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

Nature Biotechnology volume 38, pages 1265–1273 (2020)Cite this article

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

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Abstract

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 (https://github.com/kirkeby-lab).

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.

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Acknowledgements

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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Author notes

  1. These authors contributed equally: Pedro Rifes, Marc Isaksson.

Authors and Affiliations

  1. Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark

    Pedro Rifes, Gaurav Singh Rathore, Patrick Aldrin-Kirk, Oliver Knights Møller & Agnete Kirkeby

  2. The Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen, Copenhagen, Denmark

    Pedro Rifes, Gaurav Singh Rathore, Patrick Aldrin-Kirk, Guido Barzaghi & Agnete Kirkeby

  3. Department of Biomedical Engineering, Lund University, Lund, Sweden

    Marc Isaksson & Thomas Laurell

  4. Department of Experimental Medical Science, Lund University, Lund, Sweden

    Marc Isaksson, Malin Parmar & Agnete Kirkeby

  5. The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark

    Julie Lee, Kristoffer Lihme Egerod, Dylan M. Rausch & Tune H. Pers

  6. Wallenberg Center for Molecular Medicine, Lund University, Lund, Sweden

    Agnete Kirkeby

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Contributions

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). https://doi.org/10.1038/s41587-020-0525-0

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