Mar 02, 2026
Version 1
Neural organoid construction protocol V.1
- Tao Cheng1,
- Zi-Xin Jin1
- 1Zhejiang University
- LDO
Protocol Citation: Tao Cheng, Zi-Xin Jin 2026. Neural organoid construction protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx8qrwv8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2026
Last Modified: March 02, 2026
Protocol Integer ID: 244208
Keywords: neural organoid construction protocol, 3d human pluripotent stem cell, patterned neural tissue, human neural tube formation, advancing organoid engineering, neural tissue, polarized neuroepithelium, organoid engineering, human neurodevelopment, studying human neurodevelopment, key morphogenetic signal, vivo prediction, organized central nervous system, fgf17, central nervous system, human neurulation
Abstract
Using a 3D human pluripotent stem cell (hPSC) aggregate fusion assay, we demonstrate that FGF17 is sufficient to initiate human neurulation, driving the formation of polarized neuroepithelium with spatially organized central nervous system (CNS) identities. This finding provides direct functional validation of our in vivo predictions and establishes FGF17 as a key morphogenetic signal in human neural tube formation. More broadly, our work introduces a straightforward and versatile strategy for generating patterned neural tissues, offering a new platform for studying human neurodevelopment and advancing organoid engineering.
Materials
- H9 human pluripotent stem cells (hPSCs, WiCell, WA09)
- Matrigel-coated plates
- mTeSR1 medium
- ReLeSR
- pAAVS1-PURO-IRES-tdTomato-Tre-FGF17 plasmid
- pAAVS1-TALEN-L plasmid
- pAAVS1-TALEN-R plasmid
- Nucleofector® Solution HSC1
- Amaxa® Human Stem Cell Nucleofector® Starter Kit
- Program A-023 in Amaxa Nucleofector II
- Y-27632 (Selleckchem, S1049)
- Puromycin
- Accutase (Sigma-Aldrich, A6964)
- Aggregation Medium (TeSR-E6 + 10 μM Y-27632)
- 96-well U-bottom low-attachment plates
- E6 medium
- Doxycycline (DOX)
Troubleshooting
Cell culture
H9 human pluripotent stem cells (hPSCs, WiCell, WA09) and our constructed iFGF17 H9 line were maintained on Matrigel-coated plates in mTeSR1 medium at 37°C and 5% CO₂. Cultures were passaged every 3-4 days using ReLeSR. Briefly, cells were incubated with 200 µL ReLeSR for 1 minute at room temperature. The reagent was then aspirated, and the plate was incubated for an additional 3-4 minutes at 37°C before dissociating colonies by adding 500 µL of fresh mTeSR1. Cells were subsequently replated at a 1:5 to 1:10 split ratio. All experiments used cells below passage 60.
Construction of inducible iFGF17 H9 cell lines
To generate iFGF17 H9 lines, we used a Tet-On system. A plasmid (pAAVS1- PURO-IRES-tdTomato-Tre-FGF17) was constructed containing a continuous expression of PuroR and tdTomato, as well as a tetracycline-responsive element (TRE) driving the expression of full-length FGF17 cDNA. H9 cells were co-transfected with pAAVS1-PURO-IRES-tdTomato-Tre-FGF17, pAAVS1-TALEN-L and pAAVS1-TALEN-R plasmid using the Nucleofector® Solution HSC1 from Amaxa® Human Stem Cell Nucleofector® Starter Kit and program A-023 in Amaxa Nucleofector II. Post-transfection, cells recovered in mTeSR1 medium supplemented with 10 µM Y-27632 (Selleckchem, S1049). After 24 hours, the medium was replaced with fresh mTeSR1 supplemented with 0.5µg/mL Puromycin. At ~80% confluence, cells were dissociated with Accutase (Sigma-Aldrich, A6964) and plated at low density (~500 cells per well of a 6-well plate) to allow colony formation. Individual colonies were picked and expanded for further culture and experimentation.
Construction of a FGF17-induced human embryoid
Human H9 hESCs and iFGF17 hESCs were cultured on Matrigel in mTeSR1 until they reached ~40% confluence, a stage optimal for embryoid body (EB) formation. For induction, cells were dissociated with Accutase on day 0 and seeded in Aggregation Medium (TeSR-E6 + 10 µM Y-27632) into 96-well U-bottom low-attachment plates. Seeding densities were 1,000 cells/well for H9 and 500 cells/well for iFGF17 hESCs. After 24 hours, a single aggregate from each cell line was manually co-transferred into a new well containing 80 µL of E6 medium to promote fusion. From days 2 to 4, the medium was supplemented with 10 ng/mL doxycycline (DOX) to induce FGF17 expression. DOX was withdrawn on day 4, and the fused EBs were cultured in base E6 medium for continued development.
Protocol references
Luo, T. et al. Establishing dorsal-ventral patterning in human neural tube organoids with synthetic organizers. Cell stem cell 32, 1071-1086 e1078, doi:10.1016/j.stem.2025.04.011 (2025).