Method for preparing cerebellar organoids

Abstract

The present invention relates to a method for preparing cerebellar organoids, comprising: (A) a selective differentiation step into hindbrain tissue, in which embryoid bodies formed from pluripotent stem cells are cultured in the presence of a hindbrain tissue induction medium containing an inhibitor of endogenous WNT secretion and a WNT signaling agent; (B) a selective differentiation step into dorsal hindbrain tissue, in which the embryoid bodies are cultured in the presence of a dorsal hindbrain tissue induction medium containing an inhibitor of the sonic hedgehog (SHh) signaling pathway; and (C) a basolateral-apical polarization step, in which polarization of the embryoid bodies is induced in the presence of a basolateral-apical induction medium containing an extracellular matrix-based hydrogel.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing cerebellar organoids. [Background technology]

[0002] Organoids, also known as "organ analogs" or "organ analogs," are organ-specific cell aggregates produced by 3D culture of cells isolated from stem cells or organ-derived cells, and then aggregation and recombination. Organoids contain specific cells of the model organ, reproduce specific organ functions, and can be spatially organized to resemble the actual organ.

[0003] In particular, organoids that mimic most human organs can be created using pluripotent stem cells, which can differentiate into any cell type in the human body. Among these, brain organoids are attracting much attention as an in vitro culture model for mimicking various intractable central nervous system diseases, elucidating their pathogenesis, and developing treatments.

[0004] For example, Lancaster et al. prepared cerebral organoids using human induced pluripotent stem cells (Non-Patent Document 1). However, cerebral organoids prepared in this way have the problem that various organs such as the cerebrum, midbrain, and retina are mixed in a single organoid and are of different sizes.

[0005] Most intractable brain diseases occur specifically in specific brain regions, and organoids that have the characteristics of other brain regions that are not affected by the target disease or that have a mixture of characteristics from various regions cannot accurately mimic the brain disease. Therefore, in order to utilize brain organoids in research into the pathogenesis of intractable brain diseases through modeling and research into the development of treatments, it is essential to develop a technology to generate region-specific brain organoids that can mimic specific brain regions with high purity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Madeline A. Lancaster, Juergen A. Knoblich, Generation of Cerebral Organoids from Human Plurpotent Stem Cells, Nat Protoc.2014 October;9(10):2329-2340. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a method for producing cerebellar organoids from embryoid bodies derived from pluripotent stem cells.

[0008] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned above will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0009] One embodiment of the present invention includes: (A) a selective differentiation step into hindbrain tissue, in which embryoid bodies formed from pluripotent stem cells are cultured in the presence of a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agent; (B) A selective differentiation step into dorsal hindbrain tissue, in which the embryoid bodies are cultured in a dorsal hindbrain tissue induction medium containing a sonic hedgehog (SHh) signaling inhibitor; and (C) A method for preparing cerebellar organoids is provided, comprising a basolateral-apical polarization step in which polarization of the embryoid bodies is induced in the presence of a basolateral-apical induction medium containing an extracellular matrix-based hydrogel. [Effects of the Invention]

[0010] According to one embodiment of the present invention, when using the method for preparing cerebellar organoids, cerebellar organoids that mimic the cerebellar region with high purity can be produced, which can be used to contribute to the development of research into the pathogenesis of cerebellar-related diseases and research into the development of treatments. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of forebrain region-specific gene expression depending on the concentration of a WNT signaling agent in a hindbrain tissue induction medium according to an example. [Figure 2] FIG. 2 shows the results of hindbrain region-specific gene expression depending on the concentration of a WNT signaling agent in a hindbrain tissue induction medium according to an example. [Figure 3] FIG. 3 shows the results of spinal cord region-specific gene expression depending on the concentration of a WNT signaling agent in a hindbrain tissue induction medium according to an example. [Figure 4] FIG. 4 shows the results of expression of dorsal hindbrain region-specific genes depending on the concentration of the SHh signaling inhibitor in the dorsal hindbrain tissue induction medium of the example. [Figure 5] FIG. 5 shows the developmental state of embryoid bodies on day 7 after selective differentiation into dorsal hindbrain tissue depending on the concentration of the SHh signaling inhibitor in the dorsal hindbrain tissue induction medium of the Example. [Figure 6] FIG. 6 shows the chronological process of producing cerebellar organoids according to an embodiment, starting from embryoid body formation (Day 0). [Figure 7] FIG. 7 shows the developmental process of embryoid bodies during the production process of cerebellar organoids according to an embodiment, observed through a phase-contrast microscope. [Figure 8] FIG. 8 shows the results of immunofluorescence staining of cerebellar organoids on the 35th day (Day 35) from the day on which embryoid bodies were formed by the cerebellar organoid preparation method according to the embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below.

