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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402–406 (2009).

  2. 2.

    , , , & Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell Rep. 3, 1140–1152 (2013).

  3. 3.

    , , & Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proc. Natl. Acad. Sci. USA 108, 19240–19245 (2011).

  4. 4.

    et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572 (2013).

  5. 5.

    et al. Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases. J. Med. Chem. 42, 5120–5130 (1999).

  6. 6.

    et al. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J. Neurochem. 76, 173–181 (2001).

  7. 7.

    et al. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat. Biotechnol. 30, 715–720 (2012).

  8. 8.

    et al. Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. Neuron 77, 440–456 (2013).

  9. 9.

    , , , & Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat. Neurosci. 15, 477–486 S1 (2012).

  10. 10.

    et al. The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901. Bioorg. Med. Chem. Lett. 18, 6501–6504 (2008).

  11. 11.

    et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).

  12. 12.

    et al. Derivation of Diverse Hormone-Releasing Pituitary Cells from Human Pluripotent Stem Cells. Stem Cell Reports 6, 858–872 (2016).

  13. 13.

    et al. Specification of functional cranial placode derivatives from human pluripotent stem cells. Cell Rep. 5, 1387–1402 (2013).

  14. 14.

    & The role of FGF/Erk signaling in pluripotent cells. Development 137, 3351–3360 (2010).

  15. 15.

    , , , & Disrupted ERK signaling during cortical development leads to abnormal progenitor proliferation, neuronal and network excitability and behavior, modeling human neuro-cardio-facial-cutaneous and related syndromes. J. Neurosci. 32, 8663–8677 (2012).

  16. 16.

    et al. FGF signalling inhibits neural induction in human embryonic stem cells. EMBO J. 30, 4874–4884 (2011).

  17. 17.

    , , , & Molecular logic of neocortical projection neuron specification, development and diversity. Nat. Rev. Neurosci. 14, 755–769 (2013).

  18. 18.

    et al. Wnt signalling regulates adult hippocampal neurogenesis. Nature 437, 1370–1375 (2005).

  19. 19.

    Wnt signaling in the vertebrate central nervous system: from axon guidance to synaptic function. Cold Spring Harb. Perspect. Biol. 4, (2012).

  20. 20.

    et al. Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proc. Natl. Acad. Sci. USA 112, E2725–E2734 (2015).

  21. 21.

    et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).

  22. 22.

    et al. Area-specific reestablishment of damaged circuits in the adult cerebral cortex by cortical neurons derived from mouse embryonic stem cells. Neuron 85, 982–997 (2015).

  23. 23.

    et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat. Commun. 6, 6626 (2015).

  24. 24.

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

  25. 25.

    et al. Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo. Stem Cells 28, 955–964 (2010).

  26. 26.

    , , & Lineage-dependent circuit assembly in the neocortex. Development 140, 2645–2655 (2013).

  27. 27.

    et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011).

  28. 28.

    et al. Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease. Cell Stem Cell 15, 653–665 (2014).

  29. 29.

    , , , & L3MBTL1 deficiency directs the differentiation of human embryonic stem cells toward trophectoderm. Stem Cells Dev. 20, 1889–1900 (2011).

  30. 30.

    et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

  31. 31.

    & Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).

Download references


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


  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


  1. Search for Yuchen Qi in:

  2. Search for Xin-Jun Zhang in:

  3. Search for Nicolas Renier in:

  4. Search for Zhuhao Wu in:

  5. Search for Talia Atkin in:

  6. Search for Ziyi Sun in:

  7. Search for M Zeeshan Ozair in:

  8. Search for Jason Tchieu in:

  9. Search for Bastian Zimmer in:

  10. Search for Faranak Fattahi in:

  11. Search for Yosif Ganat in:

  12. Search for Ricardo Azevedo in:

  13. Search for Nadja Zeltner in:

  14. Search for Ali H Brivanlou in:

  15. Search for Maria Karayiorgou in:

  16. Search for Joseph Gogos in:

  17. Search for Mark Tomishima in:

  18. Search for Marc Tessier-Lavigne in:

  19. Search for Song-Hai Shi in:

  20. Search for Lorenz Studer in:


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.

Integrated supplementary information

Supplementary information

About this article

Publication history





Further reading