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Human neural tube morphogenesis in vitro by geometric constraints

Nature volume 599, pages 268–272 (2021)Cite this article

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Abstract

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior–posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

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Fig. 1: A reproducible human stem-cell model of neural-tube morphogenesis.
Fig. 2: Formation of a neural and non-neural ectoderm bilayer.
Fig. 3: Towards modelling neural tube defects.
Fig. 4: Neural plate size determines neural tube shape.

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Data availability

Source data are provided with this paper. scRNA-seq data have been deposited and are publicly available in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) under accession GSE173492. Source data are provided with this paper.

Code availability

The Matlab code for the simulation used in this manuscript is deposited in GitHub (https://github.com/heitormegale/Neural-fold-simulation).

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Acknowledgements

S.J.S. acknowledges NIH grant R21 HD099598-0. E.K. acknowledges the Human Frontier Science Program for the long-term postdoctoral fellowship (LT000629/2018-L). A.H.K. acknowledges support of the National Science Foundation Graduate Research Fellowship Program (grant no. 1650114). E.D.S. acknowledges NSF grant 2013131. A.W. acknowledges Welch Foundation grant no C-2021. S.M.K.G. was recipient of an Early Postdoc Mobility grant and a Postdoc Mobility grant from the Swiss National Science Foundation (P2ZHP3_174753 and P400PB_186800). B.I.S. acknowledges NSF Physics grant 1707973. K.S.K. acknowledges support from Dr Miriam and Sheldon G. Adelson Medical Research Foundation. We thank Y. Huang and the Gladstone institute for sharing CTR2 hiPS cell line, and Brivanlou laboratory and the Rockefeller University for sharing RUES2 embryonic and reporter lines. We thank UCLA Technology Center for Genomics and Bioinformatics for sequencing of 10X libraries. We thank the Streichan and Kosik laboratory members, as well as the UCSB Stem Cell Center and associate director C. Arnold for fruitful discussions and advice. Finally, we thank A. M. Tayar for valuable comments on the manuscript.

Author information

Author notes

  1. These authors contributed equally: Eyal Karzbrun, Aimal H. Khankhel

Authors and Affiliations

  1. Department of Physics, University of California Santa Barbara, Santa Barbara, CA, USA

    Eyal Karzbrun, Heitor C. Megale, Boris I. Shraiman & Sebastian J. Streichan

  2. Kavli Institute for Theoretical Physics, Santa Barbara, CA, USA

    Eyal Karzbrun & Boris I. Shraiman

  3. Biomolecular Science and Engineering, University of California Santa Barbara, Santa Barbara, CA, USA

    Aimal H. Khankhel & Sebastian J. Streichan

  4. Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, CA, USA

    Stella M. K. Glasauer, Yofiel Wyle & Kenneth S. Kosik

  5. Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, CA, USA

    Stella M. K. Glasauer & Kenneth S. Kosik

  6. Systems Synthetic and Physical Biology Program, Rice University Houston, Houston, TX, USA

    George Britton

  7. Department of Biosciences, Rice University Houston, Houston, TX, USA

    Aryeh Warmflash

  8. Department of Bioengineering, Rice University Houston, Houston, TX, USA

    Aryeh Warmflash

  9. Center for Studies in Physics and Biology, The Rockefeller University, New York, NY, USA

    Eric D. Siggia

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Contributions

E.K. and S.J.S. designed research. E.K., A.H.K., S.M.K.G., Y.W. and G.B. performed experiments. E.K., A.H.K. and H.C.M., analysed the data. A.W. and E.D.S. provided valuable information on data interpretation. E.K., H.C.M. and B.I.S. developed and analysed the physical model. S.M.K.G. and K.S.K. performed single-cell sequencing experiment and analysis. E.K., A.H.K. and S.J.S. wrote the manuscript. S.J.S. supervised the project.

Corresponding authors

Correspondence to Eyal Karzbrun or Sebastian J. Streichan.

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The authors declare no competing interests.

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Peer review information Nature thanks Gioele La Manno, Matthias Lutolf and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 hPS cell-derived neural tube protocol.

