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Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors

Abstract

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types, including neurons. However, differentiation of hPSCs with extrinsic factors is a slow, step-wise process, mimicking the protracted timing of human development. Using a small-molecule screen, we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at >75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors, including tetrodotoxin (TTX)-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development1, suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain.

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Figure 1: LSB3i-treated hPSCs rapidly acquire a nociceptor phenotype within 12 d.
Figure 2: LSB3i-treated hPSCs accelerate via a neural crest intermediate into mature bipolar nociceptors with an action potential.
Figure 3: Gene expression of LSB3i nociceptors.
Figure 4: Functional characterization of mature LSB3i nociceptors.

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References

  1. Bystron, I., Rakic, P., Molnar, Z. & Blakemore, C. The first neurons of the human cerebral cortex. Nat. Neurosci. 9, 880–886 (2006).

    Article  CAS  Google Scholar 

  2. Zhang, X.Q. & Zhang, S.C. Differentiation of neural precursors and dopaminergic neurons from human embryonic stem cells. Methods Mol. Biol. 584, 355–366 (2010).

    Article  CAS  Google Scholar 

  3. Elkabetz, Y. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22, 152–165 (2008).

    Article  CAS  Google Scholar 

  4. Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004).

    Article  CAS  Google Scholar 

  5. Saha, K. & Jaenisch, R. Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell 5, 584–595 (2009).

    Article  CAS  Google Scholar 

  6. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  Google Scholar 

  7. Kim, D.S. et al. Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev. 6, 270–281 (2010).

    Article  CAS  Google Scholar 

  8. Zhou, J. et al. High-efficiency induction of neural conversion in hESCs and hiPSCs with a single chemical inhibitor of TGF-beta superfamily receptors. Stem Cells 1741–1750 (2010).

    Article  CAS  Google Scholar 

  9. Yu, P.B. et al. BMP type I receptor inhibition reduces heterotopic [corrected] ossification. Nat. Med. 14, 1363–1369 (2008).

    Article  CAS  Google Scholar 

  10. Zhang, X. et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7, 90–100 (2010).

    Article  CAS  Google Scholar 

  11. Lee, M.K., Tuttle, J.B., Rebhun, L.I., Cleveland, D.W. & Frankfurter, A. The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis. Cell Motil. Cytoskeleton 17, 118–132 (1990).

    Article  CAS  Google Scholar 

  12. Sun, L. 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).

    Article  CAS  Google Scholar 

  13. Bennett, C.N. et al. Regulation of Wnt signaling during adipogenesis. J. Biol. Chem. 277, 30998–31004 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Gerdes, J., Schwab, U., Lemke, H. & Stein, H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer 31, 13–20 (1983).

    Article  CAS  Google Scholar 

  16. Hendzel, M.J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997).

    Article  CAS  Google Scholar 

  17. Sun, Y. et al. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nat. Neurosci. 11, 1283–1293 (2008).

    Article  CAS  Google Scholar 

  18. Gerrero, M.R. et al. Brn-3.0: a POU-domain protein expressed in the sensory, immune, and endocrine systems that functions on elements distinct from known octamer motifs. Proc. Natl. Acad. Sci. USA 90, 10841–10845 (1993).

    Article  CAS  Google Scholar 

  19. Marmigere, F. & Ernfors, P. Specification and connectivity of neuronal subtypes in the sensory lineage. Nat. Rev. Neurosci. 8, 114–127 (2007).

    Article  CAS  Google Scholar 

  20. Papapetrou, E.P. et al. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. Proc. Natl. Acad. Sci. USA 106, 12759–12764 (2009).

    Article  CAS  Google Scholar 

  21. Aoki, Y. et al. Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. Dev. Biol. 259, 19–33 (2003).

    Article  CAS  Google Scholar 

  22. Lee, G. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25, 1468–1475 (2007).

    Article  CAS  Google Scholar 

  23. George, L., Chaverra, M., Todd, V., Lansford, R. & Lefcort, F. Nociceptive sensory neurons derive from contralaterally migrating, fate-restricted neural crest cells. Nat. Neurosci. 10, 1287–1293 (2007).

    Article  CAS  Google Scholar 

  24. Schlosser, G. & Northcutt, R.G. Development of neurogenic placodes in Xenopus laevis. J. Comp. Neurol. 418, 121–146 (2000).

    Article  CAS  Google Scholar 

  25. Schlosser, G. Induction and specification of cranial placodes. Dev. Biol. 294, 303–351 (2006).

    Article  CAS  Google Scholar 

  26. Woolf, C.J. & Ma, Q. Nociceptors–noxious stimulus detectors. Neuron 55, 353–364 (2007).

    Article  CAS  Google Scholar 

  27. Fasano, C.A., Chambers, S.M., Lee, G., Tomishima, M.J. & Studer, L. Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell 6, 336–347 (2010).

    Article  CAS  Google Scholar 

  28. Ma, Q., Fode, C., Guillemot, F. & Anderson, D.J. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 13, 1717–1728 (1999).

