Microglia-rich brain organoids

Abstract

The present invention relates to a method for producing microglia-rich brain organoids, comprising the steps of incubating primitive-like macrophages with brain organoids between about 15 and about 30 days old in a low-adhesion cell culture vessel in a brain organoid medium containing CSF-1 to produce microglia. The present invention also relates to microglia-rich brain organoids obtained by the method as described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to Singapore application number 10201902893S filed on March 29, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes. Field of the Invention

[0003] The present invention relates generally to the field of cell culture. In particular, the present invention relates to the use of a method for generating microglia-replete brain organoids. Background of the Invention

[0005] Microglia are the resident immune cells of the central nervous system. These resident brain macrophages account for 5-10% of the total cells found in the brain, and play an important role in brain development, homeostasis and pathology. Microglia monitor (survey) the brain and contribute to innate immune response when damaged. In addition, microglia constantly interact with neuronal synapses, contributing to synaptic remodeling and axon growth. However, because of the lack of a suitable human model system that reenacts microglia in vivo development, most of the research on microglia is carried out using animal models. However, the human model system will be extremely important for understanding how microglia affect human central nervous system development, physiology and pathology.

[0006] With new advances in stem cell technology, it is now possible to produce three-dimensional brain organoids derived from human induced pluripotent stem cells (iPSCs), which simulate the human fetal brain to a certain extent. These brain organoids have been shown to form neuroepithelial rosettes, which are similar to the developing cerebral cortex. In addition, different subtypes of intermediate progenitor cells (TBR2-positive) and cortical neurons (TBR1-positive, CTIP-positive, SATB2-positive, and CUX1-positive) were also observed in brain organoids. Transcriptomics studies further showed that the neuronal cell types produced in these organoids are similar to their endogenous counterparts in the human fetal brain. However, despite these similarities, existing brain organoids have not been able to fully reproduce the human fetal brain because most of them lack microglia necessary for brain development and maturation. With regard to the appearance of microglia in these organoids, the methods used to produce brain organoids have obtained inconsistent results. This hinders microglial research aimed at deciphering the role of human microglia in early brain development.

[0007] Therefore, there is a need to develop methods for generating brain organoids containing microglia. SUMMARY OF THE INVENTION

[0009] In one aspect, a method for generating microglia-sufficient brain organoids is provided, comprising the steps of incubating primitive-like macrophages with brain organoids between about 15 and about 30 days old in a brain organoid medium containing CSF-1 in a low attachment cell culture vessel to generate microglia.

[0010] In another aspect, provided are microglia-sufficient brain organoids obtained by the methods described herein.

[0011] In another aspect, a method for screening compounds that target microglia function is provided, comprising the steps of contacting a microglia-sufficient brain organoid as described herein with the compound and analyzing a predetermined property of the microglia.

[0012] In another aspect, a method for screening a compound for treating a neurodegenerative disease is provided, comprising the steps of contacting a microglia-sufficient brain organoid as described herein with the compound and analyzing a predetermined property of the microglia.

[0013] In another aspect, a kit is provided which, when used in the methods as described herein, comprises a cerebral organoid culture medium comprising CSF-1 together with instructions for use.

[0014] In another aspect, a method of isolating one or more predetermined cell populations from a microglia-sufficient brain organoid is provided, comprising the steps of:

[0015] a) Incubate the microglia-rich brain organoids in digestion solution at 37°C for approximately 30 minutes;

[0016] b) mechanically agitating the microglia-rich brain organoids from step a);

[0017] c) heat treating the microglia-rich brain organoids from step b) at 1400 rpm at 37° C. for 10 minutes;

[0018] d) mechanically agitating the microglia-rich brain organoids from step c);

[0019] e) incubating the microglia-rich brain organoids from step d) at room temperature to allow debris to settle;

[0020] f) removing the digestion fluid;

[0021] g) isolating one or more predetermined cell populations from the digestion fluid.

[0022] definition

[0023] The term "organoid" as used in the context of the present application refers to a three-dimensional cell structure that simulates the organization and function of an organ. Organoids are composed of tissue-specific cell types, which are self-organized by cell sorting and spatially restricted lineage orientation (commitment). Organoids can be derived from stem cells, such as embryonic stem cells or induced pluripotent stem cells. The terms "brain organoids" and "cerebral organoids" are used interchangeably in this specification and refer to organoids with anatomical features similar to the brain. It is generally understood that brain organoids or cerebral organoids are composed of various cell types of the brain. These cell types may have different developmental potentials, and some cell types are less differentiated than other cell types.

[0024] The term "microglia", as used herein, refers to a class of glial cells involved in immune system regulation within the central nervous system. These cells are constitutive cells of the central nervous system present in the brain. Microglia constitute 20% of the total glial cell population in the brain. Microglia play a key role in the development of a healthy central nervous system and are involved in the onset, progression and elimination of diseases affecting the central nervous system. It is known that microglia selectively colonize the proliferation zone of the cortex and engulf neural precursor cells. Microglia are also known for their ability to engulf beta-amyloid peptides. The term "iMicro", as used herein, refers to cells that mimic microglia found in vivo. iMicro can be produced by co-culturing primitive macrophages or primitive-like macrophages with brain organoids. The terms "iMicro" and "microglia-like cells" are interchangeable. It is generally understood that microglia-like cells are substantially similar to microglia in terms of phenotype, genotype and function. Similar to microglia, microglia-like cells are able to engulf beta-amyloid peptides and extend their dendrites after mechanical damage. Microglia-like cells also exhibit an amoeba-like morphology in brain organoids, which is a typical morphology exhibited by microglia in the fetal brain in the early stages of brain development. Microglia-like cells also express microglia-specific markers, such as TMEM119, P2RY12, Sall1, and Merk. The term "microglia-rich brain organoids" as used herein refers to brain organoids containing microglia or microglia-like cells. Microglia-rich brain organoids can be forebrain organoids, midbrain organoids, or hindbrain organoids. Forebrain organoids can be hypothalamic organoids.

[0025] The term "stem cell" as used herein refers to a cell that can self-replicate and can differentiate into a higher degree of specialized cells. Stem cells may include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Pluripotent stem cells are cells that can differentiate into any layer of the three germ layers (endoderm, mesoderm, or ectoderm). "Embryonic stem cells" are pluripotent stem cells of the inner cell mass of a blastocyst. "Induced pluripotent stem cells" are pluripotent stem cells artificially obtained from non-pluripotent cells, which can be adult somatic cells. Stem cells as used herein may include, but are not limited to, humans, non-human primates, mice, and avian stem cells.

[0026] The term "differentiation" as used herein refers to a developmental process by which a cell develops further along a developmental path than its immediate precursor cell. A differentiated cell refers to a cell of a more highly specialized cell type that is derived from a cell of a less specialized cell type during the cell differentiation process. A differentiated cell is a cell that has a more defined position within a cell lineage.

