Method for producing dorsal brain organoid and use thereof

Abstract

To provide a technique that facilitates the differentiation of cells into dorsal brain organoids.SOLUTION: A method for producing dorsal brain organoids includes the use of at least one of a cAMP activator and a cAMP analog, in the process of generating dorsal brain organoids from pluripotent stem cells. A dorsal brain organoid production kit comprises at least one substance selected from a cAMP activator and a cAMP analog.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods for producing dorsal brain organoids. [Background technology]

[0002] Brain organoids produced from iPS cells, etc., are used as 3D cell culture models that mimic various brain regions in regenerative medicine research, etc. Conventionally, a method for inducing dorsal brain organoids that mimic the cerebral cortex has been known in which an SMAD inhibitor and a WNT inhibitor are used in the early differentiation stage to induce the production of neural cells in the anterior dorsal side (e.g., Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Cell Stem Cell. 2019 Mar 7;24(3):487-497. Summary of the Invention [Problem to be solved by the invention]

[0004] According to the method described in Non-Patent Document 1, the present inventors have found that, even if cells with a specific disease are induced to differentiate into dorsal brain organoids, they may be unintentionally induced to differentiate into ventral brain organoids. WNT signaling and SHH signaling play an important role in the creation of a dorsal brain organoid model that mimics the cerebral cortex. Therefore, in disease iPS cells with abnormalities in these signaling mechanisms, it may not be possible to induce differentiation into dorsal brain organoids even using the method described in Non-Patent Document 1. For this reason, the present disclosure aims to provide a technology that can easily induce differentiation of cells into dorsal brain organoids. [Means for solving the problem]

[0005] The present disclosure can be realized in the following forms.

[0006] (1) According to one embodiment of the present disclosure, a method for producing dorsal brain organoid is provided. In the method for producing dorsal brain organoid, at least one of cAMP activator and cAMP analogue is used in the process of producing dorsal brain organoid from pluripotent stem cells. According to the method for producing dorsal brain organoid of this embodiment, it is easy to induce differentiation of cells into dorsal brain organoid.

[0007] (2) In the method for producing dorsal brain organoid described in (1) above, said cAMP activator can be forskolin.According to this form of the method for producing dorsal brain organoid, it can further facilitate cell differentiation into dorsal brain organoid.

[0008] (3) In the method for producing dorsal brain organoid described in (1) or (2), said cAMP analogue can be 8-bromo-cAMP.According to this form of the method for producing dorsal brain organoid, it can further facilitate cell differentiation into dorsal brain organoid.

[0009] (4) In the method for producing dorsal brain organoids according to any one of the above (1) to (3), the concentration of the cAMP activator in the medium for producing the dorsal brain organoids from the pluripotent stem cells may be 200nM or more and 10μM or less. According to this form of the method for producing dorsal brain organoids, it is possible to easily induce differentiation of cells into dorsal brain organoids while suppressing cytotoxicity.

[0010] (5) In the method for producing dorsal brain organoids according to any one of the above (1) to (4), the concentration of the cAMP analogue in the medium for producing the dorsal brain organoids from the pluripotent stem cells may be 1 μM or more and 10 μM or less. According to this form of the method for producing dorsal brain organoids, it is possible to easily induce differentiation of cells into dorsal brain organoids while suppressing cytotoxicity.

[0011] (6) In the method for producing dorsal brain organoids according to any one of (1) to (5) above, the substance may be used within 3 days after the start of the process. According to this form of the method for producing dorsal brain organoids, it is possible to further facilitate differentiation of cells into dorsal brain organoids.

[0012] (7) In the method for producing dorsal brain organoids according to any one of the above (1) to (6), the period of using the substance in the above step can be 6 days or more.According to the method for producing dorsal brain organoids of this form, it can further facilitate differentiation of cells into dorsal brain organoids.

