Efficient and stable brain-like organ establishment method and application thereof

Abstract

The invention relates to an efficient and stable brain-like organ establishment method and application. Specifically, the invention provides a method for efficiently differentiating human pluripotent stem cells (hPSCs) into brain-like organs by virtue of brain-like nerve induction. The invention also provides a brain-like organ disease model. The method disclosed by the invention has the characteristics of high efficiency and stability, and the brain-like organ obtained by the method disclosed by the invention has better consistency. The method disclosed by the invention can be used for researching neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of neurobiology, and in particular to an efficient and stable method for establishing brain-like organs and its application in the research and treatment of neurodegenerative diseases. Background Art

[0002] The concept of organoids comes from in vitro cell self-assembly. As early as 1907, Wilson discovered that sponge tissue could be reassembled and form complete tissues after being decomposed. Subsequently, similar reassembly phenomena were found in simple organisms such as hydrozoans, Alpinia corals, starfish, and cnidarians such as palmate polyps. When vertebrate tissues or organs, such as skin tissue or kidney tissue, are broken up and dispersed, cells can self-assemble and reorganize into tissues with characteristic structures similar to the original organs in in vitro culture. These studies have shown that some cells in tissues and organs have the ability to self-assemble independently, and the structures formed that resemble the original tissues and organs are called "organoids."

[0003] The researchers isolated embryonic neural cells in vitro and found that the neuroepithelial cells can spontaneously aggregate and assemble into a neural rosette structure, which represents the neural tube in the embryonic period in vivo. In the study of avian retinal cell reassembly, it was found that retinal stem cells can spontaneously aggregate and form a neural rosette structure, and also have the ability to proliferate and differentiate into a variety of retinal cells. This discovery is of great significance for understanding "self-assembly", indicating that the cells that undergo self-assembly have the ability to spontaneously aggregate and reorganize, as well as the ability to proliferate and differentiate. Similarly, embryonic neural progenitor cells can not only proliferate to form a neural rosette structure in in vitro culture, but also have the ability to differentiate into neurons.

[0004] Although early studies used neural stem cells for 3D culture and simulated the brain's development process to a certain extent through self-renewal and self-assembly, the differentiation of brain organoids has the problem of system instability. For organoids cultured for a long time in vitro, in previous studies, due to the lack of sufficient oxygen and nutrients inside, cell necrosis and apoptosis are prone to occur inside the long-term cultured brain organoids. Using brain organoid technology, long-term culture simulates Alzheimer's disease, but the characteristics of Alzheimer's disease simulated by existing technologies are not typical enough.

[0005] In view of the above problems and shortcomings, there is an urgent need in this field to develop an efficient, stable and rapid method for preparing brain-like organs for the study of neurodegenerative diseases. Summary of the invention

[0006] The purpose of the present invention is to provide an efficient and stable method for establishing brain-like organs.

[0007] In a first aspect of the present invention, a brain organoid neural precursor cell population is provided, wherein the brain organoid neural precursor cell population can be differentiated into a brain organoid, and the brain organoid neural precursor cell population expresses molecular markers SOX2 and PAX6;

[0008] Among them, ≥95% of the cells in the neural precursor cell population of the brain organoid express SOX2, and ≥95% of the cells express PAX6.

[0009] In another preferred embodiment, the brain organoid neural precursor cell population also expresses neuronal markers selected from the following group: TUJ1, MAP2 and NEUN.

[0010] In another preferred embodiment, 20%-70% of the cells in the neural precursor cell population of the brain organoid express neuronal markers.

[0011] In another preferred embodiment, the number of neural rosettes in the neural precursor cell population of the brain organoid is increased.

[0012] In a second aspect of the present invention, a method for preparing neural precursor cells of brain organoids is provided, comprising the steps of:

[0013] (a) providing a pluripotent stem cell;

[0014] (b) culturing the pluripotent stem cells in suspension to form hES cell clusters;

[0015] (c) treating the hES cell clusters with a brain organoid culture medium for 1 to 2 days to obtain embryoid bodies (EBs), wherein the brain organoid culture medium contains a signaling pathway inhibitor;

[0016] (d) Inducing and culturing the embryoid bodies for 16 days using a neural differentiation culture medium to obtain neural progenitor cells of brain organoids, wherein the neural differentiation culture medium contains a signaling pathway inhibitor.

[0017] In another preferred embodiment, in step (a), the pluripotent stem cells are derived from mammals.

[0018] In another preferred embodiment, in step (a), the pluripotent stem cells are derived from humans.

[0019] In another preferred embodiment, the pluripotent stem cells are selected from embryonic stem cells or induced pluripotent stem cells (iPSC).

[0020] In another preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0021] In another preferred embodiment, step (b) includes: digesting the pluripotent stem cells to form a single cell suspension for suspension culture.

[0022] In another preferred embodiment, in step (b), accutase is used for digestion.

[0023] In another preferred embodiment, the embryoid body has one or more characteristics selected from the following group:

[0024] More than 50%, preferably more than 75%, and most preferably more than 98% of the cells express pluripotency markers.

[0025] In another preferred embodiment, the pluripotency markers include: OCT4, NANOG and SOX2.

[0026] In another preferred embodiment, in step (c), the signal pathway inhibitor is the ROCK signal pathway inhibitor Y27632.

[0027] In another preferred embodiment, in step (c), the brain organoid culture medium is STEM FLEX culture medium containing Y27632.

[0028] In another preferred embodiment, in step (d), the neural differentiation culture medium contains one or more signal pathway inhibitors.

[0029] In another preferred embodiment, in step (d), the signal pathway inhibitor is selected from the following group: BMP inhibitor, TGF-beta inhibitor, or a combination thereof.

[0030] In another preferred embodiment, the BMP inhibitor is Dorsomorphin.

[0031] In another preferred embodiment, the TGF-beta inhibitor is SB431542.

[0032] In another preferred embodiment, in step (d), the method further comprises inducing the embryoid bodies using N2 culture medium.

[0033] In another preferred embodiment, in step (d), the N2 culture medium does not contain vitamin A (VA).

[0034] In another preferred embodiment, in step (d), the N2 culture solution contains the following components:

[0035] In another preferred embodiment, the brain organoid neural precursor cells have the following characteristics:

[0036] The morphological and structural characteristics of brain-like organs are highly repeatable and consistent, with more than 60%, preferably more than 78%, and optimally more than 85% of the cells expressing neural precursor cell markers; and more than 20%, preferably more than 50% of the cells expressing neuronal markers.

[0037] In another preferred embodiment, the brain organoid precursor cell marker is selected from: SOX2, PAX6.

[0038] In another preferred embodiment, the neuronal marker of brain organoid precursor cells is TUJ1.

[0039] In another preferred embodiment, the brain organoid neural precursor cells have one or more characteristics selected from the following group:

[0040] (1) 60%-99% of cells expressed the neural precursor marker SOX2;

[0041] (2) 78%-99% of cells expressed the neural precursor marker PAX6;

[0042] (3) 20%-70% of the cells expressed the neuronal marker TUJ1.

[0043] In a third aspect of the present invention, a brain organoid is provided, wherein the brain organoid is differentiated from the neural precursor cells of the brain organoid described in the first aspect of the present invention.

[0044] In another preferred embodiment, the brain organoid has one or more characteristics selected from the following group:

[0045] (1) less than 30%, preferably less than 20%, and most preferably less than 15% of the cells in the brain organoid express neural progenitor cell markers;

[0046] (2) more than 10%, preferably more than 30%, and most preferably more than 60% of the cells in the brain organoid express mature neuronal markers;

[0047] (3) expression of presynaptic membrane protein (SYP) and postsynaptic density protein (PSD95);

[0048] (4) Forming a neural network with electrophysiological functions.

[0049] In another preferred embodiment, the brain organoid precursor cell marker is SOX2.

