WO2021235848A1 - Method for preparing organoid including oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, organoid prepared by same method, and use thereof - Google Patents

Abstract

The present invention relates to a method for preparing an organoid including oligodendrocytes from neural stem cells that are specialized for differentiation into oligodendrocytes, an organoid prepared by the method, and use thereof and, more particularly, the present invention relates to a method for preparing an organoid including oligodendrocytes from neural stem cells that are specialized for differentiation into oligodendrocytes, an organoid including oligodendrocytes, prepared by the method, a method for screening for a therapeutic agent for a demyelinating disease, the method comprising a step of analyzing whether myelinization has occurred or not after treatment of the organoid with a candidate drug, and a cell therapeutic agent composition for preventing or treating a demyelinating disease, comprising, as an active ingredient, the organoid including oligodendrocytes or oligodendrocytes isolated from the organoid.

Description

Method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, organoids prepared by the method, and uses thereof

The present invention relates to a method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, the organoid prepared by the method, and uses thereof.

As the average human lifespan increases due to the development of medical science and technology, and as the centenarian era is approaching, personalized cell therapy is emerging as a fundamental essential element that will lay the foundation for human longevity. Among various Central Nervous System (CNS) diseases, demyelinating disease is caused by abnormalities in the oligodendrocytes that form the myelin sheath and is caused by damage to the nerve cells. As a disease that causes various neurological diseases, transplantation of oligodendrocyte precursor cells (OPC) that can differentiate into oligodendrocytes is emerging as a major treatment.

Neural Stem Cells (NSCs) are multipotent stem cells capable of differentiating into three cells: nerve cells, astrocytes, and oligodendrocytes. And a method for obtaining cells through cross-differentiation has been developed, but it is known that the ability to differentiate into oligodendrocytes is remarkably low, making it unsuitable as a cell therapy for demyelinating diseases.

However, it is known that the amount of oligodendrocytes in the human brain is very small, so autografting is difficult. In actual clinical trials, a clear effect through transplantation was not proven.

Accordingly, many researchers are trying to develop drugs that can induce remyelination through drug screening to develop drugs that can treat demyelinating diseases, but search for drug candidates and preclinical Various animal and cell models (2D) used to evaluate the drug efficacy of each stage do not reflect the environment-cell interactions in the human body and complex intracellular signaling pathways, making it difficult to develop new drugs.

Therefore, for the development of effective drugs for demyelinating diseases, there is a need to develop a technology capable of rapidly and effectively inducing the formation of organoids and remyelination including oligodendrocytes.

Accordingly, the present inventors have established human neural stem cells (pre-OPCs) specialized to oligodendrocytes from human embryonic stem cells and dedifferentiated stem cells, and through this, a 3D culture method in a medium that induces organoid formation and differentiation. The present invention was completed by developing and establishing a method capable of forming organoids containing oligodendrocytes within a short period of time.

Accordingly, an object of the present invention is to provide a method for preparing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes.

Another object of the present invention is to provide an organoid comprising oligodendrocytes prepared by the method of the present invention.

Another object of the present invention is to provide a method for screening a therapeutic agent for demyelinating disease, comprising the step of analyzing whether myelination is formed or not after treating the organoid comprising oligodendrocytes of the present invention with a candidate drug will be.

Another object of the present invention is to provide an organoid comprising oligodendrocytes of the present invention or a cell therapy composition for the prevention or treatment of demyelinating diseases, comprising, as an active ingredient, an organoid comprising oligodendrocytes or oligodendrocytes isolated from the organoid. will do

In order to achieve the above object, the present invention, (1) human pluripotent stem cells (Human pluripotent stem cell; hPSC) LIF (Leukemia Inhibitory Factor), SB431542 compound and CHIR 99021 by culturing in LSC medium containing the compound Differentiating into Primitive NSCs (pNSCs); (2) culturing the primitive neural stem cells in an FEP medium containing bFGF, EGF and Purmorphamine to differentiate them into ventral rosette type NSCs (vrNSCs); (3) Differentiation of the ventral rosette-type neural stem cells into pre-Oligodendrocyte Precursor Cells (pre-OPC) specialized for differentiation into oligodendrocytes by culturing the ventral rosette-type neural stem cells in FPP medium containing bFGF, PDGF and Purmorphamine making; and (4) RA (retinoic acid)-containing medium for neural stem cells specialized for differentiation into oligodendrocytes; a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin; And forskolin (forskolin), ascorbic acid and T3 (3,3',5-triiodo-L-thyronine) containing medium; cultured while sequentially replacing the medium to form an organoid containing oligodendrocytes It provides a method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, comprising the steps of.

In one embodiment of the present invention, the medium includes DMEM/F12, sodium bicarbonate, vitamin A-free B-27 supplement, N-2 supplement, penicillin-streptomycin, L-glutamine and non-essential amino acids. It may be used as a basal medium.

In one embodiment of the present invention, the neural stem cells specialized for differentiation into oligodendrocytes may express Oligodendrocyte transcription factor 2 (OLIG2) and NKX2.2 (NK2 Homeobox 2).

In one embodiment of the present invention, the organoid may further include neurons and astrocytes differentiated from neural stem cells specialized for differentiation into oligodendrocytes.

The present invention also provides an organoid comprising oligodendrocytes prepared by the method of the present invention.

The present invention also provides a method for screening a therapeutic agent for a demyelinating disease, comprising the step of treating the organoid containing oligodendrocytes of the present invention with a candidate drug and then analyzing whether or not myelination is formed.

In one embodiment of the present invention, when the myelination is promoted by the treatment with the candidate drug, the method may further include determining the candidate drug as a therapeutic agent for demyelinating disease.

The present invention also provides a cell therapy composition for preventing or treating demyelinating disease, comprising the organoid comprising oligodendrocytes of the present invention or oligodendrocytes isolated from the organoid as an active ingredient.

In one embodiment of the present invention, the composition may be to promote myelination regeneration.

In one embodiment of the present invention, the demyelinating disease is acute disseminated demyelination, Leber hereditary optic neuropathy, demyelinating polyneuropathy, and Charcot Marie Tooth It may be selected from the group consisting of a bottle (Charcot Marie Tooth).

