In Vitro Modeling of Down Syndrome Neurogenesis Using Human-Induced Pluripotent Stem Cells

Summary

This protocol outlines a methodology for recapitulating Down syndrome (DS) impaired neurogenesis using DS human iPSCs. The protocol identified biphasic cell cycle defect as the cause of impaired neurogenesis in Down syndrome. It provides a robust platform for understanding cellular and molecular mechanisms underlying the abnormal neurogenesis associated with DS.

Abstract

Down syndrome (DS), caused by an extra copy of chromosome 21, is a leading cause of intellectual disability. One of the key factors contributing to this intellectual disability is impaired neurogenesis observed from fetal stages onwards. To study these neurodevelopmental abnormalities, human-induced pluripotent stem cells (hiPSCs) generated using cells obtained from DS patients provide a valuable and relevant model. Here, a comprehensive protocol is described for recapitulating DS-impaired neurogenesis observed during DS fetal stages. This protocol utilizes a pair of DS-hiPSCs having three copies of chromosome 21 and its isogenic euploid hiPSCs having two copies of chromosome 21. Importantly, the protocol described here recapitulates DS-impaired neurogenesis and found that biphasic cell cycle defect, i.e., reduced proliferation of DS neural progenitor cells (NPC) during the early phase of the neurogenic stage followed by increased proliferation of DS NPC during the late phase of the neurogenic stage is the cause of DS impaired neurogenesis. Increased proliferation of DS NPC during the late phase of the neurogenic stage leads to delayed exit from the cell cycle, causing reduced generation of post-mitotic neurons from DS NPCs. This protocol includes detailed steps for the maintenance of hiPSCs, their differentiation into neural lineages displaying biphasic cell cycle defect during the neurogenic stage, and the subsequent validation of reduced neural differentiation in DS cells. By following this methodology, researchers can create a robust experimental system that mimics the neurodevelopmental conditions of DS, enabling them to explore the specific alterations in brain development caused by trisomy 21.

Introduction

Down syndrome (DS), or trisomy 21, is the most common chromosomal abnormality and the leading cause of intellectual disability (ID)¹. Impaired neurogenesis during DS fetal development is one of the causes of intellectual disability in DS². Human DS fetal studies show a reduction in brain weight and volume, reduced neurons, increased astrocytes^3,4, and abnormal distribution of neurons in layers II and IV^5,6. Additionally, the second phase of cortical development, i.e., the emergence of lamination, is both delayed and disorganized in DS⁷.

Neurodevelopment defects in DS have been studied mostly using mice models of DS such as Ts65Dn, Ts1Rhr, and Ts1cje⁸. However, these mice models were not able to fully recapitulate various phenotypes observed in DS studies due to physiological and developmental differences between mice and humans^9, which led to failed clinical trials¹⁰. The invention of induced pluripotent stem cells^11,12 provided an opportunity to model Down syndrome neurological impairment using cells derived directly from individuals with DS. However, earlier attempts to model DS neurodevelopmental defects using human iPSCs met with inconsistent results and could not fully explain neurodevelopmental defects observed in DS fetal brain sections^13,14,15,16. For instance, a report published by Shi et al. found DS-related Alzheimer's phenotypes but reported no difference in DS neurogenesis compared to euploid controls¹⁵. Similarly, Weick et al. reported reduced synaptic activity but normal neurogenesis in DS compared to euploid controls¹⁶. However, normal neurogenesis in DS reported in these publications was not consistent with observation from DS fetal brain sections. Later, a report by Hibaoui et al. reported reduced neurogenesis in DS, which was consistent with the observation from the DS fetal brain section¹⁴. However, this report and another recent report described the reduced proliferation of DS NPCs as the cause of reduced neurogenesis in DS^14,17. However, only reduced proliferation of DS NPCs could not explain increased astroglial cells and delayed emergence of lamination during DS fetal brain development.

