Mar 02, 2026
Reconstruction of human embryonic trajectory reveals developmental origins of the central nervous system
- Cheng Taoldo1,
- Zi-Xin Jin1,
- Min Gao2,
- Pengfei Xu1
- 1Zhejiang University;
- 2Guizhou Medical University
- LDO
Protocol Citation: Cheng Taoldo, Zi-Xin Jin, Min Gao, Pengfei Xu 2026. Reconstruction of human embryonic trajectory reveals developmental origins of the central nervous system. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj1eobvk5/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: March 02, 2026
Last Modified: March 02, 2026
Protocol Integer ID: 244239
Keywords: 3d human pluripotent stem cell, patterned neural tissue, reconstruction of human embryonic trajectory, developmental origins of the central nervous system, human neurodevelopment, human embryonic trajectory, human neural tube formation, neural tissue, polarized neuroepithelium, studying human neurodevelopment, advancing organoid engineering, neurodevelopment, organoid engineering, key morphogenetic signal, developmental origin, 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)
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
1. 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).
2. Xue, X. et al. A patterned human neural tube model using microfluidic gradients. Nature 628, 391-399, doi:10.1038/s41586-024-07204-7 (2024).
3. Sun, Y. etal. Bioengineering innovations for neural organoids with enhanced fidelity and function. Cell stem cell 32, 689-709, doi:10.1016/j.stem.2025.03.014 (2025).
Acknowledgements
We would like to thank the members of Laboratory of Development and Organogenesis (LDO) at Zhejiang University for helpful suggestions and discussions.