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 Art

[0002] Organoids, also called "organ analogs" or "organ-like structures", are organ-specific cell aggregates produced by three-dimensional culture, re-aggregation and recombination of cells isolated from stem cells or organ-derived cells. Organoids contain specific cells of the modeled organ, reproduce the specific functions of the organ, and can achieve spatial organization into a morphology similar to that of an actual organ.

[0003] In particular, organoids that mimic most human organs can be prepared using pluripotent stem cells, which can differentiate into any cell type in the human body. Among them, brain organoids have attracted much attention as an in vitro culture model for mimicking various refractory central nervous system diseases, elucidating pathogenesis, and developing treatment methods.

[0004] For example, Lancaster et al. prepared brain organoids from human induced pluripotent stem cells (Non-Patent Document 1). However, the brain organoids prepared by this method have problems that various brain regions such as cerebrum, midbrain and retina are mixed in a single organoid, and the sizes vary.

[0005] Most refractory brain diseases are specifically occurred in specific brain regions. Organoids that have characteristics of other brain regions unrelated to the onset site of the target disease, or have mixed characteristics of multiple regions, cannot accurately mimic brain diseases. Therefore, in order to apply brain organoids to the research on the pathogenesis of refractory brain diseases through modeling, the research on development of treatment methods and the like, it is necessary to develop a technology for preparing site-specific brain organoids that can mimic specific brain regions with high purity. Prior Art Documents Non-Patent Documents

[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. [Overview of the Initiative] [Problems that the invention aims to solve]

[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 this invention aims to solve are not limited to those mentioned above, and other problems not mentioned 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 is a selective differentiation step into hindbrain tissue, comprising (A) culturing embryoid bodies formed from pluripotent stem cells in the presence of a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agonist, (B) A selective differentiation step to dorsal hindbrain tissue, in which the embryoid body is cultured in a dorsal hindbrain tissue induction medium containing a sonic hedgehog (SHh) signaling pathway inhibitor, and (C) A method for preparing cerebellar organoids is provided, comprising a basal-to-tip polarization step, in which polarization of the embryoid body is induced in the presence of a basal-to-tip inducing medium containing an extracellular matrix-based hydrogel. [Effects of the Invention]

[0010] According to one embodiment of the present invention, when using a method for preparing cerebellar organoids, it is possible to manufacture cerebellar organoids that mimic cerebellar regions with high purity, and these can be used to contribute to the advancement of research on the pathogenesis of cerebellar-related diseases and the development of treatment methods. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the expression results of forebrain region-specific genes in the hindbrain tissue induction medium of the example, according to the concentration of the WNT signaling agonist. [Figure 2] Figure 2 shows the expression results of hindbrain region-specific genes in hindbrain tissue induction medium according to the concentration of the WNT signaling agonist in the examples. [Figure 3] Figure 3 shows the expression results of spinal cord site-specific genes in response to the concentration of WNT signaling agonists in the hindbrain tissue induction medium of the example. [Figure 4] Figure 4 shows the expression results of dorsal hindbrain region-specific genes in the dorsal hindbrain tissue induction medium of the example, according to the concentration of the SHh signaling pathway inhibitor. [Figure 5] Figure 5 shows the developmental state of embryoid bodies on day 7 that underwent selective differentiation into dorsal hindbrain tissue according to the concentration of the SHh signaling pathway inhibitor in the dorsal hindbrain tissue induction medium of the example. [Figure 6] Figure 6 shows the manufacturing process of the cerebellar organoid according to the example, in chronological order from embryoid body formation (Day 0). [Figure 7] Figure 7 shows the developmental process of embryoid bodies during the manufacturing process of cerebellar organoids in the example, as observed through a phase-contrast microscope. [Figure 8] Figure 8 shows the results of observing cerebellar organoids using immunofluorescence staining on Day 35, the 35th day after embryoid bodies were formed using the cerebellar organoid preparation method according to the example. [Modes for carrying out the invention]

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

[0013] While various modifications can be made to the present invention and it can have various embodiments, specific embodiments are illustrated 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 specific embodiments, and includes all modifications, equivalents and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, when it is determined that a detailed description of related known techniques may obscure the gist of the present invention, the detailed description thereof will be omitted.