In recently published work, a human iPSC-based DS-impaired neurogenesis model showing reduced neurogenesis was developed. This model found that impaired neurogenesis in DS is due to biphasic cell cycle defects during the neurogenic stage (the stage during which neural progenitor cells are generated from pluripotent stem cells). During the first phase in the neurogenic stage, DS NPCs exhibit reduced proliferation compared to isogenic euploid neural cells, followed by increased proliferation of DS NPCs compared to isogenic euploid cells in the late phase of the neurogenic stage¹⁸.

In this manuscript, a step-by-step detailed protocol for the differentiation of Down syndrome hiPSCs and its isogenic euploid hiPSCs into cortical neurons has been described. The overall goal of this method is to provide a detailed, step-by-step protocol for differentiating a pair of DS hiPSCs and its isogenic euploid hiPSCs into cortical neurons with a focus on modeling the neurogenesis defects associated with DS. This protocol is designed to offer a robust and reproducible system for investigating the cellular and molecular mechanisms underlying abnormalities causing DS-impaired neurogenesis.

The rationale behind the development of this protocol is to allow the differentiation of pluripotent stem cells into cortical neurons by utilizing principles of developmental neurobiology, thereby allowing the identification of phenotypes that arise due to disease/disorder. It aimed to take a minimalistic approach to neural differentiation of iPSCs by avoiding compounds like cAMP or DAPT, which may mask disease phenotypes arising due to defects in the Ca⁺⁺ channel or NOTCH pathway, respectively. Similarly, the use of Ascorbic acid, BDNF, and GDNF was also avoided, which may mask other neurological disease-related phenotypes by potentiating neurogenesis.

The advantages of this technique over alternative methods lie in providing robust recapitulation of neurological phenotypes observed in DS fetal brain sections. Of note, compared to mouse models, the human iPSC-based system eliminates cross-species differences, providing a more relevant model for studying human-specific neurodevelopmental processes⁹ but until now has failed to recapitulate DS impaired neurogenesis observed in DS fetal stages onwards. Further, the use of isogenic pairs of hiPSCs reduces variability and enhances the reliability of observed phenotypic differences. This protocol will be of particular interest to researchers studying neurodevelopmental disorders and human neurogenesis. It is especially relevant for those seeking to model human-specific aspects of DS or those interested in developing therapeutic interventions targeting the neurogenesis defects associated with trisomy 21.

Protocol

The following protocol was followed with two pairs of Down syndrome and its isogenic euploid human iPSCs. One pair was generated using the retroviral mediated delivery method of reprogramming^19, and a second pair (NSi003-A and NSi003-B) was generated using the non-integrating Sendai virus delivery method²⁰. The protocol broadly consists of two stages: The neurogenic stage (Stage 1) and the neural differentiation stage (Stage 2). Further, two phases based on the differences in the proliferation of Down syndrome and isogenic euploid cell lines, i.e., the Early and Late phases, are observed in the neurogenic stage. The details of the reagents, media, and equipment used in this study are listed in the Table of Materials.