(a) Scheme showing stem-cell derived neural tube protocol. (b) Scheme showing shape-controlled ECM pattern deposited on glass surface. (c) Seeding of hPS cells onto micropatterns results in two-dimensional cultures which are restricted to the micropattern geometry. ZO1-GFP indicates tight epithelia. (d) Adding 4% Matrigel to the media results in three-dimensional stem-cell cultures containing a single large lumen. (e) The hPS cell lumen forms a tight biochemical niche. hPS cell reporter line endogenously expressed tight junction protein ZO1 tagged with GFP (AICS-0023) is used. The 3D culture is exposed to 10kda dextran tagged with Texas Red fluorophore. Horizontal sections show that tight junctions are localized to the inner surface facing the lumen (green). Dextran is visible outside the tissue, but the lumen is devoid of dextran (red). (f) The fluorescence of dextran is quantified inside and outside the lumen, showing a 10-60 fold decrease in the lumen dextran concentration. Data are presented as mean values +/- SD. (N = 9). (g) Plot of the relative fraction of closed lumens as a function of Matrigel concentration, after 24hrs incubation. Data are presented as mean values +/- SD. Total N = 88 samples. (h) Scheme showing micropatterned hPS cell before and after exposure to Matrigel. (i) Vertical and (j) horizontal sections of pluripotent markers NANOG, OCT4 and SOX2, as well as epithelial marker ZO1. Images taken before and after lumen formation. (k) Horizontal section of an array of 2D (left) and 3D (right) stem-cell cultures immunostained with OCT4. (l) Lumen formation success rate as a function of micropattern size. Total N  = 510 samples. (m) Segmentation of single nuclei is used to count the number of cells in each sample. Each nucleus is labeled with a different color. (n) Total cell number in each sample (green curve) scales linearly with pattern area while cell density (black curve) remains constant. Data are presented as mean values +/- SD. Total N = 194 samples. Scale bars are 50μm (d,e,i,j), and 500μm (k).

Source data

Extended Data Fig. 2 In vitro morphogenesis Road Map.

(a) Schematic representation of in vitro morphogenesis in four differentiation protocols. Observed cell markers are indicated. (i) Exposure to BMP, without neural induction, results in an amnion-like tissue containing cells from three germ layers without formation of a neural fold. (ii) Neural tube morphogenesis is observed when neural induction is followed by exposure to BMP4. (iii) Homogenous expression of forebrain markers is observed under exposure to neural induction without BMP. (iv) Dorsoventral axis formation is observed in response to SHH activation (SAG) and WNT downregulation (IWP2). (b) Experimental timeline for the four protocols. Neural induction media includes N2 supplement and TGF-β inhibitor SB-431542 (SB). (c-f) Vertical sections of immunostained samples from the four differentiation protocols. (g) Horizontal section close to the glass surface and (h) an array of 20 samples. FOXA2+ cells always appear at the midline. Scale bar is 50μm (c,f-h), 50μm (d,e).

Extended Data Fig. 3 Folding morphogenesis is reproducible.

(a) Horizontal sections of 16 circular cultures from a single array exhibit stereotypic fate-patterning and morphology. (b) Bar plot showing success rate of neural tissue folding morphogenesis, in 13 experiments, with total N = 100 samples. Data are presented as mean values +/- SD. (c) Neural opening area as a function of time. Data are presented as mean values +/- SD. N = 3. (d) Neural closure period in the stem-cell system compared to chick, mouse, and human. (e) Images of six samples at 96hrs showing closed neural tissue (red, N-Cadherin) covered by non-neural ectoderm (cyan, E-cadherin). Samples were generated on rectangle  shaped micropatterns. Scale bar is 500μm (a), 50μm in (c,e).

Source data

Extended Data Fig. 4 Folding morphogenesis is not observed in 2D micropatterned cultures.

(a) Induction of neural pattern formation in 3D stem-cell cultures with a lumen triggers folding morphogenesis. In contrast triggering pattern formation in 2D micropatterned cultures does not trigger folding morphogenesis. Vertical sections of a 3D immunostained sample (top) and 2D immunostained sample (bottom). Imaged 72hrs after BMP. (b) Vertical section at 36hrs post BMP, and before folding. The neural tissue thickness and width are indicated. (c) Quantification of neural tissue thickness and width as a function of micropattern size analyzed 36hrs after BMP exposure. Data are presented as mean values +/- SD, Total N = 23. (d) Vertical section of immunostained samples during neural fold morphogenesis. Arrows indicate hinge points. (e) Distance between hinge points as a function of time after BMP exposure. Data are presented as mean values +/- SD, N = 3. Scale bars are 50μm.

Source data

Extended Data Fig. 5 Neural hinge points and apical surface characterization.