[0027] The term "neural progenitor cells" (NPCs) as used herein refers to immature cells of the nervous system that can differentiate into neurons and glial cells. NPCs express phenotypic markers unique to the neural cell lineage, including CD271, Nestin, PAX6, SOX1, SOX2, VIM, and HES2. NPCs can be present in brain organoids along with other cell types such as neurons and glial cells. Co-NPCs refer to NPCs of brain organoids co-cultured with iMacs.

[0028] For the purpose of the present application, the term "cell culture" refers to such a process, and cells are grown in vitro under a controlled culture medium simulating their natural environment by the process. Cell culture conditions are different for various cell types, but can be composed of a suitable container with one or more substrates or one or more culture mediums, and the substrate or culture medium provide the necessary nutrients required for cell growth and regulate the biochemical environment. Two or more cell types can be hatched together in co-cultivation, wherein the two or more cells are maintained in a condition suitable for their co-growth. Two or more cell types can grow on the same surface or on different surfaces. When two or more cell types are co-cultivated or hatched in identical cell cultures, cells can interact, and thus a secretory soluble medium (mediator) produced by a cell can interact with another cell.

[0029] The term "macrophage" in the context of this specification refers to specialized cells that are involved in detecting, engulfing and killing bacteria and other harmful organisms. Macrophages play an important role in immunity and immune response. Primitive macrophages are produced from early red bone marrow progenitor cells in the yolk sac and further differentiate into microglia. The term "iMac" as used in this article refers to cells that simulate primitive macrophages found in vivo. The terms "iMac" and "primitive-like macrophages" can be used interchangeably. iMac or primitive-like macrophages can be derived from pluripotent cells, such as iPSC or ESC. It is generally understood that primitive-like macrophages are substantially similar to primitive macrophages in terms of phenotype, genotype and function. Similar to primitive macrophages, primitive-like macrophages show phagocytic ability, and release proinflammatory cytokines such as TNF-α, IL-1β and IL-6 after LPS stimulation. The term "co-iMac" as used in this article refers to iMac co-cultured with brain organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings, in which:

[0032] Figure 1 shows the characterization of iMac (primitive-like macrophages derived from iPSC) in brain organoids. A) A schematic overview showing the generation of microglia-rich brain organoids. In B), brain organoids were co-cultured with iMac for 18 days, transparentized with benzyl alcohol and benzyl benzoate (BABB) and imaged as a whole in 3D. The boxed area is highlighted on the right. Arrows, iMac clusters on organoids. C) Sections and immunohistochemistry of co-cultured brain organoids show the presence of iMac in organoids. The boxed area is highlighted on the right. In D), Ki67 immunohistochemical staining shows that some iMac in organoids are Ki67 positive, indicating their ability to proliferate in organoids. E) GFP-positive iMacs are shown to extend their dendrites toward neural damage induced by two-photon laser ablation. F) GFP-positive iMacs are shown to move actively in organoids and some of them contain Ab1-42 peptide-TAMRA, indicating their ability to inspect (survey) organoids and phagocytose peptides. The boxed area is highlighted on the right.

[0033] Figure 2 shows that iMacs limit organoid growth and the number of neural progenitor cells (NPCs). A) Shows representative 3D images of brain organoids at day 44 cultured for 18 days in the absence and presence of iMacs. Changes in organoid size were measured over time in the absence and presence of iMacs. B) Shows flow cytometry data showing markers used to distinguish iMacs, NPCs, neurons, and glial cells from organoids. The number and % of each cell type that makes up each organoid were obtained from flow cytometry.

[0034] Figure 3 shows that iMac promotes axonogenesis in organoids. A) shows a schematic overview of the single-cell RNA-seq experimental procedure. B) shows a t-distributed random neighbor embedding (t-SNE) diagram showing cells of organoids cultured in the absence of iMac (organoids only) and in the presence of iMac (co-culture). Markers are used in the figure to identify 4 major groups (NPCs, neurons, iMacs, and mesenchymal cells). The box is an iMac cell cluster from co-culture and highlighted on the right. C) The results of GO analysis of upregulated genes in co-cultured NPCs (co-NPCs) and co-cultured neurons (co-neurons) are shown, revealing axon development and regeneration pathways. D) is a volcano map showing that co-NPCs upregulate proteins (PRPH and DPYSL4) related to axon outgrowth. In E), immunostaining data show that co-NPCs have more and longer axons than NPCs alone. F) is a volcano map showing that co-cultured iMacs (co-iMacs) upregulate genes involved in neurogenesis and axon development. G) shows the results of GO analysis of upregulated proteins in co-iMac, revealing axon guidance pathways.

[0035] Figure 4 shows iMacs and embryonic microglia expressing PLIN2 and lipid droplets. A) is a volcano plot showing genes involved in lipid droplet formation and lipid export that are upregulated in co-iMacs. B) shows live imaging of co-iMacs containing lipid droplets. C) shows co-iMacs expressing lipid droplets and PLIN2 in organoids. Arrow, overlap between PLIN2 and lipid droplets in iMacs. D) shows microarray data showing that PLIN2 is only expressed in microglia in the early stages of mouse brain development. E) shows immunostaining showing lipid droplets and PLIN2 expressed in mouse embryonic microglia (E13.5). F) shows immunostaining showing lipid droplets and PLIN2 expressed in human embryonic microglia (15 weeks).

[0036] Figure 5 shows that iMac affects the lipid content and metabolism of cells in organoids. A) A volcano plot is depicted showing changes in the expression levels of genes and proteins involved in glycolysis, OXPHOS, and cholesterol biosynthesis in co-NPCs. B) Results of GO analysis are shown, indicating that the cholesterol biosynthesis pathway is the most significantly downregulated pathway in co-NPCs. C) More lipid droplets are found in co-cultured organoids. D) Flow cytometry data are shown, showing that co-NPCs contain higher lipid content than NPCs alone. E) Data from mass spectrometry-based lipidomics are shown, showing higher levels of CE, DAG, and TAG in co-NPCs.

[0037] Figure 6 shows that there is cholesterol transport from iMacs to cells in organoids. A) Depicts flow cytometry data and immunostaining showing that iMacs take up BODIPY-CE and store it in lipid droplets. B) Depicts flow cytometry data showing BODIPY-CE found in cells of organoids after 7 days of co-culture with iMacs. C) Shows co-NPCs containing BODIPY-CE sorted from organoids.

[0038] Figure 7 The results showed that accutase and collagenase treatment released more viable cells from organoids compared to accutase treatment alone. Accutase has been commonly used for digestion of organoids in previous studies. Organoids co-cultured with iMacs for 15-21 days were treated with accutase alone or with accutase and collagenase. The use of accutase together with collagenase allowed the isolation of more iMacs and other cell types from organoids using fluorescence-activated cell sorting (FACS) for bulk-RNA-seq and other downstream experiments, such as single-cell analysis and 2D culture of NPCs.