1. Down syndrome hiPSCs and isogenic euploid hiPSCs culture and maintenance

1. Feeder plating for hiPSCs
   NOTE: Feeder quality and density are extremely important for good quality of human iPSCs culture.
   1. Add 2 mL of 0.1% gelatin to coat a 6-well plate for at least 1-2 h at 37 °C before seeding with feeder cells.
   2. Keep the gelatin-coated plate at room temperature in the cell culture hood for at least 30 min before plating the cells.
   3. Warm mouse embryonic fibroblast (MEF) media to be used for revival in bead bath/water bath.
   4. Aliquot 5 mL of MEF media into a 15 mL centrifuge tube.
   5. Take out a vial of feeder cells from the liquid nitrogen tank (LN2 tank).
      NOTE: Feeder cells were derived by treating E13.5 Mouse embryonic fibroblasts with 10 µg/mL Mitomycin C for 3 h followed by 2-3 times washing with DPBS¹⁸. Feeder cells can be used directly or frozen in an LN2 tank for future use. All mice were used after Institutional Animal Ethics Committee approval.
   6. Keep the vial at 37 °C in the water bath using a floating rack until a small piece of ice remains.
   7. Take the vial inside the biosafety cabinet and sterilize it with alcohol before putting it inside the biosafety cabinet.
   8. Add 1 mL of warm MEF media (see Table of Materials) drop by drop.
   9. Take out the media from the vial gently and pour drop by drop into a 15 mL centrifuge tube containing MEF media.
   10. Centrifuge at room temperature for 5 min at 200 x g.
   11. Aspirate the supernatant using a vacuum aspiration system (VAS).
   12. Add 1 mL of MEF medium and gently pipette up and down 3-4 times.
   13. Take the cell count using a hemocytometer. Dead cells were excluded using Trypan Blue.
   14. Plate 3.1-3.3 x 10⁵ live feeder cells per well of a 6-well plate or 2 x 10⁶ cells per 6-well plate in MEF medium.
   15. Keep the plate inside the CO₂ incubator at 37 °C with 85% humidity overnight. Shake the plate inside the CO₂ incubator front-back and sideways 2-3 times to achieve a homogenous distribution of feeder cells.
2. Revival of Down syndrome hiPSCs and isogenic Euploid hiPSCs
   NOTE: Warm the required volume of hiPSC media in a 37 °C bead bath.
   ​Human induced pluripotent stem cells (hiPSC) revival medium: Freshly add 25 ng/mL Basic Fibroblast Growth Factor (bFGF) and 10 µM Rock inhibitor (RI) (Y-27632 2HCl) in hiPSC medium.
   1. Remove MEF media from the feeders well and give one wash to the feeders by gently adding 1 mL/well of warm DMEM/F12 from the sides of the well. Aspirate the medium and add 2 mL/well warm hiPSC revival medium. Leave the plate inside the incubator for at least 2 h before plating hiPSCs.
   2. Take out a vial of hiPSCs.
   3. Keep the vial on a floating rack in a 37 °C water bath until a small piece of ice remains.
   4. Before taking the vial inside the biosafety cabinet, sterilize it with 70% alcohol.
   5. Add 1 mL of warm hiPSC revival medium.
   6. Take out the media from the vial gently and pour drop by drop into a 15 mL centrifuge tube containing 5 mL of hiPSCs revival media.
   7. Centrifuge at room temperature for 2 min at 100 x g.
   8. Aspirate the supernatant using VAS and dislodge the pellet by gently tapping the tube.
   9. Pour 500 µL of hiPSC medium with 10 µM of RI and pour over the feeder plate containing hiPSC revival medium. Label the plate with the cell's name, passage number, researcher's name, and date.
   10. Observe the hiPSC colonies under an inverted microscope at 4x magnification; there should be clumps of cells.
   11. While keeping the plate inside the CO₂ incubator, shake the plate front-back and sideways 2-3 times to achieve a homogenous distribution of cells. Do not disturb the plate for 24 h.
   12. The following day, some colonies may look adhered to feeders, and some would be floating. Without aspirating any media, add fresh hiPSCs medium supplemented with 25 ng/mL bFGF and 10 µM of RI.
   13. On the second day after revival, most of the colonies would be attached to the surface. Change complete medium with hiPSC medium with 25 ng/mL bFGF. In the following days, hiPSC colonies will be visible.
       NOTE: It takes at least a week for hiPSCs to revive and make colonies with smooth, well-defined borders, uniform cell morphology, and a dense colony center.