    Article  CAS  Google Scholar 

  29. Marmigere, F. & Ernfors, P. Specification and connectivity of neuronal subtypes in the sensory lineage. Nat. Rev. Neurosci. 8, 114–127 (2007).

    Article  CAS  Google Scholar 

  30. Dib-Hajj, S.D. et al. Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons. FEBS Lett. 462, 117–120 (1999).

    Article  CAS  Google Scholar 

  31. Renganathan, M., Cummins, T.R. & Waxman, S.G. Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J. Neurophysiol. 86, 629–640 (2001).

    Article  CAS  Google Scholar 

  32. Jarvis, M.F. et al. A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc. Natl. Acad. Sci. USA 99, 17179–17184 (2002).

    Article  CAS  Google Scholar 

  33. Caterina, M.J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997).

    Article  CAS  Google Scholar 

  34. North, R.A. The P2X3 subunit: a molecular target in pain therapeutics. Curr. Opin. Investig. Drugs 4, 833–840 (2003).

    CAS  PubMed  Google Scholar 

  35. Kitao, Y., Robertson, B., Kudo, M. & Grant, G. Neurogenesis of subpopulations of rat lumbar dorsal root ganglion neurons including neurons projecting to the dorsal column nuclei. J. Comp. Neurol. 371, 249–257 (1996).

    Article  CAS  Google Scholar 

  36. Dorsky, R.I., Moon, R.T. & Raible, D.W. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370–373 (1998).

    Article  CAS  Google Scholar 

  37. Lee, H.Y. et al. Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells. Science 303, 1020–1023 (2004).

    Article  CAS  Google Scholar 

  38. Cornell, R.A. & Eisen, J.S. Delta/Notch signaling promotes formation of zebrafish neural crest by repressing Neurogenin 1 function. Development 129, 2639–2648 (2002).

    CAS  PubMed  Google Scholar 

  39. Molliver, D.C. et al. IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life. Neuron 19, 849–861 (1997).

    Article  CAS  Google Scholar 

  40. Ibanez, C.F. & Ernfors, P. Hierarchical control of sensory neuron development by neurotrophic factors. Neuron 54, 673–675 (2007).

    Article  CAS  Google Scholar 

  41. Luo, W. et al. A hierarchical NGF signaling cascade controls Ret-dependent and Ret-independent events during development of nonpeptidergic DRG neurons. Neuron 54, 739–754 (2007).

    Article  CAS  Google Scholar 

  42. Gascon, E. et al. Hepatocyte growth factor-Met signaling is required for Runx1 extinction and peptidergic differentiation in primary nociceptive neurons. J. Neurosci. 30, 12414–12423 (2010).

    Article  CAS  Google Scholar 

  43. Chen, C.L. et al. Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain. Neuron 49, 365–377 (2006).

    Article  CAS  Google Scholar 

  44. Kramer, I. et al. A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification. Neuron 49, 379–393 (2006).

    Article  CAS  Google Scholar 

  45. Yoshikawa, M. et al. Runx1 selectively regulates cell fate specification and axonal projections of dorsal root ganglion neurons. Dev. Biol. 303, 663–674 (2007).

    Article  CAS  Google Scholar 

  46. Placantonakis, D.G. et al. BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells 27, 521–532 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Hendrikx (SKI Flow Cytometry Core lab), A. Viale (SKI Genomics Core lab), E. Tu (SKI stem cell facility), and M. Tomishima (SKI stem cell facility) for excellent technical support. We also thank R. McKernan for support of functional analysis, M. Postlethwaite for assistance with electrophysiology, C. Benn for help in gene expression analysis and S. Kriks for assistance with hPSC culturing. The work was supported in part through grants NS066390 from National Institute of Neurological Disorders and Stroke/US National Institutes of Health (NIH) and C026447 from New York State Stem Cell Science (NYSTEM) to L.S., R01DA024681 from the National Institute on Drug Abuse/NIH to S.-H.S., PO1NS048120 from the National Institute of Mental Health/NIH to S.-H.S., and C026399 from NYSTEM to S.M.C.

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Authors

Contributions

S.M.C., experimental design, characterization experiments and manuscript; Y.Q., chemical screen to identify 3i; Y.M. and G.L., SOX10GFP BAC transgenic hPSC line generation and culturing; X.-J.Z. and L.N., initial LSB3i electrophysiology; J.B., L.C., E.S. and P.W., electrophysiology and calcium imaging experiments, PRPH characterization and manuscript; S.-H.S., electrophysiology experimental design; L.S., experimental design and manuscript.

Corresponding authors

Correspondence to Stuart M Chambers or Lorenz Studer.

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Competing interests

J.B., L.C., E.S. and P.W. are employees of Neusentis, Pfizer Global R&D.

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Supplementary Figs. 1-15 and Supplementary Tables 1-2 (PDF 4200 kb)

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Chambers, S., Qi, Y., Mica, Y. et al. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol 30, 715–720 (2012). https://doi.org/10.1038/nbt.2249

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