[0015] The terms used in the present application are merely used to describe specific embodiments, and are not intended to limit the present invention. An expression in the singular includes plural expressions unless the context clearly dictates otherwise.

[0016] In the present invention, terms such as "comprising" or "having" are intended to specify the presence of the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and it should be understood that they do not exclude in advance 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 provides a method for preparing a cerebellar organoid, comprising: (A) a step of selectively differentiating embryoid bodies formed from pluripotent stem cells into hindbrain tissue, which comprises culturing said embryoid bodies in the presence of a hindbrain tissue induction medium containing an endogenous WNT secretion inhibitor and a WNT signaling agonist; (B) a step of selectively differentiating said embryoid bodies into dorsal hindbrain tissue, which comprises culturing said embryoid bodies in the presence of a dorsal hindbrain tissue induction medium containing a Sonic Hedgehog (SHh) signaling pathway inhibitor; and (C) a basal-apical polarization step of inducing polarization of said embryoid bodies in the presence of a basal-apical induction medium containing an extracellular matrix-based hydrogel.

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

[0019] According to one embodiment of the present invention, said step (A) of selective differentiation into hindbrain tissue may induce the differentiation of said embryoid bodies into neuroectoderm, and at the same time uniformly induce patterning into hindbrain tissue throughout said embryoid bodies.

[0020] In one embodiment of the present invention, the endogenous WNT secretion inhibitor may be a substance that suppresses the secretion of WNT signaling molecules spontaneously expressed in the embryoid body. The endogenous WNT secretion inhibitor may control the uncontrollable heterogeneous expression of the WNT signaling pathway by suppressing the activation of the WNT signaling pathway by WNT proteins spontaneously expressed in the embryoid body. Therefore, by applying the endogenous WNT secretion inhibitor in combination with a WNT signaling agonist described later, the embryoid body may be selectively and uniformly differentiated into the hindbrain. The endogenous WNT secretion inhibitor may include a substance that can perform the mechanism 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 spontaneously expressed WNT signaling molecules, while simultaneously not affecting the activity of WNT signaling agonists. That is, the endogenous WNT secretion inhibitor effectively controls the heterogeneous formation of the WNT signaling pathway by suppressing the process by which endogenous WNT proteins spontaneously expressed in embryoid bodies are transformed into a form secreted extracellularly from the endoplasmic reticulum. Conversely, substances such as IWR-1-endo may cause problems by suppressing signal transduction in lower cascades of the WNT signaling pathway, thereby reducing the effectiveness of WNT signaling agonists described later.

[0021] In 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 a 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 a 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, in 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 body may be effectively suppressed. Specifically, if the concentration of the endogenous WNT secretion inhibitor is below the range, the effect of controlling the heterogeneous expression of the uncontrollable WNT signaling pathway is minimal, and the formation of a heterogeneous WNT signaling pathway may occur. Conversely, if the concentration of the endogenous WNT secretion inhibitor exceeds the range, a decrease in cell division and / or a decrease in cell viability may occur due to excessive suppression of the WNT signaling pathway.

[0022] In one embodiment of the present invention, the WNT signaling agonist may uniformly confer hindbrain specificity to the entire embryoid body. That is, as described above, while the production of uncontrolled WNT signaling substances in the embryoid body is suppressed through the endogenous WNT secretion inhibitor, the WNT signaling agonist may uniformly adjust the WNT signal intensity throughout the entire embryoid body to provide uniform hindbrain specificity throughout the embryoid body.

[0023] In one embodiment of the present invention, the WNT signaling agonist may comprise 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 comprise 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 comprise a substance that performs the mechanism of a GSK-3 beta inhibitor, and specifically comprises at least one selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, Kempaworn, 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 agonist in the hindbrain tissue induction medium may be optimized to induce selective differentiation to the hindbrain region. Specifically, the concentration of the WNT signaling agonist for inducing selective differentiation of the embryoid body to the hindbrain region 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 shown by CHIR99021 at a concentration range greater than 1 μM and 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 shown by CHIR99021 at a 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 of the LMX1A and LMX1B genes. Furthermore, according to one embodiment of the present invention, the hindbrain tissue induction medium may contain CHIR99021 in a concentration range of the WNT signaling agonist. If the concentration of the WNT signaling agonist is within the range, the expression of hindbrain region-specific genes (e.g., LMX1A and / or LMX1B genes) may be significantly increased, and the embryoid body may be uniformly differentiated into a cell population having hindbrain specificity. If the concentration of the WNT signaling agonist is lower than the range, the expression of forebrain region-specific genes other than hindbrain genes (e.g., FOXG1, LHX2, and / or DLX2 genes) may be increased. Furthermore, if the concentration of the WNT signaling agonist exceeds the range, the expression of spinal cord region-specific genes other than hindbrain genes (e.g., HOXB4, HOXC9, and / or HOXA1 genes) may be increased.

