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

Method for preparing organoid including oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, organoid prepared by same method, and use thereof Download PDF

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WO2021235848A1
WO2021235848A1 PCT/KR2021/006248 KR2021006248W WO2021235848A1 WO 2021235848 A1 WO2021235848 A1 WO 2021235848A1 KR 2021006248 W KR2021006248 W KR 2021006248W WO 2021235848 A1 WO2021235848 A1 WO 2021235848A1
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oligodendrocytes
stem cells
organoid
neural stem
differentiation
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French (fr)
Korean (ko)
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유승권
윤원진
오동훈
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고려대학교 산학협력단
주식회사 스템랩
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system

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  • 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.
  • CNS Central Nervous System
  • demyelinating disease is caused by abnormalities in the oligodendrocytes that form the myelin sheath and is caused by damage to the nerve cells.
  • CNS Central Nervous System
  • transplantation of oligodendrocyte precursor cells (OPC) that can differentiate into oligodendrocytes is emerging as a major treatment.
  • Neural Stem Cells 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.
  • 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.
  • pre-OPCs human neural stem cells
  • the present invention was completed by developing and establishing a method capable of forming organoids containing oligodendrocytes within a short period of time.
  • 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.
  • 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
  • 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.
  • the neural stem cells specialized for differentiation into oligodendrocytes may express Oligodendrocyte transcription factor 2 (OLIG2) and NKX2.2 (NK2 Homeobox 2).
  • Oligodendrocyte transcription factor 2 OLIG2
  • NKX2.2 NK2 Homeobox 2
  • 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.
  • 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.
  • the composition may be to promote myelination regeneration.
  • 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).
  • the organoid may be an organoid treated with a myelination inducing drug.
  • 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.
  • 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
  • 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
  • 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
  • TUJ1 + astrocytes
  • GFAP + astrocytes
  • MBP + oligodendrocytes
  • LSC denotes LIF, SB431542 and CHIR99021
  • FPP denotes FGF2, PDGF-AA and Purmorphamine
  • FSK denotes Forskolin
  • T3 denotes 3,3',5-triiodo-L-thyronine
  • AA denotes ascorbic acid.
  • 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 ⁇ m 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 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 S100 ⁇ (green) and GFAP (red), and the dotted line shows the separated spheroids it has been shown
  • 3C shows the time-dependent S100 ⁇ 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
  • 3E is an enlarged fluorescence image of spheroids formed from pre-OPCs derived from
  • 3F shows a monolayer differentiated fluorescence image, confirmed by staining GFAP (red) and S100 ⁇ (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.
  • 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
  • 4J shows the results of transplantation of oligodendrocytes isolated from spheroids formed from pre-OPCs treated with Miconazole into mice
  • 4K shows the results of transplantation with an electron microscope. It shows the results of confirming the formation of myelin by observation.
  • FIG. 5 is a schematic diagram illustrating a method for establishing spheroids (organoids) including oligodendrocytes from pre-OPCs according to the present invention.
  • Demyelination 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.
  • 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.
  • human oligodendrocyte progenitor cells that can be cultured have not yet been established.
  • 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.
  • 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;
  • pre-OPC pre-Olig
  • hPSCs human pluripotent stem cells
  • LIF Leukemia Inhibitory Factor
  • SB431542 a compound SB431542
  • CHIR 99021 a compound CHIR 99021 to differentiate into primitive NSCs (pNSCs).
  • 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).
  • LIF Leukemia Inhibitory Factor
  • SB431542 compound the CHIR 99021 compound
  • 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.
  • 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.
  • BM basal medium
  • 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.
  • 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.
  • LIF Leukemia Inhibitory Factor
  • SOX1 are markers of primitive neural stem cells.
  • vrNSCs ventral rosette type NSCs
  • 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.
  • 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.
  • 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.
  • 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. .
  • pre-OPC Precursor Cells
  • 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.
  • 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.
  • 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.
  • RA retinoic acid
  • a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin a medium containing platelet-derived growth factor (PDGF), insulin like growth factor (IGF1), hepatocyte growth factor (HGF) and forskolin
  • RA retinoic acid
  • PDGF platelet-derived growth factor
  • IGF1 insulin like growth factor
  • HGF hepatocyte growth factor
  • forskolin ascorbic acid and T3 (3,3',5-triiodo-L-thyronine) containing medium
  • Neural stem cells specialized for differentiation into oligodendrocytes are cultured while sequentially replacing three types of media to prepare brain organoids including differentiated oligodendrocytes.
  • 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.
  • RA retinoic acid
  • 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.
  • PDGF Platinum-derived growth factor
  • IGF1 Insulin like growth factor
  • HGF Hepatocyte growth factor
  • 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.
  • tranylcypromine is mono, which is an enzyme that normally degrades norepinephrine in nerve terminals. It inhibits amine oxidase (MAO).
  • MAO amine oxidase
  • 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
  • the method for rapidly producing an organoid containing oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes is a method for rapidly producing ventral rosette-type neural stem cells using bFGF, PDGF and Purmorphamine.
  • pre Oligodendrocyte Precursor Cell pre-OPC
  • pre-OPC Oligodendrocyte Precursor Cell
  • RA retinoic acid
  • PDGF platelet-derived growth factor
  • IGF1 insulin like growth factor
  • HGF hepatocyte growth factor
  • Sorkolin 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.
  • pNSCs primitive NSCs
  • vrNSCs ventral rosette type NSCs
  • 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
  • glia glial cells
  • the expression of BLBP a marker of glial progenitor cells
  • S100 ⁇ and GFAP which are typical markers of astrocytes
  • TH and GABA which are markers of mature neurons
  • astrocytes and neurons were differentiated and coexisted in addition to oligodendrocytes.
  • the types of organoids differentiated by each method were different.
  • 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.
  • 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.
  • MBP a marker of dendritic glial cells
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • the method may further include determining the candidate drug as a therapeutic agent for demyelinating disease.
  • 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.
  • 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.
  • 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.
  • myelin a lipoprotein
  • myelin a lipoprotein
  • accurate and fast nerve impulses It plays an important role in transmitting neural impulses.
  • 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.
  • 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
  • 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.
  • oligodendrocytes may have functional properties, that is, they may have the ability to myelinate neurons and the like.
  • oligodendrocyte precursor or “oligodendrocyte precursor cell” refers to a cell capable of generating progeny comprising oligodendrocytes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a mammal refers to a mammal that is the subject of treatment, observation or experimentation, and preferably refers to a human.
  • 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.
  • 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. noisy 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.
  • pNSCs cells expressing PAX6 and SOX1
  • pre-OPCs cells expressing mesenchymal progenitors, OLIG2 and NKX2.2
  • LSC medium LIF
  • pre-OPC cells were cultured using FPP medium (containing FGF2, PDGF-AA and Purmorphamine).
  • FPP medium containing FGF2, PDGF-AA and Purmorphamine.
  • Embedded neurospheres were prepared with 543 ⁇ g/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).
  • DMEM/F12 (Thermo) with 1% penicillin-streptomycin, 1% L-glutamine (Thermo Fisher Scientific), 1% non-essential amino acids (Thermo Fisher Scientific) and 5 ⁇ g/ml insulin (Sigma-Aldrich) Fisher Scientific, Waltham, MA, USA) was cultured for 3 days in basal medium.