In one embodiment of the present invention, the organoid may be an organoid treated with a myelination inducing drug.

In one embodiment of the present invention, the myelination inducing drug may be T3 (3,3',5-triiodo-L-thyronine), miconazole (Miconazole) or benztropine (Benztropine).

The method for producing an organoid containing oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes according to the present invention has the effect of producing an organoid containing oligodendrocytes in a short time, and dehydration There is an effect that can be usefully used as a cell therapy for the treatment of primary diseases. In addition, the organoid containing oligodendrocytes prepared by the method of the present invention can be used for screening drugs having remyelination-inducing efficacy in vitro, so it can be usefully used to prepare a 3D platform for new drug development. there is an effect

1 shows the process of establishing pre-OPCs from hESCs, culture conditions, characteristics of pre-OPCs, and the results of confirming the characteristics of oligodendrocytes differentiated from pre-OPCs. 1A shows pre-OPCs from hESCs. The shape and culture conditions for each step to establish are schematic diagrams, 1B is the culture conditions for establishing pNSCs, 1C is the result of confirming the characteristics of the established pNSCs, 1D is the culture for establishing pre-OPCs The conditions are shown, 1E is the confirmation of the characteristics of the established pre-OPCs, 1F is the culture conditions to confirm the differentiation ability of the established pre-OPCs, 1G is the characteristics of the astrocytes differentiated from the pre-OPCs analysis, and 1H shows the results of confirming the characteristics of oligodendrocytes differentiated from pre-OPCs.

Figure 2 shows the results for spheroid formation from primitive neural stem cells (pNSC) and neural stem cells (pre-OPC) specialized for differentiation into oligodendrocytes, 2A is spheroid formation and neurons from pNSC and pre-OPC Each step for differentiation into (TUJ1 +), astrocytes (GFAP +), and oligodendrocytes (MBP +) is shown as a schematic diagram, and the meaning of each abbreviation is as follows. LSC denotes LIF, SB431542 and CHIR99021, FPP denotes FGF2, PDGF-AA and Purmorphamine, FSK denotes Forskolin, T3 denotes 3,3',5-triiodo-L-thyronine, and AA denotes ascorbic acid. will be. 2B and 2C show the results of confirming the characteristics of pNSCs and pre-OPC by immunofluorescence staining for PAX6, SOX1, OLIG2 and NKX2.2, respectively. Figures 2D and 2E show the morphology of pNSC (D) and pre-OPC (E) on days 0 and 3, and 2F and 2G show cell differentiation in Matrigel droplets according to differentiation time for 4 weeks, embedding Previously, pNSC and pre-OPC were shown to form 200-300 μm neurospheres. 2H shows fluorescence images for each spheroid formed from pNSC and pre-OPC after 2 weeks of Matrigel embedding, and the dotted line indicates the VZ-like region. 2I and 2J show fluorescence images for each spheroid formed from pNSC (passage 1 and passage 15) and pre-OPC after 4 weeks of matrigel embedding, 5-HT is 5-hydroxytryptamine, ChAT is choline acetyltransferase, HB9 is MNX1, OPC is oligodendrocyte progenitor cell, pNSC is primitive neural stem cell, TH is tyrosine hydroxylase.

3 is a culture result of spheroids established from pre-OPC for rapid production of astrocytes, 3A is a spheroid fragment established from pre-OPC in TUJ1 (green) and BLBP (brain lipid binding protein; red) Shows the results confirmed by the fluorescence staining method, 3B shows the fluorescence images for each section after matrigel embedding, the results of confirming the expression levels of S100β (green) and GFAP (red), and the dotted line shows the separated spheroids it has been shown 3C shows the time-dependent S100β or GFAP-positive cell population by flow cytometry, 3D shows an enlarged fluorescence image after culturing spheroids established from pre-OPC for 6 weeks, arrows indicate elongated astrocytes, Arrowheads indicate star-shaped astrocytes, respectively, and 3E is an enlarged fluorescence image of spheroids formed from pre-OPCs derived from H9-hESCs (nos. 6 and 10) and hiPSCs (nos. 18), 3F shows a monolayer differentiated fluorescence image, confirmed by staining GFAP (red) and S100β (green), and organoids from the conventional two-dimensional culture method (2D) and pre-OPCs according to the present invention through flow cytometry. It shows a comparison of the differentiation rate into astrocytes according to the culture method (3D).

Figure 4 shows the results of confirming that the culture of spheroids formed from pre-OPC enables rapid production of oligodendrocytes, 4A is Matrigel embedding 4 weeks after, SOX10 (red) staining of spheroids One fluorescence image result, 4B and 4C show the 4th week spheroids (B) and 6th week spheroids (C) established from pre-OPC. Another marker of oligodendrocytes, OLIG2, PDGFRa, O4 or MBP 4D is a comparative analysis of the expression level of marker genes of oligodendrocytes through qPCR, 4E and 4F are the 8-week spheroids established from pre-OPC stained with MBP, SOX10 or TUJ1. Shows the results, 4G shows the results of staining the spheroids formed from pre-OPC treated with DMSO, T3, Benztropine or Miconazole, respectively, with TUJ1 and MBP, 4H and 4I are DMSO, T3, Benztropine or Miconazole treatment The results of quantitative comparative analysis of the myelination degree are shown, 4J shows the results of transplantation of oligodendrocytes isolated from spheroids formed from pre-OPCs treated with Miconazole into mice, and 4K shows the results of transplantation with an electron microscope. It shows the results of confirming the formation of myelin by observation.

5 is a schematic diagram illustrating a method for establishing spheroids (organoids) including oligodendrocytes from pre-OPCs according to the present invention.

Demyelination disease (demyelinating disease) is an intractable nervous system disease that appears due to the absence of oligodendrocytes throughout the central nervous system. Conventional research methods for treating this disease have been conducted to establish and treat oligodendrocytes from pluripotent stem cells. Although progress has been made, it has not been possible to establish a cell therapy drug that shows efficacy in clinical practice. In particular, oligodendrocytes are known to be present in very small amounts in the cerebrum and spinal cord, and the differentiation rate of oligodendrocytes from neural stem cells constituting the central nervous system to oligodendrocytes This is known to be low, so there are many difficulties in establishing oligodendrocytes from higher-stage stem cells including pluripotency. In addition, despite many studies, human oligodendrocyte progenitor cells that can be cultured have not yet been established.