[0050] In another preferred embodiment, the brain organoid mature neuron marker is selected from: MAP2, NEUN, and CTIP2.

[0051] In a fourth aspect of the present invention, a method for culturing a brain organoid as described in the third aspect of the present invention is provided, comprising:

[0052] (a) providing a pluripotent stem cell;

[0053] (b) culturing the pluripotent stem cells in suspension to form hES cell clusters;

[0054] (c) treating the hES cell clusters with a brain organoid culture medium for 1 to 2 days to obtain embryoid bodies, wherein the brain organoid culture medium contains a signaling pathway inhibitor;

[0055] (d) inducing and culturing the embryoid bodies for 16 days using a neural differentiation culture medium to obtain neurospheres containing neural progenitor cells, wherein the neural differentiation culture medium contains a signaling pathway inhibitor;

[0056] (e) maturing the neurospheres for 14 to 74 days to obtain brain organoids.

[0057] In another preferred embodiment, the step (e) comprises the following sub-steps:

[0058] (e1) culturing the neurospheres in N2B27 culture medium for 14 days;

[0059] (e2) Cultivating the cells in N2B27 medium containing Vitamin A (N2B27(VA+)) for 60-150 days.

[0060] The N2B27 culture medium is a mixture of two basic culture media, DMEM / F12 and Neuralbasal, in a ratio of 1:1, and is supplemented with NEAA, Glutamax, N2 supplement (Gibco), and B27 supplement (Gibco) without Vitamin A.

[0061] The N2B27 (VA+) culture medium is a mixture of two basic culture media, DMEM / F12 and Neuralbasal, in a ratio of 1:1, and NEAA, Glutamax, N2 supplement (Gibco), and B27 supplement (Gibco) containing Vitamin A are added.

[0062] In another preferred embodiment, in step (a), the pluripotent stem cells are derived from mammals.

[0063] In another preferred embodiment, in step (a), the pluripotent stem cells are derived from humans.

[0064] In another preferred embodiment, the pluripotent stem cells are selected from embryonic stem cells or induced pluripotent stem cells (iPSC).

[0065] In another preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0066] In another preferred embodiment, step (b) includes: digesting the pluripotent stem cells to form a single cell suspension for suspension culture.

[0067] In another preferred embodiment, in step (b), accutase is used for digestion.

[0068] In another preferred embodiment, in step (c), the signal pathway inhibitor is the ROCK signal pathway inhibitor Y27632.

[0069] In another preferred embodiment, in step (c), the brain organoid culture medium is STEM FLEX culture medium containing Y27632.

[0070] In another preferred embodiment, in step (d), the neural differentiation culture medium contains one or more signal pathway inhibitors.

[0071] In another preferred embodiment, in step (d), the signal pathway inhibitor is selected from the following group: BMP inhibitor, TGF-beta inhibitor, or a combination thereof.

[0072] In another preferred embodiment, the BMP inhibitor is Dorsomorphin.

[0073] In another preferred embodiment, the TGF-beta inhibitor is SB431542.

[0074] In another preferred embodiment, in step (d), the method further comprises inducing the embryoid bodies using N2 culture medium.

[0075] In another preferred embodiment, in step (d), the N2 culture medium does not contain vitamin A (VA).

[0076] In another preferred embodiment, in step (d), the N2 culture medium is DMEM / F12 supplemented with NEAA, Glutamax, and N2 supplement (Gibco).

[0077] In the fifth aspect of the present invention, there is provided a use of the brain organoid precursor cell population as described in the first aspect of the present invention or the brain organoid as described in the third aspect of the present invention for preparing a pharmaceutical composition for preventing or treating neurodegenerative diseases.

[0078] In another preferred embodiment, the neurodegenerative disease includes: Alzheimer's disease.

[0079] In the sixth aspect of the present invention, there is provided a use of the brain organoid precursor cell population as described in the first aspect of the present invention or the brain organoid as described in the third aspect of the present invention for constructing a neurodegenerative disease research model.

[0080] In another preferred embodiment, the neurodegenerative disease includes: Alzheimer's disease.

[0081] In a seventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0082] (1) an effective amount of a population of neural progenitor cells of the brain organoid as described in the first aspect of the present invention or a brain organoid as described in the third aspect of the present invention; and

[0083] (2) A pharmaceutically acceptable carrier.

[0084] In another preferred embodiment, the dosage form of the pharmaceutical composition includes injection.

[0085] In another preferred embodiment, the pharmaceutically acceptable carrier is a biocompatible carrier.

[0086] In another preferred embodiment, the biocompatible carrier includes gelatin, collagen, acellular matrix, hydrogel, layered double hydroxide nanoparticles.

[0087] In the eighth aspect of the present invention, a method for constructing a neurodegenerative disease research model is provided, the method comprising using the brain organoid neural precursor cell population as described in the first aspect of the present invention or the brain organoid as described in the third aspect of the present invention to construct a neurodegenerative disease research model.

[0088] In another preferred embodiment, the method includes therapeutic and non-therapeutic methods.

[0089] In another preferred embodiment, the neurodegenerative disease includes: Alzheimer's disease.

[0090] In the ninth aspect of the present invention, a method for preventing or treating a neurodegenerative disease is provided, the method comprising administering to a subject in need thereof the neural precursor cell population of the brain organoid described in the first aspect of the present invention or the brain organoid described in the third aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention.

[0091] In another preferred embodiment, the neurodegenerative disease includes: Alzheimer's disease.

[0092] The various technical features described in detail can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 A shows a schematic diagram of the differentiation process of brain organoids. Figure 1 B shows a bright field image of D2 EB, Figure 1 C shows immunofluorescence staining to identify the expression of pluripotency markers OCT4, NANOG, and SOX2.

[0094] Figure 2 A shows a schematic diagram of the use of different culture media during the D0-D2 cell aggregation process. Figure 2 B is a bright field image of the developing brain organoids at D2, D8, and D16, where the scale bar is 500 μm. Figure 2 C shows immunofluorescence staining of brain organoids grown in different cell culture media until D16, with PAX6 marking neural progenitor cells and TUJ1 marking neurons. Scale bars, 100 μm. Figure 2 D shows the Figure 2 C Statistical analysis results of the proportion of PAX6-positive cells. Figure 2 E shows the Figure 2 C. Statistical analysis results of the number of neural rosettes formed.

[0095] Figure 3 A shows the bright field image of the brain organoid on day 16, where the scale bars are all 100 μm. Figure 3 B shows immunofluorescence staining to identify SOX2-positive neural progenitor cells and TUJ1-positive neurons, where the scale bars are 100 μm.

[0096] Figure 4 A shows the size changes of brain organoids on days 30, 90, and 150 of culture. Figure 4 B is immunofluorescence staining showing SOX2-positive neural progenitor cells and MAP2-positive mature neurons in the cerebral organoids on the 30th day. Figure 4 C shows mature neurons expressing NEUN in the cerebral organoid at day 30. Figure 4 D shows the stratified features of the brain organoids cultured on days 60 and 90, with deep-layer neurons positive for CTIP2 and neural progenitor cells positive for SOX2. Figure 4 E shows the electrical activity of brain-like organs at 30, 60 and 90 days of high-density MEA detection, including the average discharge rate, average amplitude and activated electrode rate. Figure 4 F shows immunofluorescence staining to identify the presynaptic membrane protein SYP and the postsynaptic density protein PSD95. Figure 4 G shows the neural network of the brain organoid at day 90.

