Abstract
Dorsoventral (DV) patterning of the spinal cord (SC) is orchestrated by morphogen gradients that specify distinct neural progenitor domains, the dorsal roof plate (RP), ventral floor plate (FP), and delaminating neural crest cells (NCCs). While foundational insights into SC patterning have been gained from animal models, key aspects - such as the role of retinoic acid (RA), dynamics of NCC lineage development, and human-specific features - remain poorly understood due to limitations in existing in vivo and in vitro models. Here, we present a human pluripotent stem cell (hPSC)-derived SC model, termed microfluidic SC-like structures (µSCLSs), generated by applying bioengineered, antiparallel morphogen gradients via a microfluidic platform over micropatterned hPSC colonies. The µSCLS robustly recapitulates complete DV patterning with human specific transcriptional signatures. Using this platform, we uncover a previously unrecognized RA-BMP signaling crosstalk that could explain conflicting reports on the role of RA in SC DV patterning. We further demonstrate lineage-specific ventral migration of NCCs in response to chemoattractant cues, enabling direct visualization and mechanistic dissection of sensory vs. other fate trajectories. This controllable, reproducible, and human-relevant model provides a powerful system for probing human SC development, neural crest biology, and disease modeling with unprecedented resolution.
Similar content being viewed by others
Data availability
Data supporting findings of this study are available within the article and its Supplementary Information files and from the corresponding authors upon request. The scRNA-seq data generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE300459. All source data for graphs included in the paper are available in the online version of the paper. Source data are provided with this paper.
Code availability
R, Python, and MATLAB scripts used in this work are available from the lead contact upon reasonable request.
References
Gouti, M., Metzis, V. & Briscoe, J. The route to spinal cord cell types: a tale of signals and switches. Trends Genet. 31, 282–289 (2015).
Sagner, A. & Briscoe, J. Establishing neuronal diversity in the spinal cord: a time and a place. Development 146, dev182154 (2019).
Andrews, M. G., Kong, J., Novitch, B. G. & Butler, S. J. New perspectives on the mechanisms establishing the dorsal-ventral axis of the spinal cord. Curr. Top. Dev. Biol. 132, 417 (2019).
Newbern, J. M. Molecular control of the neural crest and peripheral nervous system development. Curr. Top. Dev. Biol. 111, 201 (2015).
Barth, K. A. et al. Bmp activity establishes a gradient of positional information throughout the entire neural plate. Development 126, 4977–4987 (1999).
Yu, W., McDonnell, K., Taketo, M. M. & Bai, C. B. Wnt signaling determines ventral spinal cord cell fates in a time-dependent manner. Development 135, 3687–3696 (2008).
Liem, K. F., Tremml, G., Roelink, H. & Jessell, T. M. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82, 969–979 (1995).
Zagorski, M. et al. Decoding of position in the developing neural tube from antiparallel morphogen gradients. Science 356, 1379–1383 (2017).
Briscoe, J. & Small, S. Morphogen rules: design principles of gradient-mediated embryo patterning. Development 142, 3996–4009 (2015).
Chamberlain, C. E., Jeong, J., Guo, C., Allen, B. L. & McMahon, A. P. Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning. Development 135, 1097–1106 (2008).
Rekler, D., Ofek, S., Kagan, S., Friedlander, G. & Kalcheim, C. Retinoic acid, an essential component of the roof plate organizer, promotes the spatio-temporal segregation of dorsal neural fates. Development https://doi.org/10.1242/dev.202973 (2024).
Novitch, B. G., Wichterle, H., Jessell, T. M. & Sockanathan, S. A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron 40, 81–95 (2003).
Diez del Corral, R. & Morales, A. V. Retinoic acid signaling during early spinal cord development. J. Dev. Biol. 2, 174–197 (2014).
Wilson, L., Gale, E., Chambers, D. & Maden, M. Retinoic acid and the control of dorsoventral patterning in the avian spinal cord. Dev. Biol. 269, 433–446 (2004).
Soldatov, R. et al. Spatiotemporal structure of cell fate decisions in murine neural crest. Science 364, eaas9536 (2019).
Furlan, A. et al. Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla. Science 357, eaal3753 (2017).
Morikawa, Y. et al. BMP signaling regulates sympathetic nervous system development through Smad4-dependent and -independent pathways. Development 136, 3575 (2009).
Saito, D., Takase, Y., Murai, H. & Takahashi, Y. The dorsal aorta initiates a molecular cascade that instructs sympatho-adrenal specification. Science 336, 1578–1581 (2012).
Belmadani, A. et al. The chemokine stromal cell-derived factor-1 regulates the migration of sensory neuron progenitors. J. Neurosci. 25, 3995 (2005).