  • the droplets were transferred to an orbital shaker and incubated for an additional 1 week in basal medium supplemented with 0.5 ⁇ M 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 ⁇ M forskolin (Tocris) were replaced with basal medium.
  • basal medium supplemented with 0.5 ⁇ M 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 ⁇ M forskolin (Tocris) were replaced with bas
  • the droplets were then infused with glue consisting of 10 ⁇ M forskolin (Tocris), 60 ng/ml 3,3′,5-triiodo-L-thyronine (T3, Sigma-Aldrich) and 20 ⁇ g/ml ascorbic acid (Peprotech) and basal medium. They were cultured in glial differentiation medium for 4 weeks.
  • glue consisting of 10 ⁇ M forskolin (Tocris), 60 ng/ml 3,3′,5-triiodo-L-thyronine (T3, Sigma-Aldrich) and 20 ⁇ g/ml ascorbic acid (Peprotech) and basal medium. They were cultured in glial differentiation medium for 4 weeks.
  • 4-week spheroids were treated with DMSO (Sigma-Aldrich), 60 ng/ml T3 (Sigma-Aldrich), 1 ⁇ M benztropine (Sigma-Aldrich) or 1 ⁇ M miconazo
  • 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 ⁇ m. 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.
  • mice 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.
  • 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 S100 ⁇ 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).
  • 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.
  • the gene expression level it was normalized based on the expression level of GAPDH as an internal control.
  • the primers used for qPCR are shown in Table 3 below.
  • 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.
  • pre-OPCs neural stem cells
  • hPSCs human pluripotent stem cells
  • pluripotent stem cells were prepared in basal medium (BM) [DMEM/F12, 543 ⁇ g/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.
  • BM basal medium
  • LIF Leukemia Inhibitory Factor
  • 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 ⁇ M Purmorphamine]. After 8 days of culture, rosette-shaped colonies appeared.
  • 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).
  • 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 S100 ⁇ , which are representative markers of astrocytes (see FIG. 1G).
  • 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 ).
  • 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.
  • pre-OPCs neural stem cells
  • pNSCs primitive neural stem cells
  • 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.
  • RA culture medium containing a
  • PNSCs, pNSCs and pre-OPCs neural stem cells 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 ).
  • PIGFs, pNSCs primitive neural stem cells
  • pre-OPCs neural stem cells 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 ).
  • FIGS. 2B and 2C neural stem cells specialized to oligodendrocytes
  • VZ Ventricular zone
  • 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.
  • pre-OPCs neural stem cells
  • oligodendrocytes oligodendrocytes into glial cells
  • BLBP a marker of glial progenitor cells
  • organoids spheroids
  • pNSC primitive neural stem cells
  • FIG. 3D 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).
  • 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).
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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.

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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

희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법, 상기 방법으로 제조된 오가노이드 및 이의 용도Method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes, organoids prepared by the method, and uses thereof
본 발명은 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법, 상기 방법으로 제조된 오가노이드 및 이의 용도에 관한 것이다.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.
의료 및 과학기술의 발달로 인간의 평균수명이 늘어나면서 100세를 넘는 센터내리언(centenarian) 시대가 다가옴에 따라 개인 맞춤형 세포치료제는 인간의 장수에 밑거름이 될 근본적인 필수요소로 대두되고 있다. 다양한 중추신경계(Central Nervous System, CNS) 질환 중, 탈수초성 질환(Demyelinating disease)은 미엘린 수초(Myelin Sheath)를 형성하는 희소돌기아교세포(Oligodendrocyte)에 이상이 생겨 신경세포(Neuron)의 손상으로 인해 다양한 신경질환을 유발하는 질환으로, 희소돌기아교 세포로 분화할 수 있는 희소돌기아교 전구세포(Oligodendrocyte Precursor Cells, OPC)를 이식하는 방법이 주요 치료법으로 대두되고 있다.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.
신경줄기세포(Neural Stem Cells, NSC)는 신경세포, 성상교세포(Astrocyte), 희소돌기아교세포로 3개의 세포로 분화가 가능한 다분화능 줄기세포로 신경질환을 치료할 수 있는 치료제로 각광을 받고 있으며, 분화 및 교차분화를 통하여 세포를 수득할 수 있는 방법이 개발되었지만, 희소돌기아교세포로 분화할 수 있는 능력이 현저히 낮아 탈수초성 질환의 세포 치료제로서는 적합하지 않은 것으로 알려져 있다.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.
하지만, 인간의 뇌 안에는 희소돌기아교세포의 양이 극히 적은 것으로 알려져 있어 자가이식이 어렵고, 외부에서 이식에 사용될 세포를 얻고자 배아줄기세포 및 역분화 줄기세포를 통한 분화 연구가 진행되고 있지만 아직까지 실제 임상시험에서 이식을 통한 뚜렷한 효과는 입증되지 않았다.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.
이에 많은 연구진들은 탈수초성 질환을 치료할 수 있는 약물(Drug)을 개발하고자 약물 스크리닝(Drug screening)을 통하여 재수초(Remyelination) 형성을 유도할 수 있는 약물을 개발하고자 노력하고 있지만 약물후보의 탐색, 전임상단계의 약물효능 평가를 위해 사용되는 다양한 동물 및 세포 모델(2D)이 인체 내 환경-세포간 상호작용 및 세포내 복잡한 신호전달 경로를 반영하지 못하여 신약개발의 어려움을 겪고 있다.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.
따라서 탈수초성 질환에 대한 효과적인 약물 개발을 위해, 희소돌기아교세포를 포함하는 오가노이드 및 재수초 형성을 신속하고 효과적으로 유도할 수 있는 기술의 개발이 필요한 실정이다.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.
이에, 본 발명자들은 인간 배아 줄기세포 및 역분화 줄기세포로부터 희소돌기아교세포로 특화된 인간 신경줄기세포(pre-OPCs)를 확립하였고, 이를 통해 오가노이드 형성 및 분화를 유도하는 배지에서의 3D 배양방법을 개발하여 단 기간 내에 희소돌기아교세포가 포함된 오가노이드를 형성할 수 있는 방법을 확립함으로써 본 발명을 완성하였다.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.
따라서 본 발명의 목적은 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법을 제공하는 것이다.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.
본 발명의 다른 목적은 본 발명의 방법으로 제조된 희소돌기아교세포를 포함하는 오가노이드를 제공하는 것이다.Another object of the present invention is to provide an organoid comprising oligodendrocytes prepared by the method of the present invention.
본 발명의 또 다른 목적은 본 발명의 희소돌기아교세포를 포함하는 오가노이드에 후보약물을 처리한 후, 수초 형성 여부를 분석하는 단계를 포함하는, 탈수초 질환의 치료제를 스크리닝하는 방법을 제공하는 것이다.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.
