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Norepinephrine (NE), also known as noradrenaline (NA), is mainly produced by NE neurons in the locus coeruleus (LC) of the central nervous system (CNS). There are 10,000–30,000 LC-NE neurons in non-human primates and 20,000–50,000 in humans1,2 projecting to nearly the entire brain3 through their elaborate axonal branches. The NE system is involved in arousal, wakefulness, memory, focus and attention and ‘fight or flight’ reaction4. Dysregulation of the NE system is linked to many neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), congenital central hypoventilation syndrome (CCHS), sleep disorders, attention-deficit/hyperactivity disorder (ADHD), anxiety and depression4,5,6,7,8.
Central NE neurons are heterogenous, having different developmental origins, anatomical locations, connectivity and function9,10. LC-NE neurons, the main NE nucleus in the CNS, originate from the dorsal hindbrain rhombomere 1 (r1)9,10,11, whereas other NE nuclei develop from r2–r5 (ref. 9). LC-NE neurons often degenerate at an early stage of many neurodegenerative diseases4,8, although it is not clear why they are vulnerable and how their degeneration contributes to disease pathogenesis. Hence, the LC-NE system is an emerging target for revealing pathogenesis of, and developing therapeutics for, many neurological disorders8,12,13,14,15.
Studies on the LC-NE system are hindered by the lack of readily available authentic NE neurons, especially of human origin. Immortalized ‘NE-like’ cell lines (for example, PC12 and SH-SY5Y) express some related gene profiles and produce the NE neurotransmitter but do not recapitulate developmental processes and often contain mutations16, limiting their utility. Forced expression of NE-related transcription factors Phox2b or Phox2a generates neurons with NE phenotypes from mouse embryonic stem cells (mESCs) but is less effective in inducing expression of the NE neuronal markers tyrosine hydroxylase (TH) and dopamine β hydroxylase (DBH) in cells derived from human pluripotent stem cells (hPSCs)17. It is not known whether hPSC-derived or mESC-derived cells possess NE neuron functionality. BMP7 plays a positive role in mouse NE neuron generation but has a negative effect in human cells17, suggesting potential species difference in NE fate determination. It is currently unclear how human NE neurons, especially LC-NE neurons, are specified. Consequently, generation of functional human LC-NE neurons has not been achieved.
In this Article, we found that bone morphogenic proteins (BMPs) and transforming growth factor-beta (TGFβ), known to induce NE neuron development in the rodent brain and from mESCs17, do not have an obvious effect on the generation of NE neurons from hPSCs. Notably, we established that ACTIVIN A, another member of the TGFβ superfamily, is important in regulating neurogenesis and the NE fate of the hPSC-specified r1 progenitors. This finding enabled generation of 40–60% functional LC-NE neurons that resemble their in vivo counterparts from one embryonic stem cell (ESC) line (H9) and two induced pluripotent stem cell (iPSC) lines (W24B and W24M).
Specification of dorsal hindbrain r1 progenitors from hPSCs
LC-NE neurons originate from progenitors in the dorsal hindbrain r1 during embryonic development9. Progenitors in the r1 segment express EN1/2 and GBX2 but not OTX2 (forebrain and midbrain marker) or HOXA2 (hindbrain marker from r2)18 (Fig. 1a). We first differentiated human embryonic stem cells (hESCs, H9 line) to neuroepithelial cells in the presence of BMP receptor inhibitor DMH1 (2 µM) and TGFβ receptor inhibitor SB431542 (2 µM)19. With an increasing concentration of WNT agonist CHIR99012 for 6 d during neuroepithelial specification (Fig. 1b), the differentiating cells downregulated the expression of OTX2 at the mRNA level (Fig. 1c). Concurrently, midbrain markers (EN1, EN2 and PAX2) were upregulated. At CHIR99021 concentrations above 0.6 µM, these midbrain transcription factors began to downregulate at the mRNA level, whereas the hindbrain markers (HOXA2 and GBX2) were upregulated (Fig. 1c). The NE-related transcription factors ASCL1 (also known as MASH1), PHOX2A and PHOX2B increased in response to increasing CHIR99021 doses (Extended Data Fig. 1a). This pattern of gene expression was confirmed by immunocytochemistry, showing expression of EN1 in the majority of the cells, whereas few were positive for OTX2 or HOXA2 at 1.0 µM (Fig. 1d). Flow cytometry also revealed the shift of the OTX2-expressing cell population in response to increasing concentrations of CHIR99021 (Extended Data Fig. 1b). Thus, we selected 1.0 µM CHIR99021 to pattern the hindbrain r1 identity from hPSCs, which matches a previous study using 1.4 µM CHIR99021 to pattern the hindbrain r2–r3 region from hPSCs20.
Fig. 1: Specification of dorsal hindbrain (r1) neuroepithelia.
a, Schematic representation of LC location along the embryonic forebrain, midbrain and hindbrain and their corresponding homeodomain transcription factors. b, Experimental design to pattern hindbrain r1 region from hPSCs during the first 6 d of neural induction. c, Expression of forebrain, midbrain and hindbrain genes under a series of CHIR99021 (CHIR) concentrations. Data are shown as mean ± s.e.m. n = 3 biologically independent samples for each condition. d, Immunostaining for OTX2, EN1 and HOXA2 in day 6 cells when treated with 1.0 µM CHIR99021 in the presence of SB431542 and DMH1. Scale bar, 50 µm. e, Schematic representation of the hindbrain r1 domains along the dorsal to ventral subdomains and their corresponding transcription factors. f, Immunostaining for PAX7, SOX1 and SOX2 at day 6 from cells treated with 1.0 µM CHIR99021 in the presence of SB431542 and DMH1. HO, Hoechst. Scale bar, 50 µm. g, Quantification of SOX2-, SOX1- and PAX7-expressing cells at day 6 when treated with 1.0 µM CHIR99021. Data are shown as mean ± s.d. n = 5 biologically independent samples for each condition.
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LC-NE neurons are located bilaterally at the 4th ventricle; most of their progenitors were generated from the PAX7+ lineage in mice, and a small population (9%) was generated from the PAX7− cells in mice10 (Fig. 1e). Indeed, over 80% of the neuroepithelial cells at day 6, marked by SOX1 and SOX2, were PAX7 positive (Fig. 1f,g). Thus, the differentiated cells possess the dorsal r1 identity.
Specification of NE fate from r1 progenitors by ACTIVIN A
The development of NE neurons in LC depends on the activation of Ascl1, Phox2a and Phox2b in a stage-dependent manner21,22,23. Ascl1 is expressed in the progenitors, whereas Phox2a and Phox2b are expressed in post-mitotic precursors21. Immunocytochemical analysis of the dorsal r1 progenitors indicated that very few cells (
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