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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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.
The authors declare no competing interests
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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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