Method for producing suprachiasmatic nucleus organoid

Abstract

The present invention addresses the problem of providing a method for producing a suprachiasmatic nucleus cell organoid. The problem is solved by a method for producing a suprachiasmatic nucleus organoid comprising: (1) a step for culturing pluripotent stem cells in an intermediate medium between an undifferentiation maintenance medium and a nerve differentiation medium; (2) a step for culturing the cells obtained in step (1) in a nerve differentiation medium; and (3) a step for performing an operation for forming a cell mass prior to the start of step (2), wherein (A) the number of raw material cells seeded in order to form one cell mass is 6000 or more, and (B) when the start of step (2) is taken as day 0, the medium contains a sonic hedgehog signaling pathway agonist from day 3 onward.

Description

Method for producing suprachiasmatic nucleus organoids

[0001] The present invention relates to a method for producing suprachiasmatic nucleus organoids, etc.

[0002] In modern society, as lifestyles become more diverse, disruption of circadian rhythms is becoming an increasing problem. For example, night shifts, shift changes, long-distance travel, etc. can cause a mismatch between the sleep-wake rhythm and the external light cycle, leading to sleep disorders caused by disruption of the circadian rhythm. Such sleep disorders increase the risk of various diseases, including lifestyle-related diseases, cancer, and mental illness.

[0003] In mammals, the suprachiasmatic nucleus (SNU) is the center of circadian rhythm. Therefore, regulating the circadian rhythm in the SNU may improve sleep disorders and reduce the risk of the above-mentioned diseases. However, the mechanism by which the SNU controls circadian rhythm remains largely unknown.

[0004] Proc Natl Acad Sci US A. 2008 Aug 19;105(33):11796-801.Nature. 2011 Nov 9;480(7375):57-62.

[0005] The inventors focused on the fact that obtaining suprachiasmatic nucleus organoids would enable further elucidation of the circadian rhythm control mechanism and could lead to the development of drugs to control circadian rhythm disorders.

[0006] Non-Patent Document 1 reports a method for inducing hypothalamic neurons from mouse ES cells, but this method does not induce the suprachiasmatic nucleus. Non-Patent Document 2 reports a method for inducing the pituitary gland by simultaneously inducing the hypothalamus and oral epithelium, but this method does not induce the suprachiasmatic nucleus.

[0007] An objective of the present invention is to provide a method for producing suprachiasmatic nucleus cell organoids.

[0008] In light of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by a method for producing suprachiasmatic nucleus organoids, which comprises: (1) culturing pluripotent stem cells in an intermediate medium between an undifferentiated maintenance medium and a neural differentiation medium; (2) culturing the cells obtained in step (1) in a neural differentiation medium; and (3) performing a cell mass formation operation before the start of step (2), wherein: (A) 6,000 or more raw cells are seeded to form one cell mass; and (B) if the start of step (2) is considered day 0, the medium contains a sonic hedgehog signaling pathway active substance from day 3 onward. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. A method for producing a suprachiasmatic nucleus organoid, comprising: (1) culturing pluripotent stem cells in an intermediate medium between an undifferentiated state maintenance medium and a neural differentiation medium; (2) culturing the cells obtained in step (1) in a neural differentiation medium; and (3) performing an operation to form a cell cluster before the start of step (2), wherein: (A) 6,000 or more raw material cells are seeded to form one cell cluster, and (B) when the start of step (2) is considered day 0, the culture medium contains a substance acting on the sonic hedgehog signaling pathway from day 3 onward.

[0010] Item 2. The production method according to Item 1, wherein the intermediate medium in step (1) is a medium in which the concentration of the undifferentiation maintenance factor is lower than the concentration in the undifferentiation maintenance medium.

[0011] Item 3. The method according to Item 2, wherein the undifferentiated maintenance factor is an FGF-MAPK signal inhibitor and / or a Wnt signal activator.

[0012] Item 4. The production method according to any one of Items 1 to 3, wherein the starting cells in step (3) are 9,000 or more.

[0013] Item 5. The production method according to any one of Items 1 to 4, wherein the operation in step (3) is seeding cells in a culture well and culturing them in suspension.

[0014] Item 6. The production method according to any one of Items 1 to 5, wherein the culture in the medium containing the substance acting on the sonic hedgehog signaling pathway is continued for 4 to 15 days.

[0015] Item 7. The production method according to any one of Items 1 to 6, wherein the culture in step (2) is for 4 to 15 days.

[0016] Item 8. The production method according to any one of Items 1 to 7, wherein the concentration of the substance acting on the Sonic hedgehog signaling pathway in the medium is 0.5 μM or more.

[0017] Item 9. The production method according to any one of Items 1 to 8, wherein the intermediate medium in step (1) is a medium in which the concentration of an undifferentiation maintenance factor is lower than the concentration in an undifferentiation maintenance medium, the undifferentiation maintenance factor is an FGF-MAPK signal inhibitor and / or a Wnt signal activator, the number of starting cells in step (3) is 9,000 or more, the operation in step (3) involves seeding the cells in small wells and culturing them in suspension, the culture in the medium containing the substance acting on the Sonic hedgehog signaling pathway is from day 4 to day 15, the culture in step (2) is from day 4 to day 15, and the concentration of the substance acting on the Sonic hedgehog signaling pathway in the medium is 0.5 mM or more.

[0018] Item 10. A manufacturing method according to any one of Items 1 to 9, further comprising (3) culturing the cell mass obtained in step (2) in a neural differentiation medium under hyperoxic conditions. Item 11. A suprachiasmatic nucleus organoid in which the proportion of suprachiasmatic nucleus cells is 5% or more.

[0019] Item 12. The suprachiasmatic nucleus organoid according to Item 11, wherein the proportion of suprachiasmatic nucleus cells is 10% or more.

[0020] Item 13. A suprachiasmatic nucleus organoid obtained by the production method according to any one of Items 1 to 10.

[0021] Item 14. A method for screening for a circadian rhythm regulating agent, comprising: (X) a step of contacting the suprachiasmatic nucleus organoid according to any one of Items 11 to 13 with a test substance; and (Y) a step of evaluating the circadian rhythm of the suprachiasmatic nucleus organoid.

[0022] Item 15. A circadian rhythm regulating agent having the effect of changing the circadian rhythm of the suprachiasmatic nucleus organoid according to Item 11 or 12.

[0023] According to the present invention, it is possible to provide a method for producing suprachiasmatic nucleus cell organoids, suprachiasmatic nucleus cell organoids, a method for screening circadian rhythm regulators, and the like.

