A Robust and Reproducible Protocol for Neural Tube Organoid Generation from Single Mouse Embryonic Stem Cells

Summary

Three-dimensional neural tube organoids (NTOs) derived from mouse embryonic stem cells are valuable tools to study the central nervous system during early development. Here, a step-by-step demonstration of an optimized protocol for cultivating NTOs in vitro, providing stable culture conditions and troubleshooting strategies for reliable, reproducible NTO generation is presented.

Abstract

The development of mammals is a highly complex process, characterized by the necessity for precise concentration- and time-dependent signaling for correct pattern formation and morphogenesis. Despite considerable technological advancements and knowledge gathered, numerous aspects of mammalian development remain elusive. When examining the entire organism, it becomes challenging to disentangle the effects of individual pathways or the mechanism by which external stimuli guide the interference of surrounding tissues and factors.In addressing this complexity, three-dimensional (3D) in vitro models such as organoids have emerged as valuable tools. Organoids, derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), exhibit tissue-like features that closely resemble their in vivo counterparts in terms of expression patterns and functionality. Importantly, they offer accessibility for manipulation and extensive biological studies within a controlled experimental setting. Despite originating from pluripotent cultures, organoid systems often exhibit heterogeneity and substantial variability, limiting their utility when studying complex and intricate biological questions. Therefore, there is a pressing need for detailed protocols aimed at harmonizing procedures that result in high-quality reproducible data, reduction of materials used, and which importantly permit the investigation of convoluted phenomena. In this context, an optimized protocol for the cultivation of neural tube organoids (NTOs) in vitro is presented here. By producing stable culture conditions and offering comprehensive troubleshooting strategies, this protocol enables the reliable and reproducible generation of NTOs, which serve as an adequate model to study relevant scientific questions, including the mechanisms of neural induction, patterning, and early central nervous system development.

Introduction

Mammalian development, starting from a single totipotent stem cell, is a complex process that involves the induction of pattern formation and morphogenesis^1,2. This development requires tightly regulated cellular programs to manage dynamic interactions between cells and their environments. Stem cells have the intrinsic ability to sense, integrate, and respond to systemic and local signals including morphogen gradients³, mechanical boundaries⁴, cellular proliferation, and environmental remodeling⁵. External spatiotemporal cues also drive multicellular responses, initially, in homogenous tissues, to ensure the precise formation of complex structures. Stem cells, distinguished by their self-renewal^6,7 and differentiation capabilities, play a crucial role in embryonic development and adult tissue repair, with main types including embryonic stem cells (ESCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs).

ESCs are extracted from the inner cell mass (ICM) of a blastocyst and are typically cultured in a two-dimensional (2D) environment on feeder cells or extracellular matrix using specific culture media to maintain their pluripotency⁸. In recent years, the need for more realistic cell culture conditions and technologies increased drastically, thus three-dimensional (3D) cell and tissue models were established to study fundamental biomedical processes⁹. Especially in developmental biology, these 3D models such as organoids offer a robust method for modeling tissue morphogenesis and organogenesis ex vivo⁹. Organoids form through self-organization of proliferating stem cells that spontaneously develop into complex 3D structures through symmetry breaking and pattern formation. This process is similar, though not identical to what is observed in vivo, and mostly occurs without external guidance but driven by internal dynamics and interactions¹⁰. Since organoids closely mimic the structure and function of actual organs, they can provide a more accurate representation of mammalian biology compared to traditional 2D cell cultures¹¹. In addition, one of the primary benefits is the ethical advantage, as organoids reduce the need for animal models, addressing concerns related to animal testing^12,13. Furthermore, organoids offer a platform for high-throughput drug screening, improving the prediction of drug efficacy and toxicity. However, there are also several challenges associated with using organoids. While they are more realistic than 2D cultures, they do not fully replicate the complexity and interactions of entire organs within a living organism¹⁴. Additionally, there can be significant variability observed between organoids, even when derived from the same type of cells, leading to inconsistencies in experimental results¹⁵. Thus, culturing organoids in a robust and reproducible manner is essential and requires advanced technical skills, specialized equipment, and detailed protocols.

