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Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient

A Publisher Correction to this article was published on 11 June 2020

This article has been updated


The study of brain development in humans is limited by the lack of tissue samples and suitable in vitro models. Here, we model early human neural tube development using human embryonic stem cells cultured in a microfluidic device. The approach, named microfluidic-controlled stem cell regionalization (MiSTR), exposes pluripotent stem cells to signaling gradients that mimic developmental patterning. Using a WNT-activating gradient, we generated a neural tissue exhibiting progressive caudalization from forebrain to midbrain to hindbrain, including formation of isthmic organizer characteristics. Single-cell transcriptomics revealed that rostro-caudal organization was already established at 24 h of differentiation, and that the first markers of a neural-specific transcription program emerged in the rostral cells at 48 h. The transcriptomic hallmarks of rostro-caudal organization recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. Thus, MiSTR will facilitate research on the factors and processes underlying rostro-caudal neural tube patterning.

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Fig. 1: Design of the MiSTR cell culture system and establishment of a WNT signaling gradient in hESC-derived tissue.
Fig. 2: Neural patterning along the rostro-caudal axis in day 14 MiSTR tissue.
Fig. 3: Single-cell transcriptomics of dorsal and ventral MiSTR tissues.
Fig. 4: Single-cell transcriptomics of early MiSTR patterning and temporal dissection of MiSTR regionalization.

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Data availability

scRNAseq data is deposited in the Gene Expression Omnibus with accession number GSE135399. scRNAseq code used for analysis is supplied as Supplementary Code in html and .rmd files and can also be found on GitHub (

Change history

  • 11 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


  1. Nordstrom, U., Jessell, T. M. & Edlund, T. Progressive induction of caudal neural character by graded Wnt signaling. Nat. Neurosci. 5, 525–532 (2002).

    Article  Google Scholar 

  2. Kiecker, C. & Lumsden, A. Compartments and their boundaries in vertebrate brain development. Nat. Rev. Neurosci. 6, 553–564 (2005).

    Article  CAS  Google Scholar 

  3. Kiecker, C. & Lumsden, A. The role of organizers in patterning the nervous system. Annu Rev. Neurosci. 35, 347–367 (2012).

    Article  CAS  Google Scholar 

  4. Ribes, V. & Briscoe, J. Establishing and interpreting graded Sonic Hedgehog signaling during vertebrate neural tube patterning: the role of negative feedback. Cold Spring Harb. Perspect. Biol. 1, a002014 (2009).

    Article  Google Scholar 

  5. Meinhardt, A. et al. 3D reconstitution of the patterned neural tube from embryonic stem cells. Stem Cell Rep. 3, 987–999 (2014).

    Article  Google Scholar 

  6. Demers, C. J. et al. Development-on-chip: in vitro neural tube patterning with a microfluidic device. Development 143, 1884–1892 (2016).

    Article  CAS  Google Scholar 

  7. Bagley, J. A., Reumann, D., Bian, S., Levi-Strauss, J. & Knoblich, J. A. Fused cerebral organoids model interactions between brain regions. Nat. Methods 14, 743–751 (2017).

    Article  CAS  Google Scholar 

  8. Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).

    Article  CAS  Google Scholar 

  9. Cederquist, G. Y. et al. Specification of positional identity in forebrain organoids. Nat. Biotechnol. 37, 436–444 (2019).

    Article  CAS  Google Scholar 

  10. Kirkeby, A. et al. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 1, 703–714 (2012).

    Article  CAS  Google Scholar 

  11. Jeon, N. L. et al. Generation of solution and surface gradients using microfluidic systems. Langmuir 16, 8311–8316 (2000).

    Article  CAS  Google Scholar 

  12. Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  Google Scholar 

  13. Dupe, V. et al. In vivo functional analysis of the Hoxa-1 3’ retinoic acid response element (3’RARE). Development 124, 399–410 (1997).

    CAS  Google Scholar 

  14. Strate, I., Min, T. H., Iliev, D. & Pera, E. M. Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system. Development 136, 461–472 (2009).

    Article  CAS  Google Scholar 

  15. Yang, L. et al. Analysis of FGF-dependent and FGF-independent pathways in otic placode induction. PLoS ONE 8, e55011 (2013).

    Article  CAS  Google Scholar 

  16. La Manno, G. et al. Molecular diversity of midbrain development in mouse, human, and stem cells. Cell 167, 566–580 (2016).

    Article  Google Scholar 

  17. Pijuan-Sala, B. et al. A single-cell molecular map of mouse gastrulation and early organogenesis. Nature 566, 490–495 (2019).

    Article  CAS  Google Scholar 

  18. Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017).

    Article  CAS  Google Scholar 

  19. Kanton, S. et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574, 418–422 (2019).

    Article  CAS  Google Scholar 

  20. Stoeckius, M. et al. Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 19, 224 (2018).

    Article  CAS  Google Scholar 

  21. Brafman, D. & Willert, K. Wnt/beta-catenin signaling during early vertebrate neural development. Dev. Neurobiol. 77, 1239–1259 (2017).

