to be crucial for progenitor fate determination^3,4,5,6,7. PC has been shown crucial for cell mechanisms that are required for normal cerebral cortical development, including NSPC expansion and commitment^8,9,10,11,12 as well as apicobasal polarity of radial glial scaffold supporting neuronal migration¹³. In addition, PC are required during interneurons tangential migration to the cortical plate^14,15. Finally, a role for the PC has been proposed in the establishment of synaptic connections of neurons in the cerebral cortex^16,17. Altogether, these findings argue for a crucial role of PC at major steps of cerebral cortical development^18,19 and raise the need to investigate their involvement in the pathological mechanisms underlying anomalies of cerebral cortical development.

Recent studies have largely improved our understanding of important cellular and molecular differences between cortical development in human and animal models, emphasizing the need to develop human model systems. In this view, human induced pluripotent stem cells (hIPSCs) represent a promising approach to study disease pathogenesis in a relevant genetic and cellular context. Adherent two-dimensional (2D) cell-based models or neural rosettes contain NSPCs similar to those seen in the developing cerebral cortex, which become organized into rosette-shaped structures showing correct apicobasal polarity^20,21,22. Furthermore, the three-dimensional (3D) culture system allows the generation of dorsal forebrain organoids that recapitulate many features of human cerebral cortical development^23,24,25,26. Those two complementary cell-based modeling approaches offer exciting perspectives to dissect the involvement of PC during normal and pathological development of the cerebral cortex.

Here, we provide detailed protocols for the generation and characterization of neural rosettes and derived NSPCs as well as dorsal forebrain organoids. We also provide detailed protocols to analyze the biogenesis and function of PC present on NSPCs by testing the transduction of the Sonic Hedgehog pathway and analyzing the dynamics of crucial molecules involved in this pathway. To take advantage of the 3D organization of the cerebral organoids, we also set up a simple and cost-effective method for 3D imaging of in toto immunostained cerebral organoids allowing rapid acquisition, thanks to a light sheet microscope, of the entire organoid, with high resolution enabling to visualize PC on all types of neocortical progenitors and neurons of the whole organoid. Finally, we adapted immunohistochemistry on 150 µm free-floating sections with subsequent clearing and acquisition using resonant scanning confocal microscope allowing high-resolution image acquisition, which is required for the detailed analysis of PC biogenesis and function. Specifically, 3D-imaging software allows 3D-reconstruction of PC with subsequent analysis of morphological parameters including length, number, and orientation of PC as well as signal intensity measurement of ciliary components along the axoneme.