[0024] The results of immunostaining organoids obtained in Test Example 1-1 with terminal differentiation markers of the suprachiasmatic nucleus (Six6, VIP, AVP) are shown (Test Example 1-2). The results of clock gene expression analysis of organoids obtained in Test Example 1-1 are shown (Test Example 1-4). The organoids obtained in Test Example 1-1 were transplanted into mice with SCN destruction, and the presence or absence of mouse movement was measured before and after transplantation. This shows an actogram (Test Example 1-5). The organoids obtained in Test Example 1-1 were transplanted into mice with SCN destruction, and the presence or absence of mouse movement was measured before and after transplantation. This shows a chi-square periodogram (Test Example 1-5). Brightfield images (Bright), fluorescence images (showing Six3 expression), and autofluorescence images (Auto) of a cell cluster on Day 13 are shown (Test Example 2). Brightfield images, fluorescence images (showing Six3 expression), and autofluorescence images of a cell cluster on Day 48 are shown (Test Example 3). Brightfield images of a cell cluster on Day 13 are shown (Test Example 4).

[0025] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0026] 1. Method for Producing Suprachiasmatic Nucleus Organoids In one aspect, the present invention relates to a method for producing suprachiasmatic nucleus organoids (sometimes referred to herein as the "production method of the present invention"), which comprises the steps of (1) culturing pluripotent stem cells in an intermediate medium between an undifferentiated maintenance medium and a neural differentiation medium, (2) culturing the cells obtained in step (1) in a neural differentiation medium, and (3) performing a procedure to form cell clusters before the start of step (2), wherein (A) 6,000 or more raw cells are seeded to form one cell cluster, and (B) if the start of step (2) is considered day 0, the culture medium contains a sonic hedgehog signaling pathway active substance from day 3 onwards. This is described below.

[0027] <Step (1)> Pluripotent stem cells are stem cells that have the ability to differentiate into all cell lineages belonging to the three germ layers (endoderm, mesoderm, and ectoderm) (pluripotency) and also have the ability to proliferate. Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, and pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. ES cells are preferred as pluripotent stem cells in terms of the efficiency of induction into suprachiasmatic nucleus organoids and the properties of the resulting suprachiasmatic nucleus organoids. In one embodiment of the present invention, preferred pluripotent stem cells are iPS cells, more preferably human iPS cells, because they can be obtained without destroying embryos, eggs, etc. during the production process.

[0028] Methods for producing iPS cells are known in the art, and iPS cells can be produced by introducing reprogramming factors into any somatic cells. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol. , 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y.et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0029] Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specific examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0030] The intermediate medium has the compositional characteristics of the undifferentiated maintenance medium used to maintain the undifferentiated state of pluripotent stem cells before subjecting them to step (1) and the compositional characteristics of the neural differentiation medium used in step (2), and is a medium that can acclimate pluripotent stem cells to a dedifferentiation environment (culture in neural differentiation medium). This "acclimation" can improve the induction efficiency of suprachiasmatic nucleus organoids and the properties of the resulting suprachiasmatic nucleus organoids.

[0031] The undifferentiated state maintenance medium is a medium that is capable of maintaining the undifferentiated state of pluripotent stem cells for a certain period of time (e.g., 3 days or more, 7 days or more, 14 days or more, or 28 days or more), and is not particularly limited as long as it is.

[0032] The undifferentiated state maintenance medium contains an undifferentiated state maintenance factor that has the effect of maintaining the undifferentiated state of pluripotent stem cells. Examples of factors for maintaining undifferentiated state include FGF-MAPK signal inhibitors (typically PD0325901), Wnt signal activators (GSK3 inhibitors, typically CHIR99021; other examples include Bromoindirubin-3'-oxime (BIO), Kenpaullone, etc.), LIF, BMP-4, 6-bromoindirubin-3'-oxime, CDK8 / 19i, Emricasan, polyamines, Trans-ISRIB Chroman 1, Epiblastin A, GF 109203X, Go6983, IQ-1, PD173074, SU5402, PD184352, PD98059, SB202190, SB216763, sodium butyrate, SP600125, Surfen, U0126, and WH-4-023. The undifferentiated cell maintenance factor may be one type alone or a combination of two or more types.

[0033] The concentration of the undifferentiation maintenance factor in the undifferentiation maintenance medium is not particularly limited as long as it is a concentration that can maintain the undifferentiation state. For example, the concentration of the FGF-MAPK signal inhibitor is preferably 0.2 to 4 μM, more preferably 0.5 to 2 μM, and the concentration of the Wnt signal activator is preferably 0.5 to 10 μM, more preferably 1.5 to 5 μM.

[0034] As used herein, the medium can be prepared as a basal medium, such as a medium used for culturing animal cells. Examples of basal media include Glasgow's Minimal Essential Medium (GMEM), IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's medium, Neurobasal Medium (Life Technologies), and mixtures thereof. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc., and may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc.

[0035] As the undifferentiated state maintenance medium, commercially available media prepared by adding necessary components can also be used, such as AK02N and AK03N from the StemFit (registered trademark) series.

[0036] The neuronal differentiation medium used in step (2) is not particularly limited, as long as it is a medium that can release the undifferentiated state of cells and induce neuronal differentiation.

[0037] The neural differentiation medium does not contain factors for maintaining undifferentiation, which are contained in the undifferentiated state maintenance medium, and this allows the cells to be released from the undifferentiated state and induced to differentiate toward neural differentiation.

[0038] As the basal medium for neural differentiation, preferred examples include IMDM medium, F12 medium, etc., and more preferred examples include a mixed medium thereof.

[0039] The neural differentiation medium is preferably a serum-free medium. In this case, the neural differentiation medium may contain a reducing agent, a serum replacement component, and the like. Preferred reducing agents include thioglycerol, 2-mercaptoethanol, and the like, and preferred serum replacement components include BSA, and the like. More preferably, the medium contains a combination of a reducing agent and a serum replacement component. The concentration of the reducing agent (particularly thioglycerol) is preferably 100 to 1000 μM, more preferably 300 to 600 μM, and the concentration of BSA is preferably 1 to 20 mg / ml, more preferably 2 to 10 mg / ml.

[0040] Furthermore, it is preferable that the neural differentiation medium does not contain insulin. The method of the present invention can be applied to human cells even when a neural differentiation medium that does not contain insulin is used.

[0041] The intermediate medium is typically a medium in which the concentration of the undifferentiation maintenance factor is lower than that in the undifferentiation maintenance medium. The concentration of the undifferentiation maintenance factor in the intermediate medium is preferably lower, and can be, for example, 70 / 100 or less, 60 / 100 or less, 50 / 100 or less, 40 / 100 or less, 30 / 100 or less, 20 / 100 or less, 10 / 100 or less, 5 / 100 or less, 2 / 100 or less, or 1 / 100 or less of the concentration of the corresponding undifferentiation maintenance factor in the undifferentiation maintenance medium, and the intermediate medium may not contain the undifferentiation maintenance factor.

[0042] The intermediate medium is preferably a medium having the same composition as the undifferentiation maintenance medium except that the concentration of the undifferentiation maintenance factor is reduced (the change in the concentration of the components is within ±20%, preferably within ±10%, more preferably within ±5%, and even more preferably within ±1%), or is a mixed medium of the undifferentiation maintenance medium and the neural differentiation medium.

[0043] In one embodiment of the present invention, the concentration of the undifferentiated maintenance factor in the intermediate medium can be reduced in a stepwise manner.