[0013] (8) According to another embodiment of the present invention, a kit for producing dorsal brain organoid is provided. This kit for producing dorsal brain organoid comprises at least one of cAMP activator and cAMP analogue. According to this embodiment of the kit for producing dorsal brain organoid, it is easy to induce differentiation of cells into dorsal brain organoid.

[0014] The present disclosure can be realized in various forms, for example, in the form of a method for inducing dorsal brain organoids, dorsal brain organoids, etc. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a process diagram showing a method for producing dorsal brain organoids. [Diagram 2] FIG. 1 is an explanatory diagram showing the results of immunostaining in Experiment 1. [Diagram 3] FIG. 1 is an explanatory diagram showing the results of immunostaining in Experiment 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] According to one embodiment of the present disclosure, a method for producing dorsal brain organoids is provided. Figure 1 is a process diagram showing the method for producing dorsal brain organoids. In the method for producing dorsal brain organoids, first, at least one of a cAMP activator and a cAMP analog, a medium, and pluripotent stem cells are prepared (step P110). In the method for producing dorsal brain organoids, at least one of a cAMP activator and a cAMP analog is used in the step of producing dorsal brain organoids from pluripotent stem cells (step P120).

[0017] In the present disclosure, the term "cAMP activator" refers to a substance that activates cAMP (cyclic adenosine monophosphate). cAMP is a type of cyclic nucleotide in which one molecule of phosphate is bound as a diester to the 3'-hydroxyl group and 5-hydroxyl group of adenosine ribose. cAMP is known as one of the secondary information transmitters (second messengers) that transmit information from outside the cell. Examples of cAMP activators include, but are not limited to, substances that activate proteins for synthesizing cAMP in cells, and G protein-coupled receptors that are coupled to stimulatory G proteins. Examples of substances that activate proteins for introducing cAMP into cells include, but are not limited to, forskolin, guanylyl imidodiphosphate lithium, and the like. The cAMP activator is preferably a substance that activates proteins for introducing cAMP into cells, and more preferably forskolin is used.

[0018] In the present disclosure, the term "cAMP analog" refers to a substance that has a structure similar to that of cAMP and a function similar to that of cAMP. Examples of cAMP analogs include, but are not limited to, 8-bromo-cAMP, dibutyryl-cAMP (Bucladesine), 6-Bnz-cAMP, Sp-2-Cl-cAMPs, etc. cAMP analogs are also called cAMP derivatives.

[0019] The present inventors have focused on the fact that cAMP activators and cAMP analogs control the cleavage activity of Gli3 in the SHH signaling pathway, and have found that they can be used to create brain organoids using a three-dimensional culture model. Here, the G protein-coupled receptor "GPR161" is localized in the primary cilium, produces cAMP, a second messenger, upon ligand stimulation, and is known to negatively control the Sonic Hedgehog (SHH) signaling pathway via Gli3. The present inventors considered that in SHH signaling pathway-enhanced cells such as GPR161-deficient cells, a lack of cAMP causes insufficient induction of dorsal brain organoids. They have also found that the addition of at least one of a cAMP activator and a cAMP analog to the culture medium changes the differentiation of ventral and dorsal brain organoids. That is, in the process of preparing dorsal brain organoids from pluripotent stem cells, it was found that by using at least one of cAMP activators and cAMP analogs, cells can be easily induced to differentiate into dorsal brain organoids, and the present invention was completed. The estimated mechanism by which cAMP activators and cAMP analogs can easily induce cells to differentiate into dorsal brain organoids is that by adding a substance that can activate the cAMP signal, the function of the insufficient cAMP can be compensated for, and as a result, the SHH transduction pathway can be negatively controlled, making it easier to induce differentiation into dorsal brain organoids.

