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 mu....

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-w....

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