[0044] Prior to the step (3) described below, the culture is preferably adhesion culture, in which cells are adhered to the substrate of a culture vessel and cultured. For example, adhesion culture can be performed by coating the substrate of the culture vessel with a cell adhesion molecule such as an extracellular matrix (specifically, for example, Matrigel (BD), type I collagen, type IV collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or a combination thereof) before use in culture.

[0045] Regarding culture conditions, the culture temperature is not particularly limited, but is, for example, 30 to 40°C, preferably about 36 to 38°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0046] The culture period is, for example, 1 to 7 days, preferably 1 to 5 days, more preferably 2 to 5 days, even more preferably 2 to 4 days, and even more preferably 3 to 4 days.

[0047] <Step (2)> The neural differentiation medium is as described in <Step (2)> above.

[0048] By culturing in a neuronal differentiation medium, the cell clusters formed in step (3) described below undergo neuronal differentiation and begin to express suprachiasmatic nucleus markers such as Six3.

[0049] Regarding culture conditions, the culture temperature is not particularly limited, but is, for example, 30 to 40°C, preferably about 36 to 38°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0050] The culture period is, for example, 4 to 20 days, preferably 4 to 15 days, more preferably 5 to 15 days, even more preferably 5 to 13 days, and even more preferably 6 to 13 days.

[0051] <(B) Sonic hedgehog signaling pathway active substance> In the production method of the present invention, if the start of step (2) is considered to be day 0 (day 0), the medium contains a sonic hedgehog signaling pathway active substance from before day 3 (day 3). That is, the medium (neuronal differentiation medium, or neuronal differentiation medium and intermediate medium) used during the period from any day before day 3 (addition start date) to any day after day 3 (addition end date) contains a sonic hedgehog signaling pathway active substance. This promotes neuronal differentiation and can improve the induction efficiency of suprachiasmatic nucleus organoids and the properties of the resulting suprachiasmatic nucleus organoids.

[0052] A sonic hedgehog signaling (hereinafter sometimes referred to as Shh) pathway agonist is a substance that can enhance signal transduction mediated by Shh. Examples of Shh signaling pathway agonists include proteins belonging to the Hedgehog family (e.g., Shh and Ihh), Shh receptors, Shh receptor agonists, Purmorphamine, and SAG (Smoothened Agonist; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]-benzo[b]thiophene-2-carboxamide). The preferred Shh signaling pathway agonist is SAG.

[0053] The Sonic hedgehog signaling promoting activity of SAG can be determined by methods well known to those skilled in the art, for example, a reporter gene assay focusing on the expression of the Gli1 gene (Oncogene (2007) 26, 5163-5168).

[0054] The concentration of SAG in the medium is, for example, 0.001 μM or more, preferably 0.01 μM or more, more preferably 0.1 μM or more, even more preferably 0.2 μM or more, and even more preferably 0.5 μM or more. The concentration is preferably 0.2 to 5 μM, more preferably 0.5 to 2 μM, and even more preferably 0.7 to 1.5 μM. When a substance acting on the Sonic hedgehog signaling pathway other than SAG is used, it is desirably used at a concentration that exhibits Sonic hedgehog signaling promoting activity equivalent to the above-mentioned concentration of SAG.

[0055] The starting date for adding the sonic hedgehog signaling pathway active substance is preferably day -6 to day 3, more preferably day -4 to day 3, even more preferably day -2 to day 3, even more preferably day 0 to day 3, especially preferably day 1 to day 3, especially even more preferably day 2 to day 3, and particularly preferably day 2.

[0056] The day on which the addition of the sonic hedgehog signaling pathway active substance is completed is, for example, from day 4 to day 100, preferably from day 4 to day 20, more preferably from day 4 to day 15, even more preferably from day 5 to day 13, and even more preferably from day 6 to day 13, and particularly preferably at the end of step (2).

[0057] <Step (3)> The timing of the operation to form cell aggregates is not particularly limited as long as it is performed before the start of step (2). In one embodiment of the present invention, the timing is not particularly limited as long as it is performed between the start of step (1) and the start of step (2), but is preferably day -6 to day 0, more preferably day -5 to day 0, even more preferably day -4 to day 0, and even more preferably day -3 to day 0.

[0058] When performing adhesion culture, before the operation of forming cell clusters, the cells are detached from the culture substrate according to or in accordance with a known method, and dispersed in the medium by pipetting or the like.

[0059] The procedure for forming cell clusters is not particularly limited, as long as it allows cells to aggregate to form cell clusters with a diameter of 100 μm or more (preferably 200 μm or more, and particularly preferably 500 μm or more). Examples of such procedures include seeding cells on a culture substrate and culturing them in suspension, and centrifuging the cell suspension, with the former being particularly preferred.

[0060] When cells are seeded on a culture substrate for suspension culture, the dispersed cells can be seeded in a relatively large culture vessel, such as a 10 cm dish, to simultaneously form multiple cell aggregates in a single culture vessel. However, this results in variations in the size of each aggregate. Therefore, for example, if a certain number of dispersed cells are placed in a culture well (e.g., each well of a multi-well plate (U-bottom or V-bottom) such as a 96-well microplate) and cultured statically, the cells will rapidly aggregate to form a single aggregate in each well. By recovering these aggregates from multiple wells, a uniform population of aggregates can be obtained.

[0061] The culture vessel is preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels that can be used include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as laminin, entactin, collagen, and gelatin, or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment). Examples of non-cell-adhesive culture vessels that can be used include those whose surfaces have been artificially treated to reduce cell adhesion (e.g., ultrahydrophilic treatment with MPC polymers, low protein adsorption treatment, etc.).

[0062] The time from the operation to form cell clusters to the formation of cell clusters is preferably within 3 days, more preferably within 2 days, even more preferably within 1 day, and particularly preferably 12 to 24 hours. The conditions for the operation to form cell clusters (for example, the area and shape of the culture bottom) can be adjusted so that this time is achieved.

[0063] After the cell aggregates are formed, suspension culture is carried out as is in steps (1) and (2).

[0064] <(A) Number of raw cell masses> In the production method of the present invention, the number of raw cell masses seeded to form one cell mass in step (3) is 6000 or more. This promotes neural differentiation and improves the efficiency of induction into suprachiasmatic nucleus organoids and the properties of the resulting suprachiasmatic nucleus organoids.

[0065] The starting cells are preferably 9000 or more, more preferably 12000 or more, even more preferably 15000 or more, and even more preferably 20000 or more. There is no particular upper limit, and it is, for example, 200000, 100000, or 80000.

[0066] <Step (4)> The production method of the present invention preferably further comprises the step (4) of culturing the cell mass obtained in step (2) in a neural differentiation medium, which allows further maturation of the suprachiasmatic nucleus organoids.

[0067] Step (4) is preferably carried out under high-oxygen conditions. Specifically, in step (4), the cells are preferably cultured in an incubator with a high-oxygen atmosphere. The high-oxygen concentration is not particularly limited as long as it is higher than the oxygen concentration in the atmosphere, but is preferably 25 to 60%, more preferably 30 to 50%, and particularly preferably 35 to 45%.

