CN-122012395-A - 3D human brain organoid culture method containing blue-spotted nucleus noradrenergic neurons

CN122012395ACN 122012395 ACN122012395 ACN 122012395ACN-122012395-A

Abstract

The invention relates to a method for culturing three-dimensional () 3D human brain organoids containing blue-spotted nuclear noradrenergic neurons, which comprises (a) culturing pluripotent stem cells to obtain embryoid bodies, (b) inducing the embryoid bodies from (a) into neuroepithelial tissues and hindbrain tissues, (c) inducing differentiation of the hindbrain tissues from (b) to obtain hindbrain tissues containing blue-spotted nuclear noradrenergic neurons, and (D) culturing the hindbrain tissues containing blue-spotted nuclear noradrenergic neurons from (c) to obtain 3D human brain organoids containing blue-spotted nuclear noradrenergic neurons. The invention develops a novel culture medium containing various small molecules, successfully cultures three-dimensional human brain organoids containing blue-band nuclear norepinephrine neurons, and provides a novel in-vitro three-dimensional cell model for the subsequent research on the functional action of blue-band nuclear norepinephrine in a nervous system.

Inventors

Zhang Wendiao
WANG XUEJING
TANG BEISHA
TAN SHAOHUA
WU JIAYI
Meng Qingtuan
JIANG YUYAN
WAN JUAN
WU HENG
XIAO ZIJIAN
LIU YONG

Assignees

南华大学附属第一医院

Dates

Publication Date: 20260512
Application Date: 20250826

Claims (10)