본 발명의 또 다른 목적은 본 발명의 희소돌기아교세포를 포함하는 오가노이드 또는 상기 오가노이드에서 분리된 희소돌기아교세포를 유효성분으로 포함하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물을 제공하는 것이다.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
상기와 같은 목적을 달성하기 위해 본 발명은, (1) 인간 만능성 줄기세포(Human pluripotent stem cell; hPSC)를 LIF(Leukemia Inhibitory Factor), SB431542 화합물 및 CHIR 99021 화합물을 포함하는 LSC 배지로 배양하여 원시 신경줄기세포(Primitive NSC; pNSC)로 분화시키는 단계; (2) 상기 원시 신경줄기세포를 bFGF, EGF 및 퍼모프아민(Purmorphamine)을 포함하는 FEP 배지로 배양하여 복측 로제트 타입 신경줄기세포(Ventral rosette type NSC; vrNSC)로 분화시키는 단계; (3) 상기 복측 로제트 타입 신경줄기세포를 bFGF, PDGF 및 퍼모프아민(Purmorphamine)를 포함하는 FPP 배지로 배양하여 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre Oligodendrocyte Precursor Cell; pre-OPC)로 분화시키는 단계; 및 (4) 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포를 RA(retinoic acid) 함유 배지; PDGF(Platelet-derived growth factor), IGF1(Insulin like growth factor), HGF(Hepatocyte growth factor) 및 포스콜린(forskolin) 함유 배지; 및 포스콜린(forskolin), 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine) 함유 배지;로 순차적으로 배지를 교체하면서 배양하여 희소돌기아교세포를 포함하는 오가노이드를 형성하는 단계를 포함하는, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법을 제공한다.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.
본 발명의 일실시예에 있어서, 상기 배지는 DMEM/F12, 중탄산나트륨, 비타민 A가 없는 B-27 보충제, N-2 보충제, 페니실린-스트렙토마이신, L-글루타민 및 비필수아미노산을 포함하는 배지를 기초배지로 사용하는 것일 수 있다.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.
본 발명의 일실시예에 있어서, 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포는 OLIG2(Oligodendrocyte transcription factor 2) 및 NKX2.2(NK2 Homeobox 2)를 발현하는 것일 수 있다.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).
본 발명의 일실시예에 있어서, 상기 오가노이드에는 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 분화된 신경세포 및 성상교세포를 더 포함할 수 있다.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.
또한 본 발명은 본 발명의 방법으로 제조된 희소돌기아교세포를 포함하는 오가노이드를 제공한다.The present invention also provides an organoid comprising oligodendrocytes prepared by the method of the present invention.
또한 본 발명은 본 발명의 희소돌기아교세포를 포함하는 오가노이드에 후보약물을 처리한 후, 수초 형성 여부를 분석하는 단계를 포함하는, 탈수초 질환의 치료제를 스크리닝하는 방법을 제공한다.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.
본 발명의 일실시예에 있어서, 상기 후보약물 처리에 의해 수초 형성이 촉진되는 경우, 상기 후보약물을 탈수초 질환의 치료제로 판단하는 단계를 더 포함할 수 있다.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.
또한 본 발명은 본 발명의 희소돌기아교세포를 포함하는 오가노이드 또는 상기 오가노이드에서 분리된 희소돌기아교세포를 유효성분으로 포함하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물을 제공한다.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.
본 발명의 일실시예에 있어서, 상기 조성물은 수초화 재생을 촉진시키는 것일 수 있다.In one embodiment of the present invention, the composition may be to promote myelination regeneration.
본 발명의 일실시예에 있어서, 상기 탈수초 질환은 급성 파종성 탈수초(acute disseminated demyelination), 레버 유전성 시각 신경병증(Leber hereditary optic neuropathy), 탈수초성 다발성 신경병증(demyelinating polyneuropathy) 및 샤르코 마리 투스병(Charcot Marie Tooth)으로 이루어진 군으로부터 선택되는 것일 수 있다.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).
본 발명의 일실시예에 있어서, 상기 오가노이드는 수초 형성 유도약물이 처리된 오가노이드일 수 있다.In one embodiment of the present invention, the organoid may be an organoid treated with a myelination inducing drug.
본 발명의 일실시예에 있어서, 상기 수초 형성 유도약물은 T3(3,3’,5-triiodo-L-thyronine), 미코나졸(Miconazole) 또는 벤즈트로핀(Benztropine)일 수 있다.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).
본 발명에 따른 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법은 짧은 시간 안에 희소돌기아교세포가 포함된 오가노이드를 제조할 수 있는 효과가 있으며, 탈수초성 질환의 치료를 위한 세포치료제로 유용하게 사용할 수 있는 효과가 있다. 뿐만 아니라 본 발명의 방법으로 제조된 희소돌기아교세포를 포함하는 오가노이드는 시험관 내에서 재수초 형성 유도 효능을 갖는 약물의 스크리닝에도 사용할 수 있어 신약개발을 위한 3D 플랫폼 기반의 마련에도 유용하게 사용될 수 있는 효과가 있다.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
도 1은 hESCs 로부터 pre-OPCs를 확립하는 과정, 배양조건 및 pre-OPCs의 특성과 pre-OPCs로부터 분화된 희소돌기아교세포의 특성을 확인한 결과를 나타낸 ㄱ것으로, 1A는 hESCs 로부터 pre-OPCs를 확립하기 위한 각 단계별 모양 및 배양조건을 모식도로 나타낸 것이고, 1B는 pNSCs를 확립하기 위한 배양조건을 나타낸 것이며, 1C는 확립된 pNSCs 의 특성을 확인한 결과이고, 1D 는 pre-OPCs를 확립하기 위한 배양조건을 나타낸 것이며, 1E는 확립된 pre-OPCs의 특성을 확인한 것이고, 1F는 확립된 pre-OPCs의 분화능을 확인하기 위한 배양조건을 나타낸 것이며, 1G는 pre-OPCs로부터 분화된 성상세표의 특성을 분석한 것이고, 1H는 pre-OPCs로부터 분화된 희소돌기아교세포의 특성을 확인한 결과를 나타낸 것이다.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.
도 2는 원시 신경줄기세포(pNSC) 및 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre-OPC)로부터 스페로이드 형성에 대한 결과를 나타낸 것으로, 2A는 pNSC 및 pre-OPC으로부터 스페로이드의 형성 및 뉴런(TUJ1 +), 성상세포 (GFAP +), 희소돌기아교세포(MBP +)로의 분화를 위한 각 단계를 모식도로 나타낸 것이며, 각 약어의 의미는 다음과 같다. LSC는 LIF, SB431542 및 CHIR99021를 나타낸 것이고, FPP는 FGF2, PDGF-AA 및 Purmorphamine를 나타낸 것이고, FSK는 Forskolin, T3는 3,3’,5-triiodo-L-thyronine을, AA는 ascorbic acid를 나타낸 것이다. 도 2B 및 2C는 각각 pNSCs 및 pre-OPC의 특징을 PAX6, SOX1, OLIG2 및 NKX2.2에 대한 면역형광염색법으로 확인한 결과를 나타낸 것이다. 도 2D 및 2E는 0일과 3일째의 pNSC(D) 및 pre-OPC(E)의 형태를 확인한 것이고, 2F 및 2G는 4주 동안 분화 시간에 따른 Matrigel droplets 내에서의 세포 분화를 확인한 것이며, 임베딩 이전에 pNSC 및 pre-OPC는 200~300um의 신경구를 형성한 것으로 나타났다. 2H는 마트리겔 임베딩 2주 후, pNSC 및 pre-OPC로부터 형성된 각 스페로이드에 대한 형광이미지를 나타낸 것으로, 점선 표시 영역이 VZ-like 영역을 나타낸 것이다. 2I 및 2J는 마트리겔 임베딩 4주 후, pNSC(passage 1 and passage 15) 및 pre-OPC로부터 형성된 각 스페로이드에 대한 형광이미지를 나타낸 것이며, 5-HT는 5-hydroxytryptamine, ChAT는 choline acetyltransferase, HB9는 MNX1, OPC는 oligodendrocyte progenitor cell, pNSC는 primitive neural stem cell을, TH는 tyrosine hydroxylase를 나타낸 것이다.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.