The nervous system is a complex network responsible for coordinating actions and processing sensory information by sending and receiving signals throughout the body, thereby controlling and coordinating all required functions, including movement, sensation, thinking, and autonomic functions such as heartbeat and digestion. The precursor for the central nervous system, consisting of the brain and spinal cord, is the neural tube. It is crucial to understand how neural structures form and function within early neural tube development to obtain insights into various neurological disorders and developmental abnormalities. Neurodevelopmental disorders are challenging to study in vivo, especially when they originate from human subjects due to ethical concerns. Neural organoids, such as brain organoids^16,17, have significantly enhanced the understanding of nervous system development and disease by providing versatile, accessible models that closely mimic human brain tissue. These three-dimensional structures, derived from pluripotent stem cells (PSCs), replicate various aspects of brain development, including the formation of distinct brain regions, cellular diversity, and complex neural networks^16,18,19. By allowing researchers to observe developmental stages in real-time and study key regulatory mechanisms, these organoids offer deep insights into normal brain development. Brain organoids also enable the modeling of neurodevelopmental¹⁹ and neurodegenerative diseases²⁰, such as microcephaly^16,21, Zika virus infection²², autism spectrum disorders²³, and others, elucidating disease mechanisms and identifying potential therapeutic targets. Additionally, brain organoids serve as valuable platforms for drug testing and development, facilitating the assessment of drug efficacy and toxicity in a human brain-like environment and accelerating personalized medicine approaches. Moreover, they provide a controlled setting to study gene-environment interactions, enhancing the understanding of complex diseases resulting from the interplay of genetic and environmental factors. However, since neural organoids, like brain organoids, are typically formed from aggregates of cells, they often exhibit significant cellular heterogeneity and variability in experimental outcomes, which in turn influences the interpretability of the generated data, posing a considerable challenge for researchers¹⁷. For this reason, protocol optimization and precise quality control criteria are critical for the attenuation of valuable data that adequately represent the in vivo condition.

Meinhardt and colleagues²⁴ made a crucial impact by first reporting a clonal neural organoid protocol mimicking early neural tube development, formed from single embryonic stem cells (ESCs) that drastically minimizes inter-organoid heterogeneity. This innovation allows researchers to study developmental processes and mechanisms, as well as neural patterning, in a precise and defined environment with exceptional resolution.Until now, many laboratories have developed neural organoids, mostly of mouse or human origin, that mimic different parts of the neural tube and its development, as summarized in²⁵. However, based on available information, the protocol established by Meinhardt et al.²⁴ is the only method where NTOs reliably form from a single cell, self-organize, and efficiently pattern into dorsoventral fates following a single, global pulse of retinoic acid (RA) without the addition of other ventralizing factors such as Sonic Hedgehog Agonist (SAG)²⁶, or the usage of microfluidic chambers that provide spatial information due to gradient formation. The protocol provided here was adapted from Meinhardt et al.²⁴ as provided in Krammer et al.²⁶. In this protocol, single mouse ESCs are seeded in a cell culture matrix and exposed to defined neurodifferentiation conditions. Within two days, these cells clonally expand and epithelialize, forming a 3D round-to-ellipsoid structure with a single lumen and apically-basally stratified cells surrounding it. On day 2, a globally applied 18 h pulse of the signaling molecule RA enhances neural differentiation, posteriorizes the NTOs from midbrain to hindbrain/cervical spinal cord levels, and induces the formation of a single, functional floorplate by day 6, which patterns the NTO from ventral to dorsal.

The optimized protocol offers several key advantages. It enables robust epithelialization within the first three days and generates relatively small, spherical tissues of approximately 200 µm in diameter, each containing a single lumen. The organoids arise clonally from single cells and are maintained in a serum-free, chemically defined culture medium. Within six days, gradual neural differentiation from pluripotent cultures is achieved, resulting in organoids that display an anterior midbrain identity by day 6 and dorsoventrally patterned hindbrain- or cervical spinal cord-like structures after a global RA pulse²⁶.

Several aspects have been adapted and differ from the original protocol^22,25 to enhance reproducibility and throughput. These include modified ESC culture conditions, a reduced number of input cells, an improved differentiation medium, and an adjusted differentiation protocol that omits Noggin supplementation during days 0-2 (already used in²⁶). Furthermore, seeding density has been optimized for use in 96-well plates, allowing for high-throughput analysis. Together, these refinements have led to a 40% increase in NTO formation and patterning efficiency, from initially ~40%²⁴ to now ~80%²⁶ in the updated version.