Accordingly, the present inventors have established human neural stem cells that are highly differentiated into oligodendrocytes, and using this, it is possible to treat demyelinating diseases by identifying that organoids containing oligodendrocytes can be formed with high efficiency in a short period of time. It is possible to provide an effective cell therapy agent, and further established a method for screening a therapeutic agent for demyelinating disease using the organoid.

Specifically, the present invention, (1) human pluripotent stem cells (Human pluripotent stem cells; hPSC) LIF (Leukemia Inhibitory Factor), SB431542 compound, and CHIR 99021 compound by culturing in LSC medium containing the compound Primitive neural stem cells (Primitive NSC) ; pNSC); (2) culturing the primitive neural stem cells in an FEP medium containing bFGF, EGF and Purmorphamine to differentiate them into ventral rosette type NSCs (vrNSCs); (3) Differentiation of the ventral rosette-type neural stem cells into pre-Oligodendrocyte Precursor Cells (pre-OPC) specialized for differentiation into oligodendrocytes by culturing the ventral rosette-type neural stem cells in FPP medium containing bFGF, PDGF and Purmorphamine making; and (4) RA (retinoic acid)-containing medium for neural stem cells specialized for differentiation into oligodendrocytes; a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin; And forskolin (forskolin), ascorbic acid and T3 (3,3',5-triiodo-L-thyronine) containing medium; cultured while sequentially replacing the medium to form an organoid containing oligodendrocytes It is characterized by providing a method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, comprising the steps of.

In more detail, the method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes according to the present invention will be described as follows.

First, (1) human pluripotent stem cells (hPSCs) were cultured in a medium containing Leukemia Inhibitory Factor (LIF), a compound SB431542, and a compound CHIR 99021 to differentiate into primitive NSCs (pNSCs). make it

The human pluripotent stem cells refer to stem cells having the ability to differentiate into various cells constituting the human body, and collectively refer to stem cells capable of differentiating into almost all types of cells constituting endoderm, mesoderm, and ectoderm.

The human pluripotent stem cells are differentiated into primordial neural stem cells by culturing them in a specific medium, wherein the medium is a medium containing the Leukemia Inhibitory Factor (LIF), the SB431542 compound and the CHIR 99021 compound (ie, LSC medium).

The Leukemia Inhibitory Factor (LIF) used in the present invention is one of the members belonging to the interleukin-6 cytokine family, and is known as a factor involved in cell proliferation, differentiation and survival. In addition, the SB431542 acts as an ALK5 (Activin Receptor-Like Kinase-5) inhibitor to induce rapid differentiation and improve chromosomal stability. The CHIR 99021 is a GSK (glycogen synthase kinase) inhibitor that targets GSK1/2, an upstream molecule of GSK1/2 involved in the GSK signaling process. The CHIR 99021 is represented by aminopyrimidine.

All of the medium used in the present invention is a basal medium (BM: basal medium) as a basal medium, and a medium containing specific components in the basal medium was used in each cell culture step.

The basal medium refers to a medium containing DMEM/F12, sodium bicarbonate, vitamin A-free B-27 supplement, N-2 supplement, penicillin-streptomycin, L-glutamine and non-essential amino acids, an embodiment of the present invention Contains DMEM/F12, 543 ug/ml Sodium Bicarbonate, 1 x Vitamin A-Free B-27 Supplement, 1 x N-2 Supplement, 1% Penicillin-Streptomycin, 1% L-Glutamine and 1% Non-Essential Amino Acids basal medium was used. In addition, differentiation of the human pluripotent stem cells into primitive neural stem cells was induced by culturing human pluripotent stem cells in LSC medium containing the Leukemia Inhibitory Factor (LIF), the SB 431542 compound and the CHIR 99021 compound in the basal medium. After culture, the differentiated cells were confirmed for expression of PAX6 and SOX1, which are markers of primitive neural stem cells.

Next, (2) culturing the differentiated primitive neural stem cells to differentiate them into ventral rosette type NSCs (vrNSCs).

At this time, for differentiation into the ventral rosette-type neural stem cells, the differentiated primitive neural stem cells are cultured in FEP medium containing bFGF, EGF and Purmorphamine.

The rosette-type neural stem cells refer to neural stem cells in the initial stage of the neural differentiation process of human embryonic stem cells, and the rosette-type neural stem cells have a columnar radial shape. In addition, the rosette-type neural stem cells are composed of cells expressing early neuroectodermal markers such as PAX6 and SOX1, and can differentiate into various neuronal cells and glial cells.

In particular, in the present invention, a FEP medium containing bFGF, EGF and Purmorphamine was used for differentiation into ventral rosette-type neural stem cells. added medium.

The permorphamine is a purine compound and is known to be involved in the Shh signaling system. The permorphamine is also used to induce dedifferentiation into neural stem cell-like cells.

When differentiation into ventral rosette-type neural stem cells is completed, the next step is (3) differentiating the ventral rosette-type neural stem cells into pre-Oligodendrocyte Precursor Cells (pre-OPC) specialized for differentiation into oligodendrocytes. .

At this time, the culture medium for the differentiation is cultured using an FPP medium containing bFGF, PDGF and Purmorphamine. The FPP medium refers to a medium containing bFGF, PDGF and Purmorphamine in the basal medium.

As a result of culturing using the FPP medium, neural stem cells specialized for differentiation into oligodendrocytes were obtained from ventral rosette-type neural stem cells. Expression of markers OLIG2 and NKX2.2 was confirmed.

Also, in the present invention, the neural stem cells specialized for differentiation into oligodendrocytes refer to neural stem cells whose fate is determined or predetermined to be differentiated into oligodendrocytes.