Protocol

1. Generation of 2D hIPS cell-based models of neocortical development

1. Neural rosette formation
   1. Start with hIPSC cultures harboring large regular colonies, exhibiting less than 10% differentiation and no more than 80% confluency.
   2. Rinse the hIPSCs with 2 mL of PBS.
   3. Add 2 mL of NSPC induction medium supplemented with the Rock inhibitor (NIM + 10 µM of Y-27632).
   4. Manually dissect each hIPSC colony from one 35 mm dish using a needle to cut each colony precisely in horizontal and vertical directions to create a checker-board pattern dividing each colony into equal clusters.
   5. Detach the colony using a cell scraper and transfer them onto an ultra-low-attachment 35 mm dish.
   6. Let them float overnight (ON) in a humidified incubator at 37 °C and 5% CO₂, so that they can form embryoid bodies (EBs).
      NOTE: Embryoid bodies (EBs) are defined as floating spheroid clusters or aggregates of hIPSCs.
   7. Transfer the EBs into poly-L-ornithine/laminin (PO/lam)-coated 35 mm dishes in 2 mL NIM + 10 µM of Y-27632.
   8. Daily refresh NSPC induction medium (NIM without Y-27632) until the formation of neural rosettes that takes approximately 12 to 14 days. Check under the microscope.
      ​NOTE: After this step neural rosettes can be expanded, differentiated, and processed for immunostaining analysis or dissociated to get isolated neural stem and progenitor cells (NSPCs).
2. Neural rosette expansion and early differentiation
   1. Cut neural rosettes in a grid-like pattern with a needle and dislodge the clusters using a cell scraper.
   2. Transfer the rosettes into a 4-well plate containing PO/lam-coated glass coverslip (4-5 rosettes/well) in 0.5 mL of NSPC maintenance medium (NMM).
   3. Incubate the plate in a humidified incubator at 37 °C and 5% CO₂.
   4. Refresh NMM every other day until day 20.
   5. From Day 20, culture neural rosettes in 0.5 mL of cytokine depleted NMM to allow differentiation. Refresh the medium every 2-3 days.
   6. At Day 30 and Day 40 (10 or 20 days of differentiation), fix the rosettes with 0.5 mL of 4% PFA, 20 min, RT.
3. PC biogenesis and function analysis on isolated NSPCs
   1. NSPC dissociation
      1. Cut neural rosettes from step 1.1.8 in a grid-like pattern with a needle and dislodge the clusters using a cell scraper. Transfer the cells into PO/lam-coated 35 mm dishes in 2 mL of NMM and incubate in a humidified incubator at 37 °C and 5% CO₂.
      2. Refresh NMM every other day until confluency (approximately 5-7 days).
      3. After scraping away the large, clear cells surrounding neural rosettes, pick them manually and transfer them onto fresh PO/lam-coated 35 mm dishes to enrich for NSPCs and deplete non-neural cell types.
      4. After one or two manual passages (repeat step 1.3.1.3 if needed) required to remove all contaminating cell types, remove the medium and rinse with PBS.
      5. Add 300 µL of 0.05% trypsin and incubate for 5 min at 37 °C until most cells detach.
      6. Add 2 mL of the medium containing 10% FBS (DMEM + 10% FBS) to inactivate the trypsin. Collect the cell suspension in a 15 mL conical tube. Gently pipette the cell suspension up and down three times to break up the cell clumps.
      7. Centrifuge at 300 x g for 5 min.
      8. Carefully aspirate the supernatant and resuspend the cells in NMN.
      9. Seed the dissociated NSPCs as single cells (1 x 10⁵ cells/cm²) in NMM onto PO/lam-coated dishes and incubate them in a humidified incubator at 37 °C and 5% CO₂.
      10. Expand NSPCs in NMM by changing the medium every other day.
          NOTE: NSPCs are seeded at high density to avoid differentiation.
   2. PC biogenesis analysis
      1. Seed dissociated NSPCs at 100,000 cells/cm² and incubate them in a humidified incubator at 37 °C and 5% CO₂ in NMM for 2 days.
      2. Aspirate the medium and starve NSPCs in cytokine depleted medium (NSPC starvation medium, Supplemental Table 1) for 48 h.
      3. Fix NSPCs with 4% PFA for 20 min at RT.
      4. Wash twice with PBS for 5 min at RT before immunostaining.
   3. PC function analysis: SHH signaling pathway
      1. Seed dissociated NSPCs at 100,000 cells/cm² on PO/lam-coated 8-well Chamber Slides (300 µL) or T25 dishes (5 mL) and incubate in a humidified incubator at 37 °C and 5% CO₂ in NMM for 2 days.
      2. Starve NSPCs in NSPC starvation medium (300 µL per well of 8-well chamber slide and 5 mL in T25 flask) for 48 h.
      3. To induce the SHH pathway, incubate the NSPCs with the starvation medium supplemented with recombinant SHH (100 ng/mL) or SAG (500 nM); (150 µL per well of an 8-well chamber slide and 2 mL in T25 flask) for 24 h.
      4. Fix NSPCs cultured on 8-well Chamber Slides in 250 µL of 4% PFA per well for 20 min at RT and wash them twice with 250 µL of PBS for 5 min at RT before immunostaining analysis.
      5. Remove the medium and wash NSPCs cultured in T25 dishes with PBS.
      6. Add 500 µL of 0.05% trypsin and incubate for 5 min at 37 °C until the cells detach.
      7. Add 2 mL of medium containing 10% FBS (DMEM + 10% FBS) to inactivate the trypsin and collect the cell suspension in a 15 mL conical tube.
      8. Centrifuge at 300 x g for 5 min and carefully aspirate the supernatant to obtain NSPC pellets that can be cryopreserved at -80 °C before RNA extraction and RT-PCR analysis.