[0013] While the present invention is susceptible to various modifications and embodiments, specific embodiments are shown by way of example in the drawings and will be described in detail in the detailed description.

[0014] However, it should be understood that the present invention is not limited to the specific embodiment, but includes all modifications, equivalents, and alternatives included within the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related publicly known technology may obscure the gist of the present invention, the detailed description will be omitted.

[0015] The terms used in this application are merely used to describe specific embodiments and are not intended to limit the present invention. The singular expressions include the plural expressions unless the context clearly indicates otherwise.

[0016] In the present invention, the terms "comprise" or "have" and the like are intended to specify the presence of any feature, number, step, operation, component, part, or combination thereof described in the specification, and should be understood not to preclude the presence or possibility of addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0017] One embodiment of the present invention includes: (A) a selective differentiation step into hindbrain tissue, in which embryoid bodies formed from pluripotent stem cells are cultured in the presence of a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agent; (B) A selective differentiation step into dorsal hindbrain tissue, in which the embryoid bodies are cultured in a dorsal hindbrain tissue induction medium containing a sonic hedgehog (SHh) signaling inhibitor; and (C) A method for preparing cerebellar organoids is provided, comprising a basolateral-apical polarization step in which polarization of the embryoid bodies is induced in the presence of a basolateral-apical induction medium containing an extracellular matrix-based hydrogel.

[0018] According to one embodiment of the present invention, the embryoid bodies formed from the pluripotent stem cells through the selective differentiation step (A) into hindbrain tissue may be differentiated into cell aggregates that mimic hindbrain characteristics with high purity. Specifically, the cell aggregates that mimic hindbrain characteristics may be organoids that mimic hindbrain characteristics with high purity. The cell aggregates or organoids that mimic hindbrain characteristics with high purity may refer to a state in which the inclusion of tissues that exhibit characteristics of other brain regions such as the cerebrum, striatum, and midbrain is minimized.

[0019] According to one embodiment of the present invention, the selective differentiation step (A) into hindbrain tissue may induce differentiation of the embryoid body into neuroectoderm while simultaneously inducing uniform patterning of the embryoid body into hindbrain tissue throughout the embryoid body.

[0020] According to one embodiment of the present invention, the endogenous WNT secretion inhibitor may be a substance that inhibits the secretion of a WNT signaling substance naturally expressed in the embryoid body. The endogenous WNT secretion inhibitor may suppress the activation of the WNT signaling system by WNT proteins spontaneously expressed in the embryoid body, thereby controlling the uncontrollable, heterogeneous expression of the WNT signaling system. Therefore, by applying the endogenous WNT secretion inhibitor in combination with a WNT signaling agonist described below, the embryoid body may selectively and uniformly differentiate into the hindbrain. The endogenous WNT secretion inhibitor may include a substance that can function as a porcupine O-acyltransferase (PORCN) inhibitor. Specifically, the endogenous WNT secretion inhibitor may include at least one selected from the group consisting of IWP-2, LGK-974, ETC-159, GNF6231, WNT-C59, and WNT974. More specifically, the endogenous WNT secretion inhibitor may include IWP-2. More specifically, the endogenous WNT secretion inhibitor can effectively neutralize the activity of the WNT signaling pathway by suppressing the secretion of naturally expressed WNT signaling substances, without affecting its activity when treated with a WNT signaling agonist. That is, the endogenous WNT secretion inhibitor effectively controls the heterogeneous formation of the WNT signaling pathway by inhibiting the process in which endogenous WNT proteins spontaneously expressed in embryoid bodies are transformed from the endoplasmic reticulum into a form that is secreted extracellularly. Conversely, substances such as IWR-1-endo may reduce the effectiveness of WNT signaling agonists, as described below, by suppressing signaling in the lower cascades of the WNT signaling pathway.

[0021] According to one embodiment of the present invention, the hindbrain tissue induction medium may contain an endogenous WNT secretion inhibitor in a concentration range that exhibits an effect equivalent to that of IWP-2 in the concentration range of 0.1 μM to 2 μM. Specifically, the hindbrain tissue induction medium may contain an endogenous WNT secretion inhibitor in a concentration range that exhibits an effect equivalent to that of IWP-2 in the concentration range of 0.5 μM to 2 μM, 0.5 μM to 1.5 μM, or 0.8 μM to 1.2 μM. Furthermore, according to one embodiment of the present invention, the hindbrain tissue induction medium may contain IWP-2 in the concentration range of the endogenous WNT secretion inhibitor. When the concentration of the endogenous WNT secretion inhibitor is within the above range, the formation of the WNT signaling pathway spontaneously expressed in the embryoid bodies may be effectively suppressed. Specifically, when the concentration of the endogenous WNT secretion inhibitor is below the above range, the effect of uncontrollable heterogeneous expression regulation of the WNT signaling pathway is minimal and may result in the formation of a heterogeneous WNT signaling pathway, whereas when the concentration of the endogenous WNT secretion inhibitor exceeds the above range, excessive inhibition of the WNT signaling pathway may result in reduced cell division and / or reduced cell viability.