[0097] Figure 5A shows the immunofluorescence staining results of presynaptic membrane protein (SYP) and postsynaptic density protein (PSD95) in brain organoids in FAD and healthy control groups. Figure 5 B shows the statistical analysis results of the presynaptic membrane protein SYP. Figure 5 C shows the results of statistical analysis of unilateral PSD95 in the postsynaptic density. Figure 5 D shows the formation of neural networks in FAD brain organoids detected by high-density MEA at D90. Figure 5 E shows AT8-labeled phosphorylated TAU protein in D90 brain organoids. Figure 5 F shows the Figure 5 Statistical analysis results of AT8 fluorescence area in E. Figure 5 G shows the distribution of 6E10 in D30 brain organoids. Figure 5 H shows the distribution of 6E10 in D60 brain organoids. Figure 5 I shows the distribution of 6E10 in D90 brain organoids. Figure 5 J shows the Figure 5 G. Figure 5 H and Figure 5 Statistical analysis results of 6E10 fluorescence area in I. DETAILED DESCRIPTION

[0098] After extensive and in-depth research, the inventors unexpectedly found that by treating pluripotent stem cells with different brain organoid culture media, the pluripotent stem cells can be efficiently and quickly differentiated into uniform brain organoids through brain organoid precursor cell groups. The neural precursor cell group can be directed and induced to differentiate into brain organoids. Compared with the prior art, the present invention can efficiently obtain uniform neural precursor cells of brain organoids, thereby inducing brain organoids with excellent performance.

[0099] Specifically, a highly efficient technical method for preparing specific neural progenitor cells using human pluripotent stem cells (PSCs) as starting materials and then differentiating them into mature brain-like organs was developed for the first time. Based on this, the present invention was completed.

[0100] the term

[0101] As used herein, the term "pluripotent stem cells (PSC)" refers to a type of cell that has both self-renewal and multidirectional differentiation potentials, including but not limited to: (1) embryonic pluripotent stem cells, and (2) pluripotent stem cells derived from somatic cell reprogramming.

[0102] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to a type of stem cell that has been transformed from somatic cells into a state of multipotential differentiation through a reprogramming process. Its multipotency is manifested in the ability to differentiate into three germ layers and multiple cell types.

[0103] As used herein, the term "differentiation" refers to the process by which stem cells transform into a target cell type in a specific context.

[0104] As used herein, the term "differentiation efficiency" refers to the frequency of transformation of pluripotent stem cells into target cell types in a specific context. For example, a differentiation efficiency of 90% means that at least 90% of the differentiated cell population is the target cell type.

[0105] As used herein, the term "organoid" refers to a miniaturized and, in some cases, simplified form of an organ produced in three dimensions in vitro that displays realistic and anatomically correct microanatomy. They are derived from one or more cells from tissues, embryonic stem cells, or induced pluripotent stem cells that are able to self-organize in three-dimensional culture due to, for example, their self-renewal and differentiation capabilities.

[0106] As used herein, the term "embryoid body" refers to a three-dimensional aggregate of pluripotent stem cells. The pluripotent cell types comprising embryoid bodies include, but are not limited to, embryonic stem cells (ESC) derived from the blastocyst stage of an embryo, such as mice (mESC), primates, and people (hESC) from sources. In addition, embryoid bodies can be formed by embryonic stem cells derived from alternative techniques, including somatic cell nuclear transplantation or somatic cell reprogramming to produce induced pluripotent stem cells (iPS). Similar to ES embryonic stem cells cultured in monolayer form, embryonic stem cells in embryoid bodies undergo differentiation and cell specialization along three germ lines - endoderm, ectoderm, and mesoderm - it includes all somatic cell types (which are all cell types in vivo, excluding germline cells).

[0107] As used herein, the term "neuronal lineage embryoid bodies" refers to embryoid bodies in which pluripotent stem cells have the potential to generate or become neurons.

[0108] As used herein, the term "midbrain", also known as the mesencephalon, refers to the portion of the brain located within the brainstem and above the hindbrain below the cerebral cortex.

[0109] As used herein, the term "inhibitor" refers to a molecule or compound that can reduce, downregulate, or in some cases completely stop the activity of a target molecule. Inhibitors are usually characterized and named by their target; for example, compounds that bind to an enzyme and thereby reduce its activity are called enzyme inhibitors. Inhibitors can be reversible or irreversible, meaning that with respect to binding to its target, the target binding can be subsequently destroyed (reversible) or not destroyed (irreversible). For example, the inhibition of a target molecule can be competitive, anti-competitive, non-competitive, or mixed. In contrast, molecules or compounds that are known to increase, upregulate, or initiate the activity of a target molecule are called activators.

[0110] As used herein, the term "stem cell" refers to an undifferentiated biological cell that can differentiate into more specialized cells and can divide (by mitosis) to produce more stem cells. Stem cells are present in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are separated, for example, from the inner cell mass of the blastocyst; and adult stem cells, which are present in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by supplementing adult tissues. In the developing embryo, stem cells can differentiate into any of the three major germ layers (called ectoderm, endoderm, and mesoderm) present in the early stages of embryonic development - but also keep the normal renewal of regenerated organs such as blood, skin, or intestinal tissues.

[0111] Three common, available sources of autologous adult stem cells in the human body are: bone marrow, which needs to be extracted by harvesting cells, usually from the femur or iliac crest; adipose tissue (lipid cells), which needs to be extracted by liposuction; and blood, which needs to be extracted, usually through an apheresis machine. Stem cells can also be taken from umbilical cord blood after birth. Of all the stem cell types, autologous collection involves the least risk. By definition, autologous cells are obtained from one's own body. Adult stem cells are frequently used in various medical treatments (e.g., bone marrow transplants). It is now possible to artificially grow and transform (differentiate) stem cells into specialized cell types with characteristics consistent with cells of various tissues, such as muscle or nerves.

[0112] Any mammalian stem cell can be used according to the inventive method disclosed herein, including but not limited to stem cells separated from umbilical cord blood, placenta and other sources. Stem cells can be separated from any mammalian species, such as but not limited to mice, rats, rabbits, guinea pigs, dogs, cats, pigs, sheep, cattle, horses, monkeys and people. In one example, stem cells are obtained from people. Stem cells can include pluripotent cells, which are cells with complete differentiation diversity, self-renewal and can remain dormant or static in tissues. Stem cells can also include pluripotent cells or directed progenitor cells. In one example, the method disclosed herein is carried out without using human embryonic stem cells. According to the present invention, other types of pluripotent cells can be used to replace human embryonic stem cells. In another example, the method disclosed herein is carried out on induced pluripotent stem cells. In another example, non-human embryonic stem cells are used to carry out the method disclosed herein.

[0113] As used herein, the term "pluripotent stem cell" refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm, for example, the inner gastric mucosa, gastrointestinal tract, and lungs develop from the endoderm; mesoderm, for example, muscle, bone, blood, and urogenital structures develop from the mesoderm; or ectoderm, for example, epidermal tissue and the nervous system develop from the ectoderm. However, it is worth noting that cell pluripotency is considered a continuum, ranging from fully pluripotent cells that can form every cell of the embryonic body, such as embryonic stem cells and induced pluripotent stem cells, to incomplete or partial pluripotent cells that can form cells of all three germ layers but may not have all the characteristics of fully pluripotent cells.

[0114] Cell culture is the process of cell growth under controlled conditions simulating its natural environment but outside its natural environment.Cell culture conditions vary according to cell type, but the required artificial environment is usually composed of a suitable container with one or more substrates or one or more mediums providing essential nutrients (such as amino acids, carbohydrates, vitamins and minerals) and growth factors, hormones and gases (usually CO2 and / or O2) required for cell growth and one or more mediums regulating the physiological and chemical environment (for example, pH buffering, osmotic pressure, temperature, humidity, etc.).Most cells require a surface or artificial matrix (also referred to as adhesion or monolayer culture) to grow thereon, while other cells can float freely in culture medium (also referred to as suspension culture), usually under stirring (for example, rolling bottle culture, etc.).In practice, the term "cell culture" refers to, compared with other types of culture such as plant tissue culture, fungal culture, microbial culture (bacterial culture) and cell culture as a host for virus replication, culture cells from multicellular eukaryotic organisms, especially animal cells and diseased human tissues (for example, HeLa cells, PC3 cells and HEK293T cells).