[0068] Step (4) can also be performed by semi-aerobic culture, for example, by placing cell clusters on Millicell or Transwell, immersing the bottom surface in expansion medium or maturation medium, and exposing the top surface to air.

[0069] Step (4) preferably includes a step of culturing using an expansion medium as the neural differentiation medium, and a step of culturing using a maturation medium as the neural differentiation medium.

[0070] As the basal medium for the expansion medium, preferably, DMEM medium, F12 medium, etc. are used, and more preferably, a mixed medium thereof is used.

[0071] The expansion medium is preferably a serum-free medium. In this case, the neural differentiation medium contains a serum replacement component. Examples of the serum replacement component include KSR. The concentration of KSR is preferably 5-20%, more preferably 7-15%.

[0072] The culture period in the expansion medium is, for example, 4 to 20 days, preferably 4 to 15 days, more preferably 5 to 15 days, even more preferably 5 to 13 days, and even more preferably 6 to 13 days.

[0073] As the basal medium for the maturation medium, preferably, DMEM medium, F12 medium, etc. are used, and more preferably, a mixed medium thereof is used.

[0074] The maturation medium is preferably a serum-free medium. In this case, the neural differentiation medium contains serum replacement components. Examples of serum replacement components include N2 supplement, B27 supplement, and the like, and more preferably a combination thereof. The concentration of N2 supplement is preferably 0.2-5%, more preferably 0.5-2%, and the concentration of B27 supplement is preferably 0.5-10%, more preferably 1-3%.

[0075] The culture period for culturing in a maturation medium is, for example, 4 to 400 days.

[0076] In step (4), the cells can be cultured in a medium containing a substance that acts on the Sonic hedgehog signaling pathway, or in a medium that does not contain a substance that acts on the Sonic hedgehog signaling pathway.

[0077] By the manufacturing method of the present invention, it is possible to obtain suprachiasmatic nucleus organoids with a high proportion of suprachiasmatic nucleus cells.The proportion is, for example, 1% or more, preferably 5% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 18% or more.The upper limit is not particularly limited, and is, for example, 50%, 40%, or 30%.The proportion can be measured according to the following Test Examples 1-3.

[0078] By the manufacturing method of the present invention, it is possible to obtain suprachiasmatic nucleus organoids whose constituent cells are all brain tissue cells.Cerebral tissue cells include, for example, excitatory nerves, inhibitory nerves, meninges, glial cells, etc.Constituent cells can be determined according to the following Test Example 1-3.

[0079] 2. Screening Method In one aspect, the present invention relates to a method for screening for circadian rhythm regulators, comprising: (X) a step of contacting a suprachiasmatic nucleus organoid, in which the proportion of suprachiasmatic nucleus cells is 5% or more (preferably 10% or more, more preferably 15% or more, and even more preferably 18% or more), or a suprachiasmatic nucleus organoid obtained by the production method of the present invention, with a test substance; and (Y) a step of evaluating the circadian rhythm of the suprachiasmatic nucleus organoid. This will be explained below.

[0080] Examples of test substances include cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.

[0081] Test substances can also be obtained using any of the many approaches to combinatorial library technology known in the art, including (1) biological libraries, (2) synthetic library technology using deconvolution, (3) "one-bead one-compound" library technology, and (4) synthetic library technology using affinity chromatography selection. While the biological library technology using affinity chromatography selection is limited to peptide libraries, the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be stored in solution (see Houghten (1992) Bio / Techniques 13: 412-21) or on beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9), or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application No. 2002103360).

[0082] Step (X) can be carried out, for example, by adding a test substance to a medium containing suprachiasmatic nucleus organoids. The concentration of the test substance in the medium can be appropriately set depending on the type of test substance, etc.

[0083] The period for which the contact with the test substance is maintained is not particularly limited, but can be, for example, 1 hour to 14 days.

[0084] Step (Y) can be carried out, for example, by measuring the expression level of clock genes in the suprachiasmatic nucleus organoids and evaluating their fluctuations, oscillations, or persistence. The measurement and evaluation can be carried out according to or in accordance with known methods.