[0022] According to one embodiment of the present invention, the WNT signaling agent may confer hindbrain region specificity uniformly throughout the embryoid body. That is, as described above, the endogenous WNT secretion inhibitor suppresses uncontrollable production of WNT signaling substances in the embryoid body, while the WNT signaling agent may regulate WNT signal intensity uniformly throughout the embryoid body, thereby achieving uniform hindbrain specificity throughout the embryoid body.

[0023] According to one embodiment of the present invention, the WNT signaling agonist may include at least one of a protein-based WNT signaling agonist and a compound-based WNT signaling agonist. Specifically, the protein-based WNT signaling agonist may include at least one selected from the group consisting of Wnt Family Member 1 (WNT-1), Wnt Family Member 3A (WNT-3a), and R-spondin-1 (RSPO1). The compound-based WNT signaling agonist may also include a substance that exhibits the mechanism of GSK-3 beta inhibition, specifically at least one selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, Kenpaullone, AR-AO144-18, TDZD-8, SB216763, BIO, TWS-119, and SB415286. Specifically, the WNT signaling agonist may be CHIR99021.

[0024] According to one embodiment of the present invention, the concentration of the WNT signaling agent in the hindbrain tissue induction medium may be optimized to induce selective differentiation into the hindbrain. Specifically, the concentration of the WNT signaling agent for inducing selective differentiation of the embryoid bodies into the hindbrain may be as follows:

[0025] According to one embodiment of the present invention, the hindbrain tissue induction medium may contain a WNT signaling agonist in a concentration range that exhibits an effect equivalent to that of CHIR99021 in the concentration range of more than 1 μM to less than 5 μM. Specifically, the hindbrain tissue induction medium may contain a WNT signaling agonist in a concentration range that exhibits an effect equivalent to that of CHIR99021 in the concentration range of 1.5 μM to 4.5 μM, 1.5 μM to 4 μM, 1.5 μM to 3 μM, or 1.5 μM to 2.5 μM. More specifically, the hindbrain tissue induction medium may contain the WNT signaling agonist at a concentration that specifically expresses at least one gene selected from LMX1A and LMX1B. Furthermore, according to one embodiment of the present invention, the hindbrain tissue induction medium may contain CHIR99021 in the concentration range of the WNT signaling agonist. When the concentration of the WNT signaling activator is within the above range, the expression of hindbrain-specific genes (e.g., LMX1A and / or LMX1B genes) may be effectively and significantly increased, and the embryoid bodies may be differentiated into a uniform cell population with hindbrain specificity. When the concentration of the WNT signaling activator is lower than the above range, the expression of forebrain-specific genes other than the hindbrain (e.g., FOXG1, LHX2, and / or DLX2 genes) may be increased. When the concentration of the WNT signaling activator is higher than the above range, the expression of spinal cord-specific genes other than the hindbrain (e.g., HOXB4, HOXC9, and / or HOXA1 genes) may be increased.

[0026] According to one embodiment of the present invention, at least one gene of LMX1A and LMX1B may be expressed in the selective differentiation step (A) into hindbrain tissue.

[0027] According to one embodiment of the present invention, the pluripotent stem cells may be human-derived embryonic stem cells or human-derived induced pluripotent stem (iPS) cells. The pluripotent stem cells refer to pluripotent stem cells that can differentiate into three germ layers: endoderm, mesoderm, and ectoderm, and may include embryonic stem cells, induced pluripotent stem cells (iPS), and adult stem cells that have the above abilities.

[0028] According to one embodiment of the present invention, the selective differentiation step (A) into hindbrain tissue may include: a1) forming embryoid bodies from pluripotent stem cells in an embryoid body formation medium; and a2) adding a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agent to the embryoid body formation medium.

[0029] According to one embodiment of the present invention, the medium for embryoid body formation may be a liquid medium that allows for 3D culture of the pluripotent stem cells. Furthermore, the medium for embryoid body formation may contain various additives known for culturing pluripotent stem cells depending on the purpose.

[0030] According to one embodiment of the present invention, the hindbrain tissue induction medium may contain known culture media and additives that enable the culture of embryoid bodies, and may further contain the endogenous WNT secretion inhibitor and the WNT signaling agent.

[0031] According to one embodiment of the present invention, the volume of the hindbrain tissue induction medium added may be 0.1 to 2 times the volume of the embryoid body formation medium. More specifically, the volume of the hindbrain tissue induction medium added may be the same as the volume of the embryoid body formation medium.