Here (Figure 1), a detailed description of an optimized protocol is provided that allows for a robust and reproducible generation of NTOs in cell culture matrix. The protocol encompasses all stages of NTO handling, from the initial seeding of ESCs for differentiation assays to subsequent maintenance, fixation, and analysis. It also includes guidelines for quality control through daily visual assessment of NTO morphology, as well as critical characterization of organoid identity using antibody staining to verify correct patterning and neural differentiation. In addition, comprehensive troubleshooting approaches are offered, allowing a critical assessment in every step of the protocol, which is necessary to ensure the reproducibility and reliability of experimental protocols. These detailed instructions help to identify and resolve potential issues, minimize errors, and optimize outcomes. This is crucial for maintaining the integrity of scientific experiments and enabling other scientists to successfully replicate the work and draw comprehensive conclusions from the data generated, thereby advancing the field.

Figure 1: Overview of the protocol. (A) Passaging ESCs (see step 1.1).(B) Aliquoting cell culture matrix and seeding single ESCs for differentiation into NTOs (see step 1.2). (C) Aliquoting RA and RA treatment (see step 2). (D) RA removal and routine of daily medium replacement (see step 3). This figure was created with BioRender.com. Please click here to view a larger version of this figure.

Protocol

NOTE: Check cells and NTOs daily for quality assessment before any further procedure (medium replacement, treatment, fixation, etc.). All procedures requiring direct exposure of cells to air must be performed within a biosafety cabinet to ensure sterility and prevent contamination of the culture.