3. Passaging Down syndrome hiPSCs and isogenic Euploid hiPSCs on feeders
   NOTE: All the reagents should be warm before starting. Avoid over-pipetting of cells. Handle cells very gently.
   1. Passage hiPSCs when most of the colonies start merging into each other or when there is the emergence of too many differentiated colonies. Generally, splitting needs to be performed within 5-7 days after the first passage.
   2. Revive a vial of feeders one day before splitting.
   3. Give one wash to feeders with 1 mL/well of warm DMEM/F12 and add 2 mL/well of warm hiPSCs medium supplemented with 25 ng/mL bFGF. Leave the plate inside the CO₂ incubator at 37 °C for at least 2 h before plating hiPSCs.
   4. Observe the plate with hiPSCs for differentiated colonies and remove the differentiated colony by scrapping using a 200 µL pipette under a stereomicroscope.
   5. Give one wash to hiPSCs with 1 mL/well warm DMEM/F12.
   6. Pour 1 mL/well of 1 mg/ml collagenase solution. Keep the plate inside the incubator for 8-10 min, and then observe under an inverted microscope for the loose edges of colonies.
   7. Aspirate the collagenase and give 2 washes of DMEM/F12. Then add 1 mL/well hiPSC Medium.
   8. Gently cut colonies horizontally and vertically 8-10 times in a well using the tip of a 2 mL syringe. Observe the colonies under an inverted microscope to check if most of the colonies have been cut to the appropriate size.
   9. If colonies are cut sufficiently, gently lift the colonies using a cell lifter.
   10. Gently pipette the colonies in the well using a 1 mL tip, 5-8 times, depending on the size of the colonies.
   11. Collect all the cells into a 15 mL centrifuge tube and centrifuge at room temperature for 2 min at 100 x g.
   12. Aspirate the supernatant gently using a pipette, dislodge the cell pellet by gently tapping the tube, and add 200 µL of hiPSC medium with 25 ng/mL bFGF and 10 µM of RI. Pipette 2-3 times with the 200 µL pipette and add 300 µL more hiPSC medium with 25 ng/mL bFGF and 10 µM of RI.
   13. Now gently plate these cells over the feeders containing hiPSC medium + 25 ng/mL bFGF. One well can be split into 1:2 or 1:3, depending on the number of colonies present after removing differentiated colonies. Label the plate with the cell's name, passage number, researcher's name, and date.
   14. While keeping the plate inside the CO₂ incubator, shake the plate front-back and sideways 2-3 times to achieve a homogenous distribution of cells.
   15. The next day, give one gentle wash with warm DMEM/F12 and add 2.5 mL of hiPSC medium with 25 ng/mL bFGF.
4. Freezing Down syndrome hiPSCs and isogenic Euploid hiPSCs from feeders
   NOTE: Over-pipetting of human iPSCs should be avoided during the freezing procedure.
   1. Split hiPSCs when they are still in their log phase, i.e., 4-5 days after passage.
   2. Observe the plate with hiPSCs for differentiated colonies and remove the differentiated colonies using a 200 µL pipette under a stereomicroscope.
   3. Give one wash to hiPSCs with 1 mL/well warm DMEM/F12.
   4. Add 1 mL/well of 1 mg/mL collagenase solution. Keep the plate inside the incubator for 8-10 min, and then observe under an inverted microscope for lifting edges of colonies.
   5. Aspirate the collagenase and give 2 washes of DMEM/F12. Then add 1 mL/well hiPSC Medium.
   6. Gently lift the colonies using a cell lifter.
   7. Gently pipette the colonies in the well using a 1 mL tip 5-8 times, depending on the size of the colonies.
   8. Collect all the cells into a 15 mL centrifuge tube and centrifuge at room temperature for 2 min at 100 x g.
   9. Aspirate the supernatant gently using a pipette, dislodge the cell pellet by gently tapping on the tube, and add 1 mL of hiPSC Medium without antibiotic supplemented with 25 ng/mL bFGF as per the number of wells. Dispense cells into cryovials.
   10. Add hiPSC Medium without antibiotic supplemented with 25 ng/mL bFGF and 20% DMSO in the cryovials to achieve a 10% final concentration of DMSO.
   11. Put the cryovials in a frosty having an isopropanol bath to provide slow freezing.
   12. Keep the frosty at -80 °C overnight. Transfer the vials the next day to the LN2 tank.