[0032] According to one embodiment of the present invention, the selective differentiation step (A) into hindbrain tissue may further include a step a3) of removing a portion of the whole medium and adding the hindbrain tissue induction medium every 1 to 3 days. Specifically, the step a3) may involve removing an equal amount of the hindbrain tissue induction medium added in step a2) and adding an equal amount of the hindbrain tissue induction medium approximately every 2 days. This may allow the embryoid bodies to be supplied with nutrients, allowing the endogenous WNT secretion inhibitor and the WNT signaling agonist to effectively act.

[0033] According to one embodiment of the present invention, the selective differentiation step (A) into hindbrain tissue may be carried out for a period of 4 to 10 days. Specifically, the selective differentiation step (A) into hindbrain tissue may be carried out for a period of 5 to 8 days, a period of 5 to 7 days, or a period of about 6 days.

[0034] According to one embodiment of the present invention, through the selective differentiation step (B) into dorsal hindbrain tissue, the embryoid bodies derived from pluripotent stem cells may be differentiated into cell aggregates that mimic dorsal hindbrain characteristics with high purity. Specifically, the cell aggregates that mimic dorsal hindbrain characteristics may be organoids that mimic dorsal hindbrain characteristics with high purity. The cell aggregates or organoids that mimic dorsal hindbrain characteristics with high purity may refer to a state in which the inclusion of tissues that exhibit characteristics of other brain regions such as the cerebrum, striatum, and midbrain is minimized.

[0035] According to one embodiment of the present invention, the selective differentiation step (B) into dorsal hindbrain tissue may induce differentiation of the embryoid body into neuroectoderm while simultaneously inducing uniform patterning of the embryoid body into dorsal hindbrain tissue throughout the embryoid body.

[0036] According to one embodiment of the present invention, the embryoid bodies derived from the pluripotent stem cells may be embryoid bodies differentiating into hindbrain tissue. Specifically, in the selective differentiation step (B) into dorsal hindbrain tissue, embryoid bodies differentiating into hindbrain tissues such as the pons and cerebellum may be induced to be specific to dorsal hindbrain tissue during the differentiation process, thereby inducing them to have higher cerebellar specificity.

[0037] According to one embodiment of the present invention, the selective differentiation step (B) into dorsal hindbrain tissue may include a step of adding the dorsal hindbrain tissue induction medium to a medium in which the embryoid bodies derived from the pluripotent stem cells are cultured. Specifically, the medium in which the embryoid bodies derived from the pluripotent stem cells are cultured may contain a hindbrain tissue induction medium. Specifically, the selective differentiation step (B) into dorsal hindbrain tissue may be performed in a manner overlapping with the treatment of embryoid bodies differentiating into a specific tissue. More specifically, the selective differentiation step (B) into dorsal hindbrain tissue may be performed in a manner overlapping with a selective differentiation step into hindbrain tissue using a hindbrain tissue induction medium. In this case, the hindbrain tissue induction medium may contain an inhibitor of endogenous WNT secretion and a WNT signaling agonist.

[0038] According to one embodiment of the present invention, the SHh signaling inhibitor may be a substance that inhibits the SHh signaling pathway that is naturally activated in some cells within the embryoid body. The SHh signaling inhibitor may inhibit the activation of the SHh signaling pathway by proteins that generate SHh signals spontaneously expressed within the embryoid body, thereby inhibiting heterogeneous and uncontrollable activation of the SHh signaling pathway. In other words, the inhibition of endogenous SHh signals by the SHh signaling inhibitor may cause the embryoid body to selectively and uniformly differentiate into dorsal hindbrain tissue.

[0039] According to one embodiment of the present invention, the SHh signaling inhibitor may include at least one selected from the group consisting of cyclopamine, itraconazole, robotnikinin, cerulenin, and GANT 61. Specifically, the SHh signaling inhibitor may be cyclopamine.