1. Maintenance, passaging, and seeding mouse ESCs for NTO differentiation

1. Passaging
   1. Maintain ESCs on cell-culture dishes or wells of a 6-well plate pre-coated with 0.15% gelatin for 30 min at 37 °C.
   2. Passage ESCs every 2-3 days, with daily medium replacement from day 2 onward.
   3. Pre-warm all reagents (Medium, PBS, enzyme solution) at a minimum of 21 °C and a maximum of 37 °C before use.
   4. Check cells under phase-contrast microscope (see representative images, Figure 2). Ensure that colonies have an appropriate size and proper morphology (Figure 2A), while also confirming that they adhere to the bottom of the dish.
      NOTE: Discard the culture if more than 10% of mouse ESCs are differentiated (Figure 2B), and prepare a freshly thawed culture, which you should passage at least twice before seeding ESCs for a differentiation assay. Experienced researchers can judge by visual assessment (see also Mulas et al.²⁸) or via antibody staining for marker expression (e.g., OCT4, see also Ying et al.²⁹).
   5. Open the lid of the culture dish/ plate, slightly tilt it and carefully aspirate medium. Keep cells out of the incubator for as short a time as possible. Keep cells without medium as briefly as possible to avoid drying out the culture.
   6. Wash colonies with 500 µL of pre-warmed PBS (add to cells in a single well of a 6-well plate), and immediately aspirate again.
      NOTE: This step is crucial because: i) ESC medium (2iLIF) carry over must be avoided prior to the differentiation assay (or NTO formation will be hindered), ii) FBS or BSA in culture medium can stop dissociation. This applies to ESCs cultured in a different ESC medium, containing FBS or BSA.
   7. Add 500 µL of pre-warmed enzyme solution and transfer cells back into the incubator.
   8. Incubate cells at 37 °C for 3 min using a timer.
   9. After the incubation time, take out the plate and check the cells under the phase-contrast microscope.
      NOTE: ESCs should have detached from the culture plate; if not, tap the dish/plate firmly but carefully (Figure 2C).
   10. Add 1 mL of N2B27 medium (Table 1) by pipetting it directly onto the cells.
       NOTE: When preparing medium (N2B27), glutamine is kept for a maximum of 5-10 min at 37 °C, and preferably at room temperature. ESCs should be washed off the plate, leaving 'empty' areas in the culture dish.
   11. Use a P1000 pipette and slightly tilt the plate so that all the colonies sticking to the culture dish can be washed off.
   12. To dissociate clumps of cells, slightly tilt the plate, place the pipette tip (P1000) at the center of the dish, press the tip against the bottom of the dish but slightly off the perpendicular axis, and flush out the cells. Repeat for five to ten times to break the cell clumps.
   13. Check that the cells are dissociated properly into single cells under the phase-contrast microscope (Figure 2D) and repeat if there are still clumps.
   14. After complete dissociation, transfer the cell suspension into a clean sterile 15 mL conical centrifuge tube, wash the empty well where cells were cultured in with 1 mL of N2B27 medium, and transfer to the same conical centrifuge tube with the cell suspension.
   15. Spin down cells for 5 min at 453 x g in an ultracentrifuge.
   16. Carefully aspirate the supernatant.
   17. Resuspend the cells in an appropriate amount of N2B27 medium (e.g., 1 mL) and count cells (see Table 2 for recommended cell numbers for differentiation assay into NTOs).
2. Differentiation of ESCs into neural tube organoids (NTOs)
   1. Aliquoting cell culture matrix
      1. Thaw the bottle of cell culture matrix overnight on ice at 4 °C (never at room temperature nor by hand thawing). Do not leave the cell culture matrix at 4 °C for longer than necessary; aliquot and freeze as soon as possible to preserve its functionality.
      2. In the biosafety cabinet, place 1.5-2 mL microcentrifuge tubes on ice to pre-cool them while keeping the cell culture matrix bottle on fresh ice.
      3. Mix the thawed cell culture matrix carefully to avoid bubble formation, as bubbles can cause detachment from the well/ dish after jellification.
      4. Aliquot cell culture matrix at the desired volume (0.5-1 mL is recommended) and freeze at -20 °C until further usage. Avoid replacing the tip between the aliquots, as room-temperature tips can cause the cell culture matrix to solidify within, leading to a significant loss. Instead, cool the pipette tips to 4 °C by storing them in the fridge before use.
   2. Seeding single cells in a cell culture matrix
      1. Quantify the number of cells needed (100 cells/µL cell culture matrix, see Table 2).
      2. Transfer the appropriate cell number (cell suspension) to a clean 15 mL conical centrifuge tube and top it up with 1 mL of N2B27.
      3. Spin down cells for 5 min at 453 x g in the centrifuge.
      4. Aspirate the supernatant and place the conical centrifuge tube on ice.
      5. Carefully resuspend the cell pellet on ice in an appropriate amount of thawed and liquid cell culture matrix while avoiding the formation of bubbles.
      6. Spread the appropriate amount of cell culture matrix-cell suspension, in a thin layer, onto a glass-bottom or plastic-bottom dish that is optimal for imaging (see Table 2, Figure 3A-C), without touching the walls of multi-well plates. Because of high surface tension, the cell culture matrix droplet can move from the center of the well to the walls if they were previously touched, which causes an inadequate distribution of cells/ NTOs.
         NOTE: If using multi-well plates: first draw a circle of cell culture matrix using a P20 (96-well plate) or P200 pipette tip before 'filling it' with the cell culture matrix-cell suspension (Figure 3B).
      7. Place the multi-well plate/ dish in the incubator at 37 °C to let the cell culture matrix solidify. Estimate 1 min per µL cell culture matrix for smaller amounts and a maximum of 15 min for 40 µL or more.
      8. After the incubation time, take out the plate and add an appropriate volume of medium (see Table 2). ESCs in the cell culture matrix must not dry out during seeding for differentiation assay into NTOs or when replacing the medium. After jellification, the medium must be applied as soon as possible. Jellification typically is complete in 1 min at 37 °C per 1 µL cell culture matrix and a maximum of 15 min for 40-100 µL cell culture matrix when spread as a thin layer.
         NOTE: To avoid drying out ESCs in the cell culture matrix, handle a maximum of 8 wells of a 96-well plate with a multichannel pipette or one 35 mm glass-bottom dish/well of any other multi-well plate. Keep cells out of the incubator for as short a time as possible.
      9. Check the seeding density and cell distribution within the cell culture matrix under the phase-contrast microscope (Figure 3C,D). The optimal seeding density is recommended (Table 2 and Figure 3). However, for certain applications, such as early analysis, a seeding density up to 10 times higher may be advantageous.
         ​NOTE: Carefully mix the cell culture matrix-cell suspension by resuspending with a P300 or P1000 pipette tip before seeding for the differentiation assay to counter cell number variability between wells/ dishes. This step is not recommended before seeding every single well to protect against cell culture matrix loss, as this can solidify and stick to the pipette tip.

Table 1: Medium components (N2B27 base, 2iLIF, N2B27diff). Please click here to download this Table.

Table 2: Information on cell number per dish, amount of cell culture matrix, volume of medium, and jellification time. Please click here to download this Table.