(a) Vertical sections of a hinge points showing changes in nuclei orientation across the hinge. (b) Neural crest membrane marker P75 appears in a layer of cells in between the neural tissue and surface ectoderm (ECAD). (c) Immunostaining of shroom3 and (d) F-actin at the neural apical surface. (e) Image projection of apical marker PKCζ and primary cilia marker ARL13B. (f) Sparse labeling of cells with CAAX-GFP. Labeled cells arise from a single clone indicating motility of cells following proliferation. (g) Live-cell imaging showing active filopodia (arrows) of neural cells as they change their position and shape over time. The continuous spectrum of colors indicates distance from glass (red 0μm -green - blue 35μm). Scale bars are 25μm (a,f) 10μm (b-e,g).

Extended Data Fig. 6 Live imaging of neural crest cells.

Time-lapse images showing motion of neural crest cells (SOX10-GFP). (a) Scheme indicating region in which live imaging was performed. (b) Low magnification image showing neural crest cells are organized in a stream. (c) Neural crest cell motion along the stream. A single cell is tracked and artificially colored in red. Scale bar 25μm.

Extended Data Fig. 7 Extracellular localization and expression during neural folding.

(a) Scheme showing ECM composition in three interfaces: surface-ectoderm/glass, surface-ectoderm/ neural-ectoderm and neural-ectoderm/glass. (b) Collagen and fibronectin fluorescence intensity compared at the three interfaces. Data are presented as mean values +/- SD. N = 3 independent samples (c) Vertical sections show fibronectin. (d) Horizontal sections near the glass interface show collagen V and fibronectin. Dashed line indicates border between neural and non-neural ectoderm. (e) UMAP projection and (f) violin plots of ECM genes and ECM modulators are localized to the surface ectoderm clusters. Average expression was 1-2 orders of magnitude higher in surface ectoderm vs neural ectoderm. (g) Vertical sections showing immunostaining of fibronectin matrix in control sample, ROCK inhibition (Y-27632, 10μM), and Novobiocin (100 μM). (h) Fluorescence intensities of immunostained fibronectin were measured at the neural/surface-ectoderm interface and at the neural/glass interface in all three experimental conditions. Immunostaining and imaging conditions were identical for all conditions. Fluorescence values were measured by averaging the raw fluorescence intensities in a region of interest in the image. The number of independent samples N is indicated within the plot. (i) Cell velocity of non-neural ectoderm cells measured by live-imaging of H2B-RFP cell line. No significant difference is observed in ROCK inhibited samples. N = 6. Data are presented as mean values +/- SD (b,h,i). Scale bars are 25 microns. Two-sided t-test was applied (b,h).

Source data

Extended Data Fig. 8 Single cell expression of key cell type markers.

(a) UMAP plots of single cell gene expression color coded by cluster identity (top) and experimental time point (bottom). Four main cell clusters are observed. (b) Temporal evolution of gene expression in the neural crest population indicate a transition from neural plate border/neural crest specification to migration and divarication. (c) Inferred cell-cycle populations for three clusters: neural ectoderm (NE), surface ectoderm (SE), and neural crest (NC). SE exhibits increase in G1 over time, and NC exhibits a decrease in G1. (d) Expression of key cell type markers from the four main clusters. (e) Sub-clustering of NE and SE. Neural cells sub-cluster mainly by experimental time and cell-cycle phase, which correspond to UMAP 1 and 2. In contrast, surface ectoderm cells sub-clusters do not cluster by cell-cycle or experimental time, suggesting additional differences in cell identity. (f) Scheme showing cellular process in each cluster. Titles are enriched GO pathways, and example genes appearing within each pathway.

Extended Data Fig. 9 Folding morphogenesis occurs in the absence of mesendoderm tissue.

(a) Experimental timeline to examine effect of neural induction on cell fate and folding morphogenesis. (b-d) Vertical (left) and horizontal (middle, right) sections showing that neural fates (NCAD) are upregulated with longer neural induction, whereas mesendoderm fates (Brachyury) are downregulated. (e,f) A small number of Brachyury+ cells (<10) is present in the protocol used to for neural tube morphogenesis. Total number of cells in the tissue is ~5000 cells. Scale bar is 50μm. Dome present in all cases.

Extended Data Fig. 10 Computational model of neural folding recapitulates in vitro morphology.