The neural stem cells specialized for differentiation into oligodendrocytes through the above process are then cultured to form organoids including oligodendrocytes. Specifically, (4) differentiation into oligodendrocytes RA (retinoic acid)-containing medium for specialized neural stem cells; a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin; and forskolin, ascorbic acid and T3 (3,3',5-triiodo-L-thyronine) containing medium; by culturing while sequentially changing the medium to form organoids containing oligodendrocytes .

Neural stem cells specialized for differentiation into oligodendrocytes are cultured while sequentially replacing three types of media to prepare brain organoids including differentiated oligodendrocytes.

Specifically, neural stem cells specialized for differentiation into oligodendrocytes are cultured in a medium containing RA (retinoic acid), wherein the medium is cultured for 7 days using a medium containing 0.5 uM of RA in the basal medium.

Then, the culture is performed again by replacing the medium with a PDGF-containing medium, that is, PDGF (Platelet-derived growth factor), IGF1 (Insulin like growth factor), HGF (Hepatocyte growth factor) and forskolin in the basal medium. Incubated for an additional 14 days using a medium to which was added.

Then, the PDGF-containing medium was again cultured using a medium in which forskolin, ascorbic acid and T3 (3,3′,5-triiodo-L-thyronine) were added to the basal medium, which is a replacement cell differentiation medium. Through this, it was possible to obtain organoids containing oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes.

Forskolin added to the medium directly activates the catalytic subunit of didenylate cyclase to increase the concentration of cAMP in the cell, and tranylcypromine is mono, which is an enzyme that normally degrades norepinephrine in nerve terminals. It inhibits amine oxidase (MAO). In addition, the ascorbic acid is one of the water-soluble vitamins and has strong antioxidant properties, and the T3 (3,3′,5-triiodo-L-thyronine) acts as a thyroid hormone and improves mood and neuropsychological functions in hypothyroidism. It is known to have an improving effect.

RA (retinoic acid) is known to interact with RAR/RXR, a receptor present in the cell nucleus, and is known to be involved in the differentiation of neurons.

In addition, the method for rapidly producing an organoid containing oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes according to the present invention is a method for rapidly producing ventral rosette-type neural stem cells using bFGF, PDGF and Purmorphamine. Differentiating into neural stem cells (pre Oligodendrocyte Precursor Cell; pre-OPC) specialized for differentiation into oligodendrocytes by culturing in FPP medium containing; and RA (retinoic acid)-containing medium for neural stem cells specialized for differentiation into oligodendrocytes; a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin; And forskolin (forskolin), ascorbic acid and T3 (3,3',5-triiodo-L-thyronine) containing medium; cultured while sequentially replacing the medium to form an organoid containing oligodendrocytes This can be done through steps.

The method according to the present invention comprises the steps of (1) to (2) above, that is, differentiating into primitive NSCs (pNSCs) and differentiating into ventral rosette type NSCs (vrNSCs). It is not necessarily performed, and any ventral rosette-type neural stem cells are differentiated into pre-OPCs by culturing the ventral rosette-type neural stem cells in the above steps (3) and (4), that is, in FPP medium, and the pre-OPC contains RA. badge; PDGF, IGF1, HGF and forskolin containing medium; and a medium containing forskolin, ascorbic acid, and T3 (3,3′,5-triiodo-L-thyronine); cultured while sequentially replacing the medium with oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes Organoids containing cells can be rapidly produced.

On the other hand, the present inventors confirmed whether the differentiation of glial cells (glia) progressed in the organoid prepared from the neural stem cells specialized for differentiation into oligodendrocytes of the present invention prepared by the above method, and immunostaining As a result of chemical analysis, the expression of BLBP, a marker of glial progenitor cells, was confirmed at the 2nd week of differentiation, and the expression of S100β and GFAP, which are typical markers of astrocytes, was also confirmed at the 3rd week of differentiation. At week 5, it was confirmed that the expression of S100β and GFAP was significantly increased.

In addition, the expression of TH and GABA, which are markers of mature neurons, was also confirmed in the organoid.

Therefore, it was confirmed that, in the organoid prepared by the method of the present invention, astrocytes and neurons were differentiated and coexisted in addition to oligodendrocytes.

In addition, in one embodiment of the present invention, the organoid prepared by the method of the present invention and the organoid prepared by the conventional 2D differentiation method from primitive neural stem cells, not neural stem cells specialized for differentiation into oligodendrocytes. And as a result of comparative analysis of the differentiation rate, it was found that the types of organoids differentiated by each method were different. In particular, the organoids formed from the neural stem cells specialized for differentiation into oligodendrocytes according to the present invention are the organoids formed from primitive neural stem cells. It was confirmed that the differentiation into neurons and oligodendrocytes proceeded faster than that of the nodal cells.

In the differentiation into oligodendrocytes, the expression of oligodendrocyte progenitor markers SOX10, OLIG2, PDGFRa and O4 was confirmed at the 4th week of differentiation in the organoid prepared by the method of the present invention, and rare at the 6th week of differentiation. The expression of MBP, a marker of dendritic glial cells, was confirmed, and it was confirmed that the expression level of the oligodendrocyte markers was significantly increased at the 8th week compared to the 4th week, and myelination was confirmed.

As a result, the present inventors found that the organoid of the present invention containing a large amount of oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes can be used as a cell therapy agent for the treatment of demyelinating diseases, and also It was found that a therapeutic agent for demyelinating disease can be screened using the organoid.

Therefore, the present invention can provide an organoid comprising oligodendrocytes prepared by the method of the present invention, wherein the organoid has a spherical cell aggregate form.

In the present invention, the "organoid" refers to a cell mass having a 3D three-dimensional structure, and the organoid can be prepared from tissue or stem cells, and can be cultured in three dimensions due to its self-renewal and differentiation ability. have. The organoid may have an environment that is allowed to interact with the surrounding environment during the cell growth process. Accordingly, 3D organoids can almost mimic or completely simulate organs that actually interact in vivo, and can be an excellent model for observing the development of therapeutic agents for diseases and the like.

Also, in the present invention, "spheroid" refers to an aggregate of cells having a spherical shape. What is referred to as a substantially spherical shape is not limited to a perfectly spherical one, and a slightly flattened shape may also be included.