2. Generation of dorsal forebrain organoids

1. Single-cell culture of hIPSCs
   1. Start with hIPSC cultures harboring large regular colonies exhibiting less than 10% differentiation and that have been passaged almost once. Ensure that the cells are no more than 80% confluent.
   2. Wash the colonies with 2 mL of PBS.
   3. Add 500 µL of Gentle Cell Dissociation Reagent (GCDR) and incubate for 5 to 7 min at 37 °C.
   4. Aspirate the GCDR and add 2 mL of mTeRS1 medium supplemented with 5 µM of Y-27632.
   5. Pipette the cells gently up and down 10 times to dissociate all colonies into single cells.
   6. Transfer the cells on vitronectin-coated dishes and incubate in a humidified incubator at 37 °C and 5% CO₂.
      ​NOTE: Perform at least 1 passage of the hIPSCs using GCDR prior to the differentiation experiment to adapt the hIPSCs to single-cell culture conditions.
2. On Day 0, allow hIPSCs to form embryonic bodies in EB medium containing a low concentration of FGF2 and a high concentration of rock inhibitor required for hIPSC survival. To do so follow the steps below.
   1. Wash the colonies with 2 mL of PBS.
   2. Add 500 µL of Gentle Cell Dissociation Reagent (GCDR) and incubate for 5 to 7 min at 37 °C.
   3. Aspirate the GCDR and add 1 mL of EB medium supplemented with 50 µM of Y-27632; Use a 1 mL pipette to gently detach the cells and transfer them to a 15 mL conical tube.
   4. Rinse once more with 2 mL of EB medium supplemented with 50 µM of Y-27632, transfer the remaining cells to the 15 mL conical tube. Immediately add 3 mL of EB medium supplemented with 50 µM of Y-27632 to the conical tube and gently homogenize the solution using a 5 mL pipette.
   5. Centrifuge the dissociated cells at 100 x g for 5 min.
   6. Gently aspirate the supernatant and resuspend the cells in 6 mL of EB medium supplemented with 50 µM of Y-27632.
   7. Centrifuge the cells at 100 x g for 5 min.
   8. Aspirate the supernatant and resuspend the cells in 1 mL of EB medium supplemented with 50 µM of Y-27632. Use a 1 mL pipette to gently dissociate the cells into a single-cell suspension.
   9. Immediately after resuspending the cells, dilute and count them.
   10. Dilute the proper volume of cell suspension (containing 900,000 cells) into 30 mL of EB medium supplemented with 50 µM of Y-27632 and 4 ng/mL of bFGF and gently mix the solution.
   11. Plate 9,000 cells/well into an ultra-low attachment 96U plate (300 µL/well). To avoid disturbing EB formation, incubate the plate in a humidified incubator at 37 °C and 5% CO₂ until Day 3 without medium change.
3. Neural induction (Dual SMAD inhibition)
   1. On Day 3, ensure EBs measure 300-400 µm and show regular borders. If both criteria are reached, replace half of the medium (150 µL) with induction medium-1 containing Dual SMAD inhibitors, leading to brightening of the contour of the EBs by Day 6 to Day 7 indicating neuroectodermal differentiation (Supplemental Table 2).
   2. On Day 4, replace 3/4 of the medium (225 µL) with induction medium-1.
   3. On Day 6 and Day 8: Refresh half of the induction medium-1 (150 µL).
4. Organoid embedding into basement membrane matrix (Day 10)
   1. On Day 10, embed EBs in growth factor reduced basement membrane matrix (BMM).
      NOTE: From this step, always use sterile scissors to cut the opening of the pipette tips to avoid damaging the organoids by repeated pipetting.
   2. First transfer around 15-17 organoids in conical tubes. Let the EBs settle and remove the medium. Add 50 µL of induction medium-2 and transfer them into a microcentrifuge tube containing 100 µL of BMM.
   3. Spread the matrix-EB mixture onto the center of a well of an ultra-low-attachment plate and separate the EBs to prevent them from fusing.
   4. Let the BMM solidify in the incubator at 37 °C for 45 min.
   5. Add 3 mL of the induction medium-2 in each well and put the plate in a humidified incubator at 37 °C and 5% CO₂.
      NOTE: Ensure that this procedure is done as quickly as possible as BMM solidifies at room temperature.
5. Stationary culture of organoids embedded into basement membrane matrix
   1. Day 12, 14: Refresh induction medium-2.
   2. Day 16: Refresh induction medium-2 and check under a microscope the expansion of neuroepithelial loops.
6. Basement membrane matrix dissociation and culture on an orbital shaker
   1. On Day 17, dissociate organoids from BMM by pipetting up and down 10 times with a 5 mL pipette and transfer them into differentiation medium-1 (without vitamin A). Incubate onto an orbital shaker at 80 rpm in a humidified incubator at 37 °C and 5% CO₂ to improve nutritional absorption.
      NOTE: Use a different incubator for stationary culture and culture onto an orbital shaker to avoid any vibrations detrimental to adherent hIPS cell growth.
   2. Day 19: Refresh differentiation medium-1.
   3. Day 21: Change differentiation medium-1 to differentiation medium-2 (with vitamin A).
   4. From Day 23 to Day 35: Refresh medium with differentiation medium-2 every 2-3 days.
   5. From Day 35 to Day 70: Refresh differentiation medium-2 with 1% BMM and refresh every 2-3 days.
   6. Collect the organoids after 28, 42, and 70 +/- 2 days of differentiation and fix them overnight at 4 °C, in 5 mL 4% PFA on a 15 mL conical tube.
   7. For further in toto or free-floating immunostaining analysis, organoids are then rinsed twice in PBS and conserved at 4 °C in PBS + 0.05% sodium azide.
   8. For immunostaining analysis on frozen sections, immerse 4% PFA fixed organoids in 10 mL 30% sucrose at +4 °C ON (or until organoids sink). Embed them into a frozen embedding matrix and store at -80°C until cryosectioning.