2. RA treatment of NTOs to enhance neural differentiation and to induce posteriorization and ventralization

1. Aliquoting retinoic acid (RA)
   1. Bring all-trans RA powder to room temperature (stored at -20 °C).
   2. Reconstitute powder in an appropriate volume of DMSO to reach a final concentration of 100 mM.
   3. Aliquot 1 µL of 100 mM RA into one 1.5 mL screw-top vial each.
   4. Add Argon gas until the tube is 'full'.
      NOTE: Since argon gas is heavier than oxygen, it will sink to the bottom of the tube to cover and preserve RA from air. This can be felt on the hands when it leaks out when the tube is full.
   5. Tightly close the vial.RA is highly sensitive to light and air.
   6. Store aliquots in the dark at -80 °C until further usage. Alternatively, reconstituted RA can be stored in 1.5 mL microcentrifuge tubes in liquid N₂.
      NOTE: Reconstituted and diluted RA can be stored in the fridge when covered with foil for a maximum of 2 days.
2. RA treatment of NTOs for 18 h (Figure 4A)
   1. Take out 1 µL of the aliquot with a concentration of 100 mM from -80 °C and keep in the dark until thawed at room temperature (e.g., in the palm of one's hand or in a tube rack in a biosafety cabinet without lights on).
      NOTE: If RA aliquots are stored in liquid N₂, immediately open the lid of the microcentrifuge tube to avoid explosion of the tube, then close again and proceed to step 2.2.2.
   2. Once thawed, add 400 µL of pre-warmed N2B27diff medium for a concentration of 250 µM using a P1000 pipette. Mix thoroughly to achieve homogeneity.
      NOTE: Alternatively, RA can be reconstituted and diluted in pure DMSO instead of N2B27diff medium, although this increases the concentration of DMSO in the culture. When preparing medium (N2B27diff), glutamine is kept for a maximum of 5-10 min at 37 °C, and preferably at room temperature.
   3. Take out plates/dishes growing NTOs from the incubator and place them into a sterile biosafety cabinet.
   4. Examine the growing NTOs under the phase-contrast microscope. The morphology of the NTOs is crucial in providing information about the viability of the culture and the efficacy of the experiment (Figure 4B-D).
   5. Remove the lid of the plate/ dish and slightly tilt the plate to the side. Aspirate the medium carefully, either using a P200 pipette or an aspirator, without disturbing the cell culture matrix. Keep NTOs without medium as briefly as possible to avoid drying out of the culture and/ or the cell culture mix.
   6. Add an appropriate volume of N2B27diff supplemented with 250 nM RA solution (see Table 2), cover with the lid, and transfer the NTOs back into the incubator.
      NOTE: Sometimes, especially if the RA was not mixed completely, precipitates can form that are harder to dissolve in normal N2B27diff. In this case, discard the aliquot and make fresh RA aliquots because these precipitates have an unknown and uncontrolled concentration of RA.

3. RA removal and routine of daily medium replacement of NTOs

1. Transfer the culture plate from the incubatorto the sterile biosafety cabinet.
2. Remove the lid and slightly tilt the plate to the side.
3. Carefully aspirate N2B27diff containing RA, either using a P200 pipette or aspirator, without touching the cell culture matrix.
4. Add pre-warmed PBS (use enough volume to cover the cell culture matrix droplet or as referred to in Table 2), then tilt the plate again and carefully remove it to wash out the leftover RA.
5. Add an appropriate volume of N2B27diff (as referred to in Table 2), by pipetting the medium along the wall of the well/dish to avoid damaging the cell culture matrix droplet.
6. Replace the medium daily following steps 3.1-3.3, then gently add fresh pre-warmed N2B27diff by pipetting the medium onto the wall of the well/ dish before returning the NTOs back in the incubator. Direct and vigorous pipetting or addition of cold medium/ PBS onto the cell culture matrix droplet can cause its breakage and detachment from the plate/dish.
   NOTE: When perturbing pathways by using small molecules or recombinant proteins, it is important to replace the medium daily. If drugs are to be applied for longer than one day, drug solutions need to be prepared freshly daily or at least every two days to ensure protein/ drug stability and the continued presence of correct concentrations.

4. Fixation of NTOs

NOTE: NTOs are fixed as described by Krammer et al.²⁶. Depending on the antibody to be subsequently used, PFA from different sources (store-bought, in-house, etc.) can be required.