In addition, the present invention can provide a method for screening a therapeutic agent for a demyelinating disease, comprising the step of treating the organoid comprising oligodendrocytes according to the present invention with a candidate drug and then analyzing whether myelination is formed. .

Specifically, when the myelination is promoted by the treatment with the candidate drug, the method may further include determining the candidate drug as a therapeutic agent for demyelinating disease.

In an embodiment of the present invention, it was confirmed that the myelination-inducing drugs were treated on the organoids according to the present invention, and the degree of myelination was comparatively analyzed to select the drug having the best myelination ability.

Therefore, the organoid of the present invention can be used not only to screen for drugs having myelination or remyelination enhancing activity, but also to analyze the efficacy of the drug.

In addition, the present invention can provide a cell therapy composition for preventing or treating demyelinating disease, comprising an organoid comprising oligodendrocytes or oligodendrocytes isolated from the organoid as an active ingredient.

Most of the nerve fibers in the brain are surrounded by many layers of tissue composed of a lipoprotein called myelin (myelin), and because the myelin performs a similar role as an insulator, accurate and fast nerve impulses ( It plays an important role in transmitting neural impulses. When these myelin sheaths are damaged or destroyed by local damage, immune disorders, infection, nutritional deficiency, drugs, or unknown causes, permanent damage to the inner nerve fibers is induced. Therefore, if the myelin is damaged in the body, remyelination proceeds to restore it, but often the remyelination fails and proceeds to demyelination. Demyelination diseases caused by such demyelination include multiple sclerosis, acute disseminated demyelination, Leber hereditary optic neuropathy, and Guillain-Barré syndrome. (Guillain-Barre syndrome), demyelinating polyneuropathy, and Charcot Marie Tooth disease.

Therefore, in order to promote remyelination or inhibit demyelination when myelination is damaged, the development of a new therapeutic agent capable of recruiting oligodendrocyte precursor cells and differentiating the collected progenitor cells into oligodendrocytes is difficult. necessary.

In this regard, the organoids prepared by the method of the present invention rapidly differentiate between oligodendrocyte progenitor cells and oligodendrocytes and contain a large amount of oligodendrocytes, and thus various demyelination diseases caused by demyelination. It can be expected to be usable as an effective therapeutic agent for

In the present invention, the "oligodendrocytes" and "oligodendrocyte precursor cells" refer to cells of the oligodendrocyte lineage, that is, neural progenitor cells that ultimately generate oligodendrocytes, oligodendrocyte progenitor cells. , and mature and myelinated oligodendrocytes, which may be cells differentiated in the organoid prepared by the method of the present invention.

Also, oligodendrocytes may have functional properties, that is, they may have the ability to myelinate neurons and the like. Also, "oligodendrocyte precursor" or "oligodendrocyte precursor cell" refers to a cell capable of generating progeny comprising oligodendrocytes.

Therefore, the organoid comprising the oligodendrocytes of the present invention or the oligodendrocytes isolated from the organoid promotes myelin regeneration through an abnormality in the myelin myelin of the nervous system, particularly a demyelinating disease accompanied by destruction of myelin. disease) can be used as a cell therapy composition for the treatment of diseases.

In the present invention, the term "cellular therapeutic agent" refers to cells and tissues isolated from humans, cultured, and manufactured through special manipulation, and is a drug (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention. Or, through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells in other ways to restore the function of the tissue, these cells can be used in the treatment, diagnosis and prevention of diseases. Drugs used for that purpose.

The "treatment" refers to any action in which the symptoms of a disease are improved or beneficial by administration of the cell therapy agent.

The administration route of the cell therapy composition of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, may be administered intradermally, but is not limited thereto.

The composition may be formulated in a suitable form together with a pharmaceutical carrier commonly used for cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In addition, the composition may be administered by any device capable of transporting a cell therapy agent to a target cell.

The cell therapy composition of the present invention may contain a therapeutically effective amount of the cell therapy agent for the treatment of a disease.

In addition, the “therapeutically effective amount” refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, physician or other clinician. This includes an amount that induces amelioration of the symptoms of the disease or disorder being treated.

It is apparent to those skilled in the art that the cell therapy agent included in the composition of the present invention will change depending on the desired effect. Therefore, the optimal content of the cell therapy agent can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient , administration time, administration route and secretion rate of the composition, treatment period, and drugs used at the same time may be adjusted according to various factors. In consideration of all of the above factors, it is important to include an amount that can obtain the maximum effect with a minimum amount without side effects. For example, the daily dose of cells corresponding to the cell therapy agent of the present invention is 1.0×10 ⁴ to 1.0×10 ¹⁰ cells/kg body weight, preferably 1.0×10 ⁵ to 1.0×10 ⁹ cells/kg body weight once Alternatively, it may be administered in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the dosage may be any It is not intended to limit the scope of the present invention in any way.

In addition, in the treatment method of the present invention, the composition comprising the cell therapy agent of the present invention as an active ingredient may be rectal, intravenous (iv), intra-arterial, intra-peritoneal, intramuscular, intrasternal, transdermal, topical, or intraocular. Alternatively, it may be administered in a conventional manner via an intradermal route.

The present invention provides a treatment method comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the present invention. As used herein, the term mammal refers to a mammal that is the subject of treatment, observation or experimentation, and preferably refers to a human.

In addition, in the present invention, the organoid further treated with a myelination inducing drug may be used as a cell therapeutic agent, and the myelination inducing drug is not limited thereto, but T3 (3,3',5-triiodo- L-thyronine), miconazole (Miconazole) or benztropine (Benztropine) may be.

As described above, the method for producing an organoid including oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes according to the present invention can be produced in a short time in a three-dimensional organoid form, and the existing 20 weeks or more It was found to be significantly more effective than the two-dimensional method required, and the organoid of the 3D spherical body according to the present invention contains not only oligodendrocytes but also neurons and astrocytes, so that the brain functions closer to the actual human brain. Noid can be provided.