[0115] Cell culture media for cell culture generally contain at least the following items: a carbon or carbohydrate source (e.g., glucose or glutamine) as an energy source; amino acids for protein synthesis; vitamins to promote cell survival and growth; a balanced salt solution, generally a mixture of various ions to maintain optimal osmotic pressure within the cell and as a cofactor for various cofactor-mediated reactions (e.g., cell adhesion, enzymatic reactions, etc.); a pH indicator (e.g., phenol red; indicating a pH change from neutral to alkaline or acidic, generally indicating the presence of nutrient depletion, contamination, accumulation of necrotic cells, etc.) and a buffer (e.g., bicarbonate or HEPES buffer) to maintain the desired pH in the culture medium. In addition to the components listed above, cell culture media can and are generally modified by those skilled in the art to their desired requirements in cell culture. For example, fetal bovine serum or bovine serum is required for the growth and maintenance of certain cell lines in vivo, but some cell lines do not need to be used, and in other cell lines, such as when serum-starved cells are required for cytokine analysis, it is avoided. As defined in the art, a "defined medium" (also called a "chemically defined medium" or a "synthetic medium") is a cell culture medium in which all chemicals used are known and no yeast, animal, or plant tissue is present.

[0116] Minimal media are those cell culture media that contain the minimum nutrients that may be used for colony growth and are usually free of amino acids, and are usually used for "wild-type" microbial growth. Minimal media can also be used to select or counter-select recombinants or conjugates. Minimal media usually contain a carbon source, which can be a sugar such as glucose or a less energy source such as succinate; various salts, which may vary depending on the bacterial species and growth conditions; these usually provide essential elements such as magnesium, nitrogen, phosphorus and sulfur to allow bacteria to synthesize proteins and nucleic acids; and water. Supplemental minimal media are minimal media containing a single selected agent, usually an amino acid or a sugar. This supplement allows the cultivation of auxotrophic recombinants of, for example, a specific line.

[0117] Selective medium is used only for the growth of selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic (such as ampicillin or tetracycline), the antibiotic can be added to the medium to prevent the growth of other cells that are not resistant. A medium lacking amino acids such as proline is used with, for example, Escherichia coli that cannot synthesize it, and is commonly used to map bacterial chromosomes before the advent of genomics. Selective growth medium is also used for cell culture to ensure the survival or proliferation of cells with certain characteristics such as antibiotic resistance or the ability to synthesize certain metabolites. Typically, the presence of a specific gene or allele of a gene gives cells the ability to grow in a selective medium. In this case, the gene is referred to as a marker. Selective growth medium for eukaryotic cells typically contains neomycin to select cells that have been successfully transfected with a plasmid carrying a neomycin resistance gene as a marker. Ganciclovir is an exception to this rule because it is used to specifically kill cells carrying their respective markers, i.e., herpes simplex virus thymidine kinase (HSV TK).

[0118] Standard environmental growth conditions for cells in cell culture are typically a temperature of 37° C., a CO2 content of 5%, and a humidity of 95%. All of these conditions can be achieved and maintained by using, for example, an incubator. If desired, cell culture media can also be used to create certain conditions in cells, for example, starvation of cells prior to a particular treatment or analytical procedure that requires the cells to be starved or to be deprived of certain components of the culture medium (e.g., serum-free medium).

[0119] Cell culture can be performed in a two-dimensional (2D) or three-dimensional (3D) setup. Examples of two-dimensional cell culture are, but are not limited to, cell culture in any standard cell culture vessel such as a petri dish, a 6-well plate, a 96-well plate, a culture flask, and a roller bottle.

[0120] There are a variety of platforms that can be used to promote the growth of three-dimensional cell structures, including but not limited to scaffold systems, such as hydrogel matrices and solid scaffolds; and scaffold-free systems, such as low-adhesion plates, nanoparticle-promoted magnetic suspensions, and hanging drop plates. Therefore, in one example, the method disclosed herein is a three-dimensional method. In another example, the hydrogel is, but not limited to, natural and synthetic materials, such as animal extracellular matrix, extract hydrogels, protein hydrogels, peptide hydrogels, polymer hydrogels, and wood-based nanocellulose hydrogels. In essence, the hydrogels used for three-dimensional cell culture are generally composed of interconnected pores with high water retention, which enables efficient transportation of substances such as nutrients and gases.

[0121] Cell differentiation is the process that describes the change of a cell from one cell type to another. Most commonly, a cell changes into a more specialized type. Differentiation occurs many times during the development of a multicellular organism as it changes from a simple fertilized egg to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and produce fully differentiated daughter cells during tissue repair and normal cell renewal. For example, some differentiation occurs in response to antigen exposure. Differentiation significantly changes the size, shape, membrane potential, metabolic activity, and response to signals of the cell. These changes are primarily attributed to highly controllable modifications in gene expression and are the study of the field of epigenetics. With a few exceptions, cell differentiation almost never involves changes in the DNA sequence itself. Therefore, despite having the same genome, different cells can have very different physical characteristics that are usually separated in time.

[0122] There are multiple levels of cell potential, i.e., the state of a cell or the ability to differentiate into other cell types. Greater potential indicates that a greater number of cells can be derived from the state of the cell. Cells that can differentiate into all cell types (including placental tissue) are called totipotent cells. In mammals, only the fertilized egg and subsequent blastomeres are totipotent, while in plants, many differentiated cells can become totipotent through simple laboratory techniques. Cells that can differentiate into all cell types of adult organisms are called pluripotent cells. Such cells are also called meristems in higher plants and embryonic stem cells in animals. The potential of a cell can be induced and in some cases even reversed, i.e., changes in cell differentiation from more differentiated (i.e., closer to the final terminal cell type) to less differentiated (i.e., closer to totipotent or cells). For example, it has been shown that viral induction expression of four transcription factors Oct4, Sox2, c-Myc and Kfl4 (also called Yamanaka factors) is sufficient to produce pluripotent (iPS) cells from adult fibroblasts. Pluripotent cells are cells that can differentiate into a variety of different but closely related cell types. Oligopotent cells are more restricted in their differentiation capacity than pluripotent cells, but can still differentiate into several closely related cell types. Finally, unipotent cells can differentiate into only one cell type but are capable of self-renewal. In cytopathology, the level of cell differentiation serves as a measure of, for example, cancer progression. "Grade" is a marker of the degree of cell differentiation in a tumor.

[0123] There are three basic types of cells in mammals: germ cells, somatic cells, and stem cells. 14) Each of the cells has its own copy or multiple copies of the genome, except for certain cell types, such as red blood cells, which lack a nucleus in their fully differentiated state. Most cells are diploid, meaning they have two copies of each chromosome. Such cells, called somatic cells, make up the majority of the human body, such as skin and muscle cells. Cells differentiate to specialize for different functions.

[0124] The germ line is any cell line in an organism that produces gametes - eggs and sperm - and is therefore continuous over generations. Stem cells, on the other hand, have the ability to divide indefinitely and give rise to specialized cells, which are best described in the context of human development.

[0125] Development begins when a sperm fertilizes an egg and produces a single cell that has the potential to form an entire organism. Within a short period of time after fertilization, this cell divides into identical cells. In humans, about four days after fertilization and after several cycles of cell division, these cells begin to specialize, forming a hollow ball of cells called a blastocyst. The blastocyst has an outer layer of cells and a cluster of cells inside the hollow ball, called the inner cell mass. The cells of the inner cell mass go on to form nearly every different tissue present in the human body. Although the cells of the inner cell mass can form nearly every cell found in the human body, they cannot form an organism. These cells are called pluripotent cells.