[0020] The concentration of cAMP activator in the medium for preparing dorsal brain organoid from pluripotent stem cells is not particularly limited, but from the viewpoint of facilitating efficient differentiation induction of cells into dorsal brain organoid, it is preferably 100nM or more, more preferably 200nM or more, and even more preferably 400nM or more. In addition, from the viewpoint of suppressing cytotoxicity, it is preferably 10μM or less, more preferably 5μM or less, even more preferably 2.5μM or less, even more preferably 2μM or less, even more preferably 1.5μM or less, and especially more preferably 1μM or less. The concentration of cAMP activator may be, for example, 200nM or more and 10μM or less in order to suppress cytotoxicity while facilitating differentiation induction into dorsal brain organoid.

[0021] The concentration of the cAMP analog in the medium for preparing dorsal brain organoids from pluripotent stem cells is not particularly limited, but from the viewpoint of facilitating efficient differentiation induction of cells into dorsal brain organoids, it is preferably 500 nM or more, more preferably 1 μM or more, even more preferably 1.5 μM or more, and even more preferably 2 μM or more. In addition, from the viewpoint of suppressing cytotoxicity, it is preferably 10 μM or less, more preferably 8 μM or less, even more preferably 6 μM or less, even more preferably 3 μM or less, and even more preferably 2.5 μM or less. The concentration of the cAMP analog may be, for example, 1 μM or more and 10 μM or less in order to suppress cytotoxicity while facilitating differentiation induction into dorsal brain organoids. In addition, the concentration of the cAMP activator or the cAMP analog may be changed during the culture period.

[0022] The culture period of the cells, that is, the culture period of the process of preparing dorsal brain organoids from pluripotent stem cells, is not particularly limited, but from the viewpoint of suppressing the decrease in the induction efficiency to dorsal brain organoids, it is preferably 10 days or more, more preferably 14 days or more. In addition, the period of culture in a medium containing a cAMP activator or a cAMP analog during the culture period is not particularly limited, but from the viewpoint of suppressing the decrease in the induction efficiency to dorsal brain organoids, it is preferably 6 days or more, more preferably 14 days or more. In other words, in the process of preparing dorsal brain organoids from pluripotent stem cells, the period during which at least one of the cAMP activator and the cAMP analog is used is preferably 6 days or more, more preferably 14 days or more. This period may be a continuous period or may be an intermittent period, but from the viewpoint of suppressing the decrease in the induction efficiency to dorsal brain organoids, it is preferable that it is a continuous period. From the viewpoint of economy, in the process of producing dorsal brain organoids from pluripotent stem cells, the period during which at least one of the cAMP activator and the cAMP analog is used is preferably 50 days or less, more preferably 40 days or less. In addition, it is preferable that the cAMP activator or the cAMP analog is not added after the 30th day of cell culture when it is not desired to mature the nerve, for example, when the purpose is to observe stem cells. In addition, the timing of using the cAMP activator or the cAMP analog during the cell culture period is not particularly limited, but it is preferable to use it by the 3rd day after the start of the process of producing dorsal brain organoids from pluripotent stem cells, more preferably by the 2nd day after the start of the process, and even more preferably from the start of the process. In addition, the timing of using the cAMP activator or the cAMP analog during the cell culture period may be, for example, the timing of changing the medium to Organoid differentiation media1 after starting the cell culture using Induction media.In the present specification, the induction media refers to a medium containing at least LDN-193189 (e.g., Nacalai, 19396), SB-431542 (e.g., Funakoshi, 13031) and XAV939 (e.g., Cayman Chemical, 13496). In addition, in the present specification, the organoid differentiation media 1 refers to a medium containing at least N2 supplement (e.g., Funakoshi, AR009) and NeuroBrew21 without Vitamin A (e.g., Miltenyi, 130-097-263) (NeuroBrew is a registered trademark). In addition, during the culture period, a cAMP activator and a cAMP analog may be used in combination.

