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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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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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.

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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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