(a) Vertex model scheme. Vertices are connected by Hookean springs of length \({\triangle r}_{i}\). \({\vec{r}}_{i}^{a}\) and \({\vec{r}}_{i}^{b}\) are the apical and basal vertices (x and z) of cell \(i\). Neural cells have additional myosin dependent positive apical line tension, and there is a negative line tension between neural and non-neural basal surfaces (see Supplementary Notes details). (b) Time sequence of simulation showing initial cell pattern (neural tissue in red, non-neural in blue), followed by neural bending, folding and hinge formation, and closure. Each time unit corresponds to 1000 steps in the simulation. Apical lines in the neural domain strongly contract due to myosin, but never reach zero values. (c) Energy of the tissue reduces over time and reaches a minimum. (d) Interface radius and (e) Number of cells on the interface as a function of time. Both reach a steady-state value. (f) Phase diagram of the dimensionless parameters interface energy (\(\epsilon \)), neural contractility (\(\alpha \)). Each point represents a simulation that reached a steady state. Color code represents the final configuration of the simulations: neural fold closed (red), formation of neural/non-neural interface without closure (blue), and no interface formation (black). Shaded colors were drawn by hand to highlight regime boundaries. (g) Final configuration images (steady state shapes) from the simulation in different regions i) high neural apical contractility and high interface energy leads to a closed fold; ii) Low neural apical contractility and high interface energy leads to the formation of the interface but an open fold; iii) High neural apical contractility and low interface energy leads to a flat tissue. (h) The interface radius of curvature as a function of neural apical contractility. (i) The number of cells in an interface as a function of interface energy. (j) Bar plots showing the neural apical curvature, neural apical area, and interface size for the three conditions whose final configuration are shown in g. (k) Three simulations with a 40 non-neural cells, and a varying number of neural cells N = 10,30,40. Left: images of the simulation at time zero. Right: Images of the simulation at the hinge formation stage. Images were taken at this stage for consistency with experimental data (Fig. 4d, 72hrs). We observe the formation of a single hinge point for N = 10, and two lateral hinges for N = 30,40.

Source data

Supplementary information

Supplementary Information

This file contains a Supplementary Note on the physical model of neural folding.

Reporting Summary

Supplementary Table 1

This table contains top differentially expressed genes for cell clusters identified by scRNA-seq.

Supplementary Table 2

This table contains GO Analysis for cell clusters identified by scRNA-seq.

Supplementary Table 3

This table contains a list of media and reagents used in this work.

Supplementary Table 4

This table contains a list of primary and secondary antibodies used in this work.

Supplementary Video 1

Interkinetic nuclear migration. Live-cell imaging of interkinetic nuclear migrations. Time-lapse microscopy video using a 40× objective in the region of the neural fold from 72–94 h post BMP using a live reporter cell line (RUES2, H2B-RFP).

Supplementary Video 2

Neural progenitors. Live-cell imaging of CAAX-RFP reporter line mixed into a background non-fluorescent cell line. Color indicates distance from glass (red 0 μm–blue 35 μm). 72 h post-BMP. Elongated neural progenitor cells are observed exhibiting motion and filopodia. Frame rate: one frame every 30 min.

Supplementary Video 3

Primary cilia. 63× image slices of a fixed stem cell-derived neural tube at 120 h post-BMP. Apical marker PKCζ (red), and primary cilia marker ARL13B (yellow) are shown at the apical surface of neural tissue region.

Supplementary Video 4

Surface ectoderm cell migration. Live-cell imaging of H2B-RFP reporter line showing directional migration of non-neural cells in parallel to the culture long axis. Frame rate: one frame every 25 min.

Supplementary Video 5

Neural crest cells 3D. Three-dimensional view of a fixed stem cell-derived neural tube at 96 h post-BMP. Neural crest (SOX10) and non-neural ectoderm (ECAD) cells shown.

Supplementary Video 6

Neural crest migration. Live-cell imaging of SOX10-GFP reporter cell line showing a neural crest cell migrating and proliferating. Frame rate: one frame every 10 min.

Supplementary Video 7

Live-cell imaging of H2B-RFP reporter line demonstrating cell motility in control sample 72 h post-BMP. Frame rate: one frame every 5 min.

Supplementary Video 8

Live-cell imaging of H2B-RFP reporter line demonstrating cell motility in ROCK-inhibited sample. 72 h post-BMP. Frame rate: one frame every 5 min.

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Karzbrun, E., Khankhel, A.H., Megale, H.C. et al. Human neural tube morphogenesis in vitro by geometric constraints. Nature 599, 268–272 (2021). https://doi.org/10.1038/s41586-021-04026-9

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