Article | Published:

Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells

Nature Biotechnology volume 35, pages 154163 (2017) | Download Citation

Abstract

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

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Acknowledgements

We would like to thank G. Wahl (Salk Institute) and R. Jaenisch (Whitehead Institute) for sharing plasmids, F. Zhang (MIT) for sharing the TALE-toolbox, and G. Ciceri and G. Cederquist (MSKCC) for their valuable input on experimental design and feedback on the manuscript. We thank M. Sheldon (Rutgers University) for sharing protocol for fibroblast preparation. This work was supported in part through grants from the Starr Foundation (L.S. and A.H.B.) and grants NS084334 and R01NS072381(L.S.) and by NYSTEM contracts C030137 (S.S., & L.S.) and C028128 (A.H.B.) and private funds from the Rockefeller University. The Molecular Cytogenetics Core Facility at MSKCC as well as other MSKCC facilities and investigators are supported by the NIH Cancer Center support grant P30 CA008748. Some of the images were obtained using instrumentation at The Rockefeller University Bio-Imaging Resource Center. The SKI Stem Cell Research Facility is supported by NYSTEM grants C029153 and C024175 and The Starr Foundation. X.-J.Z. and B.Z. were supported by NYSTEM fellowships (C026879).

Author information

Affiliations

  1. Developmental Biology, Sloan-Kettering Institute, New York, New York, USA.

    • Yuchen Qi
    • , Xin-Jun Zhang
    • , Jason Tchieu
    • , Bastian Zimmer
    • , Faranak Fattahi
    • , Yosif Ganat
    • , Nadja Zeltner
    • , Mark Tomishima
    • , Song-Hai Shi
    •  & Lorenz Studer
  2. Center of Stem Cell Biology, Sloan-Kettering Institute, New York, New York, USA.

    • Yuchen Qi
    • , Xin-Jun Zhang
    • , Jason Tchieu
    • , Bastian Zimmer
    • , Faranak Fattahi
    • , Yosif Ganat
    • , Nadja Zeltner
    • , Mark Tomishima
    • , Song-Hai Shi
    •  & Lorenz Studer
  3. Weill Cornell Graduate School, New York, New York, USA.

    • Yuchen Qi
    •  & Faranak Fattahi
  4. Laboratory of Brain Development and Repair, The Rockefeller University, New York, New York, USA.

    • Nicolas Renier
    • , Zhuhao Wu
    • , Ricardo Azevedo
    •  & Marc Tessier-Lavigne
  5. Department of Physiology, Columbia University, New York, New York, USA.

    • Talia Atkin
    • , Ziyi Sun
    •  & Maria Karayiorgou
  6. Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.

    • M Zeeshan Ozair
    •  & Ali H Brivanlou
  7. Department of Physiology and Department of Neuroscience, Columbia University, New York, New York, USA.

    • Joseph Gogos
  8. SKI Stem Cell Core facility, Sloan-Kettering Institute, New York, New York, USA.

    • Mark Tomishima

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Contributions

Y.Q.: conception and study design, hESC manipulation, differentiation and characterization, in vitro and in vivo analyses and data interpretation and writing of manuscript. X.-J.Z.: electrophysiological recordings, in vivo transplantation, data analysis, interpretation and writing of manuscript. N.R. and Z.W.: iDISCO analysis of grafted animals, data analysis, interpretation and writing of manuscript. T.A. and Z.S.: iPSC differentiation studies, in vitro functional and electrophysiological analyses. M.Z.O. and A.H.B.: generation of the CUX2-tdTomato reporter line and writing of the manuscript. J.T. and B.Z.: generation of PAX6 and SIX1 reporter lines, data analysis. F.F. and N.Z.: neural crest differentiation protocols and data analysis. Y.G.: transplantation studies. R.A.: iDISCO analysis. M.K. and J.G.: iPSC differentiation studies, data interpretation. M.T.: iPSC induction and characterization, data analysis. M.T.-L.: design and interpretation of iDISCO studies, writing of manuscript.S.-H.S.: conception and study, data analysis and interpretation, writing of manuscript. L.S.: conception and study design, data analysis and interpretation, writing of manuscript.

Competing interests

The Memorial Sloan-Kettering Cancer Center has filed a provisional patent application (US PRO 62/287821) on the methods described in the manuscript.

Corresponding author

Correspondence to Lorenz Studer.

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DOI

https://doi.org/10.1038/nbt.3777

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