2. Neuronal differentiation

NOTE: Preparation of Feeder-Conditioned Medium (FCM): Use a T-75 flask and plate 4 x 10⁶ feeder cells per flask. Next day, add 40 mL of hiPSC medium with 4 ng/mL bFGF. Collect for 7 days. Store each day tube at -20 °C for up to 1 month. Coat a 15 cm dish with 10 mL of 0.1% gelatin at 37 °C for at least 2 h before dissociating cells. Coat a 6-well plate with hESC-qualified basement membrane matrix (hereafter called "qualified matrix") 1 day before dissociating cells. When dissociating cells in the whole protocol, add 10 µM of RI to the cell detachment solution (see Table of Materials), DPBS, and plating media (FCM supplemented with 25 ng/mL bFGF). Prepare fresh feeder-conditioned medium (FCM) for every differentiation. Avoid over-pipetting of hiPSCs.

1. Day In vitro (DIV) 2: Making single cells and seeding of hiPSCs for neurodifferentiation
   1. Add 10 µM of RI into hiPSC media supplemented with 25 ng/mL bFGF, DPBS, cell detachment solution, and Feeder Conditioned Medium supplemented with 25 ng/mL bFGF. Warm all these reagents at 37 °C.
   2. Take a 6-well plate of iPSCs on feeders, remove differentiated colonies, and add fresh hiPSC media supplemented with 25ng/mL bFGF and 10 µM of RI. Keep the plate for 2 h in the incubator.
   3. Take out the iPSCs plate after 2 h and give one wash with DPBS without Ca⁺⁺ and Mg⁺⁺.
   4. Add 1 mL/well cell detachment solution (+ 10 µM of RI) and keep the plate inside the CO₂ incubator for 12-14 min.
   5. After 12-14 min, observe the cells under an inverted microscope; most of the cells start detaching from the plate. If some cells still adhere, gently tap the plate from the sides.
   6. Dilute cell detachment solution by adding 3 mL/well DPBS with Ca⁺⁺ and Mg⁺⁺ (+ 10 µM of RI). Using a 5 mL pipette, do 2-3 times gentle pipetting, avoiding bubbles to break clumps.
   7. Pass cells through a 40 µM strainer.
   8. Collect the cells into a 50 mL centrifuge tube.
   9. Centrifuge at room temperature for 5 min at 200 x g and aspirate media gently using VAS.
   10. Resuspend the cells in 10 mL of hiPSC medium (+ 25 ng/mL bFGF + 10 µM of RI). Add cells onto a 0.1% gelatin-coated 15 cm dish for 1.5 h to remove feeders since feeders have preferential adhesion to the gelatin-coated surface compared to hiPSCs.
   11. After 1.5 h, gently collect the media with cells and centrifuge at room temperature for 5 min at 200 x g. Aspirate media using VAS.
   12. Resuspend the cells in 1 mL of FCM (+ 25 ng/mL bFGF + 10 µM of RI) and take the cell count.
   13. Using FCM (+ 25 ng/mL bFGF + 10 µM of RI), make cell suspension of 30,000-50,000 cells/mL and seed 5 mL cell suspension/60 mm plate or 2 mL/well of 6-well plate or 500 µL/well of 24-well plate. Plates should be coated with a qualified matrix (see Table of Materials).
2. DIV 0: (~48 h later): Change feeder-conditioned medium to NPC media (DDM + B27 without Vitamin A + N2) (see the Table of Materials).
3. DIV 2: Add NPC medium with 0.125 µM of Dorsomorphin every alternate day till DIV 18.
4. DIV 18: Add NPC media without Dorsomorphin and replenish every alternate day till DIV 28.
5. DIV 28-30: Making single cells and seeding of NPCs for Stage 2
   1. On DIV 28, take out the plate (containing NPCs), and replace the media with NPC media (+ 10 µM of RI). Put the plate in the CO₂ incubator for 2 h.
   2. After 2 h, aspirate the media and give one wash with DPBS (without Ca⁺⁺ and Mg⁺⁺).
   3. Add 1 mL/well cell detachment solution (+ 10 µM of RI) and keep the plate inside the incubator for 14 min.
   4. After 14 min, observe the cells under an inverted microscope at 4x. Most of the cells start detaching from the plate. If some cells still adhere, gently tap the plate from the sides.
   5. Dilute the cell detachment solution by adding 3 mL/well DPBS with Ca⁺⁺ and Mg⁺⁺ (+ 10 µM of RI). Using a 5 mL pipette, do 2-3 times gentle pipetting, avoiding bubbles to break clumps.
   6. Pass through 40 µM strainer placed onto a 50 mL centrifuge tube.
   7. Centrifuge at room temperature for 5 min at 200 x g. Aspirate media gently using VAS.
   8. Resuspend the cells in DDM + B27 with vitamin A (+ 10 µM of RI).
   9. Count the live cells using trypan blue with a hemocytometer.
   10. Seed 50,000 cells/well on a qualified matrix-coated plate (1x) of the 48-well plate.
6. DIV 33: Change medium to neural differentiation medium (see the Table of Materials). Change the medium every 5 days. Keep replenishing media until DIV 85/90.

