Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs

Abstract

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.

Generation of human induced pluripotent stem cell-derived cerebral organoids for cellular and molecular characterization

“…(2016) Day 5 100 nM LDN-193189, 3 μM CHIR99021, 100 ng/mL SHH, 2 μM purmorphamine, 100 ng/mL FGF-8 Day 7 100 nM LDN-193189, 3 μM CHIR99021 Day 14 20 ng/mL BNDF, 20 ng/mL human recombinant glial-derived neurotrophic factor (GDNF), 0.2 mM ascorbic acid, 1 ng/mL human recombinant transforming growth factor beta (TGFβ), 0.5 mM cyclic AMP (cAMP) Low cell attachment plates – orbital shaker Hindbrain (Brainstem) Day 1 1 mM dorsomorphin, 10 μM SB431542, 10 μM transferrin, 5 mg/L insulin, 0.063 mg/L progesterone Low attachment multiwell plates; static Eura et al. (2020) Day 9–12 20 ng/mL FGF2, 10 μM transferrin, 5 mg/L insulin, 0.063 mg/L progesterone Day 13–18 20 ng/mL FGF2, 20 ng/mL EGF Day 19–25 ascorbic acid, cAMP, 20 ng/mL BDNF, 20 ng/mL GDNF, 20 ng/mL NT3 Hindbrain (Cerebellum) Day 1 iPSCs passaged to containers pre-coated with matrigel supplemented with 1% bovine serum albumin Matrigel plates - static Valiulahi et al. (2021) Day 1 Knockout serum replacement medium Low attachment multiwell plates; static Day 5 25% N2 medium, 2 μM purmorphamine, 2 μM RA Day 7 50% N2, 2 μM purmorphamine, 2 μM RA Day 9 75% N2, 2 μM purmorphamine, 2 μM RA Day 10–13 20 ng/mL BDNF, 2 μM purmorphamine, 2 μM RA Low cell attachment plates – orbital shaker Day 14+ 20 ng/mL BDNF …”

Functional bioengineered models of the central nervous system

“…Brainstem organoids express specific cholinergic (choline acetyltransferase (ChaT)) and noradrenergic markers (dopamine β-hydroxylase) as well as hindbrain-specific genes (zinc finger of the cerebellum 1 (ZIC1), ZIC4), and therefore likely contain pontine and medullary cells. Moreover, hindbrain organoids can be derived from human iPS cells and serotonergic cells with genetic profiles congruent with genes expressed in the raphe nuclei of the medulla 167 . Engineering-based models of the brainstem have not yet been developed.…”

Engineering Brain Organoids: Toward Mature Neural Circuitry with an Intact Cytoarchitecture

“…Moreover, the additional treatment of SHH, a ventralizing factor determining the ventral to dorsal axis of the brain, produced the ventral forebrain organoid as well ( 24 , 25 ). Until now, numerous region-specific brain organoid protocols have been introduced, resembling the midbrain ( 7 , 26 ), striatum ( 27 ), hippocampus ( 8 ), choroid plexus ( 28 ), thalamus ( 29 ), hypothalamus ( 7 ), hindbrain ( 30 ), cerebellum ( 9 ), and spinal cord ( 31 ).…”