[0026] According to one embodiment of the present invention, at least one of the LMX1A and LMX1B genes may be expressed in the selective differentiation step (A) to the 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 artificially induced pluripotent stem (iPS) cells. The pluripotent stem cells refer to pluripotent stem cells that can differentiate into the three germ layers: endoderm, mesoderm, and ectoderm, and may include not only embryonic stem cells but also artificially induced pluripotent stem cells (iPS), and adult stem cells that possess the aforementioned capabilities.

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

[0029] According to one embodiment of the present invention, the embryoid body-forming medium may be a liquid medium and can be applied as long as it enables three-dimensional culture of the pluripotent stem cells. Furthermore, the embryoid body-forming medium may contain a variety of known additives for the culture of pluripotent stem cells, depending on the purpose.

[0030] According to one embodiment of the present invention, the hindbrain tissue induction medium may include known culture media and additives capable of culturing embryoid bodies, and may further include the endogenous WNT secretion inhibitor and the WNT signaling agonist.

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

[0032] In one embodiment of the present invention, the selective differentiation step (A) to hindbrain tissue may further include a3) step, at one time point between 1 and 3 days, removing a portion of the total culture medium and adding the hindbrain tissue induction medium. Specifically, step a3) may involve removing the same amount of culture medium as that added in step a2) and adding the same amount of hindbrain tissue induction medium approximately every 2 days. Through this process, nutrients may be supplied to the embryoid body, and the endogenous WNT secretion inhibitor and the WNT signaling agonist may act effectively.

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

[0034] According to one embodiment of the present invention, through the selective differentiation step (B) to dorsal hindbrain tissue, embryoid bodies derived from pluripotent stem cells may differentiate into highly pure cell aggregates that mimic the characteristics of the dorsal hindbrain. Specifically, the cell aggregates that mimic the characteristics of the dorsal hindbrain may be highly pure organoids that mimic the characteristics of the dorsal hindbrain. The highly pure cell aggregates or organoids that mimic the characteristics of the dorsal hindbrain may represent a state in which the inclusion of tissues exhibiting 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) to dorsal hindbrain tissue may induce differentiation of the embryoid body into neuroectoderm while simultaneously uniformly inducing patterning of the entire embryoid body into dorsal hindbrain tissue.

[0036] According to one embodiment of the present invention, the embryoid body derived from the pluripotent stem cell may be an embryoid body in the process of differentiating into hindbrain tissue. Specifically, the selective differentiation step (B) into dorsal hindbrain tissue may induce the embryoid body, which is differentiating into hindbrain tissue such as the pons and cerebellum, to be specific to dorsal hindbrain tissue during the differentiation process, thereby inducing higher cerebellar specificity.

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

[0038] In one embodiment of the present invention, the SHh signaling pathway inhibitor may be a substance that plays a role in suppressing the SHh signaling pathway that is spontaneously activated in some cells of the embryoid body. The SHh signaling pathway inhibitor may suppress heterogeneous and uncontrollable activation of the SHh signaling pathway by suppressing the activation of the SHh signaling pathway by proteins that generate SHh signals spontaneously expressed in the embryoid body. That is, the embryoid body may be selectively and uniformly differentiated into dorsal hindbrain tissue by the suppression of endogenous SHh signals by the SHh signaling pathway inhibitor.

[0039] According to one embodiment of the present invention, the SHh signaling pathway inhibitor may include at least one selected from the group consisting of cyclopamine, itraconazole, robotnikinin, cerulenin, and GANT61. Specifically, the SHh signaling pathway inhibitor may be cyclopamine.