[0023] Other culture conditions (culture temperature, etc.) in the culture step may be conditions generally adopted in the culture of animal cells. For example, the cells may be cultured under an environment of 37°C and 5% CO2. As the basal medium, for example, Dulbecco's modified Eagle medium (D-MEM), Ham's F12 medium (HamF12), serum-free medium for vascular endothelial cells (Human Endothelial-SFM), Iscove's modified Dulbecco's medium (IMDM), MEMα medium, Glasgow basal medium, RPMI1640 medium, MCDB107 medium, etc. may be used, or a mixture of two or more types of basal medium, such as a mixed medium of D-MEM and Ham's F12 medium, may be used. In addition, other optional components such as serum, serum substitutes, antibiotics, and supplements may be added to the medium.

[0024] The method for producing dorsal brain organoids may include a step of confirming induction into dorsal brain organoids after the culturing step. Induction into dorsal brain organoids may be confirmed, for example, by using the expression of a marker for dorsal brain organoids or a marker for ventral brain organoids as an indicator. Markers for dorsal brain organoids include, but are not limited to, PAX6 and the like. Markers for ventral brain organoids include, but are not limited to, FOXA2, NKX2.2, SHH and the like.

[0025] The pluripotent stem cells used in the method for producing dorsal brain organoids of the present disclosure include, but are not limited to, iPS cells (artificial pluripotent stem cells) and ES cells (embryonic stem cells). As pluripotent stem cells, there are no limitations as long as they are cells that have both differentiation pluripotency and self-replicating ability. As pluripotent stem cells, it is preferable to use iPS cells from the viewpoint of availability and ethics. The origin of pluripotent stem cells includes, but is not limited to, mammals including humans, and non-human mammals such as monkeys, mice, rats, rabbits, dogs, cats, sheep, goats, horses, cows, and pigs. By using human iPS cells as pluripotent stem cells, organoids that mimic the human dorsal cerebral cortex can be produced.

[0026] According to the above-described method for producing dorsal brain organoids, the formation of ventral brain organoids is inhibited, and cells can be easily induced to differentiate into dorsal brain organoids. This method for producing does not involve genetic modification, so it can be easily used. The dorsal brain organoids produced by this method for producing can be used, for example, in research on cerebral cortex organoids. In addition, the method for producing dorsal brain organoids in the present disclosure can be used, for example, as a troubleshooting method for the induction of brain organoids, and is also expected to be used in the fields of regenerative medicine and pharmaceuticals.

[0027] According to another embodiment of the present disclosure, a kit for producing dorsal brain organoids is provided. The kit includes at least one of a cAMP activator and a cAMP analog. According to this embodiment of the kit for producing dorsal brain organoids, it is possible to easily induce differentiation of cells into dorsal brain organoids. The kit may further include pluripotent stem cells. The kit may also include a medium and other components that can be used for culturing cells, instruments and devices that can be used for culturing, instructions for use, and the like. When using this kit, any other substance may be used in addition to the substances included in the kit. EXAMPLES

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0029] 1. Method (1)Culture solution The compositions of the culture solutions used in the experiments are shown in Tables 1 to 3 below. Either forskolin or 8-bromo-cAMP was added to each culture solution. As described below, 24 EZ sphere well (Iwaki, 4820-900) or Nunclon Sphera-Treated 96U Bottom plate (ThermoScientific, Cat#174925) was used for the culture.

[0030] [Table 1]

[0031] [Table 2]

[0032] [Table 3]