3. In toto immunolabeling, clearing, and lightsheet acquisition of dorsal forebrain organoids

1. Fixation
   1. Collect the organoids in 6-well plates, remove the medium, and fix them with 2 mL of 4% PFA, at 4 °C, overnight.
   2. Perform three washes in 2 mL of PBS at room temperature.
   3. Transfer the organoids into 2 mL tubes and store at 4 °C in 2 mL of PBS + 0.05% sodium azide.
2. Permeabilization
   1. Incubate organoids in 1 mL of PBS containing 0.2% Triton X100 at RT for 1 h. Do this twice.
   2. Incubate in 1 mL of PBS containing 0.2% Triton X100 and 20% DMSO, at 37 °C, overnight.
   3. Incubate in 1 mL of PBS containing 0.1% Tween20, 0.1% Triton X100, 0.1% Deoxycholate, 0.1% NP40 and 20% DMSO, at 37 °C, overnight.
   4. Incubate in 1 mL of PBS containing 0.2% Triton-X100, at RT, for 1 h. Do this twice.
3. Blocking and immunolabeling
   1. Block in 1 mL of blocking solution (PBS, 0.2% Triton X100, 6% Donkey Serum,10% DMSO), at 37 °C, ON.
   2. Incubate with primary antibodies diluted in 250 µL of PBS, 0.2% Tween20, 0.1 µg/mL of Heparin, 5% DMSO and 3% Donkey Serum, at 37 °C, for 2 days.
   3. Wash in 1 mL of PBS containing 0.2% Tween20 and 0.1 µg/mL of Heparin, for 1 h, at 37 °C. Do this four times.
   4. Wash in 1 mL of PBS containing 0.2% Tween20 and 0.1 µg/mL of Heparin, overnight, at 37 °C.
   5. Incubate with secondary antibodies diluted in 250 µL of PBS containing 0.2% Tween 20, 0.1 µg/mL of Heparin and 3% Donkey Serum, at 37 °C, for 2 days.
   6. Wash in 1 mL of PBS containing 0.2% Tween 20 and 0.1 µg/mL of Heparin, for 1 h, on a wheel, at RT. Do this four times.
   7. Wash in 1 mL of PBS containing 0.2% Tween 20 and 0.1 µg/mL of Heparin, overnight, on a wheel, at RT.
   8. Wash in 1 mL of PBS, for 24 h, at RT.
   9. Stock at +4 °C in 1 mL of PBS and 0.05% sodium azide until clearing.
4. Clearing in TDE (2,2′ -Thiodiethanol)
   1. Incubate the organoids in 1 mL of 30% TDE (3 mL of TDE + 7 mL of PBS), for 24 h, at RT.
   2. Incubate in 1 mL of 60% TDE (6 mL of TDE + 4 mL of PBS), for 24 h, at RT.
   3. Incubate in 1 mL of 80% TDE (8 mL of TDE + 2 mL of PBS), for 24 h, at RT.
5. Organoid embedding prior to light sheet acquisition
   NOTE: Use a custom-made system; made with a 1 mL syringe with the tip cut with a scalpel.
   1. Prepare 100 mL of 4% Low-Melting Agarose in 60% TDE solution and aliquot in 2 mL tubes. Conserve at +4 °C.
   2. Prior to the organoid embedding, preheat one tube containing 1.5 mL of 4% low-melting agarose in 60% TDE in a water bath at 37 °C until it liquefies.
   3. Meanwhile, prepare a custom-made molding system made from a 1 mL syringe the tip of which is cut off using a scalpel.
   4. Pump in the syringe 600 µL of the gel solution using the plunger.
   5. Position the sample using an enlarged pipette tip the opening of which has been cut using sterile scissors.
   6. Fill in the syringe with 400 µL of gel solution so that the sample is positioned within the lower third of the syringe.
   7. Let the gel polymerize and store in 80% TDE solution at 4 °C protected from light until acquisition.
   8. For Light sheet acquisition, use a 20x objective immersed in 80% TDE solution added in the sample chamber to allow to accurately adjust to the refractive index of the clearing method.
   9. Insert the syringe containing the agarose embedded organoid in the largest sample holder designed to accommodate a 1 mL syringe the plunger of which can be operated to position the organoid in front of the objective.
      ​NOTE: Light sheet acquisition of an entire organoid takes approximately 5 to 10 min.