[0040] According to one embodiment of the present invention, the dorsal hindbrain tissue induction medium may contain an SHh signaling pathway inhibitor in a concentration range that exhibits an effect equivalent to that of cyclopamine in a concentration range greater than 10 μM and less than or equal to 30 μM. Specifically, the dorsal hindbrain tissue induction medium may contain an SHh signaling pathway inhibitor in a concentration range that exhibits an effect equivalent to that of cyclopamine in a concentration range greater than 10 μM and less than 30 μM, 12 μM and less than 30 μM, 12 μM and less than or equal to 30 μM, 15 μM and less than or equal to 25 μM, or 20 μM and less than or equal to 25 μM. More specifically, the dorsal hindbrain tissue induction medium may contain the SHh signaling pathway inhibitor at a concentration that specifically expresses at least one of the genes PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2. The dorsal hindbrain tissue induction medium may also contain cyclopamine in a concentration range equivalent to that of the SHh signaling pathway inhibitor. If the concentration of the SHh signaling pathway inhibitor is within the 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 body may differentiate into a cell population uniformly having dorsal hindbrain specificity. If the concentration of the SHh signaling pathway inhibitor is below the range, the expression of the dorsal hindbrain-specific genes described above may be at the minimum level. Furthermore, if the concentration of the SHh signaling pathway inhibitor is above the range, the excessive concentration of the SHh signaling pathway inhibitor may suppress cell division within the embryoid body, leading to the problem of the embryoid body dying before it can develop to the necessary extent.

[0041] According to one embodiment of the present invention, in the selective differentiation step (B) to dorsal hindbrain tissue, at least one gene among PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2 may be expressed.

[0042] According to one embodiment of the present invention, the selective differentiation step to dorsal hindbrain tissue may further include the step of b1) adding the dorsal hindbrain tissue induction medium to the culture medium in which the embryoid body is being cultured.

[0043] According to one embodiment of the present invention, the selective differentiation step to dorsal hindbrain tissue may be carried out in a manner that overlaps with the selective differentiation step to hindbrain tissue. Specifically, during the period in which the selective differentiation step to dorsal hindbrain tissue is carried out in a manner that overlaps with the selective differentiation step to hindbrain tissue, the dorsal hindbrain tissue induction medium may further contain the SHh signaling pathway inhibitor described above.

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

[0045] In one embodiment of the present invention, the selective differentiation step to dorsal hindbrain tissue may further include step b2) at one time point between 1 and 3 days, removing a portion of the entire culture medium and adding the dorsal hindbrain tissue induction medium. Specifically, step b2) may involve removing an amount of culture medium equal to the amount of dorsal hindbrain tissue induction medium added in step b1) approximately every 2 days, and adding the same amount of dorsal hindbrain tissue induction medium. Through this, nutrients are supplied to the embryoid body, and the SHh signaling pathway inhibitor may act effectively. Furthermore, if the selective differentiation step to hindbrain tissue is repeated, the SHh signaling pathway inhibitor may act effectively together with the endogenous WNT secretion inhibitor and the WNT signaling agonist.

[0046] According to one embodiment of the present invention, the selective differentiation step (B) to dorsal hindbrain tissue may be carried out over a period of 4 to 10 days. Specifically, the selective differentiation step (B) to dorsal hindbrain tissue may be carried out over 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) to dorsal hindbrain tissue may be performed during a period of 3 to 5 days to 4 to 10 days after the selective differentiation step (A) to hindbrain tissue has been performed. For example, if the point in time when the pluripotent stem cells are extracted and cultured is counted as day 0, an embryoid body (day 1) may be formed by culturing the cells for about 1 day, the selective differentiation step to hindbrain tissue may be performed from day 1 to about 7 to about 9 days, and the selective differentiation step to dorsal hindbrain tissue may be performed from about 3 to about 12 days.

[0048] In one embodiment of the present invention, the basal-to-tip polarization step (C) may induce the surface polarity of the embryoid body in culture to become basal, thereby polarizing the outer and inner surface regions of the embryoid body into a basal and aip plane, respectively. Through this, the embryoid body in culture may be differentiated into cerebellar-specific tissue by further development of neuroepithelial tissue. To fulfill this role, the basal-to-tip induction medium may contain a substance capable of conferring neuroepithelial tissue polarity to the embryoid body in culture. For example, the basal-to-tip induction medium may fulfill this role by containing an extracellular matrix-based hydrogel in a concentration range of 1% to 10% by volume.