[0033] (2) Organoid generation (using 24 EZ sphere well) 24 EZ sphere well was filled with 1 ml of induction media and 5 μl of Y27635 (Funakoshi, 10-2301, final concentration 50 μM) and warmed at 37°C. Then, iPS cells were seeded at 188,000 cells / well. On the second day of culture, 500 μl of culture medium was removed and 500 μl of induction media was added. On the fourth day of culture, 500 μl of culture medium was removed and 500 μl of induction media was added. On the sixth day of culture, as much culture medium as possible was removed (about 900 μl), and 50 μl of Matrigel (Corning, 354234, Corning is a registered trademark) was added so that the aggregates were contained in the Matrigel. After culturing for 20 minutes at 37°C, 1 ml of Organoid differentiation media 1 was added. On the eighth day of culture, 500 μl of Organoid differentiation media 1 solution was added. On the 10th day of culture, the organoids were transferred to 6 wells and 1 ml of Organoid Differentiation Media 2 solution was added. After this, the 6 wells were shaken and cultured using a rotator (TAITEC, Cell Shaker CS-LR). Every 3-4 days, 500 μl of culture medium was removed and 500 μl of Organoid Differentiation Media 2 solution was added. After one month, the creation of brain organoids was completed. When culturing for more than one month, Organoid Differentiation Media 2 without Forskolin was used. A CO2 incubator was used for the culture.

[0034] (3) Organoid production (Ultra low binding V bottom 96 well plate) 100μl of induction media and 0.5μl of Y27635 (final concentration 50μM) were placed in an ultra low binding V bottom 96 well plate and warmed at 37℃. Then, 3,000 iPS cells / well were seeded. On the second day of culture, 50μl of culture medium was removed and 50μl of induction media was added. On the fourth day of culture, 50μl of culture medium was removed and 50μl of induction media was added. On the sixth day of culture, as much culture medium as possible was removed (approximately 95μl) and 5μl of Matrigel was added so that the aggregates were contained within the Matrigel. After culturing for 20 minutes at 37℃, 100μl of Organoid differentiation media 1 was added. On the eighth day of culture, 50μl of Organoid differentiation media 1 solution was added. On the 10th day of culture, the aggregates were transferred to an ultra low binding 6 well plate and 1 ml of Organoid Differentiation Media 2 solution was added. After this, the 6 well plate was shaken and cultured on a rotator. Every 3-4 days, 500 μl of the culture medium was removed and 500 μl of Organoid Differentiation Media 2 solution was added. After one month, the creation of brain organoids was completed. When culturing for more than one month, Organoid Differentiation Media 2 without forskolin was used.

[0035] (4) Fixation and sectioning of organoids Organoids cultured for one month were fixed using the following method. The tip of a 1.2 ml tip (Watson, 110-805C) was cut with scissors, and the organoids in the 6-well plate were transferred to a 1.5 ml tube using a tip with a wide hole. The excess culture medium transferred to the tube with the organoids was discarded using the same tip. Then, 1 ml of 4% paraformaldehyde was added to the 1.5 ml tube. 4% paraformaldehyde was prepared by adding 4 g of paraformaldehyde (WAKO, 162-16065) to 70 ml of Nano pure H2O, adjusting the pH to 7.4 with sodium hydroxide, heating and stirring to melt, and adding Nano pure H2O to adjust the total volume to 100 ml. The 1.5 ml tube was then stored at 4°C for any time between 4 hours and overnight. Then, the paraformaldehyde was discarded, and 30% sucrose solution was added. The 30% sucrose solution was prepared by adding 15 g of sucrose (Nacalai Tesque, 30403-55) to 15 ml of PBS (10x D-PBS(-) Nacalai Tesque, 11482-15 100 ml diluted with Nano pure H2O 900 ml) and stirring to dissolve, then adding PBS to adjust the total volume to 50 ml. When the 30% sucrose solution was added, the organoids were floating on the liquid surface. The organoids were stored at 4°C for any period of time of 4 hours or more. After that, it was confirmed that the organoids had sunk to the bottom of the tube, and they were transferred to a base mold (As One, M475-2) using a tip with a wide hole. The excess 30% sucrose that had been transferred to the mold together with the organoids was discarded as much as possible. Then, OCT compound (Sakura Finetech Japan, 4583) was inserted and frozen in a -80°C freezer for 10 minutes or more. Using a freezing microtome (Leica CM3050), 16-micrometer-thick sections were prepared and attached to slides (Matsunami Glass Industry, Frontier Coat Slides FRC-01). The slides were stored in a -20°C freezer until use.