[0085] In addition, evaluation of the circadian rhythm of the suprachiasmatic nucleus organoids in step (Y) can be performed, for example, by measuring the fluctuations, oscillations, or persistence of expression levels of receptors expressed in the suprachiasmatic nucleus organoids, as well as the responsiveness of those receptors (e.g., responsiveness in the presence / absence of a test substance, or responsiveness to a ligand in the presence / absence of a test substance). Receptors are not particularly limited as long as they are expressed by suprachiasmatic nucleus organoids, but for example, in descending order of expression, Bsg, Rora, Prnp, Cd47, Gria2, Rack1, Nrxn1, Epha5, Thra, Ogfrl1, Rorb, Nrxn2, Gabbr1, Sqstm1, Atp6ap2, Ntrk2, Slc22a17, Grin2b, Nrp2, Vldlr, Gabrb3, Edil3, Unc5c, Nr1d1, Adg rl1, Crlf2, Nlgn1, Tmem63b, Nlgn2, Grm5, Stat3, Nr2f2, Jmjd6, Bmpr2, Tm2d1, Gabrg3, Fzd3, Ntrk3, Gpr162, Nptxr, L1cam, L rp1, Kit, Adgrb3, Amfr, Nr4a1, Sorl1, Tex264, Csf2ra, Grin1, Glrb, Ptprf, Sema6a, Vipr2, Gabra2, Acvr2a, Grik5, Nr1d2, A dgrb1, Gabrb1, Gpr27, Rgmb, Lrp8, Gria4, Adipor1, Mfsd6, Unc5d, Ptch1, Cntfr, Ogfr, Palld, Plxnc1, Adgrl3, Adgra1, Sdc4 , Nr2c2, Cry2, Paqr8, Gabrg1, Ephb1, Atrn, Gabre, Igf1r, Ddr1, Gabra1, Gria1, Lgals3, Nr4a3, Grpr, Celsr2, Neo1, Cry1, Ga brg2, Ednrb, Lrp6, Extl3, Drd1, Gprc5b, Adipor2, Igf2r, Gipr, Epha4, Nrp1, Il6st, Plxnb2, Gabra3, Ryk, Insr, Gpr85, Sema 5a, Nr2f6, Ephb2, Pkd1, Bmpr1a, Nr3c1, Gpr19, Gpr158, Rtn4rl1, Npbwr1, Adgrg1, Acvr1b, Grm1, Avpr1a, Lmbr1, Smo, Plxna1,<h2 style=";text-align:left;direction:ltr">Gpr153、Sort1、Adcyap1r1、Mfge8、Grik2、Plxna2、Gabr2、Gfra4、Acvr2b、 Gfra2、Paqr9、Nlgn3、Ar、Grik1、Adgrb2、Oprl1、Rxrb、Paqr7、Unc13b、B9d1 、Grin3a、Crcp、Gabrq、Scarb1、Grik3、Amot、Styk1、Fgfr1、Arnt、Sigmar1、 Chrnb2、Unc5a、Gabrb2、Agtrap、Adgrl2、Nr1h2、Ifnar1、Ifngr2、Npy1r、Sl c39a9、Tnfrsf1a、Plxna4、Sema4d、Fzd8、Ifngr1、Il11ra1、Nrg2、P2rx4、Nr 2f1、Epha8、Ahr、Cnr1、Grin2a、Npy6r、Esrra、Abca1、Erbb4、Fzd5、Gpr22、L par6、Plxnb1、Prokr2、Ptprm、Inpp5k、Gpr45、Tpra1、Nectin1、Tgfbr1、Ptp rt、Paqr4、Mrgpre、Cr1l、Gfra1、Ackr1、Gpr176、P2ry1、Ghsr、Gpr135、Celsr 3, Pgr, Il1rap, Rgma, Rxra, Nr4a2, Slc52a2, Ephb6, Rarb, Gabra5, Gpr61, Dcbld1, Plxna3, Grid1, Hrh3, Tyro3, ​​Grik4, Srebf1, Lgr4, Epha6, Adra1b, N pr2, Dhx16, Gria3, Ghr, Ramp2, Abca7, Adrb1, Ntsr2, Tspan12, Dcc, S1pr3, Ppard, Oprk1, Pdgfra, Grid2, Dcbld2, Abhd2, Sphk2, Lifr, Rara, Gpr179, F3 、Gpr101、Gpr37l1、Cp、Adgrv1、Robo1、Lpar1、Gal、Paqr3、Nradd、Grm8、Not ch2、Epha7、Calcr、Ramp1、Grin2d、Nid1、Acvr1、Alk、Gpr75、Glra3、Notch1 、F2r、Grm3、Fzd6、Il13ra1、Nr6a1、Robo2、Npy5r、Gpr173、Gpr165、Cd44、Ss tr1、Fzd1、Gpr6、Nr2c1、Gabra4、Grm7、Ifnar2、Gpr161、Gpr17、Pglyrp1、F8、Adra1a、S1pr1、Ret、P2ry14、Cd200r2、Il3ra、Tmem63c、Adora1、Gpr146、Pgr15l、Lrp4、Glra2、Hrh1、Thrb、Fzd2、Prlr、Hpgd、Ebi3、Tspo、Lbp、Lmbr1l、Kiss1r、Egfr、Gpr12、Igsf1、Unc13c、Rtbdn、Ltk、Gpr37、Antxr1、Hspg2、Ctsh、Htr2c、Adgrg6、Gprc5c、Colec12、Plxnd1、Epha10、Tgfbr3、Reg3g、Rorc、Htr5a、Tnfrsf19、Fzd4、Pparg、Reck、Mdga1、Cd302、Fgfr3、Nr3c2、Folr1、S1pr2、Ptk7、P2rx7、Fgfr2、Grm4、Agtr1a、Chrm2、Celsr1、Ntsr1、Lgr5、Stra6、Rtn4r、Chrna3、Chrm3、Esrrg、Chrna4、Adra2a、Bmpr1b、Fas、Mrc2、Gpr26、Met、Gpr83、Pde3a、S1pr5、Unc5b、Trhr、P2rx6、Ager、Esr1、Drd2、Adgra3、Cd27、Prtg、Epha3、Gpr150、Ly96、Crhr1、Glp1r、Htr1b、Gpr21、Flt1、Sstr2、Cspg4、Il10rb、Il6ra、Adgre5、Ednra、Adora2a、Nod1、Npr1、Pth1r、Tmem116、Ephb3、Grin2c、Gpr68、Tgfbr2、Acvr1c、Gpr62、Fgfrl1、Gpr139、Npy2r、Lag3、Egf、Lpar2、Asgr1、Opn3、Kdr、Fzd9、Adgrg2、Nmur2、Cd36、Cr2、Lrp5、Htr2a、Rxrg、Cxcr4、Axl、Il4ra、Tshr、Ddr2、Tnfrsf10b、Il15ra、Ptger3、Cd200r4、Notch3、Hcrtr2、Eda2r、Sostdc1、Epor、Gpr149、Npffr1、Efemp1、Vdr、Itga2、Htr7、Il1r2、Rarg、Gpr50、Il17rd、Aim2、Crlf1、Flt3、Mchr1、Vmn2r1、Ramp3、Gpr88、Il1rapl2、Tnfrsf1b、Tacr1、Tnfrsf23、Chrnb1、Cd200r1、<h2 style=";text-align:left;direction:ltr">Cx3cr1、Gpr3、Adora2b、Trim12a、Notch4、Fzd7、Csf1r、Qrfprl、Gucy2e、Sc n7a、Il17ra、Rxfp1、Nr2e1、Il17rc、Gpr157、Trim12c、Adra2c、Gper1、Oxtr、 Ptch2、Hcrtr1、Adgrf4、Paqr6、Calcrl、Nr5a2、Ccr10、Pdgfrb、Sstr3、Plxn b3、Ackr3、Insrr、Htr1a、Lpar4、Osmr、Ppara、Grm2、Chrm4、Oprm1、Chrna5、T gfbr3l、Tmem63a、Cd74、Adrb2、Trim30d、Lgr6、F2rl3、Tacr3、Chrng、Cd14、 Qrfpr、Fcgr1、Chrna7、Hmcn2、Gpr55、Gpr35、Nfam1、Tnfrsf22、Adgra2、Cckb r、Vmn2r3、Tnfrsf4、Thbd、Adgrf5、Ntrk1、Ngfr、Cckar、Erbb2、Adgrg3、Sph k1、Fzd10、Chrm5、Fcer2a、Chrnb4、Opn1mw、F2rl2、Vmn2r56、Chrnb3、Mertk、 Procr、Ror2、Htr6、Nr1h3、Anxa9、Gpr84、Htr3a、Tlr3、Cd79a、Gucy2c、Ephb 4、Mc1r、Tnfrsf18、Gpr25、Hmcn1、Glra1、Adrb3、Slamf1、Cd40、Amhr2、Trpv1 、Eng、Antxr2、Epha2、Chrna9、Il1r1、Tlr2、Trbc2、Npr3、Lpar3、Hrh2、Htr1 d、Ifih1、Ccrl2、Gabrd、Adgrg5、Lepr、Gabrr2、Gpr34、Gpr156、Tnfrsf9、Vmn 2r57、Il17re、C3ar1、Il20ra、Pirb、Htr2b、Pecam1、Gpr82、Gpr87、Mst1r、T ek、Gpr160、Brs3、Grin3b、Nlrp6、Cd72、Edar、Ror1、Adora3、Galr1、Tlr5、Ff ar4、Ly75、Cd300lg、Crhr2、Fcrlb、C5ar2、Chrna6、Acvrl1、Gpr52、Klri2、Tn frsf13c、Cd247、P2ry2、Gprc5a、Fcamr、Oscar、Prlhr、Nod2、Chrm1、Tas1r3、<h2 style=";text-align:left;direction:ltr">Dmbt1、Pigr、Htr1f、Klrk1、Il17rb、Bdkrb1、Pear1、Chrna1、Trim30a、Adgr e1、Cd80、Fcer1g、Nr1i3、Btla、Il31ra、Ffar2、Tnfrsf11a、Galr2、Ptger4、O prd1, Kir3dl2, Il22ra1, Htr4, Prokr1, Rgr, F2, Ptgfr, F2rl1, Klrb1f, Rxfp3, Il2ra, Bdkrb2, Ackr4, Il10ra, Itga11, Rxfp2, Cubn, Ptger1, Il12rb2, M c4r、Il20rb、Asgr2、Vmn2r120、Vmn1r43、Ptger2、Rho、Npffr2、Gpr4、Fcgr2 b、Lyve1、Cysltr2、Drd5、Ccr5、Vmn2r79、P2ry12、Ccr2、Cmklr1、Mc3r、Vmn2r 113、Paqr5、P2rx2、Ahrr、Npy4r、Gucy2f、Adgrf3、Hcar2、Folr2、Mc5r、Fgfr 4、Rrh、Csf2rb、Gabrr1、Gpr152、Ptgdr、Fcgr4、Clec1a、Htr3b、Erbb3、Trpa1 、Tnfrsf8、Epha1、Fcmr、Ltb4r1、Abcc9、Agtr2、Gpbar1、Hcar1、Ptpn6、Ackr 2、Gpr63、Nmbr、P2ry6、Sstr5、Mrgprf、Ltb4r2、Drd3、Htr5b、Treml1、Gpr171 、Ifnlr1、Trim5、Casr、Klrb1a、Gpr18、Gabrp、Mrc1、Drd4、Gcgr、Il2rb、Fcn a、Ptgir、Opn1sw、Itgb2、Il18r1、Vmn2r116、Nr1i2、Pla2r1、Chrne、Hephl1、 Cxcr2、Vipr1、Sctr、Musk、Cd300lb、Sucnr1、Il12rb1、Cysltr1、Vmn2r84、P 2rx3、Lrrc19、Vmn2r86、Vmn2r27、Tlr4、Nr5a1、Cel、Uts2r、Gpr20、Chrna2、G pr39、Tas2r137、Trpv4、Klrb1c、Vmn2r29、Mtnr1a、Esrrb、Clec10a、Adgrd1、 Hnf4a、Gfral、Unc5cl、Il2rg、Pth2r、Galr3、Cd300a、Tbxa2r、Gpr182、Ighm、Ccr9, Hrh4, Il21r, Mc2r, Ly6g6e, Hjv, Gnrhr, Aplnr, Tlr6, Il1rl2, Hnf4g, Itgal, Il1rl1, Sstr4, Opn4, Ffar1, Gpr65, Glp2r, Vmn2r59, Flt4, Tas2r135, Fpr2, Trem2, Tas1r1, Gabra6, Vmn1r41, Tlr12, Gpr183, Vmn2r53, Vmn2r78, Vmn2r85, Fcgr3, Vmn2r87, Adgrf1, Vmn2r6, Il5ra, Trbc1, Fpr1, Vmn2r124, Itgb2l、F5、Gpr141、Mpl、Glra4、Fcrl1、Grm6、Rtn4rl2、Nr1h5、Tas2r108、Spn、Cd300e、Csf2rb2、Klre1、Adra2b、Klrb1、Klrd1、Slamf8、Cd4、Clec12a、G prc5d、Esr2、Pkd2l1、C5ar1、Il27ra、Cd79b、Chrna10、Gm7609、Reg3b、Gpr151、Trac、Clec4d、Csf3r、Ptafr、Tlr13、Ffar3、Cxcr6、Il12b、Vmn1r42、Cxcr 5、Vmn2r115、Tas1r2、Nlrp1b、Adgrf2、Ccr1、Vmn1r54、Vmn2r26、Trhr2、Cd3g、Tas2r143、Pglyrp4、Vmn2r5、Cnr2、Npsr1、Vmn1r40、Tie1、Adgrl4、Vmn1r5 3、Chrnd、Il13ra2、Vmn2r24、Gucy2g、Vmn2r23、Opn5、P2rx1、Il18rap、Il11ra2、Tmigd3、Tnfrsf17、Ptgdr2、Ms4a2、Lpar5、Vmn2r69、Vmn2r25、P2ry10b、 Tacr2、Nr2e3、Ccr6、Clec1b、Gpr174、Agtr1b、Vmn2r108、Pkd1l3、Il9r、Xcr1、Il7r、Vmn2r22、Taar4、Vmn1r49、Stra6l、Vmn2r54、Adra1d、Adgre4、Vmn2r 52、Gfra3、Treml4、P2ry4、Robo4、Ghrhr、Vmn2r111、Gpr119、Nr1h4、Cd200r3、Vmn2r93、Tlr7、Ccr7、Vmn2r95、Trim30b、Nmur1、Vmn2r89、Reg1、Vmn2r18、Examples of receptors include Vmn2r97, Ccr4, Vmn2r118, Vmn2r83, Cxcr1, Cd300lf, P2ry13, Cd7, Reg2, Vmn1r47, Gpr15, Cd8b1, Gprc6a, Lhcgr, Vmn2r17, and Mrgprh, as well as other receptors such as Il23r, Klrb1b, Vmn2r96, Avpr2, Cd160, Gpr155, and Nkx3-1. These receptors have been confirmed to be expressed in the suprachiasmatic nucleus organoids of the present invention.