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

[0050] According to one embodiment of the present invention, the base-to-tip surface polarization step (C) may be performed over a period of 1 to 5 days. Specifically, the base-to-tip surface polarization step (C) may be performed over 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) to hindbrain tissue may be carried out over a period of 4 to 10 days, the selective differentiation step (B) to dorsal hindbrain tissue may be carried out over a period of 4 to 10 days, and the basal-to-anterior 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 to hindbrain tissue (A), the selective differentiation step to dorsal hindbrain tissue (B), and the basal-to-anterior polarization step (C) may be carried out as processes performed through independent elemental control, with each step overlapping in order to effectively complete the patterning to the cerebellum at each step. The selective differentiation step to hindbrain tissue (A) and the selective differentiation step to dorsal hindbrain tissue (B) may be carried out with an overlapping period of 1 to 5 days. Specifically, the selective differentiation step to hindbrain tissue (A) and the selective differentiation step to dorsal hindbrain tissue (B) may be carried out with an overlapping 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 to dorsal hindbrain tissue (B) and the basal-to-anterior polarization step (C) may be carried out in an overlapping manner over a period of 1 to 5 days. Specifically, the selective differentiation step to dorsal hindbrain tissue (B) and the basal-to-anterior polarization step (C) may be carried out in an overlapping manner over 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 the cerebellar organoid may further include a cerebellar organoid maturation step, in which the embryoid body is matured into a cerebellar organoid in the presence of a cerebellar maturation medium containing (D) 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 in which the characteristics of the cerebellar organoid are completed through stepwise signaling molecule treatment. Specifically, the cerebellar organoid maturation step (D) may be a process in which cells in the embryoid body that have gone through steps (A) to (C) are treated 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 carried out for a period of 10, 20, or 30 days or more.

[0056] According to one embodiment of the present invention, the proportion of cerebellar-compatible tissue in the entire manufactured cerebellar organoid tissue may be at least 90%. Specifically, the proportion of cerebellar-compatible tissue in the entire manufactured cerebellar organoid tissue may be at least 92%, 94%, or 95%. According to one embodiment of the present invention, the cerebellar organoid is a highly pure organoid that exhibits the characteristics of cerebellar tissue, and the inclusion of tissue from other brain regions such as the cerebrum, striatum, and midbrain may be minimized. Through this, the development of cerebellar-specific neurons and glial cells may be observed through the cerebellar organoid obtained by the method for preparing cerebellar organoids according to one embodiment of the present invention. Furthermore, the highly pure cerebellar organoid according to the present invention, which exhibits the characteristics of cerebellar tissue, can be used for a variety of applications, such as research on the cerebellar development process 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 in the detailed description below. However, this should not be understood as limiting the present invention to specific embodiments, but rather as including all modifications, equivalents, and substitutions that fall within the spirit and technical scope of the present invention. In describing the present invention, if a specific description of related prior art is deemed to obscure the gist of the invention, such detailed description will be omitted. [Examples]

[0058] [Embryoform formation] (Embryoform on day 0) Pluripotent stem cells were cultured in embryoid body-forming medium as shown in Table 1 below.

[0059] [Table 1]

[0060] When the density of pluripotent stem cells reached approximately 80%, residual material adhering to the cell surface was washed using D-PBS, and each cell was separated. Furthermore, the culture medium was diluted to contain approximately 100,000 cells per 10 ml of embryoid body-forming medium, and approximately 100 μl of this medium was injected into each well of a round-bottom ultra-low adhesion surface microplate (Corning, 7007). The plates were then placed in an incubator at 37°C under a CO2 atmosphere and cultured for approximately 24 hours to form embryoid bodies.