1. A method of culturing a 3D human brain organoid comprising blue-spotted nuclear noradrenergic neurons, comprising: (a) Culturing pluripotent stem cells to obtain embryoid bodies; (b) Inducing the embryoid body from (a) into hindbrain tissue; (c) Inducing differentiation of hindbrain tissue from (b) to obtain hindbrain tissue containing blue-spotted nuclei noradrenergic neurons; (d) Culturing hindbrain tissue from (c) containing blue-spotted nuclear noradrenergic neurons to obtain a 3D human brain organoid comprising blue-spotted nuclear noradrenergic neurons; The step (a) is divided into two stages, wherein the culture medium in the first stage contains the ROCK inhibitor, and the culture medium in the second stage does not contain the ROCK inhibitor; step (b) is split into two phases, wherein the medium of the first phase comprises a SMAD inhibitor, a BMP receptor inhibitor, and a small molecule agonist of the Wnt signaling pathway; Step (c) is split into two phases, the medium of the first phase comprising ACTIVIN A, a BMP receptor inhibitor and a small molecule agonist of the Wnt signaling pathway, and the medium of the second phase being free of BMP receptor inhibitor and a small molecule agonist of the Wnt signaling pathway.
2. The method according to claim 1, wherein the culturing time of step (a) is 6 days, wherein the culturing time of step (a) is 1 to 3 days in the first stage, the culturing time of step (a) is 1 to 3 days in the second stage, the culturing time of step (b) is 5 days, wherein the culturing time of step (b) is 3 to 4 days in the first stage, the culturing time of step (b) is 1 to 2 days in the second stage, the culturing time of step (c) is 6 days, wherein the culturing time of step (c) is 1 to 3 days in the first stage, and the culturing time of step (c) is 1 to 3 days in the second stage; the incubation time in step (d) is 10-30 days.
3. The method according to claim 1, wherein the medium of the first stage of step (a) comprises a basal medium and a first specific additive factor, wherein the first specific additive factor comprises the components of the final concentration of serum replacement, 10% -20%, fibroblast growth factor, 4-8ng/ml, glutamine supplement, 1-2x, non-essential amino acid supplement, 1-2x, antibiotic, 1-2x, rock inhibitor, 20-30 μΜ, preferably wherein the medium of the second stage of step (a) comprises basal medium and a second specific additive factor, wherein the second specific additive factor comprises the components of the final concentration of serum replacement, 10% -20%, fibroblast growth factor, 4-8ng/ml, glutamine supplement, 1-2x, non-essential amino acid supplement, 1-2x, antibiotic, 1-2x.
4. The method of claim 3, wherein the ROCK inhibitor is Y27632, the basal medium is DMEM-F12, the serum replacement is KSR, the fibroblast growth factor is FGF-2/bFGF, the glutamine supplement is L-alanyl-L-glutamine dipeptide, and the non-essential amino acid supplement is NEAA; The antibiotic is penicillin-streptomycin double antibody.
5. The method of claim 1, wherein the medium of the first stage of step (b) comprises basal medium and a third specific additive factor, wherein the third specific additive factor comprises the components of glutamine supplement 1-2x, nonessential amino acid supplement 1-2x, antibiotic 1-2x, supplement for expanding undifferentiated cells 1-2x, SMAD inhibitor 2-12 μm, BMP receptor inhibitor 2-10 μm, small molecule agonist of Wnt signaling pathway 1-8 μm, preferably the supplement for expanding undifferentiated cells is N2, the SMAD inhibitor is SB431542, the BMP receptor inhibitor is DMH1, the small molecule agonist of Wnt signaling pathway is CHIR99021, preferably the medium of the second stage of step (b) comprises basal medium and a fourth specific additive factor, wherein the fourth specific additive factor comprises the components of glutamine, 1-2x, nonessential amino acid supplement 1-2x, supplement 1-10 μm, small molecule agonist of Wnt signaling pathway 1-8 μm, small molecule agonist of Wnt signaling pathway 1-2.
6. The method of claim 1, wherein the medium of the first stage of step (c) comprises basal neuron medium and a fifth specific additive factor, wherein the fifth specific additive factor comprises a glutamine supplement 1-2x, a non-essential amino acid supplement 1-2x, an antibiotic 1-2x, a supplement for expanding undifferentiated cells 0.5% -2%, a BMP receptor inhibitor 2-10 μm, a small molecule agonist of Wnt signaling pathway 1-8 μm, a blue-leaf nuclear noradrenergic cell inducer 20-40ng/ml, preferably the blue-leaf nuclear noradrenergic cell inducer ACTIVIN A, the basal neuron medium DMEM-F12/Neuronal basal, preferably the medium of the second stage comprises basal neuron medium and a sixth specific additive factor, wherein the sixth specific additive factor comprises a glutamine supplement 1-2x, a non-amino acid inhibitor 2-10 μm, a small molecule agonist of Wnt signaling pathway 1-8 μm, a blue-leaf nuclear noradrenergic cell inducer 20-40ng/ml, preferably the blue-leaf nuclear noradrenergic cell inducer ACTIVIN A, the basal neuron medium DMEM-F12/Neuronal basal, preferably the medium of the second stage comprises a basal neuron medium and a sixth specific additive, wherein the sixth specific additive factor comprises a final concentration of glutamine supplement 1-2x, a non-amino acid inhibitor 1-2x, a non-nuclear adrenergic receptor inhibitor 1-10 μm, a small molecule agonist for maintaining the blue-leaf nuclear noradrenergic cell inducer 1-8 ng, preferably the blue-leaf nuclear noradrenergic cell inducer 1-8 ng-80 ng-blue-leaf nuclear cytokine, the second blue print nucleus noradrenergic cell inducer is GDF11.
7. The method according to claim 1, wherein the medium used in the step (d) comprises a basal neuron medium and a seventh specific additive factor, wherein the seventh specific additive factor comprises the following components in final concentration, glutamine supplement, 1-2x, nonessential amino acid supplement, 1-2x, antibiotic, 1-2x, supplement for maintaining neurons, 1-2%, first blue-spot nuclear noradrenergic cell inducer, 80-160ng/ml, second blue-spot nuclear noradrenergic cell inducer, 3-8ng/ml, vitamin, 200-300 μM, brain-derived neurotrophic factor, 10-20ng/ml, glial cell-derived nerve growth factor, 10-20ng/ml, preferably, ascorbic acid, brain-derived neurotrophic factor, BDNF, and glial cell-derived nerve growth factor, GDNF.
8. A 3D human brain organoid comprising blue-spotted nuclear noradrenergic neurons cultured according to the culture method of any one of claims 1-7.
9. The use of a 3D human brain organoid comprising blue-spotted nuclear noradrenergic neurons according to claim 8 for the preparation of an in vitro model for studying the development of the nervous system and neuroimmune-related diseases.
10. A culture medium for culturing 3D human brain organoids containing blue-spotted nuclear noradrenergic neurons is characterized by comprising a culture medium of a first stage of step (a), a culture medium of a second stage of step (a), a culture medium of a first stage of step (b), a culture medium of a second stage of step (b), a culture medium of a first stage of step (c), a culture medium of a second stage of step (c) and a culture medium of step (D); Wherein the medium of the first stage of step (a) comprises a ROCK inhibitor, and the medium of the second stage of step (a) does not comprise a ROCK inhibitor; The medium of the first stage of step (b) comprises a SMAD inhibitor, a BMP receptor inhibitor, and a small molecule agonist of the Wnt signaling pathway; The culture medium of the first stage of step (c) comprises ACTIVIN A, a BMP receptor inhibitor, and a small molecule agonist of the Wnt signaling pathway, and the culture medium of the second stage of step (c) does not comprise a BMP receptor inhibitor and a small molecule agonist of the Wnt signaling pathway.