3. Immunocytochemistry (ICC)

NOTE: Add enough buffer at all stages to cover cells and avoid drying of the well while aspirating during the washing stage.

1. Take out the differentiated cell plate outside the cell culture area.
2. Aspirate the media and give a DPBS wash.
3. For fixation, add 1 mL of 4% paraformaldehyde (PFA) to the cells. Cover the plate with aluminum foil and place it on a rocker shaker at 37 °C for 30 min.
4. Aspirate PFA and give three washes each of 15 min with DPBS (without Ca⁺⁺ and Mg⁺⁺).
5. Add blocking buffer (3% BSA + 5% Donkey Serum (or sera from host animal of secondary antibody) + 0.2% Triton-X in DPBS) for 1 h at room temperature.
6. Add 250 µL/well of primary antibodies [Ki67 (1: 100 dilution), TUBB3 (1: 500 dilution), PAX6 (1: 100)] and incubate overnight at 4 °C on a rocker shaker. Dilution was done in a blocking solution.
7. Wash three times with DPBS after primary antibody incubation.
8. Add 250 µL/well of secondary antibody (diluted 1: 250 in 3% BSA + 0.2% Triton-X in DPBS) for 1 h at room temperature.
9. Wash 3 times with DPBS after secondary antibody incubation.
10. Add DAPI (1: 1000 dilution of 5 mg/mL stock in DPBS) for 15 min at room temperature. Wash three times with DPBS. Leave the last wash in the culture plate and observe under the microscope.

4. Image acquisition and analysis

1. Capture images using the fluorescence microscope at 20x resolution.
2. Capture images of Ki67, as well as TUBB3 staining images using an excitation filter of 540 nm, PAX6 using an excitation filter of 488 nm, and DAPI images using an excitation filter of 358 nm.
3. Capture images from ten different random fields for analysis.
4. Analyze ICC images using ImageJ software.
5. For TUBB3 and PAX6 analysis, perform thresholding all the images for the red and green signals, respectively, and blue signal intensity for DAPI. The mean intensity of the red pixels of TUBB3 and green pixels of PAX6 were normalized with the mean intensity of the blue pixels of DAPI.
6. For Ki67 analysis, count the cells using the automated cell counter feature in ImageJ software. Normalize the number of Ki67-positive cells with the number of DAPI-positive cells.
7. Perform statistical analysis using statistics and graphing software by comparing the two groups using unpaired t-tests. Data should be expressed as mean ± SEM from three independent experiments. A p-value of less than 0.05 is considered statistically significant.

Results

Singularized human iPSCs were seeded onto qualified matrix-coated dishes as single-cell suspensions, and differentiation was initiated by removing bFGF. To inhibit non-ectodermal differentiation, Dorsomorphin, a BMP signaling inhibitor, was added from DIV 2-18²¹. For further differentiation of progenitor cells from the neurogenic stage, single-cell suspensions were replated at low density on the qualified matrix (Figure 1) for an additional 6-10 weeks to observe early...