The organoid containing oligodendrocytes prepared by the method of the present invention can be usefully used as a cell therapy for demyelinating diseases, and can also be used as a drug development platform for rapidly and accurately screening therapeutic agents for demyelinating diseases. have.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples.

<Materials and test methods>

① Cell culture

Cultures of pNSCs cells (expressing PAX6 and SOX1) and pre-OPCs cells (expressing mesenchymal progenitors, OLIG2 and NKX2.2) were performed before embedding in Matrigel (356231, Corning, NY, USA), pNSC cells were incubated in LSC medium (LIF). , containing SB431542 and CHIR99021), and pre-OPC cells were cultured using FPP medium (containing FGF2, PDGF-AA and Purmorphamine). Considering the culture passage, pNSC cells were prepared as cells of passage 15 (close to the pre-OPCs stage). Cells were dispersed on day 0 using Accutase (EMD Millipore, Burlington, MA, USA), aliquoted on ultra-low adhesion plates (Corning), and maintained in LSC or FPP medium until embedding. The resulting neurospheres (>200 μm) were embedded in Matrigel droplets on day 4. Embedded neurospheres were prepared with 543 μg/ml sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 1× N-2 supplement (Thermo Fisher Scientific), and 1× B-27 supplement without vitamin A (Thermo Fisher). Scientific), DMEM/F12 (Thermo) with 1% penicillin-streptomycin, 1% L-glutamine (Thermo Fisher Scientific), 1% non-essential amino acids (Thermo Fisher Scientific) and 5 μg/ml insulin (Sigma-Aldrich) Fisher Scientific, Waltham, MA, USA) was cultured for 3 days in basal medium. After that, the droplets were transferred to an orbital shaker and incubated for an additional 1 week in basal medium supplemented with 0.5 μM retinoic acid (Sigma-Aldrich), the medium was incubated with 10 ng/ml PDGF-AA (Peprotech), 10 ng/ml IGF- 1 (Peprotech), 10 ng/ml NT-3 (Peprotech), 5 ng/ml HGF (Peprotech) and 10 μM forskolin (Tocris) were replaced with basal medium. The droplets were then infused with glue consisting of 10 μM forskolin (Tocris), 60 ng/ml 3,3′,5-triiodo-L-thyronine (T3, Sigma-Aldrich) and 20 μg/ml ascorbic acid (Peprotech) and basal medium. They were cultured in glial differentiation medium for 4 weeks. For subsequent screening of promyelinating compounds, 4-week spheroids were treated with DMSO (Sigma-Aldrich), 60 ng/ml T3 (Sigma-Aldrich), 1 μM benztropine (Sigma-Aldrich) or 1 μM miconazole (miconazole, Sigma). -Aldrich) for 8 weeks.

② Sample preparation and analysis

Spheroid samples are O.C.T. It was embedded with a compound (Tissue Tek; Sakura Finetek USA, Inc., Torrance, CA, USA) and frozen sectioned to a thickness of 20 μm. The sections were blocked with 2% normal donkey serum in PBS containing 0.2% Triton X-100 for 1 hour, and reacted with the designated primary antibody (Table 1 and Table 2 below) at 4°C overnight, and then at room temperature. was reacted with Alexa Fluor 488 or 594-conjugated secondary antibody (Thermo Fisher Scientific) for 1 hour. Nuclei were counterstained with 1ug/ml DAPI (Sigma-Aldrich) for 5 minutes, samples were washed 3 times with PBS and observed with an Olympus confocal laser scanning microscope. Also, transplanted mouse brain preparation and TEM analysis were prepared according to the disclosure of the Stem Cells Dev paper (2019;28:633-648) published by Yun W et al.

③ Flow cytometry

Spheroids were mechanically dissociated and collected using Accutase. After washing 3 times with cold PBS, blocking with 5% normal donkey serum for 15 min, first reacted with primary antibody against S100β and GFAP for 30 min, and then with Alexa Fluor-conjugated secondary antibody for 30 min. reacted for a minute. After performing staining, cells were fixed with 0.5% formaldehyde. Cells were permeabilized with 0.1% Triton X-100 during the blocking process. Cells were then analyzed using a FACS Verse flow cytometer (BD Biosciences, San Jose, CA, USA).

④ qPCR analysis

Quantitative PCR (qPCR) analysis was performed on cDNA three times. The negative control group included a negative blank for reverse transcription of each sample and a blank without a template. In the measurement of the gene expression level, it was normalized based on the expression level of GAPDH as an internal control. In addition, the primers used for qPCR are shown in Table 3 below.

In addition, all data were expressed as mean ± standard deviation (SD) repeated 3 to 6 times, and the results were analyzed by unpaired two-tailed Student's t test and variance test (ANOVA). P values less than 0.05 were considered statistically significant. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.

<Example 1>

Establishment of neural stem cells specialized for differentiation into oligodendrocytes

The present inventors performed experiments based on various culture systems to establish neural stem cells specialized for differentiation into oligodendrocytes. Figure 1A shows the shape and culture conditions of cells at each stage from hESCs (Human embryonic stem cells) to pre-OPCs cells. The following describes a cell culture method for the establishment of neural stem cells (pre-OPCs) specialized for differentiation from human pluripotent stem cells (hPSCs) into oligodendrocytes.

To induce differentiation, all cells derived from pluripotent stem cells were prepared in basal medium (BM) [DMEM/F12, 543 μg/ml sodium bicarbonate, 1×B-27 Supplement without vitamin A, 1×N-2 Supplement, 1% penicillin-streptomycin, 1% L-glutamine and 1% non-essential amino acids] were used as the basis for culture. In order to establish primitive neural stem cells (Primitive NSCs, pNSCs), pluripotent stem cells were cultured in LSC medium [BM, 10 ng/ml Leukemia Inhibitory Factor (LIF), containing 2 μM SB431542 and 3 μM Chir99021] differentiation conditions (Fig. see 1B). After 6 days of culture in the LSC culture medium, it was confirmed by immunochemical staining that the differentiated cells express the primitive neural stem cell markers PAX6 and SOX1 (see FIG. 1C ). In addition, primordial neural stem cells were subcultured in LSC culture medium, and thereafter, primordial neural stem cells were passaged at low density in a Matrigel-coated cell culture dish to differentiate into ventral rosette type NSCs (vrNSCs). It was cultured in FEP medium [BM, containing 20 ng/ml bFGF, 20 ng/ml EGF and 0.5 μM Purmorphamine]. After 8 days of culture, rosette-shaped colonies appeared. In addition, for the establishment of neural stem cells (pre-OPCs) specialized for differentiation into oligodendrocytes, the FEP culture medium was replaced with the FPP culture medium [BM, containing 20 ng/ml bFGF and 20 ng/ml PDGF] and cultured (Fig. 1D). Reference).