[0039] Figure 8 Shown are the sizes of organoids grown for 18 days in the absence or presence of iMac. DETAILED DESCRIPTION OF THE INVENTION

[0041] In a first aspect, the present invention relates to a method for producing microglia-rich brain organoids, comprising the steps of incubating primitive-like macrophages with brain organoids between about 15 and about 30 days old in a brain organoid culture medium containing CSF-1 in a low-adsorption cell culture container to produce microglia. The low-adsorption cell culture container can be a low-adsorption multi-well plate or an ultra-low-adsorption multi-well plate. It is generally understood in the art that a low-adsorption or ultra-low-adsorption cell culture container or culture plate is a container or culture plate that prevents cells from adsorbing to the container or culture plate. Low adsorption or ultra-low adsorption can be achieved by a variety of means, such as by coating the container or culture plate with a compound that prevents cell adsorption. In one embodiment, the low-adsorption cell culture container is Ultra-low adsorption multi-well plates.

[0042] In one embodiment, the brain organoid is about 23-29 days old from the pluripotent state. This will be understood to mean that the pluripotent cells have differentiated about 23-29 days old to produce the brain organoid. In a preferred embodiment, the brain organoid is about 26 days old from the pluripotent state.

[0043] In one embodiment, the cerebral organoid culture medium comprises about 25-100 ng / mL CSF- 1. In another embodiment, the cerebral organoid culture medium comprises 100 ng / mL CSF-1.

[0044] In one embodiment, less than about 200,000 primitive-like macrophages are cultured with the brain organoid. In another embodiment, about 150,000 or fewer primitive-like macrophages are cultured with the brain organoid.

[0045] In one embodiment, the step of culturing the primitive-like macrophages and the brain organoids is performed for at least 1 week. The primitive-like macrophages and the brain organoids can be cultured together for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. The length of the culture will depend on the desired endpoint of the experiment.

[0046] In one embodiment, culturing or co-culturing primitive-like macrophages with brain organoids results in differentiation of primitive-like macrophages into microglia. In one embodiment, culturing or co-culturing iMacs with brain organoids results in differentiation of iMacs into iMicros.

[0047] Longer co-cultures allow observation of long-term effects of co-culture under normal and diseased conditions. Longer co-cultures can be used to observe the effects of drug screening.

[0048] The differentiation of primitive macrophages into microglia can be determined by detecting the ability of microglia to respond to damage in the brain or to phagocytize beta-amyloid peptides. Differentiation into microglia can also be determined by quantifying the expression levels of microglia-specific markers such as TMEM119, P2RY12, Sall1, and Merk.

[0049] Incubation of primitive macrophages with brain organoids can affect the size of the organoids, the number and proportion of NPCs in the organoids, the maturation of NPCs in the organoids, and the lipid distribution. For example, culturing primitive macrophages with brain organoids can reduce the size of the organoids and the number of NPCs in the organoids. In one embodiment, culturing primitive macrophages with brain organoids can lead to downregulation of genes involved in cell proliferation in NPCs. These genes include, but are not limited to, TOP2A, CEMPF, CDC20, and UBE2C.

[0050] Incubation of primitive macrophages with brain organoids can promote the maturation of NPCs. In one embodiment, primitive macrophages can upregulate genes involved in axon outgrowth and neurogenesis. These genes may include, but are not limited to, IL-1b, IL10, NRG1, SEMA4C, VEGFA, and ADAM8. In another embodiment, incubation of primitive macrophages with brain organoids can result in NPCs presenting longer and more numerous axons.

[0051] Incubation or co-culture of primitive-like macrophages with brain organoids can improve lipid distribution in organoids and lipid content in NPCs. In one embodiment, incubation of primitive-like macrophages with brain organoids can result in upregulation of genes involved in lipid droplet formation and lipid export in primitive-like macrophages. These genes may include, but are not limited to, PLIN2, M1D1P1, ARL4C, SEPT9, and ABCA1. In another embodiment, NPCs in co-cultured brain organoids show higher neutral lipid content.

[0052] In one embodiment, the cells of the microglia-rich brain organoids produced by the methods described herein are dispersed and one or more predetermined cell populations are isolated. The predetermined cell populations may include, but are not limited to, neurons, neuronal progenitor cells, and glial cells.

[0053] In one embodiment, fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) is used to separate one or more predetermined cell populations.

[0054] In one embodiment, primitive-like macrophages for use in the methods described herein are generated from a first stem cell population by the following steps:

[0055] i) incubating the stem cells in a culture medium containing a GSK3 inhibitor, BMP4 and VEGF to differentiate the stem cells into cells of a mesodermal cell lineage;

[0056] ii) incubating the cells of the mesodermal cell line in a medium containing FGF-2 to differentiate the cells of the mesodermal cell line into hemangioblasts;

[0057] iii) incubating the angioblasts in a culture medium containing VEGF and FGF-2;

[0058] iv) incubating the hemangioblasts in a medium containing DKK1, SCF, FGF2, IL3 and IL6 to differentiate the hemangioblasts into hematopoietic cells;

[0059] v) incubating the hematopoietic cells in a medium containing SCF, FGF-2, IL-3 and IL-6 to induce maturation of the hematopoietic cells;

[0060] vi) incubating the mature hematopoietic cells in a medium containing CSF-1 to differentiate the mature hematopoietic cells into primitive-like macrophages.

[0061] In one embodiment, in step i), the GSK3 inhibitor is CHIR99021 and the stem cells are cultured for up to 2 days to allow the stem cells to differentiate into cells of the mesodermal lineage.

[0062] In another embodiment, in step ii), the cells of the mesodermal cell line are cultured for up to 4 days to allow the cells of the mesodermal cell line to differentiate into hemangioblasts. In yet another embodiment, steps iii) and iv) are performed over a period of up to 10 days.

[0063] In yet another embodiment, in step vi), the mature hematopoietic cells are cultured for up to 10 days to allow the mature hematopoietic cells to differentiate into primitive-like macrophages.

[0064] In one embodiment, primitive-like macrophages are generated over a period of 26 days.

[0065] In one embodiment, brain organoids for use in the methods described herein are generated from the second stem cell population by the following steps:

[0066] i) incubating the second stem cell population in a low attachment cell culture vessel to form embryoid bodies (EBs);

[0067] ii) incubating the embryoid bodies in a neural induction medium to differentiate the embryoid bodies into organoids containing neural ectoderm cells;

[0068] iii) embedding the organoids comprising neuroectodermal cells in matrigel and incubating the organoids comprising neuroectodermal cells in a brain organoid culture medium containing N2 and without vitamin A type B27, so that the organoids comprising neuroectodermal cells differentiate into organoids comprising neuroepithelial cells;

[0069] iv) incubating the organoid comprising the neuroepithelial cells with a cerebral organoid culture medium containing N2 and vitamin A type B27, so that the organoid comprising the neuroepithelial cells differentiates into the cerebral organoid.