[0086] Furthermore, in step (Y), the circadian rhythm of the suprachiasmatic nucleus organoids can be evaluated by measuring the expression levels of clock genes and evaluating their fluctuations, oscillations, or their persistence, and by measuring the fluctuations, oscillations, or persistence of the expression levels of receptors expressed in the suprachiasmatic nucleus organoids, as well as the responsiveness of those receptors (e.g., responsiveness in the presence / absence of a test substance, or responsiveness to a ligand in the presence / absence of a test substance). For example, this can be done by measuring the responsiveness of receptors to the addition of a ligand in the fluctuations in the expression levels of clock genes.

[0087] If the evaluation results of step (Y) show a change in the circadian rhythm when compared with the evaluation results of a control sample under the same conditions except that the test substance is not used, the test substance can be selected as a circadian rhythm regulator (or a candidate substance thereof).

[0088] The selected circadian rhythm regulating agent can be used as an active ingredient in agents for treating, ameliorating, or preventing diseases, disorders, etc. involving circadian rhythm. Examples of such drugs include sleeping pills.

[0089] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0090] Test Example 1. Production of suprachiasmatic nucleus organoids 1 <Test Example 1-1. Culture> Culture conditions were investigated using sustained Six3 positivity as an indicator, and the culture conditions were determined.