[0126] Pluripotent stem cells further specialize into multipotent progenitor cells, which then produce functional cells. Examples of stem cells and progenitor cells include, but are not limited to, radial glial cells (embryonic neural stem cells) that generate excitatory neurons in the fetal brain through the process of neurogenesis; hematopoietic stem cells (adult stem cells) from the bone marrow that produce red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that produce stromal cells, adipocytes, and bone cell types; epithelial stem cells (progenitor cells) that produce various types of skin cells; and muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.

[0127] As the cell blastomeres differentiate from the single-layered blastocyst of mammals into the three major layers of germ cells, ectoderm, mesoderm, and endoderm (listed from most distal (outside) to proximal (inside)), pathways guided by cell adhesion molecules composed of the four amino acids arginine, glycine, asparagine, and serine are created. The ectoderm eventually forms the organism's skin and nervous system, the mesoderm forms bone and muscle tissue, and the endoderm forms internal organs.

[0128] Each specialized cell type in an organism expresses a subset of all the genes that make up the genome of that species. Each cell type is defined by a specific pattern of expression of its regulated genes. Cell differentiation is therefore the transformation of a cell from one cell type to another, and thus involves a switch from one gene expression pattern to another. Cell differentiation during development can be understood as the result of a gene regulatory network. Regulatory genes and their cis-regulatory modules are nodes in a gene regulatory network; they receive inputs and create outputs elsewhere in the network. Some evolutionarily conserved types of molecular processes are often involved in the cellular machinery that controls these changes. The major types of molecular processes that control cell differentiation involve cell signaling. Many of the signaling molecules that transmit information from cell to cell during cell differentiation control are called growth factors. Although the details of specific signal transduction pathways vary, these pathways often share the following general steps. A ligand produced by one cell binds to a receptor in the extracellular region of another cell, inducing a conformational change in the receptor. The shape of the receptor's cytoplasmic domain changes, and the receptor acquires enzymatic activity. The receptor then catalyzes reactions that phosphorylate other proteins, activating them. The cascade of phosphorylation reactions ultimately activates dormant transcription factors or cytoskeletal proteins, which contribute to the differentiation process in the target cell. Cells and tissues can differ in competence, which is variation in their ability to respond to external signals.

[0129] Signal induction refers to a cascade of signaling events during which a cell or tissue signals to another cell or tissue to influence its developmental fate. For example, the role of the lens in eye formation in different fish species has been studied and provides a striking example of signal induction.

[0130] Other important mechanisms fall into the category of asymmetric cell divisions that result in daughter cells having different developmental fates. Asymmetric cell division can occur due to asymmetrically expressed maternal cytoplasmic determinants or due to cell signaling. In the former mechanism, different daughter cells are generated during cytokinesis due to the uneven distribution of regulatory molecules in the parental cell; the unique cytoplasm inherited by each daughter cell results in a different differentiation pattern for each daughter cell.

[0131] Since every cell, regardless of cell type, has the same genome, cell type must be determined at the level of gene expression. While regulation of gene expression can occur through cis- and trans-regulatory elements including gene promoters and enhancers, the question is how this expression pattern is maintained over multiple generations of cell divisions. It turns out that epigenetic processes play a crucial role in regulating the decision to adopt a stem cell fate, a progenitor cell fate, or a mature cell fate.

[0132] Embryonic stem cells are able to self-renew and differentiate into the desired fate depending on their location in the body. Stem cell homeostasis is maintained by epigenetic mechanisms that are highly dynamic in regulating chromatin structure as well as specific gene transcription programs. Epigenetics has been used to refer to changes in gene expression that can be inherited through modifications that do not affect the DNA sequence. For example, the mammalian epigenome undergoes comprehensive remodeling during early stem cell development, which is required to ensure that cells are restricted to the desired lineage. There is evidence that maintaining the lineage commitment of stem cells is controlled by epigenetic mechanisms, such as but not limited to the regulation of DNA methylation, histone modifications, and ATP-dependent remodeling of chromatin structure.

[0133] In molecular biology, a transcription factor (or sequence-specific DNA binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA by binding to a specific DNA sequence. This, in turn, helps regulate the expression of genes near that sequence. This is essential in cell development, for example during embryogenesis.

[0134] Transcription factors work alone or in combination with other proteins in a complex by promoting (as activators) or blocking (as repressors) the recruitment of RNA polymerase (the enzyme that performs the actual transcription of genetic information from DNA to RNA) to specific genes. A defining feature of transcription factors is that they contain at least one DNA binding domain (DBD), which attaches to a specific DNA sequence adjacent to the gene they regulate. Transcription factors are generally classified into different families based on their DNA binding domain. Other proteins, such as but not limited to coactivators, chromatin remodelers, histone acetyltransferases, histone deacetylases, kinases, and methylases, although also essential for gene regulation, lack a DNA binding domain and, therefore, do not fall within the definition of a transcription factor. Transcription factors are one of the groups of proteins that read and interpret the genetic "blueprint" in DNA. They bind to DNA and help initiate a program that increases or decreases gene transcription. Therefore, they are essential for many important cellular processes. Transcription factors are involved in many important functions and biological roles, such as but not limited to the development of organisms.

[0135] Many transcription factors in multicellular organisms participate in development. In response to stimulation, these transcription factors turn on or off the transcription of appropriate genes, which in turn allows changes in cell morphology or activity required for cell fate determination and cell differentiation. For example, the Hox transcription factor family is important for appropriate body pattern formation in the diverse organisms from fruit flies to humans. Another example is the transcription factor encoded by the sex determining region Y (SRY) gene, which plays a major role in determining human sex.

[0136] Brain organoids

[0137] In the early stages of differentiation, brain organoids are mainly composed of multiple neural tube-like structures. Neural progenitor cells with SOX2 and PAX6 as marker genes form a VZ-like structure, which is the basis for neurogenesis in brain organoids. These neural progenitor cells proliferate while migrating and differentiating outward along the top surface of the neural tube-like structure. In the early stages of brain organoid differentiation, the cells migrating to the outer layer mainly include TBR2-positive intermediate progenitor cells, forming the SVZ region; radial glial cells form the outer SVZ (oSVZ) region; neurons migrate to the outer layer along the radial glial cells, preferentially forming CTIP2-positive deep neurons, and then forming SATB2-positive shallow neurons, forming the CP region; brain organoids that have undergone a long period of differentiation can have REELIN-positive, CUX1-positive, and BRN2-positive neurons in the first, second, and third layers of the cerebral cortex (Qian et al., 2016).

[0138] Cultivation method of the present invention

[0139] The method for culturing human brain organs of the present invention comprises:

[0140] (a) providing a pluripotent stem cell;

[0141] (b) culturing the pluripotent stem cells in suspension to form hES cell clusters;

[0142] (c) treating the hES cell clusters with a brain organoid culture medium for 1 to 2 days to obtain embryoid bodies, wherein the brain organoid culture medium contains a signaling pathway inhibitor;

[0143] (d) inducing and culturing the embryoid bodies for 16 days using a neural differentiation culture medium to obtain neurospheres containing neural progenitor cells, wherein the neural differentiation culture medium contains a signaling pathway inhibitor;

[0144] (e) maturing the neurospheres for 14 to 74 days to obtain brain organoids.

[0145] In another preferred embodiment, the step (e) comprises the following sub-steps:

[0146] (e1) culturing the neurospheres in N2B27 culture medium for 14 days;

[0147] (e2) Cultivating the cells in N2B27 medium containing Vitamin A (N2B27(VA+)) for 60-150 days.

[0148] Pharmaceutical composition

[0149] The present invention provides a pharmaceutical composition containing the brain organoid of the present invention.

[0150] The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient: the brain organoid described in the present invention.