[0061] [Selective differentiation into hindbrain tissue] (embryoid bodies on days 1-4) Approximately one day after the formation of embryoid bodies as described above, we checked each well to see if one spherical embryoid body with a clean surface had formed properly. We prepared hindbrain tissue induction media containing IWP-2 as an endogenous WNT secretion inhibitor at a concentration of 1 μM, and CHIR99021 as a WNT signaling agonist at concentrations of 1 μM, 2 μM, 3 μM, and 5 μM, respectively, and added approximately 100 μl of each of these hindbrain tissue induction media to each well containing embryoid bodies.

[0062] Approximately every two days, about 100 μl of the existing culture medium was removed, and about 100 μl of the aforementioned hindbrain tissue induction medium was added. The embryoid bodies were then cultured for 7 days to confirm whether the selective differentiation of the embryoid bodies into hindbrain tissue had occurred successfully.

[0063] Figure 1 shows the expression results of forebrain region-specific genes in the hindbrain tissue induction medium of the example, according to the concentration of the WNT signaling agonist. Figure 2 shows the expression results of hindbrain region-specific genes in the hindbrain tissue induction medium of the example, according to the concentration of the WNT signaling agonist. Furthermore, Figure 3 shows the expression results of spinal cord region-specific genes in the hindbrain tissue induction medium of the example, according to the concentration of the WNT signaling agonist.

[0064] As shown in Figures 1-3, hindbrain tissue induction media containing CHIR99021 at concentrations greater than 1 μM but less than 5 μM strongly induced the expression of hindbrain region-specific genes. Furthermore, hindbrain tissue induction media containing CHIR99021 at concentrations in the 2 μM-3 μM range showed significantly stronger expression of hindbrain region-specific genes compared to those of other brain regions. Conversely, hindbrain tissue induction media containing CHIR99021 at approximately 1 μM showed stronger expression of forebrain region-specific genes compared to other brain regions, and hindbrain tissue induction media containing CHIR99021 at approximately 5 μM showed stronger expression of spinal cord region-specific genes compared to other brain regions.

[0065] Therefore, using 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 agonist at a concentration of 2 μM to 3 μM, selective differentiation of embryoid bodies into hindbrain tissue was performed as described above, and the next step was prepared.

[0066] [Selective differentiation into dorsal hindbrain tissue] (embryoid bodies on days 4-7) Dorsal hindbrain tissue induction medium was prepared by adding cyclopamine, an SHh signaling pathway inhibitor, to the aforementioned hindbrain tissue induction medium at concentrations of 0 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM. Then, starting from the 4th day after selective differentiation into hindbrain tissue, approximately 100 μl of the existing medium was removed approximately every 2 days for about 3 days, and approximately 100 μl of the dorsal hindbrain tissue induction medium prepared as described above was added each time to simultaneously induce selective differentiation of embryoid bodies into hindbrain tissue and selective differentiation into dorsal hindbrain tissue.

[0067] Figure 4 shows the expression results of dorsal hindbrain region-specific genes depending on the concentration of the SHh signaling pathway inhibitor in the dorsal hindbrain tissue induction medium of the example. Figure 5 shows the developmental state of embryoid bodies on day 7 from selective differentiation into dorsal hindbrain tissue according to the concentration of the SHh signaling pathway inhibitor in the dorsal hindbrain tissue induction medium of the example.

[0068] As shown in Figure 4, when the concentration of cyclopamine, an SHh signaling pathway inhibitor, was 20 μM or higher, the expression levels of the genes PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2, which can be identified as differentiating into dorsal hindbrain tissue, increased sharply. Furthermore, as shown in Figure 5, when the concentration of cyclopamine, an SHh signaling pathway inhibitor, was higher than 30 μM, the embryoid bodies were unable to grow further and died. Through these results, it was found that embryoid bodies can be effectively cultured into dorsal hindbrain tissue when the concentration of the SHh signaling pathway inhibitor in the dorsal hindbrain tissue induction medium is between 10 μM and 30 μM.

[0069] Therefore, using a dorsal hindbrain tissue induction medium containing cyclopamine, an SHh signaling pathway inhibitor, at a concentration of approximately 20 μM, we selectively differentiated embryoid bodies into dorsal hindbrain tissue, as described above, and prepared for the next step.

