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Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes

Nature Biotechnology volume 33, pages 89–96 (2015)Cite this article

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Abstract

Specification of cell identity during development depends on exposure of cells to sequences of extrinsic cues delivered at precise times and concentrations. Identification of combinations of patterning molecules that control cell fate is essential for the effective use of human pluripotent stem cells (hPSCs) for basic and translational studies. Here we describe a scalable, automated approach to systematically test the combinatorial actions of small molecules for the targeted differentiation of hPSCs. Applied to the generation of neuronal subtypes, this analysis revealed an unappreciated role for canonical Wnt signaling in specifying motor neuron diversity from hPSCs and allowed us to define rapid (14 days), efficient procedures to generate spinal and cranial motor neurons as well as spinal interneurons and sensory neurons. Our systematic approach to improving hPSC-targeted differentiation should facilitate disease modeling studies and drug screening assays.

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Figure 1: Large-scale examination of differentiation conditions reveals a concentration dependent Chir/RA cooperation to induce sMN progenitors.
Figure 2: Time-dependent combinatorial action of RA, Chir-99021 and SAG to efficiently specify MN progenitors with no increase of sMN generation.
Figure 3: γ-secretase inhibitors rapidly force the conversion of sMN progenitors to sMNs.
Figure 4: Rapid and efficient differentiation of functional sMNs from hPSCs.
Figure 5: Chir regulates key developmental pathways in a concentration-dependent manner to rapidly specify hindbrain cMNs.
Figure 6: Synchronous generation of neuronal subtypes for comparative studies of axon response to guidance cues.

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Acknowledgements

I-Stem is part of the Biotherapies Institute for Rare Diseases (BIRD) supported by the Association Française contre les Myopathies (AFM-Téléthon). This project was funded by AFM, INSERM and Laboratoire d'Excellence “Labex Revive” (Investissement d'Avenir; ANR-10-LABX-73). S.N. is currently supported by the “Labex Revive” and was previously supported by a fellowship from the Genopole. N.S.-M. was supported by the program INGESTEM (investissement d'avenir ANR-11-INBS-009-01). R.A.P. and V.C. are supported by ATIP-Avenir, Agence Nationale de la Recherche (ANR-12-BSV4-0021-01, ANR-13-JSV4-0002-01) and the Ville de Paris. We thank C. Varela and N. Lefort (I-STEM) for karyotyping the cell lines and L. Aubry (I-STEM) for providing the IMR90 hiPSC line. We thank H. Wichterle, F. Giudicelli, V. Ribes, E.O. Mazzoni and A. Rebsam for helpful discussion and critical review of the manuscript. We thank J.-F. Brunet for providing the Phox2b antibody, E.R. Lowry and H. Wichterle (Columbia University) for testing the sMN differentiation and for the Hb9∷GFP-Neomycin plasmid. We thank D. Furling (Institute of Myology) for providing human primary myoblasts.

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Authors and Affiliations

  1. CECS, I-STEM (Institute for Stem Cell Therapy and Exploration of Monogenic Diseases), AFM, Evry, France

    Yves Maury & Julien Côme

  2. CNRS UMR 8118, Université Paris Descartes Sorbonne Paris Cité, Paris, France

    Rebecca A Piskorowski & Vivien Chevaleyre

  3. INSERM/UEVE UMR 861, I-STEM, AFM, Evry, France

    Nouzha Salah-Mohellibi, Marc Peschanski, Cécile Martinat & Stéphane Nedelec

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  1. Yves Maury
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Contributions

Y.M., J.C., S.N., R.A.P. (electrophysiology) and N.S.-M. (real-time PCR) performed experiments. Y.M., J.C., C.M., R.A.P. and S.N. analyzed data. M.P., C.M., S.N. designed the project. S.N. and C.M. initiated the project, supervised and organized experiments. M.P. and V.C. provided resources. S.N. wrote the manuscript with input from all authors.

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Correspondence to Stéphane Nedelec.

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Maury, Y., Côme, J., Piskorowski, R. et al. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat Biotechnol 33, 89–96 (2015). https://doi.org/10.1038/nbt.3049

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