As a result, it was confirmed that the expression of OLIG2 and NKX2.2, which are representative markers of oligodendrocyte progenitor cells, significantly increased in neural stem cells (pre-OPCs) specialized for differentiation into oligodendrocytes established by the above method (FIG. 1E). Reference).

In addition, the PDGF culture medium [containing BM, 20 ng/ml PDGF, 10 ng/ml NT-3, 10 ng/ml IGF-1, 10 uM Forskolin and 60 ng/ml T3] was replaced and after 2 weeks of co-culture ( 1F), as a result of examining differentiation for 8 weeks, it was confirmed that the shape of glial cells appeared, and the cells were found to express GFAP and S100β, which are representative markers of astrocytes (see FIG. 1G). In addition, oligodendrocytes express mature markers such as O4, MBP, and MAG, and O4 in oligodendrocytes differentiated from neural stem cells (pre-OPCs) specialized for differentiation into oligodendrocytes; As it was confirmed that MBP and MAG were expressed, it was confirmed that differentiation into oligodendrocytes was made by the above method (see FIG. 1H ).

<Example 2>

Establishment of organoids from neural stem cells specialized for differentiation into oligodendrocytes

Organoids were formed from neural stem cells (pre-OPCs) specialized for differentiation into oligodendrocytes established in Example 1 according to the following method, and organoids formed from pre-OPCs and primitive neural stem cells (pNSCs) formed from Comparative analysis of organoids was performed through the following method.

First, the neural stem cells (pre-OPCs) specialized in PAX6 and SOX-1 expressing primitive neural stem cells (pNSCs) and oligodendrocytes expressing OLIG2 and NKX2.2 established in Example 1 were 4 After the formation of spheres through co-culture for a day, it was cultured for 7 days in RA culture medium (containing BM and 0.5 uM Retinoic Acids (RA)) after embedding in Matrigel. Then, after additional culture for 14 days in the PDGF culture medium, in the glial differentiation culture medium (BM, 10 uM Forskolin, 60 ng/ml T3, 20 ug/ml ascorbic acid containing) for 5 to 9 weeks depending on the experiment. cultured. A schematic diagram of this culture sequence is shown in Figure 2A. After completing the culture for 4 weeks, the characteristics of the organoids obtained from the neural stem cells specialized into primitive neural stem cells and oligodendrocytes were analyzed.

As a result, the characteristics of primitive neural stem cells (PNSCs, pNSCs) and neural stem cells (pre-OPCs) specialized for oligodendrocytes used in the above experiment were confirmed through immunochemical staining. was confirmed to express OLIG2 and NKX2.2, and neural stem cells specialized to oligodendrocytes were confirmed to express OLIG2 and NKX2.2 ( FIGS. 2B and 2C ). In addition, as a result of microscopic observation of the shape of organoids obtained from primitive neural stem cells and neural stem cells specialized into oligodendrocytes, it was confirmed that these two types of cells formed neurospheres, but their shapes were different (Fig. 2D and 2E). In addition, in the observation result 7 days after RA treatment, it was shown that the formation of large neuroepithelial buds observed in brain organoids was confirmed in the organoids formed from primitive neural stem cells, whereas the neural stem cells specialized into oligodendrocytes of the present invention were The formed organoids could observe a radially protruding process consistent with direct innervation (see Figs. 2F and 2G).

As a result of immunostaining chemistry analysis, it was found that the VZ (Ventricular zone) was largely formed in the organoids obtained from primitive neural stem cells and contained TUJ1+ immature neurons, whereas in the organoids obtained from neural stem cells specialized into oligodendrocytes, Formation of small VZ was confirmed at 2 weeks of culture (see Fig. 2H).

In addition, as a result of analyzing various neuronal markers, it was confirmed that the differentiation of organoids obtained from neural stem cells specialized into oligodendrocytes was promoted into neurons showing TH and GABA, markers of mature neurons (see Fig. 2I). ), furthermore, it was confirmed that OLIG2, a marker of motor neurons, was still expressed, whereas HB9+, a marker of motor neurons, was not observed (see FIG. 2J ).

Based on these results, the present inventors confirmed that the organoids formed from neural stem cells specialized for differentiation into oligodendrocytes according to the present invention progressed faster than the organoids formed from primitive neural stem cells.

<Example 3>

Verification of differentiation from organoids formed from neural stem cells to glial cells

Whether the differentiation of organoids obtained from neural stem cells (pre-OPCs) specialized for oligodendrocytes into glial cells (Glia) proceeded rapidly was analyzed through immunostaining chemistry.

As a result, it was confirmed that BLBP, a marker of glial progenitor cells, was expressed in organoids (spheroids) formed from pre-OPCs at the 2nd week of differentiation into glial cells (FIG. 3A), and at the 3rd week, Expression of S100β and GFAP, which are typical markers of astrocytes, was confirmed by immunostaining chemistry (FIG. 3B) and flow cytometry (FIG. 3C). On the other hand, in organoids formed from primitive neural stem cells (pNSCs), expression of these markers could not be confirmed even at the 4th week of differentiation, and the expression of S100β and GFAP was also very low at the 5th week of differentiation.

It was confirmed through immunostaining chemistry that morphologically diverse astrocytes, known as a feature of organoids, exist (FIG. 3D), and it was also confirmed that various astrocytes were mixed in gene expression (FIG. 3E).

In addition, the culture method for forming organoids from neural stem cells specialized for oligodendrocytes according to the present invention can significantly increase the differentiation efficiency from organoids to astrocytes in a shorter time than the existing 2D differentiation method ( 3F).