[0040] According to one embodiment of the present invention, the dorsal hindbrain tissue induction medium may contain an SHh signaling inhibitor in a concentration range that exhibits an effect equivalent to that of cyclopamine in the concentration range of more than 10 μM to less than 30 μM. Specifically, the dorsal hindbrain tissue induction medium may contain an SHh signaling inhibitor in a concentration range that exhibits an effect equivalent to that of cyclopamine in the concentration range of more than 10 μM to less than 30 μM, 12 μM to less than 30 μM, 12 μM to 30 μM, 15 μM to 25 μM, or 20 μM to 25 μM. More specifically, the dorsal hindbrain tissue induction medium may contain the SHh signaling inhibitor at a concentration that specifically expresses at least one gene selected from the group consisting of PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2. Furthermore, the dorsal hindbrain tissue induction medium may contain cyclopamine in the concentration range of the SHh signaling inhibitor. When the concentration of the SHh signaling inhibitor is within the above range, the expression of dorsal hindbrain-specific genes (e.g., PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and / or SKOR2) may be effectively and significantly increased, and the embryoid bodies may uniformly differentiate into a cell population with dorsal hindbrain specificity. When the concentration of the SHh signaling inhibitor is below the above range, the expression of the dorsal hindbrain-specific genes may be minimized. Furthermore, when the concentration of the SHh signaling inhibitor is above the above range, excessive concentrations of the SHh signaling inhibitor may suppress cell division within the embryoid bodies, resulting in the embryoid bodies dying before they can develop to the required extent.

[0041] According to one embodiment of the present invention, in the step (B) of selectively differentiating into dorsal hindbrain tissue, at least one gene selected from the group consisting of PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2 may be expressed.

[0042] According to one embodiment of the present invention, the step of selectively differentiating into dorsal hindbrain tissue may further include the step of b1) adding the dorsal hindbrain tissue induction medium to the medium in which the embryoid bodies are cultured.

[0043] According to one embodiment of the present invention, the selective differentiation step into dorsal hindbrain tissue may be performed in an overlapping manner with the selective differentiation step into hindbrain tissue. Specifically, during the period in which the selective differentiation step into dorsal hindbrain tissue is performed in an overlapping manner with the selective differentiation step into hindbrain tissue, the dorsal hindbrain tissue induction medium may further contain the SHh signaling inhibitor described above.

[0044] According to one embodiment of the present invention, the amount of the dorsal hindbrain tissue induction medium added may be 0.1 to 2 times the volume of the medium during culture. More specifically, the amount of the dorsal hindbrain tissue induction medium added may be the same volume as the medium during culture.

[0045] According to one embodiment of the present invention, the selective differentiation step into dorsal hindbrain tissue may further include the step of (b2) removing a portion of the entire medium and adding the dorsal hindbrain tissue induction medium every 1 to 3 days. Specifically, the step of (b2) may involve removing an amount of medium equal to the amount of dorsal hindbrain tissue induction medium added in step (b1) approximately every two days and replacing the same amount of dorsal hindbrain tissue induction medium. This may allow nutrients to be supplied to the embryoid bodies, allowing the SHh signaling inhibitor to effectively act. Furthermore, if the selective differentiation step into hindbrain tissue overlaps, the SHh signaling inhibitor may effectively act together with the endogenous WNT secretion inhibitor and the WNT signaling activator.

[0046] According to one embodiment of the present invention, the selective differentiation step (B) into dorsal hindbrain tissue may be carried out for a period of 4 to 10 days. Specifically, the selective differentiation step (B) into dorsal hindbrain tissue may be carried out for a period of 5 to 8 days, a period of 5 to 7 days, or about 6 days.

[0047] According to one embodiment of the present invention, the selective differentiation step (B) into dorsal hindbrain tissue may be carried out for a period of 4 to 10 days starting from 3 to 5 days after the selective differentiation step (A) into hindbrain tissue has been carried out. For example, if the time point when the pluripotent stem cells are extracted and cultured is counted as day 0, embryoid bodies are formed by culturing the cells for about 1 day (day 1), and the selective differentiation step into hindbrain tissue may be carried out from day 1 to about day 7 to about day 9, or the selective differentiation step into dorsal hindbrain tissue may be carried out from about day 3 to about day 12.

[0048] According to one embodiment of the present invention, the basal-apical polarization step (C) may induce the surface polarity of the embryoid bodies being cultured to be basal, thereby polarizing the outer and inner surface regions of the embryoid bodies into basal and apical regions, respectively. Through this, the embryoid bodies being cultured may differentiate into cerebellum-specific tissue by further developing neuroepithelial tissue. To achieve this function, the basal-apical induction medium may contain a substance that can impart neuroepithelial tissue polarity to the embryoid bodies being cultured. For example, the basal-apical induction medium may fulfill this function by containing an extracellular matrix-based hydrogel at a concentration ranging from 1% to 10% by volume.

[0049] According to one embodiment of the present invention, the extracellular matrix-based hydrogel may include extracellular matrix-based materials such as laminin and collagen, etc. For example, the extracellular matrix-based hydrogel may be Matrigel.

[0050] According to one embodiment of the present invention, the basal-apical polarization step (C) may be performed for a period of 1 to 5 days. Specifically, the basal-apical polarization step (C) may be performed for a period of 1 to 4 days, 2 to 4 days, or about 3 days.