[0070] In one embodiment, in step i) of the method of generating brain organoids from a second stem cell population, the low attachment cell culture container is a U-bottom culture plate and the second stem cell population is cultured for up to 6 days to form embryoid bodies (EBs).

[0071] In another embodiment, in step ii) of the method of generating brain organoids from the second stem cell population, the EBs are cultured for up to 6 days to allow the EBs to differentiate into organoids comprising neuroectodermal cells.

[0072] In yet another embodiment, in step iii) of the method for generating cerebral organoids from a second stem cell population, the organoids comprising neuroectodermal cells are cultured in a cerebral organoid culture medium containing vitamin A type B27 without for up to 4 days to differentiate the organoids comprising neuroectodermal cells into the organoids comprising neuroepithelial cells.

[0073] In another embodiment, prior to or during step iv) of the method of generating brain organoids from a second stem cell population, Matrigel is removed from the organoids comprising neuroepithelial cells.

[0074] In yet another embodiment, in step iv) of the method of generating cerebral organoids from a second stem cell population, the organoids comprising neuroepithelial cells are incubated in a cerebral organoid culture medium containing vitamin A type B27 for up to 26 days to differentiate the organoids comprising neuroepithelial cells into cerebral organoids.

[0075] In one embodiment, the organoids comprising neuroepithelial cells are incubated with shaking at 85 rpm to differentiate the organoids comprising neuroepithelial cells into brain organoids.

[0076] In one embodiment, cerebral organoids are generated from the second stem cell population over a period of about 15-30 days. In another embodiment, cerebral organoids are generated over a period of about 23-29 days. In a preferred embodiment, cerebral organoids are generated over a period of about 26 days.

[0077] In one embodiment, the first and second stem cell populations are embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or a combination thereof. In one embodiment, the first and second stem cell populations are human induced pluripotent stem cells. The first and second stem cell populations can be the same or different stem cells. In one embodiment, the first and second stem cell populations are the same stem cells.

[0078] In one aspect, the present invention provides microglia-rich brain organoids obtained by the methods described herein. Microglia-rich brain organoids may comprise microglia-like cells expressing microglia-specific markers, such as TMEM119, P2RY12, Sall1, and Merk, among others.

[0079] In another aspect, the present invention provides a method for screening compounds targeting microglia function, comprising the steps of contacting a microglia-sufficient brain organoid as described herein with the compound and analyzing a predetermined property of microglia.

[0080] In one embodiment, a method of screening for compounds targeting microglia function comprises contacting a compound with microglia-rich organoids and comparing these organoids with organoids that have not been contacted with the compound. The method may further include downstream analysis, including functional studies (neuronal electrical activity, microglial phagocytosis of amyloid-β peptides and their response to mechanical injury), single RNA sequencing, and analysis of metabolic changes that may contribute to neuronal disease.

[0081] In one aspect, the present invention provides a method for screening compounds for treating neurodegenerative diseases, comprising the steps of contacting a microglia-sufficient brain organoid as described herein with the compound and analyzing a predetermined property of the microglia.

[0082] In one embodiment, a method of screening for a compound for treating a neurodegenerative disease comprises contacting a compound with microglia-rich organoids and comparing these organoids to organoids that have not been contacted with the compound. The method may further comprise downstream analysis, including functional studies (neuronal electrical activity, microglial phagocytosis of amyloid-β peptides and their response to mechanical injury), single RNA sequencing, and analysis of metabolic changes that may contribute to neuronal disease.

[0083] The predetermined characteristic can be a morphological feature, a flow cytometer profile, a gene expression profile, a distribution pattern, or a combination thereof of microglia.

[0084] In one aspect, the invention provides a kit comprising a cerebral organoid culture medium comprising CSF-1 together with instructions for use when used in the methods as described herein.

[0085] In one embodiment, the kit as described herein further comprises

[0086] a) a first culture medium containing a GSK3 inhibitor, BMP4 and VEGF;

[0087] b) a second culture medium comprising BMP4, VEGF and FGF-2;

[0088] c) a third culture medium containing VEGF and FGF-1;

[0089] d) a fourth culture medium containing VEGF, DKK1, SCF, FGF-2, IL-3 and IL-6;

[0090] e) a fifth culture medium comprising FGF, IL6, IL3 and SCF; and

[0091] f) a sixth culture medium containing CSF-1;

[0092] to generate primitive-like macrophages from the first stem cell population; and

[0093] g) neural induction medium;

[0094] h) cerebral organoid culture medium containing N2 and without vitamin B27; and

[0095] i) Cerebral organoid culture medium containing N2 and vitamin A type B27;

[0096] to generate brain organoids from a second stem cell population.

[0097] In one aspect, the present invention provides a method for isolating one or more predetermined cell populations from microglia-sufficient brain organoids, comprising the steps of:

[0098] a) Incubate the microglia-rich brain organoids in digestion solution at 37°C for approximately 30 minutes;

[0099] b) mechanically agitating the microglia-rich brain organoids from step a);

[0100] c) heat treating the microglia-rich brain organoids from step b) at 1400 rpm at 37° C. for 10 minutes;

[0101] d) mechanically agitating the microglia-rich brain organoids from step c);

[0102] e) incubating the microglia-rich brain organoids from step d) at room temperature to allow debris to settle;

[0103] f) removing the digestion fluid;

[0104] g) isolating one or more predetermined cell populations from the digestion fluid.

[0105] In one embodiment, steps c) to f) are repeated at least two more times.

[0106] In another embodiment, the digestate is filtered and centrifuged prior to step g).

[0107] In one embodiment, one or more predetermined cell populations are separated by FACS or MACS. The one or more predetermined cell populations may include, but are not limited to, neurons, neuronal progenitor cells, glial cells, and primitive-like macrophages.

[0108] In another embodiment, the digestion solution is accutase and collagenase in a ratio of 9:1.

[0109] In one embodiment, the digestion fluid from step g) is filtered and subjected to downstream assays. Downstream assays may include, but are not limited to, flow cytometry, sorting, and immunostaining.

[0110] The present invention described illustratively in this article can be properly implemented in the absence of any element or any element, any restriction or any restriction, and the elements and restrictions are not specifically disclosed in this article. Therefore, for example, the terms "comprise", "include", "contain", etc. should be expanded and understood without restriction. In addition, the terms and expressions used herein are used as terms of description rather than terms of limitation, and it is not intended to exclude any equivalents or parts of the features shown and described when using the terms and expressions, but it is considered that various modifications within the scope of the invention of the claims are possible. Therefore, it should be understood that, although the present invention is specifically disclosed by preferred embodiments and optional features, the modifications and changes of the present invention implemented therein disclosed herein are adoptable by those skilled in the art, and it is considered that the modifications and changes are within the scope of the present invention.