    Article  Google Scholar 

  22. Metzis, V. et al. Nervous system regionalization entails axial allocation before neural differentiation. Cell 175, 1105–1118 (2018).

    Article  CAS  Google Scholar 

  23. Abu-Abed, S. et al. The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures. Genes Dev. 15, 226–240 (2001).

    Article  CAS  Google Scholar 

  24. Andoniadou, C. L. et al. HESX1- and TCF3-mediated repression of Wnt/beta-catenin targets is required for normal development of the anterior forebrain. Development 138, 4931–4942 (2011).

    Article  CAS  Google Scholar 

  25. Peng, G. & Westerfield, M. Lhx5 promotes forebrain development and activates transcription of secreted Wnt antagonists. Development 133, 3191–3200 (2006).

    Article  CAS  Google Scholar 

  26. Filipe, M., Goncalves, L., Bento, M., Silva, A. C. & Belo, J. A. Comparative expression of mouse and chicken Shisa homologues during early development. Dev. Dyn. 235, 2567–2573 (2006).

    Article  CAS  Google Scholar 

  27. Xia, Y. & Whitesides, G. M. Soft Lithography. Angew. Chem. Int. Ed. 37, 550–575 (1998).

    Article  CAS  Google Scholar 

  28. Satyanarayana, S., Karnik, R. N. & Majumdar, A. Stamp-and-stick room-temperature bonding technique for microdevices. J. Microelectromechanical Syst. 14, 392–399 (2005).

    Article  Google Scholar 

  29. Edward, J. T. Molecular volumes and the Stokes–Einstein equation. J. Chem. Educ. 47, 261 (1970).

    Article  CAS  Google Scholar 

  30. Maury, Y. et al. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat. Biotechnol. 33, 89–96 (2015).

    Article  CAS  Google Scholar 

  31. Nestorowa, S. et al. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation. Blood 128, e20–31 (2016).

    Article  CAS  Google Scholar 

  32. Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902(2019).

    Article  CAS  Google Scholar 

  33. Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).

    Article  Google Scholar 

  34. Haghverdi, L., Lun, A. T. L., Morgan, M. D. & Marioni, J. C. Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors. Nat. Biotechnol. 36, 421–427 (2018).

    Article  CAS  Google Scholar 

  35. Kempf, H. et al. Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells. Nat. Commun. 7, 13602 (2016).

    Article  CAS  Google Scholar 

  36. Funa, N. S. et al. beta-Catenin regulates primitive streak induction through collaborative interactions with SMAD2/SMAD3 and OCT4. Cell Stem Cell 16, 639–652 (2015).

    Article  CAS  Google Scholar 

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This study was supported by the Novo Nordisk Foundation (grant no. NNF18OC0030286 to A.K.), The Lundbeck Foundation (grant no. R190-2014-3904 to T.H.P.) and the following grants to A.K.: Innovation Fund Denmark (no. BrainStem 4108-00008 A), the Strong Research Environment at Lund University Multipark, the Swedish Research Council (no. 70862601/Bagadilico), The Crafoord Foundation, The Segerfalk Foundation, The Tore Nilsson Foundation, The Sven-Olof Janson Foundation and the Swedish Fund for Research Without Animal Experiments. The research leading to these results has received funding from the New York Stem Cell Foundation (M.P.), the European Research Council under the ERC Grant Agreement no. 30971 (M.P.), the Swedish Research Council (grant agreement no. 521-2012-5624, M.P.). The Novo Nordisk Foundation Center for Stem Cell Biology (DanStem) and the Novo Nordisk Foundation Center for Basic Metabolic Research (CBMR) are supported by Novo Nordisk Foundation grants (nos. NNF17CC0027852 and NNF18CC0034900, respectively). M.P. is a New York Stem Cell Foundation Robertson Investigator. We thank S. da Rocha Baez, I. Nilsson, M. Madrona, M. Heide Ankjær, H.K. Lilja-Fischer (CBMR Single-cell Omics Platform), H. Neil (DanStem Genomics Platform), J. Bulkescher (DanStem Imaging Platform) and A. Meligkova (DanStem Stem Cell Culture Platform) for excellent technical and bioinformatics assistance and for use of instruments.

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Authors and Affiliations



M.I., T.L., P.R., T.H.P., M.P., G.S.R. and A.K. designed the study. M.I., P.R., G.S.R., P.A.K., D.M.R., G.B., K.L.E., O.K.M. and J.L. performed experiments. M.I., T.L., P.R. and A.K. wrote the manuscript.

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Correspondence to Agnete Kirkeby.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Tables 3–5 and Notes.

Reporting Summary

Supplementary Table 1

Marker genes for cell clusters obtained from single-cell RNAseq analysis

Supplementary Table 2

Supplementary statistical summary of analysis

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Rifes, P., Isaksson, M., Rathore, G.S. et al. Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient. Nat Biotechnol 38, 1265–1273 (2020).

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