Kasemeier-Kulesa, J. C., McLennan, R., Romine, M. H., Kulesa, P. M. & Lefcort, F. CXCR4 controls ventral migration of sympathetic precursor cells. J. Neurosci. 30, 13078–13088 (2010).
Marklund, U. et al. Detailed expression analysis of regulatory genes in the early developing human neural tube. Stem Cells Dev. 23, 5–15 (2014).
Rayon, T., Maizels, R. J., Barrington, C. & Briscoe, J. Single-cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human-specific features. Development 148, dev199711 (2021).
Curtis, E. et al. A first-in-human, phase I study of neural stem cell transplantation for chronic spinal cord injury. Cell Stem Cell 22, 941–950.e6 (2018).
Martin, J. R. et al. Long-term clinical and safety outcomes from a single-site phase 1 study of neural stem cell transplantation for chronic thoracic spinal cord injury. Cell Rep. Med. 5, 101841 (2024).
Bydon, M. et al. Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: Phase I trial. Nat. Commun. 15, 1–11 (2024).
Zheng, Y. et al. Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche. Sci. Adv. 5, 5933–5944 (2019).
Meinhardt, A. et al. 3D reconstitution of the patterned neural tube from embryonic stem cells. Stem Cell Rep. 3, 987–999 (2014).
Ranga, A. et al. Neural tube morphogenesis in synthetic 3D microenvironments. Proc. Natl. Acad. Sci. Usa. 113, E6831–E6839 (2016).
Ogura, T., Sakaguchi, H., Miyamoto, S. & Takahashi, J. Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells. Development 145, dev162214 (2018).
Rifes, P. et al. Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient. Nat. Biotechnol. 38, 1265–1273 (2020).
Demers, C. J. et al. Development-on-chip: In vitro neural tube patterning with a microfluidic device. Dev. (Camb.) 143, 1884–1892 (2016).
Xue, X. et al. A patterned human neural tube model using microfluidic gradients. Nature 628, 391–399 (2024).
Luo, T. et al. Establishing dorsal-ventral patterning in human neural tube organoids with synthetic organizers. Cell Stem Cell 0, 1–16 (2025).
Lippmann, E. S. et al. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Rep. 4, 632–644 (2015).
Buckley, D. M., Burroughs-Garcia, J., Lewandoski, M. & Waters, S. T. Characterization of the Gbx1−/− mouse mutant: a requirement for Gbx1 in normal locomotion and sensorimotor circuit development. PLoS ONE 8, e56214 (2013).
Rondon, R. et al. Dual transcriptional activities of PAX3 and PAX7 spatially encode spinal cell fates through distinct gene networks. PLoS Biol. 23, e3003448 (2025).
Briscoe, J., Pierani, A., Jessell, T. M. & Ericson, J. A Qhomeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435–445 (2000).
Jessell, T. M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).
Lek, M. et al. A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning. Development 137, 4051–4060 (2010).
Schoenwolf, G. C., Bleyl, S. B., Brauer, P. R., Francis-West, P. H. Larsen’s Human Embryology (Elsevier, Philadelphia, 2021).
Lee, S. K., Lee, B., Ruiz, E. C. & Pfaff, S. L. Olig2 and Ngn2 function in opposition to modulate gene expression in motor neuron progenitor cells. Genes Dev. 19, 282 (2005).
Tani, S., Chung, U. il, Ohba, S. & Hojo, H. Understanding paraxial mesoderm development and sclerotome specification for skeletal repair. Exp. Mol. Med. 52, 1166–1177 (2020).
McMahon, J. A. et al. Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev. 12, 1438–1452 (1998).
Cairns, D. M., Sato, M. E., Lee, P. G., Lassar, A. B. & Zeng, L. A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3. Dev. Biol. 323, 152–165 (2008).
Delile, J. et al. Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord. Development 146, dev173807 (2019).
Xu, Y. et al. A single-cell transcriptome atlas profiles early organogenesis in human embryos. Nat. Cell Biol. 25, 604–615 (2023).
Florio, M. et al. Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex. Elife 7, e32332 (2018).
Liu, J. et al. The primate-specific gene TMEM14B marks outer radial glia cells and promotes cortical expansion and folding. Cell Stem Cell 21, 635–649.e8 (2017).
Vallstedt, A. et al. Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31, 743–755 (2001).
Calder, E. L. et al. Retinoic acid-mediated regulation of GLI3 enables efficient motoneuron derivation from human ESCs in the absence of extrinsic SHH activation. J. Neurosci. 35, 11462 (2015).
Gupta, S. et al. Deriving dorsal spinal sensory interneurons from human pluripotent stem cells. Stem Cell Rep. 10, 390 (2018).
Mahony, S. et al. Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis. Genome Biol. 12, 1–15 (2011).