[0051] According to one embodiment of the present invention, the selective differentiation step (A) into hindbrain tissue may be carried out over a period of 4 to 10 days, the selective differentiation step (B) into dorsal hindbrain tissue may be carried out over a period of 4 to 10 days, and the basal-apical polarization step (C) may be carried out over a period of 1 to 5 days.

[0052] According to one embodiment of the present invention, the selective differentiation step (A) into hindbrain tissue, the selective differentiation step (B) into dorsal hindbrain tissue, and the basolateral-apical polarization step (C) are processes that are each performed through the control of independent elements, and each step may be performed in an overlapping manner to effectively complete patterning into the cerebellum at each step. The selective differentiation step (A) into hindbrain tissue and the selective differentiation step (B) into dorsal hindbrain tissue may be performed in an overlapping manner for a period of 1 to 5 days. Specifically, the selective differentiation step (A) into hindbrain tissue and the selective differentiation step (B) into dorsal hindbrain tissue may be performed in an overlapping manner for a period of 1 to 4 days, 2 to 4 days, or about 3 days.

[0053] According to one embodiment of the present invention, the selective differentiation step (B) into dorsal hindbrain tissue and the basolateral-apical polarization step (C) may be performed in an overlapping manner for a period of 1 to 5 days. Specifically, the selective differentiation step (B) into dorsal hindbrain tissue and the basolateral-apical polarization step (C) may be performed in an overlapping manner for a period of 1 to 4 days, 2 to 4 days, or about 3 days.

[0054] According to one embodiment of the present invention, the method for preparing cerebellar organoids may further include (D) a cerebellar organoid maturation step of maturing the embryoid bodies into cerebellar organoids in the presence of a cerebellar maturation medium containing at least one additive selected from the group consisting of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cAMP, and ascorbic acid.

[0055] The cerebellar organoid maturation step (D) may be a step of completing the characteristics of cerebellar organoids through stepwise signaling substance treatment. Specifically, the cerebellar organoid maturation step (D) may be a process of treating the cells in the embryoid bodies that have undergone steps (A) to (C) to differentiate into neuroepithelial tissue suitable for cerebellar tissue so that they can be cultured as cerebellar-compatible tissue. To obtain mature cerebellar organoids, the cerebellar organoid maturation step (D) may be performed for a period of 10 days, 20 days, 30 days, or more.

[0056] According to one embodiment of the present invention, the percentage of cerebellar-compatible tissue in the entire cerebellar organoid tissue produced may be at least 90%. Specifically, the percentage of cerebellar-compatible tissue in the entire cerebellar organoid tissue produced may be at least 92%, 94%, or 95%. According to one embodiment of the present invention, the cerebellar organoids are organoids that exhibit the characteristics of cerebellar tissue with high purity, and may minimize the inclusion of tissue from other brain regions such as the cerebrum, striatum, and midbrain. Through this, the development of cerebellar-specific neurons and glial cells may be observed through the cerebellar organoids obtained by the method for preparing cerebellar organoids according to one embodiment of the present invention. Furthermore, the cerebellar organoids according to the present invention, which exhibit the characteristics of cerebellar tissue with high purity, can be used for various applications, such as research on cerebellar development and research on mimicking cerebellar-specific diseases.

[0057] The present invention may have various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail below. However, this does not limit the present invention to the specific embodiments, and it should be understood that the present invention includes all modifications, equivalents, and alternatives included within the spirit and technical scope of the present invention. In describing the present invention, if a detailed description of related publicly known technology is considered to obscure the gist of the present invention, the detailed description will be omitted. [Example]

[0058] [Embryoid body formation] (Embryoid bodies on day 0 (Day 0)) Pluripotent stem cells were cultured in an embryoid body formation medium as shown in Table 1 below.

[0059] [Table 1]

[0060] When the pluripotent stem cells reached approximately 80% confluency, they were washed with D-PBS to remove residual material from the cell surface and separated into individual cells. The cells were then diluted to approximately 100,000 cells per 10 ml of embryoid body formation medium, and approximately 100 μl of the medium containing the cells was poured into each well of a round-bottom, ultra-low attachment microplate (Corning, 7007). The cells were then placed in a 37°C, CO2-filled incubator and cultured for approximately 24 hours to form embryoid bodies.

[0061] [Selective differentiation into hindbrain tissue] (Day 1-4 embryoid bodies) Approximately one day after the formation of embryoid bodies as described above, the formation of one spherical embryoid body with a clean surface was confirmed in each well. Hindbrain tissue induction medium containing IWP-2 (1 μM) as an endogenous WNT secretion inhibitor and CHIR99021 (1 μM, 2 μM, 3 μM, and 5 μM) as a WNT signaling activator was prepared, and approximately 100 μl of the hindbrain tissue induction medium was added to each well containing the embryoid bodies.