Description

3D human brain organoid culture method containing blue-spotted nucleus noradrenergic neurons Technical Field The invention relates to a method for culturing 3D human brain organoids containing blue-spotted nuclear noradrenergic neurons. Background The 3D human brain organoid is an effective model capable of simulating early development of human brain under in vitro culture conditions, and is formed by induced differentiation of human induced pluripotent stem cells. The 3D human brain organoids which have been put into scientific research at present are human whole brain organoids, forebrain organoids, midbrain organoids, hippocampus and the like. The 3D human brain organoids that are mature in culture comprise a variety of cell types. For example, mature whole brain organoids are cultured to include neural progenitor cells, mature neurons, gabaergic neurons, glutamatergic neurons, and the like. However, current 3D human brain organoids cultured in vitro lack norepinephrine (Norepinephrine; NE) energy cells. In brain tissue, the vast majority of cells that release NE accumulate in the blue-spot nuclear (Locus coeruleus, LC) tissue. LC is present in the pontic region of the brainstem and is the major source of NE in the central nervous system (Central nervous system; CNS). LC is able to sense any danger or threat present in the external environment and regulate various basic functions of the brain, mainly by NE-able neurons projecting to other brain areas and sending signals, reminding other brain areas of impending danger, generating "fight and escape" responses, regulating sleep/wake cycle, attention and cognitive functions etc. Recent studies in mouse brain have shown that NE activates hepatic beta-adrenergic receptors, initiates glycogenolysis, increases plasma glucose, and activates Hormone sensitive fatty acids (Horone-SENSITIVE LIPASE; HSL), resulting in an increase in plasma free fatty acids (FREE FATTY ACIDS; FFA), enhancing combat or escape responses. And regulates the functions and behaviors of the mouse brain and the role in arousal through the NE-FFA-Na+ -K+ -ATPase (NKA) signal pathway, emphasizes the important role that NE can regulate the excitability of neurons through lipid signals, avoids the transition excitation of neurons and further generates neurotoxicity. In addition, NE may also interact with microglial cells in the nervous system, regulating sleep in mice. While a reduced level of NE will lead to transitional sleep and natural wake-up dysfunction. There is growing evidence that sleep disruption or sleep disorder is an important risk factor for a variety of neurodegenerative diseases, including Alzheimer's Disease (AD) and Parkinson's Disease (PD), among others. In fact, sleep disturbance clinical manifestations have been shown in parkinson's disease patients. More importantly, in post-mortem brain tissues of parkinsonism patients, blue-spot nuclear NE neurons are observed to show earlier and more serious degeneration phenomena than substantia nigra dopamine neurons, suggesting that blue-spot nuclear NE can play an important role in PD occurrence and development. Furthermore, dysfunction of NE is not only associated with neurodegenerative diseases, but also with neuropsychiatric diseases. In a study of high frequency repeated transcranial magnetic stimulation (aHF-rTMS) treatment of depression, aHF-rTMS treatment was found to significantly reduce the depression score (HDRS) in patients, wherein a significant decrease in the blue-spot nuclear function junction was positively correlated with clinical improvement in depression. In particular, the decline in functional connectivity of the blue-spotted nucleus with the left upper frontal lobe, central anterior back and posterior cerebellum is closely related to the improvement of depressive symptoms. In addition, genes associated with the connectivity of the locus coeruleus nucleus such as CNR1 (encoding CB1 cannabinoid receptor) and SST (encoding somatostatin) also undergo significant expression changes, suggesting that these genes may be involved in the regulation of depression by the locus coeruleus nucleus. In summary, NE-capable neurons in the locus coeruleus play an important role in maintaining normal functions of the nervous system, and dysfunction of the function can lead to various nervous system diseases including neuropsychiatric diseases, neurodegenerative diseases, and the like. Research on the blue-spot nuclei is also expected to provide basis for revealing the etiology of the diseases and searching for potential targeted treatments. Thus, in order to better explore the potential role of the blue-spotted nuclear NE neurons in disease in an in vitro environment, it is crucial and urgent to culture 3D human brain organoids comprising noradrenergic cells. The prior art only cultures two-dimensional noradrenergic neurons in vitro, but does not contain multiple neuron types therein. Thus, two-dimensional noradrenergic neurons fo