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Generation of serotonin neurons from human pluripotent stem cells

Nature Biotechnology volume 34, pages 89–94 (2016)Cite this article

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Abstract

Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2-3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.

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Figure 1: Specification of rostral hindbrain progenitors.
Figure 2: Specification of ventral hindbrain progenitors.
Figure 3: Generation of central serotonin neurons.
Figure 4: Functional properties of the central serotonin neurons.

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Acknowledgements

This work was partially supported by US National Institutes of Health (NIH) grants (NS045926, MH099587 and MH100031 to S.C.Z.; 1R01DK071801 to L.L.) and the National Institute of Child Health and Human Development (NICHD; P30 HD03352). The Q-Exactive Orbitrap mass spectrometer was purchased through the support of an NIH shared instrument grant (NIH–National Center for Research Resources (NCRR) S10RR029531 to L.L.). L.L. acknowledges an H.I. Romnes Faculty Research Fellowship. S.C.Z. acknowledges a Steenbock Professorship.

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Authors and Affiliations

  1. Waisman Center, University of Wisconsin, Madison, Wisconsin, USA

    Jianfeng Lu, Huisheng Liu, Cindy Tzu-Ling Huang, Mohammad Amin Sherafat, Jeffrey Jones, Melvin Ayala & Su-Chun Zhang

  2. School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA

    Xuefei Zhong, Ling Hao & Lingjun Li

  3. Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Lingjun Li

  4. Department of Neuroscience and Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA

    Su-Chun Zhang

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Contributions

J.L. designed and performed experiments, analyzed data and co-wrote the paper. X.Z., L.H., L.L. and M.A.S. processed and assessed the serotonin release samples. H.L. performed the whole-cell patch-clamp recordings. C.T.-L.H. cultured cells and performed immunocytochemistry experiments. M.A.S., J.J. and M.A. performed animal experiments. S.-C.Z. designed experiments and co-wrote the paper.

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Correspondence to Su-Chun Zhang.

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Integrated supplementary information

Supplementary Figure 1 Generation of NSCs from hPSCs in SDC medium.

(a) Q-PCR of pluripotent markers (OCT4, NANOG), neural markers (SOX1, PAX6, N-Cadherin), markers for the other germ layers (T, EOMES, SOX17) and Trophoblast marker CDX2 when cells were treated with different concentrations of CHIR99021. (b) Cells stained for pluripotent marker OCT4 (Red) and neural stem cell marker SOX1 (Green) when treated with different concentrations of CHIR99021. (c) FACS images of SOX2-expressing cells when treated with different concentrations of CHIR99021. (d) Quantification of SOX2-expressing cells in c (n = 3). Data are represented as mean ± SEM. (e) Negative staining and positive staining of HOXA3-expressing cells (Red) when treated with 1.4μM CHIR99021 or 3.0μM CHIR99021 together with 0.1μM retinoic acid (RA) respectively. This antibody stained cells up to the r2 region in mouse embryos. Scale bar: 50μm; Hoechst staining is blue; CHIR: CHIR99021.

Supplementary Figure 2 The effects of SHH and FGF4 on the rostral hindbrain NSCs.

(a) Q-PCR of regional neural markers along A-P axis before (1.4μM CHIR - SHH) or after (1.4μM CHIR + SHH) hindbrain NSCs treated with 1000ng/ml SHH. HFB: human fetal brain samples; Cells in 0μM CHIR – SHH group were used as a positive control of markers in forebrain. (b) Cells stained with NKX2.2 and OLIG2 without treatment, treated with 1000ng/ml SHH or 1000ng/ml SHH together with 10ng/ml FGF4 during the second week of differentiation, respectively. (c) Quantification of NKX2.2- or OLIG2-expressing cells in b (n = 3). Data are represented as mean ± SEM. Scale bar: 50μm; Ho: Hoechst staining; CHIR: CHIR99021.

Supplementary Figure 3 The identity of neurons derived from rostral hindbrain NSCs not treated with SHH or FGF4.

(a) Neurons (Tuj1+, Green) derived from progenitors without treatment of either SHH or FGF4 stained positively for GABA but not serotonin. (b) Quantification of GABA- or serotonin-expressing cells in a (n = 3). Data are represented as mean ± SEM. Scale bar: 50μm; Ho: Hoechst staining.

Supplementary Figure 4 Electrophysiological properties of the central serotonin neurons.

(a) Inward Na+ and outward K+ currents were triggered upon - 50mV to + 50 mV voltage steps. The initial currents were enlarged in the insert panel. (b) Action potentials were induced from - 40 pA to + 100 pA injected current steps. (c) Plotting of action potential frequency to injected currents. (d) Spontaneous excitatory (downward currents) and inhibitory (upward currents) postsynaptic currents were recorded when neurons were held at 0 mV.

Supplementary Figure 5 Full western blot images of the selected portions shown in Figure 3h.

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Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 1044 kb)

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Lu, J., Zhong, X., Liu, H. et al. Generation of serotonin neurons from human pluripotent stem cells. Nat Biotechnol 34, 89–94 (2016). https://doi.org/10.1038/nbt.3435

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