1. Thaw PFA aliquot at 4 °C overnight.
2. Bring PFA to room temperature before use.
3. If using a higher stock concentration of PFA, dilute PFA to 4% in N2B27diff medium or PBS in a chemical hood.
4. Take NTOs out of the incubator and transfer them to a chemical hood.
5. Open the lid and add 4% PFA in a ratio of 1:1 directly to the NTOs to achieve a final concentration of 2% (e.g., 100 µL of 4% PFA added to the 100 µL N2B27diff in the 96-well plate).
6. Put the lid back on andincubate NTOs for 30 min at room temperature. Set a timer, as longer fixation can impair the performance of certain antibody treatments.
   NOTE: PFA is toxic and must not be inhaled. Work carefully and with caution under the chemical hood. Some antibodies require the use of a specific PFA or shorter/ optimized fixation time.
7. After 30 min, remove the lid and slightly tilt the plate to the side, then follow steps 3.3-3.4.
8. Carefully add pre-warmed PBS (use enough volume to cover the cell culture matrix droplet or as referred to in Table 2), and incubate for 5 min at room temperature. Repeat this step 3 times to remove all left over PFA.
9. After the final wash, add an appropriate amount of PBS (see Table 2), seal the plate using parafilm, and store at 4 °C until further usage.

5. Antibody staining of NTOs

1. Antibody staining is a valuable tool in assessing the correct identity of the NTOs as well as the success of the assay/ experiment. Antibody staining can be performed as outlined in Meinhardt et al.²⁴ and Krammer et al.²⁶.
   NOTE: If NTOs appear suboptimal (Figure 4E-G) or do not show correct spatiotemporal marker expression (Figure 5 to Figure 7), discard the material as it does not meet quality standards.
2. Keep the block/ permeabilization solution (1x PBS with 1% BSA and 0.5% Triton-X) at 4 °C and adapt to room temperature before use.
3. For full 3D reconstitution and analysis, keep antibodies (1^st and 2^nd) at 4 °C for at least 1, but preferably 2-3 nights, to ensure good antibody penetration.
   NOTE: Alexa Fluor anti-561 2^nd antibody might give a stronger background signal in the cell culture matrix in the first three days of the differentiation assay.

6. Clearing of NTOs

1. Prepare for clearing²⁶
   1. Complete the antibody staining process before beginning the clearing procedure (ensure both 1^st and 2^nd antibodies are incubated and thoroughly washed out).
   2. Add propylgallate to the clearing solution (2 mg propylgallate per mL clearing solution) to minimize photobleaching of NTOs.
      NOTE: Perform the clearing 1-2 h before imaging to prevent oxidation (liquid turns from light yellow/cream into dark bright yellow or even brown) or crystallization of the clearing solution. If crystallization occurs, dissolve by adding PBS to the well.
2. Solution pH and mixing precautions
   1. Maintain the correct pH of the clearing solution to avoid rapid oxidation or, dissolving of the cell culture matrix.
   2. Avoid vigorous shaking or harsh mixing of the solution to prevent air bubble formation.

7. Analysis of NTOs

1. Microscopy preparation
   1. After completing antibody staining and whole tissue clearing, use a confocal microscope with 10-40x objectives for imaging.
2. Visual assessment
   1. Use visual assessment and manual quantification to efficiently determine whether NTOs exhibit appropriate neural marker expression and dorsoventral organization.
3. 3D analysis software
   1. For 3D analysis, utilize software such as CellProfiler^28,29, Imaris (File Converter and Viewer) or a similar tool to assess spatial markers and structural details.
4. Verification of NTO identity
   1. Confirm the correct identity of NTOs by evaluating the temporal expression of neural markers and the spatiotemporal expression of specific dorsoventral patterning markers. This step is essential to ensure that the differentiation assay has been successful before further research.
5. Characterization aspects: Refer to Figure 4, as well as Figure 6 and Figure 7 to verify the following critical aspects of culture success:
   1. Refer to Figure 4 and ZO-1 staining shown in Meinhardt et al.²⁴ to verify successful epithelialization and lumen formation.
   2. Successful neural differentiation is characterized by the downregulation of OCT4 and the upregulation of SOX1, followed by β3TUBULIN expression (detailed in²⁴). Refer to Figure 6 and Figure 7 to verify successful neural differentiation.
   3. Dorsoventral patterning is confirmed by the presence of a ventral FOXA2⁺/SHH⁺ floor plate and PAX3⁺ expression at the opposing dorsal pole (detailed ventral patterning in²⁶). Refer to Figure 6 and Figure 7 to verify successful dorsoventral patterning.