[0151] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0152] As used herein, the components of a "pharmaceutically acceptable carrier" are substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., have a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0153] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration. The dosage form of the pharmaceutical composition of the present invention is an injection, a lyophilized preparation, a stem cell preparation, and a cell model. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0154] The effective amount of the active ingredient of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, a satisfactory effect can be obtained. For example, due to the urgency of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally. Generally, the pharmaceutical composition of the present invention can be administered by local injection or the like.

[0155] The pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0156] The pharmaceutically acceptable carrier in the present invention can be a biocompatible carrier or a medically degradable tissue engineering carrier, including but not limited to: PCL, PGA, polyethylene glycol, hyaluronic acid, allogeneic bone repair materials, xenogeneic bone repair materials, gelatin, collagen, and silk. The motor neuron-like cell group or spinal cord-specific neural precursor cell in the present invention can be loaded on tissue engineering carriers of different sizes and shapes, and prepared into a biomaterial for treating a specific motor neuron-like cell group-related disease through in vitro pre-culture.

[0157] The advantages of the present invention mainly include:

[0158] (a) The brain-like organoids obtained by the present invention have good consistency, and neurons can mature in 1 month. The brain-like organoids can reach mature electrophysiological functions after 3 months of culture. This is an efficient and stable brain-like organoid culture system.

[0159] (b) The brain organoids of the present invention can be well used in the study of neurodegenerative diseases, such as Alzheimer's disease, can simulate AD-related pathological features, and can functionally demonstrate neural network damage, and can serve as a new platform for drug screening.

[0160] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0161] Example 1 hiPSCs form EBs

[0162] 1. Observe the human pluripotent stem cell clones. The cultured clones should have smooth edges and no differentiated cells. When the human pluripotent stem cells cultured in a 35mm cell culture dish grow to 70-80% density, digestion operations can be performed.

[0163] 2. Aspirate the culture medium, wash the clones with PBS, add 1 mL of Accutase to a 35 mm culture dish, and incubate at 37°C for 8-12 minutes to digest the clones into single cells.

[0164] 3. Aspirate Accutase, add 1 mL of brain organoid starting culture medium (STEM FLEX, containing 10 μM Y27632), and gently tap the side wall of the culture dish to loosen the cells; then use a pipette to gently blow 5-6 times to disperse the clones into single cells, aspirate the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm for 3 minutes.

[0165] 4. Discard the supernatant and add 1 mL of brain organoid starting culture medium (STEM FLEX, containing 10 μM Y27632) to resuspend the cells.

[0166] 5. Pipette 10 μL of cell suspension into a hemacytometer and count; if the cell density is too high, dilute it 10-20 times before counting.

[0167] 6. According to the cell counting results, single cells were inoculated into a 96V well plate at a density of 9000 cells / well, 120 μL of culture solution was added to each well, and the 96V well plate was placed in a 5% CO2, 37°C incubator.

[0168] 7. After 24 hours, observe the formation of embryoid bodies (EBs) under a microscope and change the medium. Change the medium to the starting brain organoid culture medium every day and continue culturing for 2 days.

[0169] result

[0170] hPSCs (human pluripotent stem cells) digested into single-cell state were aggregated into clusters using a 96 V-bottom well plate. After two days of culture, cell clusters of uniform size were obtained in each well. Their morphology was observed to be uniform spheres under bright field observation ( Figure 1 B).

[0171] The expression of pluripotency marker genes was analyzed by immunofluorescence staining. It was found that the cell clusters of D2 expressed OCT4, NANOG and SOX2. Human pluripotent stem cells maintained a certain degree of pluripotency under 3D culture ( Figure 1 C).

[0172] During the cell clustering process from D0 to D2, three different culture media were compared: one was the hES culture medium containing KSR commonly used in the EB stage, one was the commercial hES culture medium STEMFLEX, and the other was the commercial hES culture medium mTeSR ( Figure 2 A); It was found that the morphology and uniformity of brain organoids were different during development after being cultured in different hES culture media from D0 to D2.

[0173] The morphology of brain organoids cultured in KSR was different at D16. The morphology of brain organoids cultured in STEMFLEX was typical of neuroepithelial tissue structure with clear edges at D16. The neuroepithelial characteristics of brain organoids cultured in mTeSR were not sufficient at D16 ( Figure 2 B). By immunofluorescence staining, the PAX6 positive ratio of neural progenitor cells and the number of neural rosettes were counted, and it was found that using STEMFLEX as the culture medium from D0 to D2 had a better effect on the neural differentiation of brain organoids ( Figure 2 CE).

[0174] Example 2 Neural induction of brain organoids

[0175] 1. On the second day after step 6 in Example 1, the culture medium in the 96V well plate was aspirated and the cell culture medium was replaced with hES culture medium containing SB431542 (10 μM) and Dorosomorphin (4 μM).

[0176] 2. Change the medium every two days and continue culturing from the 2nd day to the 6th day.

[0177] 3. On the 6th day, aspirate the original culture medium, add N2 culture medium containing SB431542 (10μM) and Dorosomorphin (4μM), transfer the brain organoids in the 96V well plate to a low-adsorption suspension culture dish using a 200μL yellow pipette tip with a cut end, and culture until the 8th day.

[0178] 4. On the 8th day, replace the culture medium with N2. Change the medium every other day.

[0179] 5. On the 16th day, replace the culture medium with N2B27 and place the culture plates on a horizontal shaker (speed 50-70 rpm). Note that the culture plates should be placed in a horizontal manner to avoid affecting the gas exchange of the culture plates. Change the medium every two days.

[0180] The culture medium used in the above steps is as follows:

[0181] hES culture medium: DMEM / F12 supplemented with NEAA, Glutamax, 20% KSR, 10 μM SB431542 and 4 μM Dorosomorphin.

[0182] N2 culture medium: DMEM / F12 supplemented with NEAA, Glutamax, and N2 supplement (Gibco).

[0183] N2B27: DMEM / F12 and Neuralbasal basal culture media were mixed in a 1:1 ratio, and NEAA, Glutamax, N2supplement (Gibco), and B27 supplement (Gibco) without Vitamin A were added.

[0184] result

[0185] On the 16th day of differentiation, the cells showed the characteristics of a neuroepithelial structure with translucent edges and pseudostratified layers ( Figure 3 A). Brain organoids from different batches and different iPSC cell lines all showed consistent structural features, indicating that this method has good stability and reproducibility.

[0186] Immunofluorescence staining was used to observe the internal structural characteristics on the 16th day, and SOX2-positive neural progenitor cells were mainly distributed, which aggregated to form a neural rosette structure ( Figure 3 B), indicating that the brain organoids at this period were in a typical neural differentiation stage.

[0187] Example 3 Maturation of brain organoids

[0188] 1. On day 30, replace the culture medium of the brain organoids with N2B27 (VA+) medium containing Vitamin A and continue to culture on a horizontal shaker at 70 rpm. Change the medium every 2-3 days for long-term culture.

[0189] 2. Collect samples on the 30th, 60th and 90th days, and observe and test the maturity of the brain organoids.

[0190] 3. Use high-throughput MEA method to detect electrophysiological conditions of brain-like organs at different culture time points.

[0191] Among them, the composition of N2B27 (VA+) culture medium is a 1:1 mixture of two basic culture media: DMEM / F12 and Neuralbasal, supplemented with NEAA, Glutamax, N2 supplement (Gibco), and B27 supplement (Gibco) containing Vitamin A.