도 3은 성상세포의 신속한 생산을 위한 pre-OPC로부터 확립된 스페로이드의 배양 결과로서, 3A는 pre-OPC로부터 확립된 스페로이드 절편을 TUJ1 (green) 및 BLBP(brain lipid binding protein; red)에 대한 형광염색법으로 확인한 결과를 나타낸 것이고, 3B는 마트리겔 임배딩 후 각 절편에 대한 형광 이미지를 나타낸 것으로 S100β (green) 및 GFAP (red)의 발현 정도를 확인한 결과이고, 점선은 분리된 스페로이드를 나타낸 것이다. 3C는 시간별 S100β 또는 GFAP 양성 세포 집단을 유세포 분석기로 확인한 것이고, 3D는 pre-OPC로부터 확립된 스페로이드를 6주 동안 배양 후, 확대된 형광 이미지를 나타낸 것으로, 화살표는 길쭉한 모양의 성상세포를, 화살촉은 별 모양의 성상세포를 각각 나타낸 것이며, 3E는 H9-hESCs (nos. 6 and 10) 및 hiPSCs (nos. 18)로부터 유래된 pre-OPC에서 형성된 스페로이드의 확대된 형광 이미지를 나타낸 것이고, 3F는 단층분화된 형광 이미지를 나타낸 것으로, GFAP (red) 및 S100β (green)을 염색하여 확인한 것이며, 유세포 분석을 통해 종래 2차원적 배양방법(2D) 및 본 발명에 따른 pre-OPCs로부터 오가노이드의 배양방법(3D)에 따른 성상교세포로의 분화율을 비교한 것을 나타낸 것이다.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).
도 4는 pre-OPC로부터 형성된 스페로이드의 배양은 희소돌기아교세포의 신속한 생산을 가능하게 함을 확인한 결과를 나타낸 것으로, 4A는 마트리겔 임배딩 4주 후, 스페로이드를 SOX10(red)를 염색한 형광 이미지 결과이고, 4B 및 4C는 pre-OPC로부터 확립된 4주차 스페로이드(B) 및 6주차 스페로이드(C)를 나타낸 것으로 희소돌기아교세포의 또 다른 마커인 OLIG2, PDGFRa, O4 또는 MBP의 발현을 확인한 것이며, 4D는 qPCR을 통해 희소돌기아교세포의 마커 유전자들의 발현수준을 비교 분석한 것이고, 4E 및 4F는 pre-OPC로부터 확립된 8주차 스페로이드를 MBP, SOX10 또는 TUJ1으로 염색한 결과를 나타낸 것이며, 4G는 DMSO, T3, Benztropine 또는 Miconazole이 각각 처리된 pre-OPC로부터 형성된 스페로이드를 TUJ1 및 MBP으로 염색한 결과를 나타낸 것이고, 4H 및 4I는 DMSO, T3, Benztropine 또는 Miconazole 처리에 따른 수초 형성 정도를 정량적으로 비교 분석한 결과를 나타낸 것이며, 4J는 Miconazole을 처리한 pre-OPCs로부터 형성된 스페로이드에서 분리한 희소돌기아교세포를 쥐에 이식한 결과를 나타낸 것이며, 4K는 전자현미경으로 관찰하여 수초 형성을 확인한 결과를 나타낸 것이다.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.
도 5는 본 발명에 따른 pre-OPCs로부터 희소돌기아교세포를 포함하는 스페로이드(오가노이드)의 확립 방법을 모식도로 나타낸 것이다.5 is a schematic diagram illustrating a method for establishing spheroids (organoids) including oligodendrocytes from pre-OPCs according to the present invention.
탈수초 질환(demyelinating disease)은 중추신경계 전반에 걸쳐서 희소돌기아교세포의 부재로 나타나는 난치성 신경계 질환으로 이를 치료하기 위한 종래 연구방법은 만능성 줄기세포로부터 희소돌기아교세포를 확립하여 치료하려는 연구가 많이 진행 되었지만, 임상에서 효능을 보이는 세포치료제를 확립하지 못하였고, 특히 희소돌기아교세포는 대뇌 및 척수에서도 극히 소량 존재하는 세포로 알려져 있으며 중추신경계를 구성하는 신경줄기세포에서도 희소돌기아교세포로의 분화율이 낮다고 알려져 있어, 만능성을 포함한 상위단계의 줄기세포로부터 희소돌기아교 세포를 확립하는데 많은 어려움이 있다. 또한 많은 연구에도 불구하고 아직까지 배양 가능한 인간 희소돌기아교 전구세포를 확립하지 못하고 있다.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.
이에 본 발명자들은 희소돌기아교세포로 고도로 분화가 특화된 인간 신경줄기세포를 확립하였고, 이를 이용하여 단기간에 고효율로 희소돌기아교세포가 포함된 오가노이드를 형성할 수 있음을 규명함으로써 탈수초 질환을 치료할 수 있는 효과적인 세포치료제를 제공할 수 있고, 나아가 상기 오가노이드를 이용하여 탈수초 질환 치료제를 스크리닝할 수 있는 방법을 확립하였다.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.
구체적으로 본 발명은, (1) 인간 만능성 줄기세포(Human pluripotent stem cell; hPSC)를 LIF(Leukemia Inhibitory Factor), SB431542 화합물 및 CHIR 99021 화합물을 포함하는 LSC 배지로 배양하여 원시 신경줄기세포(Primitive NSC; pNSC)로 분화시키는 단계; (2) 상기 원시 신경줄기세포를 bFGF, EGF 및 퍼모프아민(Purmorphamine)을 포함하는 FEP 배지로 배양하여 복측 로제트 타입 신경줄기세포(Ventral rosette type NSC; vrNSC)로 분화시키는 단계; (3) 상기 복측 로제트 타입 신경줄기세포를 bFGF, PDGF 및 퍼모프아민(Purmorphamine)를 포함하는 FPP 배지로 배양하여 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre Oligodendrocyte Precursor Cell; pre-OPC)로 분화시키는 단계; 및 (4) 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포를 RA(retinoic acid) 함유 배지; PDGF(Platelet-derived growth factor), IGF1(Insulin like growth factor), HGF(Hepatocyte growth factor) 및 포스콜린(forskolin) 함유 배지; 및 포스콜린(forskolin), 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine) 함유 배지;로 순차적으로 배지를 교체하면서 배양하여 희소돌기아교세포를 포함하는 오가노이드를 형성하는 단계를 포함하는, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법을 제공함에 특징이 있다.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.
보다 상세하게 본 발명에 따른 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법을 설명하면 다음과 같다.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.
먼저, (1) 인간 만능성 줄기세포(Human pluripotent stem cell; hPSC)를 LIF(Leukemia Inhibitory Factor), SB431542 화합물 및 CHIR 99021 화합물을 포함하는 배지로 배양하여 원시 신경줄기세포(Primitive NSC; pNSC)로 분화시킨다.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
상기 인간 만능성 줄기세포는 인체를 구성하는 다양한 세포로 모두 분화할 능력을 가진 줄기세포를 말하는 것으로, 내배엽, 중배엽, 외배엽을 구성하는 거의 모든 종류의 세포로 분화할 수 있는 줄기세포를 통칭한다.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.