[0091] Undifferentiation maintenance medium (composition: Glasgow Minimum Essential Medium (GMEM, Gibco), 10% KnockOut TM Serum Replacement (KSR; Gibco), 1% fetal bovine serum (FBS), 1 mM sodium pyruvate (Gibco), 1 × MEM Non-Essential Amino Acids (Gibco), 100 μM β-mercaptoethanol (Wako, β-ME). Two inhibitors (2i) (3 μM CHIR99021 (Axon) (Wnt signaling activator) and 1 μM PD0325901 (Wako) (FGF-MAPK signaling inhibitor)) were added. ESGRO mouse leukemia inhibitory factor (mLIF, Millipore) was added to a final concentration of 2 × 10 3 Mouse ES cells (C57 / B6N Per2::Luciferase cells (Scientific Reports volume 6, Article number: 32769 (2016); Nature Protocols volume 12, pages 2513-2530 (2017)) or Six3::mVenus cells (Nature Communications volume 8, Article number: 1339 (2017))) that had been cultured in a feeder-free, adherent culture medium containing 100 μg of erythritol iodide (1.5 U / mL) were switched to an intermediate medium (undifferentiated maintenance medium minus the FGF-MAPK signal inhibitor and Wnt signal activator) and cultured for 3 days.

[0092] Cells were harvested and seeded into 96-well plates (U-bottom wells) at 30,000 cells / well (Per2::Luciferase cells: used in the experiments in Figures 1–4 and 7) or 10,000 cells / well (Six3::mVenus cells: used in the experiments in Figures 5–6) and cultured in neural differentiation medium (Iscove's modified Dulbecco's medium / Ham's F-12 1:1, 1× chemically defined lipid concentrate, penicillin / streptomycin, monothioglycerol (450 μM), and purified BSA (>99% purified by crystallization; Sigma, 5 mg / ml)) (Day 0). By Day 1, the formation of a single spherical cell cluster (approximately 500 μm in diameter) was confirmed in each well. On day 2, SAG (sonic hedgehog agonist, Enzo, LX-270-426-M001) was added to a final concentration of 1 μM, and the culture was continued until day 7.

[0093] On day 7, the cell clusters were collected and placed in neural differentiation medium (expansion medium) (composition: DMEM / F-12, GlutaMAX TM The cells were washed once with KSR1000 (KSR1000 supplement, KSR1000 10%), then seeded onto EZSpheres or other culture dishes, and suspension culture was initiated in the neural differentiation medium (expansion medium). Culture was performed in a multi-gas incubator at 40% O2 and 5% CO2 until Day 13. Medium was changed every other day.

[0094] On day 13, the medium was changed to neural differentiation medium (maturation medium) (composition: DMEM / F-12, GlutaMAX TM The medium was changed to 1% N2 supplement, 1% B27 (with Vitamin A), and 2% B27 (with Vitamin A), and the suspension culture was continued. The culture was continued in a multi-gas incubator with 40% O2 and 5% CO2. The medium was changed every other day.

[0095] Between days 20 and 30, the cell aggregates were observed under a phase-contrast microscope, and cell aggregates with transparent protrusions on their surface (fluffy aggregates) were removed. Although this varied depending on the batch, approximately 10-50% of the cell aggregates were fluffy aggregates.

[0096] The cell masses were collected and organoids were obtained.

[0097] Test Example 1-2. Immunostaining The organoids (Day 72) obtained in Test Example 1-1 were immunostained with terminal differentiation markers of the suprachiasmatic nucleus (Six6, VIP, AVP). For comparison, the suprachiasmatic nucleus of an adult mouse was also immunostained in the same manner. Immunostaining was performed according to known methods.

[0098] The results are shown in Figure 1. The organoids expressed terminal differentiation markers of the suprachiasmatic nucleus, similar to the suprachiasmatic nucleus of adult mice, and to some extent formed a regional distribution similar to that of the suprachiasmatic nucleus of adult mice.

[0099] Test Example 1-3 Single Cell RNA Seq Analysis Single cell RNA Seq analysis was performed on the organoids (Day 70) obtained in Test Example 1-1, and the proportion of suprachiasmatic nucleus cells among the organoid-constituting cells was calculated.

[0100] The specific method is as follows: Day 70 SCN Organoid 11 cell clumps were dispersed using neuronal cell dispersion medium, and large clumps were removed using Flowmi. After thorough washing, trypan blue was used to confirm that more than 80% of cells were viable. A library was then created using the Chromium v3.1 kit (10x genomics), targeting 5,000 cells. The samples were then sequenced using Illumina Novaseq and analyzed using Cellranger and Seurat.

[0101] The major populations were roughly half neurons and half glia, with the remainder consisting of ependyma, pia mater, and oligodendrocytes, representing a complete set of cells from the surface to the interior of the brain. Furthermore, there were only seven neuronal populations, with only four being major. Annotation was performed using scRNA-Seq data from the mouse hypothalamus (Romanov Nature 2020), revealing the four major populations: SCN, Arc-TIDA (dopamine neurons in the arcuate nucleus), Arc-Agrp (neurons such as Agrp in the arcuate nucleus), and Lh-Hcrt (orexin neurons in the lateral cortex), pinpointing the induction of only a small portion of the hypothalamus. The specific percentages compared to in vivo cell ratios are shown in the table below, based on the Mano Cell Report Method (https: / / www.cell.com / cell-reports-methods / pdfExtended / S2667-2375(21)00083-7).

[0102]

[0103] <Test Example 1-4. Clock gene expression analysis> The organoids obtained in Test Example 1-1 were subjected to clock gene expression analysis to evaluate the circadian oscillation sustainability.

[0104] The specific method is as follows. SCN organoids generated using Per2::Luciferase KI / KI cells were embedded in agarose gel in the bottom of a 35 mm glass-bottom dish and replaced with phenol red-free maturation medium. From day 101, 500 μM luciferin was added, and images were captured using an LCV 100 (Olympus) luminescence-modified microscope with a 20X objective and a 0.5X magnification lens. The imaging conditions were 16-bit EM gain 500, exposure 50 min, bin 3, and hourly intervals.

[0105] The results are shown in Figure 2. We found that clock gene oscillations persisted for more than 10 days. Since the peak / trough ratio remained constant, this was due to a decrease in the baseline caused by substrate consumption, etc., and no attenuation due to desynchronization was observed.

[0106] <Test Example 1-5. Transplantation analysis> The organoids obtained in Test Example 1-1 were transplanted into mice in which the suprachiasmatic nucleus had been destroyed, and the presence or absence of movement of the mice before and after transplantation was measured, and actograms and chi-square periodograms were created.

[0107] The specific method is as follows. SCN lesion and transplantation experiments were performed as previously described (Sujino Curr Biol 2006, https: / / www.sciencedirect.com / science / article / pii / S0960982203002227). SCN organoids were transplanted in place of the mouse fetal hypothalamus. Day 20 SCN organoids were placed in a semi-aerated Millicell buffer and cultured overnight in a CO2 incubator (5% CO2 20% O2). Subsequently, they were divided into 3-4 pieces using ophthalmic scissors and transplanted into SCN-lesioned mice as previously described. Wheel running rhythms were analyzed.