[0111] The invention is described herein in a general and generic manner. Narrower species and subgeneric groupings falling within the general disclosure each also form part of the present invention. This includes the general description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the removed matter is specifically described herein.

[0112] Other embodiments are within the following claims and non-limiting examples. In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual element or subgroup of elements of the Markush group.

[0113] Experimental part

[0114] Non-limiting and comparative examples of the present invention will be further described in more detail by referring to specific examples, which should not be construed as limiting the scope of the present invention in any way.

[0115] Materials and methods

[0116] Generation of human iPSC-derived macrophages

[0117] In order to produce human iMac, in the first step (day 0-6), the modified scheme of adding CHIR99021 is used to rebuild the mesoderm structure (specification) and induce human angioblast-like cells to form. Human iPSC colonies are specialized to mesoderm by incubation with BMP-4 and VEGF, and their differentiation is enhanced by incubation with CHIR99021 in the first 2 days of differentiation. Induce angioblast formation (day 2-4) by adding FGF-2 to replace CHIR99021, and then maintain (day 4-6) with VEGF and FGF-2. In the next step (day 6-10), DKK1 (inhibiting Wnt signal transduction to promote the Wnt antagonist of primitive hematopoiesis) is specially added to induce angioblasts to the finalization of hematopoietic cells. Hematopoietic cells are matured by continuing to incubate with SCF, FGF-2, IL-3 and IL-6, and the cytokine is added to promote hematopoietic maturation and CSF-1R expression (day 12-16). From day 16, terminal differentiation into human iMacs was initiated by exposure to CSF-1. In more detail, human iPSCs (HD33i) were cultured to 75% confluence and then digested with 1 mg / mL collagenase (GIBCO, 17104-019) for 20 minutes. Cells were collected by mechanical scraping to produce aggregates of 50-200 μm, centrifuged at 300 g, resuspended with mTesr1 and mixed at a ratio of 1:25 (approximately 1.0 x 10 5Cells) were passaged onto 6-well plates coated with Matrigel. Starting from the next day, all the culture media were changed every other day, and during the differentiation process, the cells were cultured in Stempro medium supplemented with the following cytokines for the next 16 days: differentiation day 0 (5 ng / mL BMP4, 50 ng / mL VEGF, and 2 uM CHIR99021), differentiation day 2 (5 ng / mL BMP4, 50 ng / mL VEGF, and 20 ng / mL FGF2), differentiation day 4 (15 ng / mL VEGF and 5 ng / mL FGF2), differentiation days 6-10 (10 ng / mL VEGF, 10 ng / mL FGF2, 50 ng / mL SCF, 30 ng / mL DKK-1 (RnD, 5439-DK), 10 ng / mL IL-6 (RnD, 206-IL), and 20 ng / mL IL-3), differentiation days 12 and 14 (10 ng / mL FGF2, 50 ng / mL SCF, 10 ng / mL IL-6 and 20ng / mL IL-3). Starting from day 16 of differentiation, cells were switched to SF-Diff supplemented with 50ng / mL CSF-1, and all media changes were performed every 3 days until day 25 of differentiation, which is when floating cells were used for experiments. Cells were also cultured in a hypoxic incubator for the first 8 days, set to 5% CO2 and 5% O2, and in a standard incubator after day 8 of differentiation. Floating cells usually appeared around day 7 of differentiation, were collected and re-seeded onto basal cells during media changes.

[0118] Generation of iPSC-derived brain organoids

[0119] Three-dimensional brain organoids were generated using the following protocol. From day 0 to day 6, embryoid bodies (EBs) were formed by culturing human iPSCs in low-adsorption U-bottom 96-well plates containing hESC medium. From day 6 to day 11, neural ectoderm formation was induced by culturing EBs in neural induction medium. From day 11 to day 15, neuroepithelial cell formation was induced by embedding organoids with matrigel and culturing them in 10 cm dishes containing brain organoid medium (containing vitamin A-free B27). From day 15 to day 26, the medium was replaced with brain organoid medium (containing vitamin A-free B27) and the 10 cm dish containing the organoids was placed on an oscillator at 85 rpm to induce brain tissue formation.

[0120] Generation of dorsal and ventral forebrain organoids and co-culture with iMacs

[0121] Dorsal forebrain organoids (hCS) were produced from human iPSC according to the scheme described in 2017 by Birey et al. At day 0, human iPSC colonies were transferred to ultra-low adsorption 10 cm culture dishes containing 15 mL hPS culture medium supplemented with Dorsomorphin (5uM), SB-431542 (10uM) and rock inhibitor (10uM). From day 2, culture medium (without Rock inhibitor) was changed every day until day 5. At day 6, culture medium was changed into neural culture medium (NM) supplemented with EGF2 (20 ng / mL) and FGF2 (20 ng / mL) for the following 19 days (the culture medium was changed every day for the first 10 days, and the culture medium was changed every other day for the latter 9 days). From day 25, FGF2 and EGF were changed into BDNF (20 ng / mL) and NT3 (20 ng / mL) (changing culture medium every other day). From day 43, NM without growth factors was used for culture medium replacement every four days. Ventral forebrain organoids (hSS) were generated from human iPSCs according to the protocol described by Birey et al. in 2017. The same protocol as for dorsal organoids was used, except that IWP-2 (5uM) was added from day 4 to day 23 while SAG (100nM) was added from day 12 to day 23. The ventral forebrain organoids were co-cultured with GFP-positive iMacs for 2 weeks and then fused with the dorsal forebrain organoids. In order to fuse the dorsal and ventral forebrain organoids, the organoids were placed in 1.5mL microcentrifuge tubes and cultured for 3 days, with the culture medium replaced on day 2. After fusion, the organoids were transferred to Iwaki plates. Matrigel was used to stabilize the organoids on the Iwaki plates. Live cell images were acquired using a confocal laser scanning electron microscope (Olympus, Japan) and analyzed using Imaris software (BITPLANE).