상기 인간 만능성 줄기세포는 특정 배지에서 배양하여 원시 신경줄기세포로 분화시키며, 이때 상기 배지는 LIF(Leukemia Inhibitory Factor), SB431542 화합물 및 CHIR 99021 화합물을 포함하는 배지(즉, LSC 배지)를 이용한다.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).
본 발명에서 사용한 상기 LIF(Leukemia Inhibitory Factor)는, 인터루킨-6 사이토카인 계열에 속하는 멤버 중 하나로서, 세포의 증식, 분화 및 생존에 관여하는 인자로 알려져 있다. 또한, 상기 SB431542는 ALK5(Activin Receptor-Like Kinase-5) 억제제로 신속한 분화를 유도하며 염색체 안정성을 향상시키는 역할을 한다. 상기 CHIR 99021은 GSK(glycogen synthase kinase) 억제제로 GSK 신호전달과정에 관여하는 GSK1/2의 업스트림(upstream) 분자인 GSK1/2를 표적으로 하는 물질이다. 상기 CHIR 99021은 아미노피리미딘(aminopyrimidine)으로 표시된다.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.
본 발명에서 사용된 모든 배지는 기초배지(BM:basal medium)를 기본배지로 하며, 기초배지에 특정 성분들을 더 포함한 배지를 세포의 각 배양 단계에서 사용하였다.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.
상기 기초배지는 DMEM/F12, 중탄산나트륨, 비타민 A가 없는 B-27 보충제, N-2 보충제, 페니실린-스트렙토마이신, L-글루타민 및 비필수아미노산을 포함하는 배지를 말하며, 본 발명의 일실시예에서는 DMEM/F12, 543 ug/ml 중탄산나트륨, 1 x 비타민 A가 없는 B-27 보충제, 1 x N-2 보충제, 1% 페니실린-스트렙토마이신, 1% L-글루타민 및 1% 비필수아미노산이 함유된 기초배지를 사용하였다. 또한, 기초배지에 상기 LIF(Leukemia Inhibitory Factor), SB 431542 화합물 및 CHIR 99021 화합물을 포함하는 LSC 배지에서 인간 만능성 줄기세포를 배양함으로써 상기 인간 만능성 줄기세포를 원시 신경줄기세포로 분화 유도하였다. 배양 후에는 분화된 세포는 원시 신경줄기세포의 마커인 PAX6 및 SOX1의 발현을 확인하였다.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.
다음으로, (2) 상기 분화된 원시 신경줄기세포를 배양하여 복측 로제트 타입 신경줄기세포(Ventral rosette type NSC; vrNSC)로 분화시킨다.Next, (2) culturing the differentiated primitive neural stem cells to differentiate them into ventral rosette type NSCs (vrNSCs).
이때 상기 복측 로제트 타입 신경줄기세포로의 분화를 위해, 상기 분화된 원시 신경줄기세포를 bFGF, EGF 및 퍼모프아민(Purmorphamine)을 포함하는 FEP 배지에서 배양한다.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.
상기 로제트 타입 신경줄기세포는 인간 배아줄기세포의 신경분화 과정의 초기단계의 신경줄기세포를 말하며, 로제트 타입은 원주형의 방사상 형태를 갖는다. 또한 상기 로제트 타입 신경줄기세포는 PAX6 및 SOX1 같은 초기 신경외배엽(neuroectodermal) 마커를 발현하는 세포로 구성되며, 다양한 뉴런 세포 및 신경교세포로 분화할 수 있다.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.
특히 본 발명에서는 복측 로제트 타입 신경줄기세포로의 분화를 위해 bFGF, EGF 및 퍼모프아민(Purmorphamine)을 포함하는 FEP 배지를 이용하였는데, FEP 배지는 기초배지에 bFGF, EGF 및 퍼모프아민(Purmorphamine)이 첨가된 배지를 말한다.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.
상기 퍼모프아민은 퓨린화합물(purine compound)로서, Shh 신호체계에 관여하는 것으로 알려져 있다. 상기 퍼모프아민은 신경줄기세포 유사세포로 역분화를 유도하기 위해 사용되기도 한다.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.
복측 로제트 타입 신경줄기세포로의 분화가 완료되면, 다음으로 (3) 복측 로제트 타입 신경줄기세포를 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre Oligodendrocyte Precursor Cell; pre-OPC)로 분화시키는 단계를 수행한다.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. .
이때 상기 분화를 위한 배지는 bFGF, PDGF 및 퍼모프아민(Purmorphamine)를 포함하는 FPP 배지를 이용하여 배양한다. 상기 FPP 배지는 기초배지에 bFGF, PDGF 및 퍼모프아민(Purmorphamine)이 포함된 배지를 말한다.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.
상기 FPP 배지를 이용하여 배양한 결과, 복측 로제트 타입 신경줄기세포로부터 희소돌기아교세포로 분화가 특화된 신경줄기세포를 얻을 수 있었고, 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포에서 희소돌기아교 전구세포의 대표적인 마커인 OLIG2 및 NKX2.2의 발현을 확인할 수 있었다.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.
또한 본 발명에서 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포는 희소돌기아교세포로 분화 운명이 결정된 또는 예정된 신경줄기세포를 의미한다.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.
상기 과정을 통해 희소돌기아교세포로 분화가 특화된 신경줄기세포는 다음으로, 희소돌기아교세포를 포함하는 오가노이드가 형성되도록 배양하는 단계를 수행하는데, 구체적으로, (4) 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포를 RA(retinoic acid) 함유 배지; PDGF(Platelet-derived growth factor), IGF1(Insulin like growth factor), HGF(Hepatocyte growth factor) 및 포스콜린(forskolin) 함유 배지; 및 포스콜린(forskolin), 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine) 함유 배지;로 순차적으로 배지를 교체하면서 배양함으로써 희소돌기아교세포를 포함하는 오가노이드를 형성한다.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 .
희소돌기아교세포로 분화가 특화된 신경줄기세포는 3 종류의 배지를 순차적으로 교체하면서 배양하여, 분화된 희소돌기아교세포를 포함하는 뇌 오가노이드를 제조한다. Neural stem cells specialized for differentiation into oligodendrocytes are cultured while sequentially replacing three types of media to prepare brain organoids including differentiated oligodendrocytes.
상세하게는, 희소돌기아교세포로 분화가 특화된 신경줄기세포를 RA(retinoic acid) 함유 배지에서 배양하는데, 이때 상기 배지는 기초배지에 0.5uM의 RA 함유 배지를 이용하여 7일 동안 배양을 수행한다.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.
이후 상기 배지를 PDGF 함유 배지로 교체하여 다시 배양을 수행하는데, 즉, 기초배지에 PDGF(Platelet-derived growth factor), IGF1(Insulin like growth factor), HGF(Hepatocyte growth factor) 및 포스콜린(forskolin)을 첨가한 배지를 이용하여 추가로 14일 동안 배양한다.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.
이후 다시 상기 PDGF 함유 배지를 교체포 분화배지인, 기초배지에 포스콜린(forskolin), 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine)가 첨가된 배지를 이용하여 배양함을 통해, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드를 얻을 수 있었다.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.