[0070] [Basal-to-apical polarization] (Embryoids on days 7-10) Approximately 2% by volume of Matrigel was added to the dorsal hindbrain tissue induction medium to prepare a basal-to-anterior plane induction medium. Then, after selective differentiation into the dorsal hindbrain tissue, approximately 100 μl of the existing medium was removed once a day for about 3 days starting from the 4th day, and approximately 100 μl of the basal-to-anterior plane induction medium prepared as described above was added to each removal, thereby simultaneously inducing selective differentiation of embryoid bodies into dorsal hindbrain tissue and basal-to-anterior plane polarization.

[0071] [Cerebellar organoid maturation] (Embryoforms from day 10 to day 34) 5 ml of cerebellar maturation medium containing brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) was placed in each well of the well plate, and the basal-to-apical polarized embryoid bodies were transferred to each well. Then, approximately 4 ml of the existing medium was removed and approximately 4 ml of new cerebellar maturation medium was added at intervals of about 2 days, and the maturation process in cerebellar organoids was carried out (Days 10-34).

[0072] Figure 6 shows the manufacturing process of the cerebellar organoid according to the example, chronologically from embryoid body formation (Day 0). Furthermore, Figure 7 shows the developmental process of the embryoid body during the manufacturing process of the cerebellar organoid according to the example, as observed through a phase-contrast microscope.

[0073] Furthermore, Figure 8 shows the results of observation of cerebellar organoids on Day 35 (35 days after embryoid body formation) using the cerebellar organoid preparation method according to the example, as observed by immunofluorescence staining. Specifically, according to the results in Figure 8, it was confirmed that the prepared cerebellar organoids contained a large number of cells expressing SKOR2, which labels cerebellar-specific Purkinje neuron precursor cells, and ATOH1, which labels cerebellar-specific granule cell precursor cells.

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 agonist. (B) A selective differentiation step to dorsal hindbrain tissue, in which the embryoid body is cultured in a dorsal hindbrain tissue induction medium containing a sonic hedgehog (SHh) signaling pathway inhibitor, and (C) A basal-to-tip polarization step, comprising inducing polarization of the embryoid body in the presence of a basal-to-tip inducing medium containing an extracellular matrix-based hydrogel, The endogenous WNT secretion inhibitor contains IWP-2 in a concentration range of 0.1 μM to 2 μM. The WNT signaling agent comprises CHIR99021 in a concentration range greater than 1 μM and less than 5 μM. A method for preparing cerebellar organoids, wherein the sonic hedgehog (SHh) signaling pathway inhibitor contains cyclopamine in a concentration range of more than 10 μM and less than or equal to 30 μM.

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

3. The method for preparing a cerebellar organoid according to claim 1, wherein at least one of the genes LMX1A and LMX1B is expressed in the selective differentiation step (A) to hindbrain tissue.

4. The method for preparing a cerebellar organoid according to claim 1, wherein at least one gene from among PAX7, OLIG3, ATOH1, BARHL1, PTF1A, and SKOR2 is expressed in the selective differentiation step (B) to dorsal hindbrain tissue.

5. The method for preparing a cerebellar organoid according to claim 1, wherein the basal-to-tip surface induction medium comprises an extracellular matrix-based hydrogel in a concentration range of 1% to 10% by volume.

6. The aforementioned selective differentiation step (A) into hindbrain tissue takes place over a period of 4 to 10 days. The aforementioned selective differentiation step (B) into dorsal hindbrain tissue is carried out over a period of 4 to 10 days. The method for preparing a cerebellar organoid according to claim 1, wherein the base-to-tip surface polarization step (C) is performed over a period of 1 to 5 days.

7. The method for preparing a cerebellar organoid according to claim 6, wherein the selective differentiation step (A) to hindbrain tissue and the selective differentiation step (B) to dorsal hindbrain tissue are carried out in a manner that overlaps over a period of 1 to 5 days.

8. The method for preparing a cerebellar organoid according to claim 6, wherein the selective differentiation step to dorsal hindbrain tissue (B) and the basal-to-anterior polarization step (C) are carried out in a manner that overlaps over a period of 1 to 5 days.

9. A method for preparing a cerebellar organoid according to claim 1, further comprising a cerebellar organoid maturation step, in which the embryoid body polarized in step (C) is matured into the cerebellar organoid in the presence of a cerebellar maturation medium comprising 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.

10. The method for preparing a cerebellar organoid 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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