[0122] Cultivation of forebrain organoids from human iPSCs

[0123] In order to produce forebrain-specific organoids, human iPSC colonies were separated with type IV collagenase 7 days after passage, washed with fresh stem cell culture medium and cultured in 15mL conical centrifuge tubes. On the 1st day, the iPSC colonies separated and washed were transferred to ultra-low adsorption 6-well plates (Corning Costar) containing 3mL stem cell culture medium (without FGF-2) plus 2μM Dorsomorphine (Sigma) and 2μM A83-01 (Tocris). On the 5th-6th day, half of the culture medium was replaced with an induction medium consisting of DMEM:F12, 1XN2 additives (Invitrogen), 10μg / mL heparin (Sigma), 1X penicillin / streptomycin, 1X non-essential amino acids, 1XGlutamax, 4ng / mL WNT-3A (R&D Systems), 1μM CHIR99021 (Cellagentech) and 1μM SB-431542 (Cellagentech). On the 7th day, the organoids were embedded in matrigel (BD Biosciences) and allowed to continue to grow in the induction medium for another 6 days. On the 14th day, the embedded organoids were mechanically separated from the matrigel onto the culture plate by using a 5mL pipette tip to pipette up and down. Typically 10-20 organoids are transferred to each well of a 12-well rotating bioreactor (SpinΩ) containing differentiation medium, and the differentiation medium is composed of DMEM:F12, 1X N2 and B27 additives (Invitrogen), 1X penicillin / streptomycin, 1X 2-mercaptoethanol, 1X non-essential amino acids, 2.5μg / mL insulin (Sigma). At day 71, the differentiation medium was changed to a maturation medium consisting of Neurobasal (Gibco), 1X B27 supplement, 1X penicillin / streptomycin, 1X 2-mercaptoethanol, 0.2mM ascorbic acid, 20ng / mL BDNF (Peprotech), 20ng / mL GDNF (Peprotech), 1ng / mL TFGβ (Peprotech), and 0.5mM cAMP (Sigma). Organoids can be grown in maturation medium for more than 110 days. All medium was replaced every other day. For static culture, the same protocol was followed to generate organoids on day 14, which were then maintained in ultra-low attachment 6-well plates (Corning Costar) with differentiation medium.

[0124] Cultivation of brain organoids from human iPSCs

[0125] To generate midbrain-specific organoids, human iPSC colonies were isolated with type IV collagenase 7 days after passage and washed with fresh stem cell culture medium in 15 mL conical centrifuge tubes. On day 1, the isolated and washed iPSC colonies were transferred to ultra-low attachment 6-well plates containing EB culture medium, which consisted of DMEM: F12, 15% Knockout serum replacement, 1X Glutamax, 1X 2-mercaptoethanol, 100nM LDN-193189, 10μM SB-431542, 100ng / mL SHH (Peprotech), 2μM Purmorphamine (Stemgent), 100ng / mL FGF-8 (Peprotech). On day 5, EB medium was gradually switched to SHH medium, which consisted of DMEM:F12, 1X N2 supplement, 1X Glutamax, 100nM LDN-193189, 3μM CHIR99021, 100ng / mL SHH, 2μM Purmorphamine, 100ng / mL FGF-8. On day 7, SHH medium was replaced with induction medium, which consisted of DMEM:F12, 1X N2 supplement, 1X Glutamax, 100nM LDN-193189, 3μM CHIR99021. On day 14, 10-20 organoids were transferred to SpinΩ containing differentiation medium consisting of Neurobasal, 1X B27 supplement, 1X Glutamax, 1X 2-mercaptoethanol, 20ng / mL BDNF, 20ng / mL GDNF, 0.2mM ascorbic acid, 1ng / mL TFGβ, and 0.5mM cAMP. The entire medium was changed every other day.

[0126] Cultivation of hypothalamic organoids from human iPSCs

[0127] In order to produce hypothalamus-specific organoids, human iPSC colonies were separated with type IV collagenase 7 days after passage and washed with fresh stem cell culture medium in 15mL conical centrifuge tubes. On the 1st day, the iPSC colonies separated and washed were transferred to ultra-low adsorption 6-well plates (Corning Costar) containing stem cell culture medium. One day later (the 2nd day), stem cell culture medium was replaced with induction medium A, which consisted of DMEM: F12, 10% Knockout serum substitute, 1X non-essential amino acids, 1X penicillin / streptomycin, 1X 2-mercaptoethanol, 1X Glutamax, 2.5μM LDN-193189 (Stemgent), 3μM SB-431542 and 450μM 1-thioglycerol (Sigma). On day 4, the culture medium was switched to induction medium B, which consisted of DMEM:F12, 10% Knockout serum replacement, 1X non-essential amino acids, 1X penicillin / streptomycin, 1X Glutamax, 1X N2 supplement, 10 ng / ml Wnt-3A, 20 ng / mL SHH, and 2 μM Purmorphamine. On day 7, 5-10 organoids were transferred to a 12-well spin bioreactor and induction medium B was replaced with differentiation medium, which consisted of DMEM:F12 / Neurobasal (1:1 ratio), 1X B27 supplement, 1X non-essential amino acids, 1X penicillin / streptomycin, 1X Glutamax, 10 ng / ml FGF-2, and 10 ng / mL CTNF (Peprotech). The culture medium was changed every other day.

[0128] Preparation of reagents for neural induction medium

[0129] Combine DMEM-F12 with 1% N2 supplement (vol / vol), 1% Glutamax supplement (vol / vol), and 1% MEM-NEAA (vol / vol). Add heparin (final concentration 1 μg mL -1 ) and filter the culture medium using a vacuum driven 0.2 μm filter unit.

[0130] Reagent preparation for brain organoid differentiation medium

[0131] For approximately 250 mL of culture medium, combine 125 mL of DMEM-F12 with 125 mL of Neurobasal, 1.25 mL of N2 supplement, 62.5 μL of insulin, 2.5 mL of Glutamax supplement, 2.5 mL of MEM-NEAA, and 2.5 mL of penicillin-streptomycin. Prepare a 1:100 dilution of 2-mercaptoethanol in DMEM-F12 and add 87.5 μL of this solution to the culture medium. Add 2.5 mL of B27 supplement.

[0132] Digestion protocol for isolating one or more desired cell populations from microglia-replete brain organoids

[0133] The organoids were placed in 1.5 mL Eppendorf tubes containing 0.6 mL digestion solution containing accutase and collagenase mixed at a ratio of 9: 1. After incubation at 37 ° C for 30 minutes, the mixture was stirred by gently pipetting 10 times using a 1 mL pipette tip to release cells from the organoids. The mixture was placed in a heating block at 1400 rpm and treated at 37 ° C for 10 minutes to further release cells from the organoids. The mixture was stirred by gently pipetting 10 times using a 1 mL pipette tip to further release cells from the organoids. The mixture was incubated at room temperature to allow the debris to settle to the bottom of the centrifuge tube, and the supernatant containing the digested cells was collected. New digestion solution was added to the centrifuge tube containing the debris. The centrifuge tube was subjected to another round of heating block treatment and pipetting to collect more single cells from the supernatant (digestion solution). The collected supernatant was filtered through 70 um filter paper, centrifuged and stained with antibodies for FACS. Neurons, neuronal progenitor cells, glial cells, and primitive-like macrophages (iMacs) can be isolated using FACS.