[0062] Approximately 100 μl of the existing medium was removed approximately every two days, and approximately 100 μl of the hindbrain tissue induction medium was added. The embryoid bodies were cultured for 7 days to confirm whether the embryoid bodies had successfully differentiated into hindbrain tissue.

[0063] Figure 1 shows the results of expression of forebrain region-specific genes depending on the concentration of a WNT signaling agent in a hindbrain tissue induction medium according to an example. Figure 2 shows the results of expression of hindbrain region-specific genes depending on the concentration of a WNT signaling agent in a hindbrain tissue induction medium according to an example. Figure 3 shows the results of expression of spinal cord region-specific genes depending on the concentration of a WNT signaling agent in a hindbrain tissue induction medium according to an example.

[0064] The results of Figures 1 to 3 confirmed that the expression of hindbrain region-specific genes was strongly induced in hindbrain tissue induction medium containing CHIR99021 in the concentration range of more than 1 μM but less than 5 μM. Furthermore, the expression intensity of hindbrain region-specific genes was significantly higher than that of genes specific to other brain regions in hindbrain tissue induction medium containing CHIR99021 in the concentration range of 2 μM to 3 μM. Conversely, the expression of forebrain region-specific genes was higher than that of other brain regions in hindbrain tissue induction medium containing CHIR99021 at approximately 1 μM, and the expression of spinal cord region-specific genes was higher than that of other brain regions in hindbrain tissue induction medium containing CHIR99021 at approximately 5 μM.

[0065] Therefore, we used a hindbrain tissue induction medium containing IWP-2 as an endogenous WNT secretion inhibitor at a concentration of 1 μM and CHIR99021 as a WNT signaling agent at concentrations of 2 μM to 3 μM to selectively differentiate embryoid bodies into hindbrain tissue as described above, in preparation for the next step.

[0066] [Selective differentiation into dorsal hindbrain tissue] (Day 4-7 embryoid bodies) Cyclopamine, an SHh signaling inhibitor, was added to the hindbrain tissue induction medium at concentrations ranging from 0 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM to prepare dorsal hindbrain tissue induction medium. Then, starting from day 4 after the selective differentiation into hindbrain tissue, approximately 100 μl of the existing medium was removed approximately every two days for approximately three days, and approximately 100 μl of the dorsal hindbrain tissue induction medium prepared as above was added to each well to simultaneously induce selective differentiation of embryoid bodies into hindbrain tissue and dorsal hindbrain tissue.

[0067] Figure 4 shows the results of dorsal hindbrain region-specific gene expression depending on the concentration of SHh signaling inhibitor in the dorsal hindbrain tissue induction medium of the Example. Also, Figure 5 shows the developmental state of embryoid bodies on day 7 after selective differentiation into dorsal hindbrain tissue depending on the concentration of SHh signaling inhibitor in the dorsal hindbrain tissue induction medium of the Example.

[0068] The results in Figure 4 confirmed that when the concentration of the SHh signaling inhibitor cyclopamine was 20 μM or higher, the expression levels of genes PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2, which indicate differentiation into dorsal hindbrain tissue, increased dramatically. Furthermore, the results in Figure 5 confirmed that when the concentration of the SHh signaling inhibitor cyclopamine was higher than 30 μM, the embryoid bodies were unable to grow any further and died. These results suggest that embryoid bodies can be effectively cultured into dorsal hindbrain tissue when the concentration of the SHh signaling inhibitor in the dorsal hindbrain tissue induction medium is higher than 10 μM and lower than 30 μM.

[0069] Therefore, we used a dorsal hindbrain tissue induction medium containing cyclopamine, an SHh signaling inhibitor, at a concentration of approximately 20 μM to selectively differentiate the embryoid bodies into dorsal hindbrain tissue as described above, in preparation for the next step.

[0070] [Basal-apical polarization] (Embryoid bodies on days 7-10 (Day 7-10)) Approximately 2% by volume of Matrigel was added to the dorsal hindbrain tissue induction medium to prepare a basolateral-apical induction medium. Starting from day 4 of the selective differentiation into dorsal hindbrain tissue, approximately 100 μl of the existing medium was removed approximately once a day for approximately 3 days, and approximately 100 μl of the basolateral-apical induction medium prepared as above was added to each medium. This simultaneously induced the selective differentiation of embryoid bodies into dorsal hindbrain tissue and basolateral-apical polarization.

[0071] [Cerebellar organoid maturation] (Embryoid bodies from Day 10 to Day 34 (Day 10-34)) Five ml of cerebellar maturation medium containing brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) was added to each well of a plate, and the basolateral-apical polarized embryoid bodies were transferred to each well. Approximately 4 ml of the medium was then removed and approximately 4 ml of new cerebellar maturation medium was added at approximately 2-day intervals to allow the maturation process of the cerebellar organoids (Days 10-34).