Therefore, the culture method capable of forming organoids from neural stem cells specialized in oligodendrocytes according to the present invention and the organoids formed by the method are characterized by rapid differentiation into astrocytes, and similar to the actual human brain. It was found that astrocytes exist in a mixture, and can be used more effectively as a cell therapy for the treatment of brain diseases.

<Example 4>

Validation of differentiation and myelination ability from organoids formed from neural stem cells to oligodendrocytes

Furthermore, the present inventors analyzed whether the differentiation into oligodendrocytes rapidly progressed in the organoids obtained from neural stem cells specialized for oligodendrocytes according to the present invention by immunostaining chemistry.

As a result, the expression of SOX10, a marker of oligodendrocyte progenitors, was confirmed at the 4th week of differentiation in organoids obtained from neural stem cells specialized for oligodendrocytes (FIG. 4A), and OLIG2, PDGFRa and O4 were expressed in SOX10-positive cells. It could be confirmed that (Fig. 4B), the expression of the oligodendrocyte marker MBP was also confirmed at the 6th week (Fig. 4C). In addition, the 8th week organoid showed increased expression of oligodendrocyte-related markers compared to the 4th week organoid (refer to FIG. 4D), and in particular, at the 8th week, in vitro myelination was formed in the organoid. It was confirmed that this was made ( FIGS. 4E and 4F ).

Furthermore, in order to confirm whether drug screening through evaluation of myelination ability is possible using the organoid culture method according to the present invention, the present inventors have previously reported that T3 (60 ng/ml), Miconazole ( As a result of treating the organoids with 1uM) and Benztropine (1uM), it was confirmed that myelination was more advanced in the group treated with the drug compared to the control group treated with DMSO (Fig. 4G), specifically Among the above drugs, it was confirmed that the most amount of myelin was formed in the organoid treated with Miconazole ( FIGS. 4H and 4I ). In addition, the present inventors isolated oligodendrocytes from organoids treated with Miconazole, and then transplanted them into non-myelinated Shiverer mice. As a result, it was confirmed that MBP+ oligodendrocytes, that is, myelin formation (see Fig. 4J), were transplanted. After 12 weeks, it was confirmed that the myelin group was formed through an electron microscope.

Therefore, based on the above results, the present inventors found that the culture method capable of forming organoids from neural stem cells specialized for differentiation into oligodendrocytes according to the present invention is oligodendrocytes in a shorter time than using primitive neural stem cells. And it was possible to differentiate into astrocytes and to evaluate the ability to form remyelination, so it was found that it could be usefully used for drug screening for the treatment of demyelinating diseases. It was found that organoids can be usefully used as cell therapy agents for the treatment of demyelinating diseases.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

1. (1) culturing human pluripotent stem cells (hPSCs) in LSC medium containing Leukemia Inhibitory Factor (LIF), SB431542 compound and CHIR 99021 compound to differentiate into primitive neural stem cells (Primitive NSC; pNSC) step;

   (2) culturing the primitive neural stem cells in an FEP medium containing bFGF, EGF and Purmorphamine to differentiate them into ventral rosette type NSCs (vrNSCs);

   (3) Differentiation of the ventral rosette-type neural stem cells into pre-Oligodendrocyte Precursor Cells (pre-OPC) specialized for differentiation into oligodendrocytes by culturing the ventral rosette-type neural stem cells in FPP medium containing bFGF, PDGF and Purmorphamine making; and

   (4) RA (retinoic acid)-containing medium for neural stem cells specialized for differentiation into oligodendrocytes; a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin; And forskolin (forskolin), ascorbic acid and T3 (3,3',5-triiodo-L-thyronine) containing medium; cultured while sequentially replacing the medium to form an organoid containing oligodendrocytes comprising steps,

   A method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes.
2. According to claim 1,

   The medium is DMEM/F12, sodium bicarbonate, vitamin A-free B-27 supplement, N-2 supplement, penicillin-streptomycin, L-glutamine and a medium containing non-essential amino acids as a basal medium, characterized in that , A method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes.
3. According to claim 1,

   The neural stem cells specialized for differentiation into oligodendrocytes are oligodendrocyte transcription factor 2 (OLIG2) and oligodendrocyte transcription factor 2) and oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, characterized in that expressing NKX2.2 (NK2 Homeobox 2). A method for producing an organoid comprising a cell.
4. According to claim 1,

   The organoid includes oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, characterized in that the organoids further include neurons and astrocytes differentiated from neural stem cells specialized for differentiation into oligodendrocytes Method for producing organoids.
5. An organoid comprising oligodendrocytes prepared by the method of any one of claims 1 to 4.
6. A method of screening for a therapeutic agent for a demyelinating disease, comprising the step of treating the organoid containing the oligodendrocytes of claim 5 with a candidate drug and then analyzing whether myelination is formed.
7. 7. The method of claim 6,

   When myelination is promoted by the treatment with the candidate drug, the method further comprising the step of determining the candidate drug as a therapeutic agent for a demyelinating disease.
8. A cell therapy composition for the prevention or treatment of demyelinating diseases, comprising the organoid comprising the oligodendrocytes of claim 5 or the oligodendrocytes isolated from the organoid as an active ingredient.
9. 9. The method of claim 8,

   The composition is a cell therapy composition for the prevention or treatment of demyelination diseases, characterized in that it promotes myelination regeneration.
10. 9. The method of claim 8,

    The demyelinating disease is acute disseminated demyelination, Leber hereditary optic neuropathy, demyelinating polyneuropathy and Charcot Marie Tooth disease. Cell therapy composition for the prevention or treatment of demyelinating disease, characterized in that selected from.
11. 9. The method of claim 8,

    The organoid is a cell therapy composition for the prevention or treatment of demyelination disease, characterized in that the organoid treated with a myelination inducing drug.
12. 12. The method of claim 11,

    The myelination inducing drug is T3 (3,3',5-triiodo-L-thyronine), miconazole (Miconazole) or benztropine (Benztropine), characterized in that the cell therapy for preventing or treating demyelination disease composition.

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