[0036] (5) Immunostaining of organoids The slide was placed in a staining tray (Kartell, #355) and washed with PBS for 5 minutes three times. Then, a line was drawn around the section on the slide using a Pap pen (Daido Sangyo, PAP-SM). By using a Pap pen, it is possible to create a hydrophobic enclosure around the section, and antibody reaction can be performed within the enclosure. The slide was placed in a humid box (a Petri dish (Corning, 150 mm x 15 mm Petri dish 351058, Corning is a registered trademark) with a moist paper towel in the corner), blocking buffer was added to the section, and the slide was left at room temperature for 1 hour. The blocking buffer was prepared with 9.7 ml of PBS, 0.3 ml of Normal Serum Block (Biolegend, 927502), and 30 μl of Triton X (Sigma, 30-5140-5). Then, the blocking buffer was removed, and the primary antibody (diluted in blocking buffer) was added to the section, and the section was left overnight at 4°C. Then, the slide was placed in a slide chamber and washed with PBS for 5 minutes three times. The slide was placed in a humid box, and a secondary antibody (diluted in blocking buffer) containing DAPI (Sigma, D9542) was added, and the section was left in the dark at room temperature for 1 hour. Then, the slide was placed in a slide chamber and washed with PBS for 5 minutes three times. Then, a cover glass (Matsunami Glass Co., Ltd., C024601) was placed on the section using a mounting material (SouthernBiotech, Fluromount-G 0100-01 (Fluromount-G is a registered trademark)), and the section was observed under a microscope (Olympus, FV3000). The primary antibodies used were FOXA2 antibody (Santa Cruz, HNF-3beta sc-101060, diluted 1:500), PAX6 antibody (Biolegend, 901301, diluted 1:500), and NKX2.2 antibody (University of Iowa Hybridoma Bank, 74.5A5-s, diluted 1:20).The secondary antibodies used were Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody (Thermofisher Scientific, Alexa Fluor 555 (Alexa Fluor is a registered trademark), A-21137, diluted at 1:500), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Thermofisher Scientific, Alexa Fluor 647 (Alexa Fluor is a registered trademark), A-21244, diluted at 1:500), and Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody (Thermofisher Scientific, Alexa Fluor 488 (Alexa Fluor is a registered trademark), A-21141, diluted at 1:500).

[0037] 2.Results <Experiment 1: Confirmation of dorsal brain organoid induction ability by cAMP activator> We confirmed the ability of cAMP activators to induce dorsal brain organoids. Wild-type iPS cells and iPS cells with GPR161 knocked out were used as cells, and forskolin was used as the cAMP activator. As described above, organoids were produced in a medium containing 500 nM forskolin. As a control, organoids were produced in the same manner using a medium without forskolin. After fixation and sectioning, immunostaining was performed. Immunostaining confirmed the expression of FOXA2, a marker for ventral brain organoids, and PAX6, a marker for dorsal brain organoids.

[0038] FIG. 2 is an explanatory diagram showing the results of immunostaining in Experiment 1. In FIG. 2, for wild-type and GPR161 knockout cells, a representative microscopic image of immunostaining using FOXA2 is shown at the top of the page, and a representative microscopic image of immunostaining using PAX6 is shown at the bottom of the page. From the results shown in FIG. 2, the following was found. That is, in the wild-type, many PAX6-positive cells were observed, and it was shown that they were induced to become dorsal brain organoids. In contrast, in GPR161 knockout cells, more FOXA2-positive cells were observed than PAX6-positive cells, and it was shown that they were induced to become ventral brain organoids. In other words, the conventional dorsal brain organoid production method did not succeed in producing dorsal brain organoids in GPR161 knockout iPS cells, and ventral brain organoids were produced. This result shows that the conventional dorsal brain organoid production method does not allow research on dorsal brain organoids in iPS cells with enhanced SHH signaling pathway. In contrast, in GPR161 knockout cells induced to differentiate in a medium containing forskolin, more PAX6-positive cells were observed than FOXA2-positive cells. This indicates that the addition of forskolin as a cAMP activator during culture can facilitate differentiation into dorsal brain organoids.