상기 배지에 첨가된 포스콜린은 다데닐산고리화효소의 촉매소단위를 직접 활성화하여 세포내 cAMP의 농도를 상승시키는 작용을 하며, 트라닐시프로민은 신경말단에서 정상적으로 노르에피네프린을 분해하는 효소인 모노아민산화효소(MAO)를 억제하는 작용을 한다. 또한, 상기 아스코르빅산은 수용성 비타민의 하나로 강한 항산화성을 가지며, 상기 T3(3,3′,5-triiodo-L-thyronine)은 갑상선 호르몬으로 작용하며 갑상선 기능 저하증에서 기분과 신경 심리학적 기능을 향상시키는 작용을 갖는 것으로 알려져 있다.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)는 세포핵 안에 존재하는 수용체인 RAR/RXR와 상호작용을 하는 것으로 알려져 있고, 신경세포의 분화에 관여하는 것으로 알려져 있다.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.
또한 본 발명에 따른 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드를 신속하게 제조하는 방법은, 복측 로제트 타입 신경줄기세포를 bFGF, PDGF 및 퍼모프아민(Purmorphamine)를 포함하는 FPP 배지로 배양하여 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre Oligodendrocyte Precursor Cell; pre-OPC)로 분화시키는 단계; 및 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포를 RA(retinoic acid) 함유 배지; PDGF(Platelet-derived growth factor), IGF1(Insulin like growth factor), HGF(Hepatocyte growth factor) 및 포스콜린(forskolin) 함유 배지; 및 포스콜린(forskolin), 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine) 함유 배지;로 순차적으로 배지를 교체하면서 배양하여 희소돌기아교세포를 포함하는 오가노이드를 형성하는 단계를 통해 수행될 수 있다.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.
본 발명에 따른 방법은 상기 (1) 내지 (2)의 방법, 즉 원시 신경줄기세포(Primitive NSC; pNSC)로 분화시키는 단계 및 복측 로제트 타입 신경줄기세포(Ventral rosette type NSC; vrNSC)로 분화시키는 단계를 반드시 수행하지 않아도 되며, 어떠한 복측 로제트 타입 신경줄기세포라도 상기 (3) 및 (4)의 단계, 즉 복측 로제트 타입 신경줄기세포를 FPP 배지로 배양하여 pre-OPC로 분화시키고, 상기 pre-OPC를 RA 함유 배지; PDGF, IGF1, HGF 및 포스콜린 함유 배지; 및 포스콜린, 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine) 함유 배지;로 순차적으로 배지를 교체하면서 배양하여, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드를 신속하게 제조할 수 있다.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.
한편, 본 발명자들은 상기 방법으로 제조된 본 발명의 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 제조된 오가노이드에 대해, 상기 오가노이드안에서 신경교세포(glia)의 분화가 진행되었는지 확인하였는데, 면역염색화학 분석 결과, 분화 2주차에 신경교 전구세포(glial progenitor cell)의 마커인 BLBP의 발현을 확인할 수 있었고 3주차에는 성상교세포(astrocytes)의 전형적인 마커인 S100β 와 GFAP의 발현도 확인할 수 있었으며, 특히 분화 5주차에는 S100β 및 GFAP의 발현이 상당히 증가되어 있는 것을 확인할 수 있었다.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.
또한, 상기 오가노이드안에는 성숙한 신경세포의 마커인 TH 및 GABA의 발현도 확인할 수 있었다.In addition, the expression of TH and GABA, which are markers of mature neurons, was also confirmed in the organoid.
따라서 본 발명의 방법으로 제조된 상기 오가노이드에는 희소돌기아교세포 이외에도 성상교세포 및 신경세포가 분화되어 함께 존재하는 것을 확인할 수 있었다.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.
뿐만 아니라, 본 발명의 일실시예에서는 상기 본 발명의 방법으로 제조된 오가노이드와 희소돌기아교세포로 분화가 특화된 신경줄기세포가 아닌 원시 신경줄기세포로부터 종래 2D 분화방법으로 제조된 오가노이드를 대상으로 형태 및 분화 속도를 비교 분석한 결과, 각 방법으로 분화되어 형성된 오가노이드의 형태가 다른 것으로 나타났고, 특히 본 발명에 따른 희소돌기아세포로 분화가 특화된 신경줄기세포로부터 형성된 오가노이드는 원시 신경줄기세포로부터 형성된 오가노이드보다 신경세포 및 희소돌기아교세포로의 분화가 더 빠르게 진행되었음을 확인할 수 있었다.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.
상기 희소돌기아교세포로의 분화는 본 발명의 방법으로 제조된 오가노이드에서 분화 4주차에 희소돌기아교 전구세포의 마커인 SOX10, OLIG2, PDGFRa 및 O4의 발현을 확인할 수 있었으며, 분화 6주차에는 희소돌기아교세포의 마커인 MBP의 발현을 확인할 수 있었고, 8주차에는 4주차에 비해 희소돌기아교세포 마커들의 발현량이 월등히 증가되어 있는 것을 확인할 수 있었고, 수초형성(myelination)이 이루어진 것을 확인할 수 있었다.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.
이러한 결과로 본 발명자들은 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 다량 포함하는 본 발명의 오가노이드를 탈수초 질환의 치료를 위한 세포치료제로 사용할 수 있음을 알 수 있었고, 또한 상기 오가노이드를 이용하여 탈수초 질환 치료제를 스크리닝할 수 있음을 알 수 있었다.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.
그러므로 본 발명은 본 발명의 방법으로 제조된 희소돌기아교세포를 포함하는 오가노이드를 제공할 수 있으며, 상기 오가노이드는 구 형상의 세포 응집체 형태를 갖는다.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.
본 발명에서 상기 "오가노이드(organoid)"는 3D 입체구조를 가지는 세포덩어리를 의미하는 것으로, 오가노이드는 조직 또는 줄기세포에서 제조될 수 있으며, 자가재생 및 분화능력으로 인해 3차원으로 배양될 수 있다. 상기 오가노이드는 세포의 성장 과정에서 주변 환경과 상호 작용하도록 허용되는 환경을 가질 수 있다. 이에 따라, 3D 오가노이드는 실제로 in vivo에서 상호 작용을 하고 있는 장기를 거의 유사 또는 완벽히 모사할 수 있고, 질병의 치료제 개발 및 등을 관찰할 수 있는 훌륭한 모델이 될 수 있다.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.
또한, 본 발명에서 "스페로이드(spheroid)"는 구 형상을 한 세포의 응집체를 의미한다. 실질적으로 구 형상이라고 칭하는 것은 완전한 구 형상의 것에 한정되는 것이 아니라, 약간 편평 형상으로 된 형태도 포함될 수 있다.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.
또한 본 발명은 본 발명에 따른 희소돌기아교세포를 포함하는 오가노이드에 후보약물을 처리한 후, 수초 형성 여부를 분석하는 단계를 포함하는, 탈수초 질환의 치료제를 스크리닝하는 방법을 제공할 수 있다.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. .
구체적으로, 상기 후보약물 처리에 의해 수초 형성이 촉진되는 경우, 상기 후보약물을 탈수초 질환의 치료제로 판단하는 단계를 더 포함할 수 있다.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.
본 발명의 일실시예에서는, 본 발명에 따른 상기 오가노이드에 수초형성 유도 약물들을 처리한 후, 수초형성 정도를 비교 분석하여, 수초 형성능이 가장 우수한 약물을 선별할 수 있음을 확인하였다.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.