[0192] result

[0193] By culturing brain organoids for a long time, the volume of brain organoids gradually increases ( Figure 4 A). We collected samples of brain organoids on the 30th, 60th, and 90th days for identification and analysis, and found that the brain organoids cultured for 30 days showed typical stratification of neural progenitor cell SOX2 and neuronal MAP2, similar to the stratification characteristics of the VZ and PP regions of the developing cerebral cortex ( Figure 4 B). In addition, the cerebral organoids on day 30 already had cells expressing the mature neuronal marker NEUN ( Figure 4 C), indicating that the brain organoids cultured in this system began to mature after about one month of culture. The brain organoids on days 60 and 90 showed clear layered characteristics of SOX2 and CTIP2, similar to the VZ and CP regions of the developing cortex ( Figure 4 D). By analyzing the electrophysiological characteristics of brain organoids through MEA, it was found that the average activation rate of electrodes gradually increased over time ( Figure 4 E). In the 90-day-old brain organoids, co-localization or close positional relationship between presynaptic membrane proteins and postsynaptic density proteins can be observed ( Figure 4F), indicating the formation of synapses between neurons in the brain-like organoids; further MEA detection of the electrical activity of the neural network revealed that the brain-like organoids had synchronous electrical signal emission, indicating the electrical activity of the neural network ( Figure 4 G).

[0194] Example 4 Establishment of brain organoid disease model

[0195] 1. Culture the iPSC cell lines FAD02-1, FAD02-3 and FAD02-4 derived from familial Alzheimer's disease (AD) patients established in our laboratory, as well as the healthy control iPSC cell lines DC60-3, DC87-3 and FC02-3.

[0196] 2. Differentiate and culture brain organoids as described in Examples 1-3.

[0197] 3. Collect FAD brain organoid samples on days 30, 60, and 90 for comparative analysis with the healthy control group. Detect classic AD pathological features: pTAU and Aβ plaques, etc.

[0198] result

[0199] The cerebral cortex, hippocampus and other related brain regions of AD will show a decrease in synaptic density (DeKosky and Scheff, 1990). The reduction of synapses is related to the cognitive impairment of AD patients (Dickson et al., 1995). Neuronal apoptosis is an important factor causing synaptic loss, and neurofibrillary tangles and Aβ plaques are also important factors affecting synaptic maintenance.

[0200] Through immunofluorescence staining experiments, the healthy group brain organoids and FAD brain organoids were analyzed to detect the expression of presynaptic membrane marker SYP and postsynaptic membrane marker PSD95.

[0201] The results showed that there were SYP-positive signals and PSD95-positive signals in the healthy control group, and the two proteins were co-localized or closely related. However, in the FAD brain organoids, it was difficult to observe the expression of PSD95 ( Figure 5 A).

[0202] The statistical analysis results showed that the proportion of SYP fluorescence signals in the healthy control group was significantly higher than that in the FAD brain organoids, and the proportion of PSD95 also tended to be higher than that in the FAD group ( Figure 5 B, C). This indicates that the neuronal synapses in the FAD brain organoids were damaged to a certain extent.

[0203] The maintenance of synapses is very important for the conduction of electrical signals between neurons and the establishment of neural networks. High-density MEA is a detection chip with high-density electrode arrangement, which is a detection method for high-throughput collection of neuronal electrical signals. MEA is an emerging cell electrophysiological detection method, and there are currently few studies on the electrophysiology of AD brain organoids. By analyzing the electrodes of synchronous discharge signals through MEA and observing the formation of neural networks, it was found that the brain organs of the healthy control group can form synchronous discharge signals with neural network characteristics ( Figure 4 G), while the formation of neural networks is difficult to observe in FAD ( Figure 5 D). This result is also consistent with the loss of synapses in FAD brain organoids. Using AT8 antibody to label phosphorylated TAU protein, neurofibrillary tangles were observed in FAD brain organoids at D90 ( Figure 5 E and F). Using 6E10 antibody to label Aβ, it was found that Aβ aggregation could be detected in FAD brain organoids as early as D30, and the fluorescence area of ​​6E10 was statistically significantly higher than that of the healthy control group. More Aβ aggregation and Aβ plaques were observed at D60 and D90 ( Figure 5 G. Figure 5 H. Figure 5 I) simulates the accumulation of Aβ plaques, a typical pathological feature in the brain of AD patients.

[0204] discuss

[0205] Although early studies used neural stem cells for 3D culture and simulated the brain development process to a certain extent through self-renewal and self-assembly, they did not reproduce the structure of the cerebral cortex layer very well. This is mainly because the isolated primary tissue cells are not suitable starting cells, and it is more appropriate to use pluripotent stem cells (Karus et al., 2014). In 2005, the Sasai team obtained neural precursor cells from mouse embryonic stem cells through serum-free suspension embryonic body culture (SFEB culture), and obtained a higher neural differentiation efficiency by adding small molecule compounds related to neural differentiation signaling pathways (Kiichi et al., 2005). Compared with the in vitro culture of mouse embryonic stem cells, the in vitro culture of human embryonic stem cells is not easy to maintain. After digestion and dispersion, human embryonic stem cells are more fragile, so it is necessary to add the ROCK signaling pathway inhibitor Y-27632 during the EB formation process to improve the survival efficiency of human embryonic stem cells (Watanabe et al., 2007). The classic method for pluripotent stem cells to form EBs is the hanging drop method. The curved surface of the drop formed by the gravity of the cell hanging drop helps embryonic stem cells to aggregate, enhances direct contact between cells, and promotes EB formation (Dang et al., 2002; Foty, 2011). In order to improve the efficiency of human embryonic stem cell EB formation, the Sasai team tried to use a U-shaped bottom low-adsorption 96-well plate, which successfully improved the efficiency of EB formation; then the researchers attached these EBs to the adherent culture dish to continue neural differentiation. Compared with previous methods, larger neural rosettes were formed, and the cavity structure characteristics of the neural tube were more prominent (Eiraku et al., 2008). The Sasai team explored the possibility of pluripotent stem cells forming 3D tissues under in vitro differentiation conditions, accumulated valuable experience, and laid an important foundation for studying the formation and cultivation of brain-like organs.

[0206] In 2013, Lancaster et al. established a method for culturing 3D brain organoids based on human pluripotent stem cells. The brain organoids cultured by this method contain the characteristics of the dorsal region and the ventral forebrain region, and can develop into tissues with brain region characteristics such as the eye, hippocampus, thalamus and midbrain. The method is to digest human induced pluripotent stem cells to form single cells, and then spontaneously aggregate to form embryoid bodies, and then induce the embryoid bodies to form neural tubes, so that they have the fate of differentiating into neural ectoderm. The differentiating brain organoids are embedded in Matrigel at an early stage (around the 14th day) and placed in a bioreactor to better absorb oxygen and nutrients and further mature. In the bioreactor, the brain organoids gradually develop. After one month of differentiation, it is observed that the interior of the brain organoids is filled with neural tube structures of varying sizes, composed of neural progenitor cells and differentiated neurons; after a differentiation time of 3 months, it can be observed that the cultured brain organoids have cortical-like structures. Through the early-appearing neuronal marker genes, the late-appearing neuronal marker genes, and the marker genes of neural progenitor cells, it can be observed that the induced brain organoids have at least three layers: the ventricular zone (VZ), the lateral ventricular zone (SVZ), and the cortical plate zone (CP) (Lancaster et al., 2013). The method of Lancaster et al. is different from the previous method of directional induction by Eiraku et al. (Eiraku et al., 2008). Lancaster et al. did not add factors that induce specific brain regions in the process of inducing neural progenitor cells, and the nerves differentiated spontaneously. In addition, the extracellular matrix Matrigel coated outside the brain organoid has an uncertain animal-derived component, which contains rich growth factors and nutrients, so that the brain organoid can develop towards different brain regions during the growth and development process, showing a certain diversity. The emergence of this method marks that researchers have truly entered the era of 3D brain organoid research.