[0072] Figure 6 shows the chronological sequence of the process for producing cerebellar organoids according to the present invention, starting from embryoid body formation (Day 0). Furthermore, Figure 7 shows the developmental process of embryoid bodies during the process for producing cerebellar organoids according to the present invention, as observed through a phase-contrast microscope.

[0073] 8 shows the results of immunofluorescence staining of cerebellar organoids on day 35 (Day 35) after embryoid body formation using the cerebellar organoid preparation method according to the embodiment. Specifically, the results of FIG. 8 confirmed that numerous cells expressing SKOR2, which labels precursor cells of cerebellar-specific Purkinje neurons, and ATOH1, which labels precursor cells of cerebellar-specific granule cells, were observed in the prepared cerebellar organoids.

Claims

1. (A) a selective differentiation step into hindbrain tissue, in which embryoid bodies formed from pluripotent stem cells are cultured in the presence of a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agent; (B) a selective differentiation step into dorsal hindbrain tissue, in which the embryoid body is cultured in the presence of a dorsal hindbrain tissue induction medium containing a sonic hedgehog (SHh) signaling pathway inhibitor; and (C) A method for preparing cerebellar organoids, comprising a basolateral-apical polarization step in which polarization of the embryoid body is induced in the presence of a basolateral-apical induction medium containing an extracellular matrix-based hydrogel.

2. The selective differentiation step (A) into hindbrain tissue comprises: a1) forming embryoid bodies from pluripotent stem cells in an embryoid body formation medium; and a2) the method for preparing the cerebellar organoid of claim 1, comprising adding a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agent to the embryoid body formation medium.

3. The method for preparing cerebellar organoids according to claim 1, wherein the endogenous WNT secretion inhibitor comprises at least one selected from the group consisting of IWP-2, LGK-974, ETC-159, GNF6231, WNT-C59 and WNT974.

4. The method for preparing cerebellar organoids according to claim 1, wherein the hindbrain tissue induction medium contains an endogenous WNT secretion inhibitor in a concentration range that exhibits an effect equivalent to that of IWP-2 in a concentration range of 0.1 μM or more and 2 μM or less.

5. The method for preparing cerebellar organoids of claim 1, wherein the hindbrain tissue induction medium comprises a WNT signaling agent in a concentration range that exhibits an effect equivalent to that exhibited by CHIR99021 in a concentration range of more than 1 μM and less than 5 μM.

6. A method for preparing cerebellar organoids as described in claim 1, wherein at least one gene selected from LMX1A and LMX1B is expressed in the selective differentiation step (A) into hindbrain tissue.

7. The method for preparing cerebellar organoids according to claim 1, wherein the dorsal hindbrain tissue induction medium contains an SHh signaling inhibitor in a concentration range that exhibits an effect equivalent to that of cyclopamine in a concentration range of more than 10 μM and less than 30 μM.

8. A method for preparing cerebellar organoids as described in claim 1, wherein in the selective differentiation step (B) into dorsal hindbrain tissue, at least one gene selected from the group consisting of PAX7, OLIG3, ATOH1, BARHL1, PTF1A and SKOR2 is expressed.

9. The method for preparing cerebellar organoids according to claim 1, wherein the basal surface-apical surface induction medium comprises an extracellular matrix-based hydrogel in a concentration range of 1% by volume or more and 10% by volume or less.

10. The selective differentiation step (A) into hindbrain tissue is carried out for a period of 4 to 10 days; The selective differentiation step (B) into dorsal hindbrain tissue is carried out for a period of 4 to 10 days; The method for preparing cerebellar organoids according to claim 1, wherein the basal-apical polarization step (C) is carried out for a period of 1 to 5 days.

11. The method for preparing cerebellar organoids according to claim 10, wherein the selective differentiation step (A) into hindbrain tissue and the selective differentiation step (B) into dorsal hindbrain tissue are performed in an overlapping manner for a period of 1 to 5 days.

12. The method for preparing cerebellar organoids according to claim 10, wherein the selective differentiation step (B) into dorsal hindbrain tissue and the basal surface-apical surface polarization step (C) are performed in an overlapping manner for a period of 1 to 5 days.

13. (D) The method for preparing the cerebellar organoid of claim 1, further comprising a cerebellar organoid maturation step of maturing the embryoid body into the cerebellar organoid in the presence of a cerebellar maturation medium containing at least one additive selected from the group consisting of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), and ascorbic acid.

14. The method for preparing cerebellar organoids according to claim 1, wherein the proportion of cerebellar-compatible tissue in the entire prepared cerebellar organoid tissue is at least 90%.

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