그러므로 본 발명의 상기 오가노이드는 수초형성 또는 재수초 형성 증진 활성을 갖는 약물을 스크리닝 하는데 사용할 수 있을 뿐만 아니라, 약물의 효능을 분석하는데 사용될 수 있다.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.
또한 본 발명은 희소돌기아교세포를 포함하는 오가노이드 또는 상기 오가노이드에서 분리된 희소돌기아교세포를 유효성분으로 포함하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물을 제공할 수 있다.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.
뇌의 신경섬유는 대부분 수초(미엘린, myelin)라는 지질단백질로 구성된 조직의 많은 층으로 둘러싸여 있는 구조를 이루고 있으며, 수초는 절연체와 비슷한 역할을 수행하기 때문에, 신경섬유를 따라 정확하고 빠른 신경 충동(neural impulse)을 전달하는데 중요한 역할을 수행하고 있다. 이러한 수초가 국소 손상, 면역 장애, 감염, 영양 결핍, 약물, 또는 알려지지 않은 원인 등에 의하여 손상, 파괴 등이 유발되게 되면, 내부에 있는 신경 섬유에 영구적인 손상이 유도된다. 따라서, 체내에서는 수초가 손상되면 이를 회복시키기 위하여 재수초화가 진행되나, 종종 재수초화가 실패하고 탈수초화로 진행되는 경우가 발생된다. 이러한 탈수초화로 인하여 발생되는 탈수초 질환(demyelinating disease)으로는 다발성 경화증(Multiple sclerosis), 급성 파종성 탈수초(acute disseminated demyelination), 레버 유전성 시각 신경병증(Leber hereditary optic neuropathy), 길랭-바레증후군(Guillain-Barre syndrome), 탈수초성 다발성 신경병증(demyelinating polyneuropathy), 샤르코 마리 투스병(Charcot Marie Tooth) 등이 있다.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.
따라서, 수초가 손상되었을 때 재수초화가 촉진되거나 또는 탈수초화를 억제시키기 위해서 희소돌기아교세포 전구체세포를 모집(recruitment)하고, 모인 전구체세포를 희소돌기아교세포로 분화시킬 수 있는 신규 치료제의 개발이 필요하다.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.
이러한 점에서 본 발명의 방법으로 제조된 오가노이드는 희소돌기아교 전구세포 및 희소돌기아교세포의 분화가 빠르게 진행되어 있어, 다량의 희소돌기아교세포를 포함하고 있으므로, 탈수초화로 인한 다양한 탈수초 질환의 효과적인 치료제로서 사용 가능할 것으로 기대할 수 있다.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
본 발명에서 상기 “희소돌기아교세포” 및 “희소돌기아교세포 전구체 세포”는 희소돌기아교세포 계통의 세포들, 즉, 궁극적으로 희소돌기아교세포를 발생시키는 신경 전구 세포, 희소돌기아교세포 전구 세포, 및 성숙 및 수초화 희소돌기아교세포를 포함할 수 있으며, 이는 본 발명의 상기 방법에 의해 제조된 오가노이드 안에서 분화된 세포일 수 있다.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.
또한 희소돌기아교세포는 기능적 특성을 가질 수 있는데, 즉, 이들은 뉴런의 수초화 능력 등을 가질 수 있다. 또한 "희소돌기아교세포 전구물질" 또는 “희소돌기아교세포 전구체 세포”는 희소돌기아교세포를 포함하는 후대를 생성할 수 있는 세포를 말한다.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.
그러므로 본 발명의 희소돌기아교세포를 포함하는 오가노이드 또는 상기 오가노이드에서 분리된 희소돌기아교세포는 수초화 재생 촉진을 통해, 신경계의 미엘린 수초에서 비정상, 특히 수초의 파괴를 수반하는 탈수초 질환(demyelinating disease)의 치료를 위한 세포치료제 조성물로 사용될 수 있다.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.
본 발명에서 상기 "세포치료제(cellular therapeutic agent)"란, 인간으로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA 규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 이러한 세포가 질병의 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다.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.
상기 "치료"는 상기 세포치료제의 투여로 질환의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.The "treatment" refers to any action in which the symptoms of a disease are improved or beneficial by administration of the cell therapy agent.
본 발명의 세포치료제 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 비경구 투여, 예를 들어, 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여될 수 있으나, 이에 제한되지는 않는다.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.
상기 조성물은 세포 치료에 일반적으로 사용되는 약제학적 담체와 함께 적합한 형태로 제형화될 수 있다. '약학적으로 허용되는'이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).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).
또한, 상기 조성물은 세포치료제가 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수도 있다.In addition, the composition may be administered by any device capable of transporting a cell therapy agent to a target cell.
본 발명의 세포치료제 조성물은 질환의 치료를 위하여 치료학적으로 유효한 양의 세포치료제를 포함할 수 있다.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.
또한 상기 “치료학적으로 유효한 양(therapeutically effective amount)”은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다.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.
본 발명의 조성물에 포함되는 세포치료제는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로 최적의 세포치료제 함량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 포함하는 것이 중요하다. 예컨대, 본 발명의 세포치료제에 해당하는 세포의 1일 투여량은 1.0×10 4 내지 1.0×10 10 세포/kg 체중, 바람직하게는 1.0×10 5 내지 1.0×10 9 세포/kg 체중을 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.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.
또한, 본 발명의 치료방법에서 본 발명의 세포치료제를 유효성분으로 포함하는 조성물은 직장, 정맥내(intravenous therapy, i.v), 동맥내, 복강내, 근육내, 흉골내, 경피, 국소, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.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.
본 발명은 포유동물에게 치료학적으로 유효한 양의 본 발명의 상기 세포치료제 조성물을 투여하는 것을 포함하는 치료방법을 제공한다. 여기에서 사용된 용어 포유동물은 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다.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.
또한 본 발명에서 상기 오가노이드는 추가로 수초 형성 유도약물이 처리된 오가노이드를 세포 치료제로 사용할 수 있으며, 상기 수초 형성 유도약물은 이에 제한되지는 않으나, T3(3,3’,5-triiodo-L-thyronine), 미코나졸(Miconazole) 또는 벤즈트로핀(Benztropine)일 수 있다. 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.
이상, 본 발명에 따른 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법은 3차원적 오가노이드 형태로 짧은 시간 안에 제조할 수 있어, 기존 20주 이상이 소요되는 2차원적 방법에 비해 월등히 효과적임을 알 수 있었고, 본 발명에 따른 3D 구형체의 오가노이드에는 희소돌기아교세포 뿐만 아니라 신경세포 및 성상교세포도 포함하고 있어, 실제 인간 뇌와 더욱 가까운 뇌 오가노이드를 제공할 수 있다. 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.
본 발명의 방법으로 제조된 희소돌기아교세포를 포함하는 오가노이드는 탈수초 질환을 위한 세포치료제로 유용하게 사용할 수 있으며 나아가 탈수초 질환의 치료제를 신속하고 정확하게 스크리닝하기 위한 약물 개발 플랫폼으로도 사용할 수 있다.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.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.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.