Results

R1 mouse ESCs were used for all representative examples in the figures presented in this manuscript. Other mentioned mouse ESCs can exhibit mild differences in epithelialization timing, neural differentiation, or patterning efficiency when seeded for NTOs, however, results should not significantly differ from the data shown here.

Only good quality ESCs (Figure 2A) seeded in the appropriate cell density (Figure 3) form good quality NTO...

Discussion

Here, several optimizing adaptations and improvements to the original protocol for generating high-quality NTOs are presented to facilitate standardization across laboratories. Key modifications include different ESC culture conditions, reduced input cell numbers, improved differentiation medium, modified differentiation protocol, and seeding density adjustments for high-throughput analysis using multi-well plates. Several aspects of the protocol are particularly critical for achieving reliable and reproducible results. ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter | Uncoated	MatTek Corporation	Cat# P35G-1.5-14-C
Accutase	Invitrogen	Cat# 00-4555-56
Albumin (BSA) Fraction V (pH 7.0)	Panreac AppliChem	Cat# A1391
All-Trans Retinoic Acid	Merck	Cat# R2625
B-27 Supplement (50X), serum free	Gibco	Cat# 17504044
Basement Membrane Matrix (Matrigel)	Corning	Cat# BDL354234	Protein concentration 10 mg/ml
CellCarrier-96 Ultra Microplates	PerkinElmer	Cat# 6055302
CellProfiler 4	Stirling et al. 2021		https://cellprofiler.org
CHIR 99021	Tocris	Cat# 4423
CUBIC-R+(N)	Matsumoto et al, 2019	N/A
Dimethylsulfoxid	Sigma Aldrich	Cat# D2650
DMEM/F-12, no glutamine	Gibco	Cat# 21331020
Dulbecco’s Phosphate-buffered saline (DPBS, 1X)	Gibco	Cat# 14190144
Eppendorf Centrifuge 5810 R	Eppendorf	Cat# 5811000015
Eppendorf Microtube 3810X	Merck	Cat# EP0030125150
Falcon Conical Centrifuge Tubes	Corning	Cat# 352095
Gelatin	Merck	Cat# G2500
L-Glutamine (200 mM)	Gibco	Cat# A2916801
HeracellVIOS 160i CO2 Incubator, 165 L	ThermoFisher Scientific	Cat# 51033557
Imaris	Oxford Instruments		https://imaris.oxinst.com
Inverted LED phase contrast microscope (CKX53)	Olympus
2-Mercaptoethanol (50 mM)	Gibco	Cat# 31350010
Monoclonal mouse anti-OCT-3/4	Santa Cruz Biotechnology	Cat# sc-5279; RRID:AB_628051	Use 1:200
Monoclonal mouse anti-PAX3	DSHB	Cat# PAX3-C; RRID:AB_528426	Use 1:100
Monoclonal rabbit anti-SHH	Cell Signalling Technologies	Cat# 2207; RRID:AB_2188191	Use 1:300
Monoclonal rabbit anti-β3-Tubulin	Cell Signalling Technologies	Cat# 5568; RRID:AB_10694505	Use 1:500
Monoclonal mouse anti-ZO-1	Invitrogen	Cat# 33-9100; RRID:AB_2533147	Use 1:200
Mouse LIF	Qkine	Cat# Qk018-1000
N-2 Supplement (100X)	Gibco	Cat# 17502048
Neurobasal Medium	Gibco	Cat# 21103049
Paraformaldehyde, reagent grade, crystalline	Merck	Cat# P6148
PD 0325901	Tocris	Cat# 4192
Penicillin-Streptomycin Solution	Gibco	Cat# 15140130
Phase-contrast microscope
Polyclonal goat anti-FOXA2	R&D	Cat# AF2400; RRID:AB_2294104	Use 1:400
Polyclonal goat anti-SOX1	R&D Systems	Cat# AF3369; RRID:AB_2239879	Use 1:200
RNA-Seq data related to Figure 6	Ishihara et al., 2022	GEO: GSE214368
SafeFAST Premium Microbiological Safety Cabinet	Dasit group FASTER	Cat# F00025200000
Sodium Pyruvate (100 mM)	Gibco	Cat# 11360070
Spinning disk confocal inverted microscope (IX3)	Olympus
Triton X-100	Sigma Aldrich	Cat# 9036-19-5