Discussion

In this work, an efficient undirected monolayer cortical neurodifferentiation protocol for an isogenic pair of Euploid hiPSCs and DS hiPSCs is described. Since the cells are grown as monolayers, they are more exposed to culture conditions, which is not possible to the same extent as using embryoid bodies for differentiating iPSCs, which are generally used in other protocols^14,16. While the utility of organoid systems is growing, monolayer-based neural differentia...

Materials

Name	Company	Catalog Number	Comments
MEF medium:
DMEM High Glucose	Gibco	11965-092
FBS	VWR	97068-085	10% final concentration
Non-Essential Amino Acids	Hyclone	SH30238.01	1X final concentration
Penicillin/Streptomycin	Hyclone	SV30010	1X final concentration
β-Mercaptoethanol	Gibco	21985-023	1X final concentration
hiPSC medium:
Knockout DMEM/F12	Gibco	12660-012
Knockout Serum Replacement (KOSR)	Gibco	10828-028	20% final concentration
Non-Essential Amino Acids	Hyclone	SH30238.01	1X final concentration
Glutamax	Gibco	35050061	1X final concentration
Penicillin/Streptomycin	Hyclone	SV30010	1X final concentration
β-Mercaptoethanol (1000X)	Gibco	21985-023	1X final concentration
DDM medium:
Knockout DMEM/F12	Gibco	12660-012
Non-Essential Amino Acids	Hyclone	SH30238.01	1X final concentration
Glutamax	Gibco	35050061	1X final concentration
Penicillin/Streptomycin	Hyclone	SV30010	1X final concentration
Albumax (10%)	Invitrogen	11020-021	0.5 X of 10% Albumax is final concentration
NPC medium:
DDM medium
N2 Supplement	Gibco	17502048	1X final concentration
B-27 Supplement (50X), minus vitamin A	Gibco	12587010	1X final concentration
Neural Differentiation medium (ND):
DDM medium			1/2 of volume
Neurobasal Medium	Gibco	21103-049	1/2 of volume
N2 Supplement	Gibco	17502048	0.5 X final concentration
B-27 Supplement (50X)	Gibco	17504044	0.5 X final concentration
Glutamax	Gibco	35050061	1X of Neurobasal medium
Penicillin/Streptomycin	Hyclone	SV30010	1X of Neurobasal medium
Antibodies and reagents for immunostaining:
Paraformaldehyde	Sigma-Aldrich	158127-500G	4%
DPBS, no calcium, no magnesium	Sigma-Aldrich	D5652
Triton-X-100 Solution	Sigma-Aldrich	X100-500ML	0.20%
BSA	Hyclone	A7979-50ML	1.00%
Purified anti-tubulin β-3 (TUBB3) (TUJ1)	BioLegend	801202	1:500 dilution
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa fluor 594 conjugate	Life Tech Invitrogen	A21203	1:250 dilution
Purified Mouse-Anti-Human Ki67	BD Pharmingen	550609	1:100 dilution
Purified anti-PAX6	BioLegends	901302	1:100 dilution
Alexa fluor 488 Donkey (anti-rabbit)	Life Tech Invitrogen	A21206	1:250 dilution
DAPI Solution (5 mg/mL)	Sigma	D9542	1:1000 dilution
Others
Cell detachment solution (Accutase)	Gibco	A11105-01	Ready to use working solution
Rock inhibitor (RI)	Sellechckem	Y27632	10 mM/ml final concentration
Dorsomorphin	Sellechckem	S7306	0.125 nM/ml final concentration
DPBS with calcium and magnisium (DPBS+ Ca, Mg)	Gibco	14040133	Ready to use working solution
DPBS without calcium and magnisium	Gibco	14190136	Ready to use working solution
Gelatin Type A	Sigma	G2500-100G	0.10%
hESC-qualified basement membrane matrix (Matrigel GFR)	Corning	356230	1 mg stock vial diluted 1:240
Trypsin 0.05%	Gibco	25300054
Trypan Blue	Gibco	15250-061	0.40%
Basic Fibrablast Growth Factor (bFGF)	Peprotech	100-18B	25 ng/ml final concentration
Collagenase	Gibco	17104-019	1mg/ml final concentration