Brainstem organoids from human pluripotent stem cells contain neural crest population

Abstract

The brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. . Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases. IntroductionThe brainstem is a posterior region of the brain between the deep structures of the cerebral hemispheres. It connects the cerebrum with the spinal cord and is divided into three parts: midbrain, pons and medulla oblongata. They contain multiple nuclei and small fiber tracts widely projecting to the cerebrum cortex, basal ganglia and other parts of the cerebrum. Brainstem functions such as alertness, heartbeat, blood pressure and respiration are considered to be more vital for life than that of the cortex. Therefore, damages to or disorders of brainstem such as infarction, hemorrhage, tumors, or any neurodegenerative diseases may lead to death.Of those, Parkinson's disease (PD) is the most well-known degenerative disease, showing progressive voluntary movement impairments, including rigidity, akinesia, tremor, and postural instability. These symptoms are caused by the loss of dopaminergic neurons at midbrain substantia nigra. In addition, non-motor symptoms, such as autonomic dysfunction, sleep disorder, and depression in PD patients, are thought to be derived from impairments of the serotonergic or noradrenergic system in the brainstem. However, the mechanisms driving these symptoms have yet to be determined. Hence, we need the development of models that can recapitulate the human midbrain and surrounding brainstem to elucidate the process of neural degeneration in this area.Recent progress on protocols for inducing organs in-a-dish (organoids) provides potentials for the modeling of various diseases (Clevers, 2016). Organoids mimic the structure of organs Eura, Matsui, Luginbühl et al.

Immune Factor, TNFα, Disrupts Human Brain Organoid Development Similar to Schizophrenia—Schizophrenia Increases Developmental Vulnerability to TNFα

“…A recent study revealed the presence of neural crest cells in brain stem organoids (Eura et al, 2019 ). The presence of neural crest cells in the cerebral organoids was not investigated in the present study.…”

Brainstem Organoids From Human Pluripotent Stem Cells

“…The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or decision to submit it for publication. This manuscript has been released as a Pre-Print at bioRxiv ( Eura et al, 2019 ).…”

Brain Organoids: Tiny Mirrors of Human Neurodevelopment and Neurological Disorders

“…For that reason, the generation of human brainstem organoids (hBSOs) is imperative to bridge the gap in understanding the complexities/intricacies of brainstem and dissect the pathology of brainstem disorders in advanced systems. To initiate the generation of hBSOs, hiPSCs/ hESCs are seeded in a cell culture medium supplemented with dorsomorphin, ROCK inhibitor, and SB431542 in culture dishes on an orbital shaker to keep the cells in suspension stage (Eura and others 2019). After 3 days of culture, the medium is supplemented with bFGF and EGF to increase the number of stem/progenitor cells.…”

“…After 3 days of culture, the medium is supplemented with bFGF and EGF to increase the number of stem/progenitor cells. This is followed by the addition of cAMP, GDNF, BDNF, NT-3, and ascorbic acid for the subsequent differentiation of stem cells into dopaminergic neurons and neural crest cells (Eura and others 2019). Over a month of culturing, hBSOs start to develop some dark granule spots termed as melanocytes.…”

“…Additionally, hBSOs also start to express other markers like FOXA2 (inducer of midbrain dopaminergic neurons), TH (mature dopaminergic marker), ChAT (cholinergic neuron marker), GBX2/OTX2 (markers that regulate the positioning of midbrain/hindbrain), and VGLUT1 and GAD67 markers for excitatory and inhibitory neurons, respectively (Eura and others 2019). At present, these hBSOs are better suited to study the molecular-cellular mechanisms driving brainstem dysfunction (in particular midbrain) and to probe effective drugs/as grafts to cure critically ill patients.…”