[0108] The results are shown in Figures 3 and 4. The rhythm that had disappeared due to destruction of the suprachiasmatic nucleus was restored after transplantation of the suprachiasmatic nucleus organoid.

[0109] Test Example 2. Production of suprachiasmatic nucleus organoids 2 The number of cells seeded on Day 0 was 3300 cells / well, 6600 cells / well, or 9900 cells / well, and the cells were cultured in the same manner as in Test Example 1, except that a SAG-free group (DMSO-added group) was also obtained.

[0110] Figure 5 shows bright-field images (Bright), fluorescent images (showing Six3 expression), and autofluorescent images (Auto) of cell clusters on Day 13. We found that if 6,000 or more raw cells were seeded to form a single cell cluster, the suprachiasmatic nucleus marker was well expressed in the organoids.

[0111] Test Example 3. Production of suprachiasmatic nucleus organoids 3 SAG was added on Day 0, Day 0.5, Day 1.0, Day 2.0, Day 3.0, or Day 5.0, and a SAG-free group (DMSO Day 0 added group) was also obtained. Culture was performed in the same manner as in Test Example 1.

[0112] Figure 6 shows bright-field, fluorescent (Six3 expression), and autofluorescent images of the cell clusters on day 48. We found that the addition of SAG before day 3 resulted in good expression of the suprachiasmatic nucleus marker in the organoids.

[0113] Test Example 4. Production of suprachiasmatic nucleus organoids 4 The number of cells seeded on Day 0 was 30,000 cells / well, 45,000 cells / well, or 60,000 cells / well, and a group using undifferentiated maintenance medium instead of intermediate medium (2i (+) maintenance) was also obtained, and a SAG non-addition group (DMSO addition group) was also obtained. Except for this, the culture was performed in the same manner as in Test Example 1.

[0114] Figure 7 shows a bright field image of the cell clusters on day 13. The morphology of the cell clusters revealed that no differentiation occurred when the undifferentiation maintenance medium was used instead of the intermediate medium.

[0115] Test Example 5. Preparation of Suprachiasmatic Nucleus Organoids. Human iPS cells (201 B7) cultured in a feeder-free adherent culture medium (composition: AK02N, Y27632 (ROCK inhibitor) 1 μM) were seeded at 10,000 or 30,000 cells / well into a 96-well plate (U-bottom wells) and cultured in undifferentiated maintenance medium supplemented with 1 μM SAG (medium A). The following day, the entire medium was transferred to a dish containing neural differentiation medium (approximately equal amounts of SAG and Y27632). The following day, half of the medium was replaced with neural differentiation medium (Y27632 and SAG 1 μM). The following day, half of the medium was replaced with neural differentiation medium (Y27632 and SAG 1 μM). The next day, the entire medium was replaced with neural differentiation medium (Y27632 / SAG 1 μM) (Day 0).

[0116] On day 11, cell clusters were collected and washed once with neural differentiation medium (expansion medium) (composition: DMEM / F12 (Gibco 10565), KSR 10%) before seeding onto EZSpheres. Suspension culture was initiated in the neural differentiation medium (expansion medium). Culture was performed in a multi-gas incubator at 40% O2 and 5% CO2 until day 23. Medium was changed every other day.

[0117] On day 23, the medium was changed to neural differentiation medium (maturation medium) (DMEM / F12 (Gibco 10565), 1X N2 supplements, 1X B27 (with Vitamin A)) and suspension culture was continued. The culture continued in a multi-gas incubator with 40% O2 and 5% CO2. The medium was changed every other day.

[0118] The organoids collected on day 26 were immunostained with a marker for the suprachiasmatic nucleus, and the expression of SCN markers was confirmed.

Claims

1. (1) A step of culturing pluripotent stem cells in an intermediate medium between undifferentiated maintenance medium and neural differentiation medium. (2) A step of culturing the cells obtained in step (1) in a neuronal differentiation medium, and (3) The process includes a step of performing an operation to form a cell aggregate before the start of step (2), (A) More than 6,000 raw cells are seeded to form one cell aggregate, and (B) if the start of step (2) is considered day 0, the culture medium contains a sonic hedgehog signaling pathway agent from day 3 onwards. A method for manufacturing suprachiasmatic nucleus organoids.

2. The manufacturing method according to claim 1, wherein the intermediate medium in step (1) is a medium in which the concentration of the undifferentiated maintenance factor is lower than the concentration in the undifferentiated maintenance medium.

3. The method for producing the product according to claim 2, wherein the undifferentiated maintenance factor is an FGF-MAPK signaling inhibitor and / or a Wnt signaling activator.

4. The manufacturing method according to claim 1, wherein the raw material cells in step (3) number 9,000 or more.

5. The manufacturing method according to claim 1, wherein the operation in step (3) is seeding cells into culture wells and performing suspension culture.

6. The manufacturing method according to claim 1, wherein the culture in the culture medium containing the sonic hedgehog signaling pathway activator is carried out for 4 to 15 days.

7. The manufacturing method according to claim 1, wherein the culture in step (2) is carried out for 4 to 15 days.

8. The manufacturing method according to claim 1, wherein the concentration of the sonic hedgehog signaling pathway agent in the culture medium is 0.001 μM or higher.

9. The intermediate medium in step (1) is a medium in which the concentration of the undifferentiated maintenance factor is lower than the concentration in the undifferentiated maintenance medium. The aforementioned undifferentiated maintenance factor is an FGF-MAPK signaling inhibitor and / or a Wnt signaling activator, the raw material cells in step (3) are 9000 or more, and the operation in step (3) is seeding cells in small wells and performing suspension culture. The culture in the medium containing the aforementioned sonic hedgehog signaling pathway activator lasts from day 4 to day 15. The culture in step (2) is from day 4 to day 15, and The concentration of the sonic hedgehog signaling pathway agent in the culture medium is 0.001 μM or higher. The manufacturing method according to claim 1.

10. Furthermore, the manufacturing method according to claim 1, further comprising the step of culturing the cell aggregate obtained in step (2) in a neuronal differentiation medium.

11. A suprachiasmatic nucleus organoid in which suprachiasmatic nucleus cells make up 1% or more of the total.

12. The suprachiasmatic nucleus organoid according to claim 11, wherein the proportion of suprachiasmatic nucleus cells is 10% or more.

13. A suprachiasmatic nucleus organoid obtained by the manufacturing method described in any one of claims 1 to 10, wherein the proportion of suprachiasmatic nucleus cells is 1% or more.

14. (X) The step of bringing the suprachiasmatic nucleus organoid described in claim 11 or 12 into contact with the test substance, (Y) A step of evaluating the circadian rhythm of the suprachiasmatic nucleus organoid, A screening method for circadian rhythm regulators, including those mentioned above.

15. A circadian rhythm regulator having the effect of altering the circadian rhythm of the suprachiasmatic nucleus organoid as described in claim 11 or 12.