[0207] In order to further improve and optimize the culture method of brain organoids and explore the characteristics of neural differentiation of brain organoids, Paola's team used the method published by Lancaster to culture brain organoids for a long time for 9 to 13 months. They found that as time went on, the types of neural cells in the brain organoids gradually became richer, with both cortical-related neural cells and retinal-related cells appearing; at the same time, a neural activity network was established (Quadrato et al., 2017). On the other hand, due to the uncertainty of Matrigel components, the culture of brain organoids increased the instability factor, which also added limitations to the precise induction of brain organoids. The bioreactor used in Lancaster's study has the function of automatically rotating and stirring the culture medium, increasing gas exchange during the culture process and providing sufficient oxygen for brain organoids. Qian et al. used 3D printing technology to design and develop a mini rotating bioreactor, SpinΩ; the culture device is the size of a 12-well plate, and the capacity of each well is sufficient for a brain organoid to grow and develop in it (Qian et al., 2016). In order to reduce the instability of the brain organoid culture process, researchers began to consider using small molecule compounds to induce brain organoids in a targeted manner. Factors specific to the brain region were added during the embryoid body culture stage for induction for 7 days, and then the brain organoids were embedded in Matrigel for another 7 days. The Matrigel wrapped around the brain organoids was removed by repeated blowing with a pipette, and then transferred to SpinΩ for culture. Since the induction factors were added during the culture process and the Matrigel wrapping time was shortened, the stability of the system was increased, and the forebrain, midbrain, and hypothalamus organoids could be obtained by induction of different factors.

[0208] Kadoshima et al. digested human pluripotent stem cells into single cells and aggregated them to form embryoid bodies. In the first 18 days of suspension culture, Wnt signaling pathway inhibitor IWR1e and TGFβ signaling pathway inhibitor SB431542 were added to differentiate pluripotent stem cells into the fate of the cerebral cortex (Kadoshima et al. 2013). After 18 days, the brain organoids were transferred to a high oxygen incubator containing 40% oxygen and 5% carbon dioxide for culture, and 1% Matrigel and 10% FBS were added to the neural differentiation culture medium, so that the brain organoids could obtain sufficient oxygen and nutrients during the long-term culture process and maintain a certain proliferation and differentiation ability. After 56 days, the neural differentiation culture medium increased the Matrigel concentration to 2% and added B27 to promote the maturation of neurons. After 3 months of culture, this method can obtain four layers of structures similar to the human cerebral cortex, namely VZ, SVZ, SP and CP areas. Paola's team optimized the above method and verified through single-cell sequencing and omics analysis that the brain-like organs differentiated by this method have high reproducibility between different batches, which to a certain extent solved the problem of instability of the brain-like organoid differentiation system (Velasco et al., 2019).

[0209] Cultivating brain organoids in vitro is a relatively long-term process. In previous studies, long-term cultivation of brain organoids is prone to cell necrosis and apoptosis due to the lack of sufficient oxygen and nutrients inside. In order to solve this problem, Giandomenico et al. cut the brain organoids cultured for about 2 months into thin slices, floated them on the culture medium, and cultured them for a long time using the liquid-air interface culture method, which can be cultured for up to 3 months (Giandomenico et al., 2019). This method has better solved the problem of cell necrosis and apoptosis inside brain organoids in previous long-term culture. In addition, the maintenance of neural stem cells plays an important role in the long-term culture of brain organoids. The proliferation, migration, cell-cell interaction and formation of neural circuits of neural stem cells play an important role in the development and maturation of brain organoids. The method of Giandomenico et al. cannot maintain neural stem cells. After long-term culture, the neural stem cells in the brain organoid slices gradually differentiated, and the brain organoids lost the VZ and SVZ structures; this may be because the brain organoids at the time of slicing have passed the optimal period of VZ and SVZ differentiation. When the brain organoids were differentiated to the stage of forming VZ and SVZ (differentiation for about 1 month), the research team of Guo-li Ming cut them into 500μm slices, took the middle part of the brain organoid slices and continued to culture them. The brain organoid slices had more sufficient contact with the culture medium and oxygen, cultured the brain organoid slices for a long time, and thinned the brain organoids regularly to keep them 500μm thick. This method successfully reduced cell apoptosis and maintained the structure of the cortical VZ and SVZ (Qian et al., 2020). This culture method allows researchers to further perform precise operations on the culture of brain organoids.

[0210] The brain-like organ obtained by the present invention has good consistency, and neurons can mature in 1 month. The brain-like organ reaches the maturity of electrophysiological function in 3 months of culture. It is an efficient and stable brain-like organ culture system. For the study of neurodegenerative diseases, it is difficult for researchers to directly use human brain tissue as experimental and research objects due to the difficulty in obtaining human brain tissue and ethical restrictions; there are also certain limitations in the use of animal models for research, that is, there are certain species differences between animals and humans, and the results of scientific research based on animal models are not easy to transform. Using iPS technology to establish pluripotent stem cells with the genetic background of patients and differentiate them into brain-like organs, and applying the brain-like organ culture method to neurodegenerative diseases, such as Alzheimer's disease, can simulate AD-related pathological characteristics and can functionally demonstrate neural network damage, which can be used as a new platform for drug screening.

[0211] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A brain organoid neural precursor cell population, characterized in that: The brain organoid neural precursor cell population can be differentiated into a brain organoid, The neural precursor cell population of the brain organoid expresses the molecular markers SOX2 and PAX6; Among them, ≥95% of the cells in the neural precursor cell population of the brain organoid express SOX2, and ≥95% of the cells express PAX6.

2. A method for preparing neural precursor cells of brain organoids, characterized in that: Includes steps: (a) providing a pluripotent stem cell; (b) culturing the pluripotent stem cells in suspension to form hES cell clusters; (c) treating the hES cell clusters with a brain organoid culture medium for 1 to 2 days to obtain embryoid bodies (EBs), wherein the brain organoid culture medium contains a signaling pathway inhibitor; (d) Inducing and culturing the embryoid bodies for 16 days using a neural differentiation culture medium to obtain neural progenitor cells of brain organoids, wherein the neural differentiation culture medium contains a signaling pathway inhibitor.

3. The method according to claim 2, characterized in that The brain organoid neural precursor cells have the following characteristics: The morphological and structural characteristics of brain-like organs are highly repeatable and consistent, with more than 60%, preferably more than 78%, and optimally more than 85% of the cells expressing neural precursor cell markers; and more than 20%, preferably more than 50% of the cells expressing neuronal markers.

4. A brain organoid, characterized in that: The brain organoid is differentiated from the neural precursor cells of the brain organoid according to claim 1.

5. A method for culturing the brain organoid according to claim 4, comprising: (a) providing a pluripotent stem cell; (b) culturing the pluripotent stem cells in suspension to form hES cell clusters; (c) treating the hES cell clusters with a brain organoid culture medium for 1 to 2 days to obtain embryoid bodies, wherein the brain organoid culture medium contains a signaling pathway inhibitor; (d) inducing and culturing the embryoid bodies for 16 days using a neural differentiation culture medium to obtain neurospheres containing neural progenitor cells, wherein the neural differentiation culture medium contains a signaling pathway inhibitor; (e) maturing the neurospheres for 14 to 74 days to obtain brain organoids.

6. A use of the brain organoid precursor cell population according to claim 1 or the brain organoid according to claim 4, characterized in that: Used for preparing a pharmaceutical composition for preventing or treating neurodegenerative diseases.

7. A use of the brain organoid precursor cell population according to claim 1 or the brain organoid according to claim 4, characterized in that: Used to construct research models for neurodegenerative diseases.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (1) an effective amount of the neural precursor cell population of the brain organoid according to claim 1 or the brain organoid according to claim 4; and (2) A pharmaceutically acceptable carrier.

9. A method for constructing a neurodegenerative disease research model, the method comprising using the brain organoid neural precursor cell population as described in claim 1 or the brain organoid as described in claim 4 to construct a neurodegenerative disease research model.

10. A method for preventing or treating a neurodegenerative disease, the method comprising administering the neural precursor cell population of the brain organoid of claim 1 or the brain organoid of claim 4, or the pharmaceutical composition of claim 8 to a subject in need thereof.

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