[0039] <Experiment 2: Confirmation of dorsal brain organoid induction ability by cAMP analogues> We confirmed the ability of cAMP analogs to induce dorsal brain organoids. iPS cells with GPR161 knocked out were used as the cells, and 8-bromo-cAMP was used as the cAMP analog. As described above, organoids were produced in medium containing 0nM, 500nM, 1μM, and 2μM 8-bromo-cAMP, and after fixing and sectioning, immunostaining was performed. Immunostaining confirmed the expression of FOXA2, a marker for the extreme ventral brain organoids, NKX2.2, a marker for the ventral brain organoids, and PAX6, a marker for the dorsal brain organoids.

[0040] FIG. 3 is an explanatory diagram showing the results of immunostaining in Experiment 2. In FIG. 3, for GPR161 knockout cells induced to differentiate in a medium containing 8-bromo-cAMP at each concentration, a representative microscopic image of immunostaining using FOXA2 is shown at the top of the page, a representative microscopic image of immunostaining using NKX2.2 is shown at the middle of the page, and a representative microscopic image of immunostaining using PAX6 is shown at the bottom of the page. From the results shown in FIG. 3, the following was found. That is, when a medium not containing 8-bromo-cAMP was used, FOXA2 expression was high, and it was shown that differentiation was induced into ventral brain organoids. As the concentration of 8-bromo-cAMP increased, the expression level of FOXA2 decreased and the expression level of PAX6 increased. Therefore, it was suggested that the addition of 8-bromo-cAMP could induce differentiation into dorsal brain organoids instead of ventral brain organoids. When a medium containing 2 μM of 8-bromo-cAMP was used, both NKX2.2 and PAX6 were expressed, which indicates that ventral and dorsal brain organoids were mixed, and that moderate dorsal induction ability was demonstrated. This suggests that 8-bromo-cAMP as a cAMP analog is also effective when creating organoids containing both dorsal and ventral brain organoids. These results show that the addition of 8-bromo-cAMP as a cAMP analog can facilitate differentiation into dorsal brain organoids.

[0041] The present invention is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present invention. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the Summary of the Invention column can be appropriately replaced or combined in order to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

Claims

1. A method for producing dorsal brain organoids, comprising: In the process of producing dorsal brain organoids from pluripotent stem cells, using at least one of a cAMP activator and a cAMP analog; A method for producing dorsal brain organoids.

2. The method for producing dorsal brain organoids according to claim 1, The cAMP activator is forskolin. A method for producing dorsal brain organoids.

3. The method for producing dorsal brain organoids according to claim 1, The cAMP analog is 8-bromo-cAMP. A method for producing dorsal brain organoids.

4. The method for producing dorsal brain organoids according to claim 1 or 2, In the medium for producing the dorsal brain organoid from the pluripotent stem cells, the concentration of the cAMP activator is 200 nM or more and 10 μM or less; A method for producing dorsal brain organoids.

5. The method for producing dorsal brain organoids according to claim 1 or 3, In the medium for producing the dorsal brain organoid from the pluripotent stem cells, the concentration of the cAMP analog is 1 μM or more and 10 μM or less; A method for producing dorsal brain organoids.

6. The method for producing dorsal brain organoids according to claim 1 or 2, Using the substance by the third day after the start of the process. A method for producing dorsal brain organoids.

7. The method for producing dorsal brain organoids according to claim 1 or 2, In the step, the substance is used for 6 days or more. A method for producing dorsal brain organoids.

8. A kit for producing dorsal brain organoids, comprising: The composition includes at least one of a cAMP activator and a cAMP analogue. A kit for producing dorsal brain organoids.

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