[0134] Example 1: Primitive-like macrophages (iMac) colonize brain organoids and differentiate into functionally active microglia-like cells (iMicro)

[0135] use Figure 1A The scheme shown, brain organoids and primitive macrophages (iMac) are produced from the same human iPSC. Microglia were observed in the human fetal brain as early as 4.5 weeks after conception. iMac was co-cultured with relatively young brain organoids (day 26) simulating the early pregnancy stage fetal brain. Co-culture was performed using organoid growth medium supplemented with 100ng / mL CSF-1, and half of the culture medium was replaced every three days. In addition, the ultra-low adsorption 24-well plate used for co-culture minimizes the adsorption of iMac on the culture plate, thereby maximizing its physical interaction with the organoid. In addition, one organoid was cultured per well, which ensured that there was enough space and nutrients for its survival and growth.

[0136] After 15 days of co-culture, iMacs were observed to colonize the surface of organoids as single cells and cell clusters ( Figure 1B ). Cross-sectional images of organoids showed that many iMacs were also found inside the organoids, indicating their ability to penetrate into the organoids ( Figure 1C Some of the iMacs found in organoids are Ki67 + , indicating its proliferation ability ( Figure 1D ).

[0137] Microglia are known for their ability to respond to inflammation or injury in the brain. To test whether iMacs respond to injury in organoids, organoids were transfected with GFP. + iMac co-cultures were then subjected to laser-induced neuronal injury. Live cell imaging revealed that GFP + After injury, iMacs began to extend their synapses toward the injury site, a typical behavior of microglia in vivo in response to neuronal damage ( Figure 1E ). Microglia are also known for their ability to phagocytose amyloid-β peptide, a pathological peptide associated with Alzheimer's disease. To test whether iMacs have the ability to phagocytose such peptides within organoids, organoids were incubated with fluorine-555-labeled amyloid-β peptide and then with GFP. + iMac co-culture. Live cell imaging showed that iMac actively moved in the organoids and was labeled with a fluorine 555 signal, indicating its ability to constantly monitor the organoids and engulf them ( Figure 1F ).

[0138] Overall, the results suggest that iMacs in organoids differentiate into microglia-like cells (iMicros) that monitor and respond to their surroundings, similar to in vivo microglia in the brain.

[0139] Example 2: Addition of iMac reduces organoid size and number of neural progenitor cells (NPCs)

[0140] To investigate the role of iMacs on organoid development, organoid growth was examined throughout co-culture. In the absence of iMacs, organoids showed continued growth. However, when co-cultured with iMacs, organoid growth was significantly reduced and they became more spherical in shape ( Figure 2A , Figure 8 To understand whether there were any changes in the composition of cells in the organoids after co-culture with iMacs, the organoids were enzymatically digested and single-cell suspensions were stained with antibodies to identify macrophages, NPCs, neurons, and glial progenitor cells by flow cytometry. Importantly, the number and proportion of NPCs in the organoids were reduced when the organoids were co-cultured with iMacs ( Figure 2B ). This is further supported by the observation that iMac-enriched regions of organoids are often devoid of NPCs.

[0141] Example 3: Addition of iMac promotes axon development in organoids

[0142] To understand how iMicro affects brain organoid growth and NPC numbers, a single-cell RNA-seq experiment was set up that allowed the determination of all transcriptomic changes in organoid cells in the presence or absence of macrophages. The 10X Genomics Chromium system, which uses an emulsion method with gel beads for rapid droplet-based encapsulation of single cells, was used on single-cell suspensions obtained from three co-cultured organoids (co-cultures), three organoids cultured in the absence of iMacs (organoids only), and iMacs cultured in the absence of organoids (iMacs only). Figure 3A ).

[0143] The transcriptomes were then subjected to cluster analysis, which allowed the identification of four separate cell clusters ( Figure 3B The cell cluster identities were annotated by correlating the expression of neuronal markers (MYT1L, DCX, NRXN1, MAP2, TUBB), NPC markers (VIM, PAX6, SOX2, HES1), mesenchymal cell markers (COL1A2, COL5A1, DCN, LUM), and macrophage markers (PTPRC, AIF1, ITGAM) ( Figure 3B ).

[0144] Gene ontology (GO) analysis was performed based on differentially expressed genes (DEGs) between NPCs from organoids only (NPCs only) and NPCs from co-cultured organoids (co-NPCs) and between neurons from organoids only (neurons only) and neurons from co-cultured organoids (co-neurons). GO analysis showed that axon development and regeneration pathways were upregulated in both co-NPCs and co-neurons ( Figure 3C This was also supported by proteomic analysis of NPCs sorted from organoids alone and co-cultured organoids, as axon growth-related proteins such as PRPH and DPYSL4 were upregulated in co-NPCs ( Figure 3D ).

[0145] To further verify whether the presence of microglia in organoids affects the potential of NPCs, co-NPCs and NPCs alone were sorted from organoids and cultured in 2D for 24 hours, and then the number and length of their axons were measured. Co-NPCs showed more and longer axons than NPCs alone ( Figure 3E ). Consistently, iMac in organoids (co-iMac) upregulated genes involved in axon outgrowth and neurogenesis, such as IL-1b, IL10, NRG1, SEMA4C, VEGFA, and ADAM8 ( Figure 3F ).

[0146] In addition, proteomic analysis of co-iMac and iMac alone ( Figure 3G) showed that co-iMac upregulates proteins involved in axon guidance.

[0147] Example 4: co-iMac and embryonic microglia express PLIN2 and lipid droplets.

[0148] DEG analysis between co-iMac and iMac alone showed that lipid droplet coating protein 2 (PLIN2), known to be involved in lipid droplet formation, was one of the most upregulated DEGs in co-iMac. In addition, other genes involved in lipid droplet formation and lipid export, such as M1D1LP1, ARL4C, SEPT9, and ABCA1, were found to be upregulated in co-iMac ( Figure 4A Live cell imaging showed that many co-iMacs contained lipid droplets ( Figure 4B ), and immunostaining showed that PLIN2 expression and lipid droplets were almost exclusively restricted to co-iMacs but not to other cell types in the organoids ( Figure 4C Similarly, lipid droplets and PLIN2 expression were observed only in microglia of mouse and human fetal brains ( Figure 4D and 4E ). However, its expression was not observed in microglia of adult mouse and primate brains (data not shown). This suggests that co-iMac and embryonic microglia may play a central role in driving metabolic changes in neuronal cells that may be necessary for brain development.

[0149] Example 5: iMac affects lipid content and metabolism of cells in organoids

[0150] Further analysis of DEGs between co-NPCs and NPCs showed that most of the DEGs were genes involved in metabolic pathways such as glycolysis, OXPHOS, and important cholesterol biosynthesis ( Figure 5A This was further verified by proteomic analysis ( Figure 5A ). Gene ontology (GO) analysis showed that the cholesterol biosynthesis pathway was the most significantly downregulated pathway in co-NPCs ( Figure 5B ).