Delás, M. J. et al. Developmental cell fate choice in neural tube progenitors employs two distinct cis-regulatory strategies. Dev. Cell https://doi.org/10.1016/J.DEVCEL.2022.11.016 (2022).
Levine, A. J. & Brivanlou, A. H. GDF3, a BMP inhibitor, regulates cell fate in stem cells and early embryos. Development 133, 209–216 (2006).
Levine, A. J., Levine, Z. J. & Brivanlou, A. H. GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses. Dev. Biol. 325, 43 (2008).
Lee, V. M. et al. Molecular events controlling cessation of trunk neural crest migration and onset of differentiation. Front. Cell Dev. Biol. 8, 506999 (2020).
Andrews, M. G. et al. BMPs direct sensory interneuron identity in the developing spinal cord using signal-specific not morphogenic activities. Elife 6, e30647 (2017).
Kalcheim, C. & Teillet, M. A. Consequences of somite manipulation on the pattern of dorsal root ganglion development. Development 106, 85–93 (1989).
Nitzan, E. & Kalcheim, C. Neural crest and somitic mesoderm as paradigms to investigate cell fate decisions during development. Dev. Growth Differ. 55, 60–78 (2013).
Karzbrun, E., Kshirsagar, A., Cohen, S. R., Hanna, J. H. & Reiner, O. Human brain organoids on a chip reveal the physics of folding. Nat. Phys. 14, 515–522 (2018).
Shibata, S. Modeling embryo-endometrial interface recapitulating human embryo implantation. Sci. Adv. 10, eadi4819 (2024).
Li, X. et al. Desktop aligner for fabrication of multilayer microfluidic devices. Rev. Sci. Instrum. 86, 75008 (2015).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Peng, T. et al. A BaSiC tool for background and shading correction of optical microscopy images. Nat. Commun. 8, 14836 (2017).
Chalfoun, J. et al. MIST: accurate and scalable microscopy image stitching tool with stage modeling and error minimization. Sci. Rep. 7, 4988 (2017).
Serra, D. et al. Self-organization and symmetry breaking in intestinal organoid development. Nature 569, 66–72 (2019).
Hao, Y. et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat. Biotechnol. 42, 293–304 (2023).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Liberzon, A. et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics 27, 1739–1740 (2011).
Liberzon, A. et al. The molecular signatures database (MSigDB) hallmark gene set collection. Cell Syst. 1, 417 (2015).
Codling, E. A., Plank, M. J. & Benhamou, S. Random walk models in biology. J. R. Soc. Interface 5, 813–834 (2008).
Mardia, K. V. & Jupp, P. E. Directional Statistics 1–432 https://doi.org/10.1002/9780470316979. (2008).
Wang, W., Chen, E. & Li, H. Truncated rank-based tests for two-part models with excessive zeros and applications to microbiome data. Ann. Appl. Stat. 17, 1663–1680 (2021).
Acknowledgements
We thank Dr. O. Reiner for providing us the Lifeact-GFP H2B-mCherry WIBR3 hESC line. This study is supported by the Michigan-Cambridge Collaboration Initiative, University of Michigan Mcubed Fund, 21st Century Jobs Trust Fund received through the Michigan Strategic Fund from the State of Michigan (Grant CASE-315037), University of Michigan Mid-career Biosciences Faculty Achievement Recognition Award, National Science Foundation of the United States (CBET 1901718, PFI 2213845, and EFMA 2422149), National Institutes of Health of the United States (R21 NS127983, R01 GM143297, and R01 NS129850), and US-Israel Binational Science Foundation (US-Israel BSF 2023009). We acknowledge the Michigan Medicine Microscopy Core for training and support in microscopy imaging, the Michigan Advanced Genomics Core for scRNA-seq service, and the Michigan Lurie Nanofabrication Facility for support in microfabrication.
Author information
Authors and Affiliations
Contributions
J.B. and J.F. conceived and initiated this project; J.B. designed, performed, and quantified most experiments, including scRNA-seq data analysis and interpretation; Y.S.K. generated SOX10::T2A-Cre lineage tracing hESC line; F.C., C.G., and S.S. helped repeat experiments; N.K. generated cytoplasmic EGFP-expressing hESC line; Z.Z. helped with CHIP-seq data analysis; A.T. quantified µSCLS growth; X.X. developed MATLAB scripts for image processing; D.H.N designed probes for RNA-FISH; P.L. helped with data interpretation and experimental designs; J.B. and J.F. wrote manuscript. J.F. supervised the study. All authors edited and approved the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Yuchuan Miao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Bok, J., Kim, Y.S., Cheng, F. et al. A controllable human spinal cord model with full dorsoventral patterning. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71162-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-71162-z
