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Production method for cerebral organoid

A manufacturing method and organoid technology, which is applied in the field of screening of preventive or therapeutic drugs, and can solve problems such as different structures and inability to apply insights to humans

Pending Publication Date: 2022-02-08
KEIO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the structures of the brains of humans and mice are different, and insights obtained from Alzheimer's disease model mice cannot be applied to humans in some cases.

Method used

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  • Production method for cerebral organoid
  • Production method for cerebral organoid
  • Production method for cerebral organoid

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0103] (Creation of Brain Organoids 1)

[0104] Cultivate pluripotent stem cells to produce brain organoids. As pluripotent stem cells, wild-type human iPS cell lines 414C2, 201B7, and RPC771, wild-type human ES cell line KhES1, and Alzheimer's disease patient-derived human iPS cell lines PS1-2, PS2-2.

[0105] It is clear that PS1-2 cells have a genetic variation in which the amino acid variation from alanine to glutamic acid (A246E) occurs at the 246th amino acid of PS1 protein in PS1 gene. In addition, it was clarified that PS2-2 cells have a gene mutation in which an amino acid mutation (N141I) from asparagine to isoleucine occurs at the 141st amino acid of the PS2 protein in the PS2 gene.

[0106] figure 1 It is a diagram showing the progress chart of brain organoid production. The culture of each pluripotent stem cell was carried out in a feeder-free format in which no feeder cells were used. First, each cell was cultured in a medium containing 10 ng / mL of FGF2 for ...

experiment example 2

[0114] (Detection of Amyloid Plaques 1)

[0115] The amyloid plaques of the brain organoids prepared in Experimental Example 1 were detected. On the 120th day of culture (Day 120, the 106th day from the start of stirring culture), each brain organoid was taken out and fixed with 4% paraformaldehyde. Next, the fixed organoids were embedded in resin and cryosections were made.

[0116] Next, each frozen section was immunostained with an anti-amyloid β antibody (Clone 6E10, BioLegend) to detect amyloid plaques. figure 2 (a) Representative photographs of the results using brain organoids prepared from the wild-type human iPS cell line 414C2 at day 120 of culture. in addition, figure 2 (b) is a representative photograph showing the results of cultured 120-day brain organoids produced by PS1-2, which is a human iPS cell line derived from an Alzheimer's disease patient. figure 2 In (b), arrows indicate amyloid plaques with a diameter of 20 μm or more.

[0117] As a result, it...

experiment example 3

[0122] (Detection of Amyloid Plaques 2)

[0123] Amyloid plaques in the brain organoids prepared in Experimental Example 1 were detected using 2-(4'-methylaminophenyl)benzothiazole (BTA-1, Sigma) instead of the anti-amyloid β antibody. BTA-1 is a derivative of thioflavin-T, and is a compound that exhibits about 50 times higher affinity to amyloid β peptide deposits than thioflavin-T.

[0124] On the 120th day of culture (Day 120, the 106th day from the start of stirring culture), each brain organoid was taken out and fixed with 4% paraformaldehyde. Next, the fixed organoids were embedded in resin and cryosections were made.

[0125] Next, each frozen section was stained with BTA-1 to detect amyloid plaques. In addition, for comparison, a control human brain tissue section (26 years old) and an Alzheimer's patient's brain tissue section (73 years old) were similarly stained with BTA-1.

[0126] Figure 4 (a) is a representative photograph showing the result of the human bra...

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Abstract

A method for producing a cerebral organoid having amyloid plaques is provided by the invention. The method comprises step (a) for forming, in the presence of an SMAD inhibitor, a germ layer body from a pluripotent stem cell having a mutation in an Alzheimer's disease-related gene; step (b) for, after step (a), embedding the germ layer body in an extracellular matrix, and three-dimensionally culturing the germ layer body in the presence of the SMAD inhibitor and a glycogen synthase kinase 3beta (GSK3beta) inhibitor, to form an organoid; and step (c) for, after step (b), removing the organoid from the extracellular matrix, and culturing the organoid in a culture medium while stirring same, wherein at least a portion of step (c) is performed in the presence of leukemia inhibitory factor (LIF).

Description

technical field [0001] The present invention relates to a method for manufacturing brain organoids. More specifically, the present invention relates to a method for producing brain organoids with amyloid plaques, a method for screening brain organoids with amyloid plaques, and a drug for preventing or treating Alzheimer's disease. This application claims priority based on Japanese Patent Application No. 2019-126266 for which it applied in Japan on July 5, 2019, and uses the content in this specification. Background technique [0002] Organoids are small organs formed by the aggregation of cells, which have similar structures and functions to organs in living organisms. In recent years, research on producing various organoids from pluripotent stem cells has been actively conducted, for example, brain organoids, intestinal organoids, liver organoids, kidney organoids, etc. have been produced. [0003] However, Alzheimer's disease is an irreversible, progressive brain disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/071C12Q1/02
CPCC12N5/0618G01N33/5082G01N2800/2821G01N2333/4709C12N2513/00C12N2506/45C12N2501/15C12N2501/727C12N2501/235C12N2501/115C12N2500/02G01N33/5088C12N5/0619C12N2501/155C12N2506/02C12N2501/415C12N2501/13C12N2501/01C12N2533/90G01N33/5023
Inventor 石井弘子冈野荣之
Owner KEIO UNIV
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