[0151] Given that high intracellular cholesterol levels usually lead to downregulation of genes involved in cholesterol biosynthesis, a study was performed to investigate whether there was any increase in lipid content in co-NPCs after co-culture with iMacs. TM 493 / 503 staining (which stains neutral lipids) showed that more lipid droplets were present in co-cultured organoids and more widely distributed within the organoids compared to organoids alone ( Figure 5C ). Next, the organoids were digested into single cells and analyzed using BODIPY TM493 / 503 staining for flow cytometry analysis. Flow cytometry data indicated that neutral lipid content was higher in co-NPCs compared to NPCs alone ( Figure 5D ). To further verify this, mass spectrometry-based lipidomics was performed to quantify lipids in co-NPCs and NPCs alone. The data showed that there were more neutral lipids, such as diacylglycerol (DAG), triacylglycerol (TAG), and cholesterol ester (CE) in co-NPCs compared to NPCs alone ( Figure 5E ). Overall, the data indicate that addition of iMac modulates lipid profiles in organoids and specifically lipid content in NPCs.

[0152] Example 6: iMac transports cholesterol to cells in organoids

[0153] Astrocytes are known to be the main cell population that synthesizes and metabolizes lipids in the adult brain. It has been shown that cultured neurons from the mammalian central nervous system require cholesterol from astrocytes to form a large number of efficient synapses. However, astrocytes are not present in the early stages of brain development. Therefore, the function described can be performed by microglia in early development. To test whether there is any cholesterol transport from co-iMac to co-NPC in organoids, iMac was coupled to a green fluorescent FL C12 cholesterol ester (BODIPY-CE) was incubated overnight and washed three times with PBS before co-culture with organoids. Immunostaining showed that iMacs took up BODIPY-CE and stored it in lipid droplets, as indicated by overlap with PLIN2 protein ( Fig. 6A ). Then, iMacs containing BODIPY-CE were co-cultured with organoids for 7 days and the organoids were analyzed by flow cytometry. Flow cytometry data showed that NPCs, neurons, and glial cells in the organoids contained BODIPY-CE ( Figure 6B To further confirm this, co-NPCs were sorted from organoids and immunostained. The images clearly showed that CE-FITC ( Figure 6C ). Overall, the data suggest that cholesterol transport from iMacs to cells in organoids occurs.

[0154] Equivalent

[0155] The above-described embodiments are presented for the purpose of illustrating the present invention, and should not be construed as imposing any limitation on the scope of the present invention. It will be apparent that many modifications and changes may be made to the specific embodiments of the present invention described above and in the embodiments without departing from the underlying principles of the present invention. The application is intended to include all these modifications and changes.

Claims

1. A method for generating microglia-rich brain organoids comprising the following steps: Microglia were generated by incubating naive-like macrophages with brain organoids between 15 and 30 days old in low-attachment cell culture vessels in CSF-1-containing brain organoid medium. a) generating the primitive-like macrophages from the first stem cell population by the following steps: i) incubating the stem cells in a culture medium containing a GSK3 inhibitor, BMP4 and VEGF to differentiate the stem cells into cells of a mesodermal cell lineage; ii) incubating the cells of the mesodermal cell line in a medium containing FGF-2 to differentiate the cells of the mesodermal cell line into hemangioblasts; iii) incubating the angioblasts in a culture medium containing VEGF and FGF-2; iv) incubating the hemangioblasts in a medium containing DKK1, SCF, FGF2, IL3 and IL6 to differentiate the hemangioblasts into hematopoietic cells; v) incubating the hematopoietic cells in a medium containing SCF, FGF-2, IL-3 and IL-6 to induce maturation of the hematopoietic cells; vi) incubating the mature hematopoietic cells in a medium containing CSF-1 to differentiate the mature hematopoietic cells into primitive-like macrophages; and b) generating the brain organoid from the second stem cell population by the following steps: i) incubating the second stem cell population in a low attachment cell culture container to form embryoid bodies; ii) incubating the embryoid bodies in a neural induction medium to differentiate the embryoid bodies into organoids containing neural ectoderm cells; iii) embedding the organoids comprising neuroectodermal cells in matrigel and incubating the organoids comprising neuroectodermal cells in a brain organoid culture medium containing N2 and without vitamin A type B27, so that the organoids comprising neuroectodermal cells differentiate into organoids comprising neuroepithelial cells; iv) incubating the organoid comprising the neuroepithelial cells with a cerebral organoid culture medium containing N2 and without vitamin A type B27, so that the organoid comprising the neuroepithelial cells differentiates into the cerebral organoid, The first stem cell population and the second stem cell population are embryonic stem cells, induced pluripotent stem cells or a combination thereof.

2. The method of claim 1, wherein the brain organoids are 26 days old.

3. The method of claim 1 or 2, wherein the cerebral organoid culture medium comprises 25-100 ng / mL CSF-1.

4. The method of claim 1 or 2, wherein 150,000 or fewer primitive-like macrophages are incubated with the brain organoids.

5. The method of claim 1 or 2, wherein the primitive-like macrophages and brain organoids are incubated together for at least 1 week.

6. The method of claim 5, further comprising the step of dispersing cells of the microglia-sufficient brain organoid and isolating one or more predetermined cell populations.

7. The method of claim 6, wherein one or more predetermined cell populations are separated using fluorescence activated cell sorting or magnetic activated cell sorting.

8. The method of claim 1, wherein the primitive-like macrophages are generated over a period of 26 days.

9. The method of claim 1, wherein the brain organoids are produced over a period of 26 days.

10. A brain organoid with sufficient microglia, obtained by the method according to any one of claims 1 to 9.

11. A method for screening compounds targeting microglial function, comprising the following steps: The microglia-sufficient brain organoid of claim 10 is contacted with the compound and a predetermined property of the microglia is analyzed.

12. A method for screening a compound for treating a neurodegenerative disease, comprising the following steps: The microglia-sufficient brain organoid of claim 10 is contacted with the compound and a predetermined property of the microglia is analyzed.

13. A kit, when used in the method according to any one of claims 1 to 9, comprising: a) a first culture medium containing a GSK3 inhibitor, BMP4 and VEGF; b) a second culture medium comprising BMP4, VEGF and FGF-2; c) a third culture medium containing VEGF and FGF-1; d) a fourth culture medium containing VEGF, DKK1, SCF, FGF-2, IL-3 and IL-6; e) a fifth culture medium comprising FGF, IL6, IL3 and SCF; and f) a sixth culture medium containing CSF-1; to generate primitive-like macrophages from the first stem cell population; and g) neural induction medium; h) cerebral organoid culture medium containing N2 and without vitamin B27; and i) Cerebral organoid culture medium containing N2 and vitamin A type B27; to generate brain organoids from a second stem cell population.

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