4. Immunostaining and clearing of free-floating sections of dorsal forebrain organoids

1. Fixation, agarose embedding, and sectioning of the organoids
   1. Collect organoids after 28, 42, and 70 +/- 2 days of differentiation in a 6-well plate and fix overnight at 4 °C in 2 mL of 4% PFA.
   2. Rinse twice in 2 mL of PBS and conserve at 4 °C in 2 mL of PBS + 0.05% sodium azide.
   3. Embed the fixed organoids into 4% low-melting agarose in a plastic embedding mold (7 x 7 mm). Carefully remove the agarose block from the plastic mold and glue it to the vibratome stage.
   4. Section the embedded organoids using a vibratome to obtain 150 µm free-floating sections transferred in a well of a 24-well plate containing 500 µL of PBS using a paintbrush to avoid damaging the sections.
      NOTE: Low-melting agarose is recommended as its melting temperature is only approximately 60 °C. Prepare 4% low-melting agarose in PBS solution and leave it to cool just above 37 °C in a water bath prior to organoid embedding.
2. Permeabilization, blocking, and immunostaining
   1. Incubate the sections in 500 µL of PBS containing 0.3% Triton X100, at RT, under agitation, for 20 min. Do this three times.
   2. Incubate the sections in 500 µL of PBS containing 0.3% Triton X100 and 5% nonfat Milk, at RT, under agitation, for 2 h.
   3. Incubate the sections in 250 µL of primary antibody solution (PBS + 0.3% Triton X100 + 1% nonfat Milk), under agitation, at +4 °C, overnight.
   4. Wash the sections in 500 µL of PBS, at RT, under agitation, for 20 min. Do this four times.
   5. Incubate the sections in 250 µL of secondary antibody solution (PBS + 0.3% Triton X100 + 1% nonfat milk), at RT, under agitation, for 2 h.
   6. Wash the sections in 500 µL of PBS, at RT, under agitation, for 20 min. Do this four times.
   7. Store the sections in 500 µL of PBS, at +4° C until clearing.
3. Clearing in TDE (2,2′ -Thiodiethanol)
   1. Incubate the sections in 500 µL of 30% and 60% TDE for 1 h each at RT, and then in 500 µL of 80% TDE overnight, at RT.
   2. Store the cleared sections in 500 µL of 80% TDE until acquisition at +4 °C.
4. Mounting of free-floating sections prior to acquisition with resonant scanning confocal
   1. Mount a free-floating section in a sealed chamber enabling to maintain the sample in 80% TDE solution and designed to adapt on a motorized XY stage of confocal microscopes.
   2. Put one round coverslip in the chamber system.
   3. Carefully transfer the cleared immunostained section using a paintbrush.
   4. Fill the chamber with 80% TDE solution.
   5. Add two standard coverslips plus one second round coverslip and a silicone seal.
   6. Screw on the screwing ring of the chamber to perfectly seal the system.

5. Light sheet and resonant scanning confocal analysis

1. Process light sheet and resonant scanning acquisitions using software that enable 3D visualization and analysis of the entire immunostained sample.
   NOTE: Such software allows to quickly open huge data to easily make snapshots and animations. It allows to move the sample in different orientations and to generate 2D views thanks to a 2D slicer tool in different orientations, XY and YZ for example.
2. For automatic detection of both centrosomes and primary cilia, use a spot wizard enabling to quantify their number in pathological versus control conditions.
3. For 3D reconstruction of PC enabling precise measurement of their length, use a filament wizard to manually fix the starting point of the PC and the software uses the fluorescence signal to reconstruct the PC precisely.

Results

2D hIPS cell-based models to study primary cilium biogenesis and function
The protocol detailed here has been adapted from previously published studies^20,21,22. This protocol allows the generation of neural rosette structures that contain neocortical progenitors and neurons similar to those seen in the developing neocortex. Detailed validation can be performed by conventional immunostaining analysis using sp...

Discussion

PC are now regarded as key organelles regulating crucial steps during normal cerebral cortical development^18,19,31 including NSPC expansion and commitment^8,9,10,11,12 as well as neuronal migration^13,14 and syn...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoéthanol (50 mM)	ThermoFisher Scientific	31350010
6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates	Corning	3471
96-well Clear Round Bottom Ultra-Low Attachment Microplate	Corning	7007
B-27 Supplement (50X), minus vitamin A	ThermoFisher Scientific	12587010
B-27 Supplement (50X), serum free	ThermoFisher Scientific	17504044
CellAdhere Dilution Buffer	StemCell Technologies	7183
DMEM/F-12, Glutamax	ThermoFisher Scientific	31331028
DMSO	ATCC	4-X
Dorsomorphin	StemCell Technologies	72102
Easy Grip 35 10mm	Falcon	353001
EDTA	ThermoFisher Scientific	15575020
EGF , 25µg	Thermofischer	PHG0315
FGF2 , 25µg	Thermofischer	PHG0264
Gentle Cell Dissociation Reagent	StemCell Technologies	7174
Insulin	ThermoFisher Scientific	12585014
KnockOut Serum	ThermoFisher Scientific	10828028
Laminin (1mg)	Thermofischer	23017015
LDN193189	StemCell Technologies	72147
Matrigel Growth Factor Reduced	Corning	354230
MEM Non-Essential Amino Acids Solution (100X)	ThermoFisher Scientific	11140050
Mowiol 4-88	Sigma Aldrich	81381-250G
mTeSR1	StemCell Technologies	85850
Neural Basal Medium	Thermofischer	21103049
Orbital shaker	Dutscher	995002
PBS	ThermoFisher Scientific	14190094
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15140122
PFA 32%	Electron Microscopy Sciences	15714
Poly-L-Ornithine (PO)	Sigma	P4957
Recombinant human BDNF 10 µg	Stem Cell Technologies	78005
Recombinant Human FGF-basic	Peprotech	100-18B
rSHH	R&D Systems	8908-SH
SAG	Santa Cruz	Sc-202814
SB431542	StemCell Technologies	72232
Stembeads FGF2	StemCulture	SB500
Sucrose	Sigma Aldrich	S7903-250G
Superfrost Plus Adhesion Slides	Thermo Scientific	J1800AMNZ
Supplément N2- (100X)	ThermoFisher Scientific	17502048
TDE 2,2’-Thiodiethanol	Sigma Aldrich	166782-500G
Vitronectin	StemCell Technologies	7180
Y-27632	StemCell Technologies	72304
Primary Antibodies
ARL13B	Abcam	Ab136648	1/200e
ARL13B	Proteintech	17711-1-AP	1/500e
CTIP2	Abcam	Ab18465	1/500e
GLI2	R&D Systems	AF3526	1/100
GPR161	Proteintech	13398-1-AP	1/100
N-Cadherin	BD Transduction Lab	610921	1/500e
P-Vimentin	MBL	D076-3	1/500e
PAX6	Biolegend	PRB-278P	1/200e
PCNT	Abcam	Ab4448	1/1000e
S0X2	R&D Systems	MAB2018	1/200e
SATB2	Abcam	Ab51502	1/200e
TBR2	Abcam	Ab216870	1/400e
TPX2	NovusBio	NB500-179	1/500e
γTUBULIN	Sigma Aldrich	T6557	1/500e
Secondary Antibodies
Donkey anti-rabbit AF488	ThermoFisher Scientific	A21206	1/500e
Goat anti-mouse AF555	ThermoFisher Scientific	A21422	1/500e
Goat anti-mouse AF647	ThermoFisher Scientific	A21236	1/500e
Goat anti-rat AF555	ThermoFisher Scientific	A21434	1/500e