<재료 및 실험방법><Materials and test methods>
① 세포배양① Cell culture
pNSCs 세포(PAX6 및 SOX1을 발현) 및 pre-OPCs 세포(중간전구세포, OLIG2 및 NKX2.2를 발현)의 배양은 Matrigel(356231, Corning, NY, USA) 포매 전, pNSC 세포를 LSC 배지(LIF, SB431542 및 CHIR99021 함유)를 이용하여 배양하였고, pre-OPC 세포는 FPP 배지(FGF2, PDGF-AA 및 Purmorphamine 함유)를 이용하여 배양하였다. 배양 계대를 고려하여 pNSC 세포는 15 계대(pre-OPCs 단계에 가까움)의 세포로 준비하였다. 세포는 Accutase(EMD Millipore, Burlington, MA, USA)를 이용하여 0일째 분산시키고 초저 부착 플레이트(Corning)에 분주한 후, 포매될 때까지 LSC 또는 FPP 배지에서 유지시켰다. 생성된 신경구(neurospheres, > 200 μm)는 4 일째에 Matrigel 액적에 임배드 시켰다. 임배드된 신경구는 543 μg/ml 중탄산나트륨(Sigma-Aldrich, St. Louis, MO, USA), 1× N-2 보충제(Thermo Fisher Scientific), 비타민 A가 없는 1×B-27 보충제(Thermo Fisher Scientific), 1% 페니실린-스트렙토마이신, 1% L-글루타민(Thermo Fisher Scientific), 1% 비필수 아미노산(Thermo Fisher Scientific) 및 5 μg/ml 인슐린 (Sigma-Aldrich)이 함유된 DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) 로 구성된 기초배지에서 3일 동안 배양하였다. 그 후, 액적을 오비탈 쉐이커로 옮기고 0.5 μM 레티노산 (Sigma-Aldrich)이 보충된 기초 배재에서 추가 1 주 동안 배양하였고, 배지를 10 ng/ml PDGF-AA(Peprotech), 10 ng/ml IGF-1(Peprotech), 10 ng/ml NT-3 (Peprotech), 5 ng/ml HGF(Peprotech) 및 10 μM forskolin(Tocris)이 함유된 기초 배지로 교체하였다. 이후 액적을 10 μM forskolin(Tocris), 60 ng/ml 3,3′,5-triiodo-L-thyronine(T3, Sigma-Aldrich) 및 20 μg/ml 아스코르빅산(Peprotech) 및 기초 배지로 구성된 아교 분화 배지(glial differentiation medium)에서 4주 동안 배양하였다. 이후 promyelinating 화합물의 스크리닝을 위해, 4 주된 스페로이드(spheroids)를 DMSO (Sigma-Aldrich), 60ng/ml T3 (Sigma-Aldrich), 1μM 벤즈트로핀(Sigma-Aldrich) 또는 1μM 미코나졸(miconazole, Sigma-Aldrich)로 8주 동안 처리하였다.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 샘플 준비 및 분석② Sample preparation and analysis
스페로이드(Spheroid) 샘플은 O.C.T. 컴파운드(Tissue Tek; Sakura Finetek USA, Inc., Torrance, CA, USA)로 임배드하였고, 20㎛ 두께로 냉동 절편화하였다. 절편들은 0.2 % Triton X-100이 포함된 PBS에서 2 % 정상 당나귀 혈청으로 1시간 동안 블록킹을 수행하였고, 지정된 1차 항체(하기 표 1 및 표 2)로 4℃에서 밤새도록 반응시킨 다음, 실온에서 1 시간 동안 Alexa Fluor 488 또는 594-접합된 이차 항체(Thermo Fisher Scientific)와 반응시켰다. 핵은 1ug/ml DAPI (Sigma-Aldrich)로 5 분 동안 대조 염색하였고, 샘플은 PBS로 3 회 세척하고 Olympus 공초점 레이저 스캐닝 현미경으로 관찰하였다. 또한 이식된 마우스 뇌 준비 및 TEM 분석은 Yun W 등에 의해 발표된 Stem Cells Dev 논문(2019;28:633-648)의 개시 내용에 따라 준비하였다.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.
Figure PCTKR2021006248-appb-img-000001
Figure PCTKR2021006248-appb-img-000001
Figure PCTKR2021006248-appb-img-000002
Figure PCTKR2021006248-appb-img-000002
③ 유세포 분석③ Flow cytometry
스페로이드를 Accutase를 이용하여 기계적으로 해리하고 수집하였다. 이후 차가운 PBS로 3회 세척하고, 5 % 정상 당나귀 혈청으로 15 분 동안 블록킹을 수행한 후, 먼저 S100β 및 GFAP에 대한 1 차 항체로 30 분 동안 반응시킨 다음, Alexa Fluor-conjugated 2 차 항체로 30 분 동안 반응시켰다. 염색 수행 후, 0.5 % 포름 알데히드로 세포를 고정시켰다. 세포는 블록킹 과정에서 0.1 % Triton X-100으로 투과성화 되었다. 이어서 세포를 FACS Verse 유세포 분석기 (BD Biosciences, San Jose, CA, USA)를 이용하여 분석하였다.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 분석④ qPCR analysis
정량적 PCR(qPCR) 분석은 cDNA를 대상으로 3회 수행하였다. 음성대조군은 각 샘플의 역전사 음성 블랭크 및 주형이 없는 블랭크를 포함하도록 하였다. 유전자 발현 수준의 측정에서 내부 대조군으로는 GAPDH의 발현 수준을 기준으로 정규화하였다. 또한 qPCR에 사용되는 프라이머는 하기 표 3에 나타내었다.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.
Figure PCTKR2021006248-appb-img-000003
Figure PCTKR2021006248-appb-img-000003
또한 모든 데이터는 3 ~ 6 회 반복 수행한 평균 ± 표준 편차 (SD)로 표시하였고, 결과는 unpaired two-tailed Student’s t 검정 및 분산 검정 (ANOVA)으로 분석하였다. 0.05 미만의 P값은 통계적으로 유의한 것으로 간주하였다. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.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.
<실시예 1><Example 1>
희소돌기아교세포로의 분화가 특화된 신경줄기세포 확립Establishment of neural stem cells specialized for differentiation into oligodendrocytes
본 발명자는 희소돌기아교세포로의 분화가 특화된 신경줄기세포를 확립하기 위해, 다양한 배양 시스템 기반의 실험을 수행하였다. 도 1A에는 hESCs(Human embryonic stem cells)로부터 pre-OPCs 세포로 확립하기까지 각 단계별 세포의 모양 및 배양조건을 나타낸 것이다. 하기는 인간 만능성 줄기세포(Human pluripotent stem cells; hPSCs)에서 희소돌기아교세포로의 분화가 특화된 신경줄기세포(pre-OPCs)의 확립을 위한 세포 배양 방법을 기술하였다.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.
분화를 유도하기 위하여 만능성 줄기세포(Pluripotent stem cells)에서 유래된 모든 세포는 기초 배지(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 및 1% non-essential amino acids 함유]를 기본으로 하여 배양하였다. 원시 신경줄기세포(Primitive NSCs, pNSCs)를 확립하기 위하여 만능성 줄기세포를 LSC 배지[BM, 10 ng/ml LIF(Leukemia Inhibitory Factor), 2 μM SB431542 및 3 μM Chir99021 함유] 분화 조건에서 배양하였다(도 1B 참조). LSC 배양액 조건에서 배양 6일 후, 분화된 세포는 원시 신경줄기세포 마커인 PAX6, SOX1을 발현하는 것을 면역화학염색법으로 확인하였다(도 1C 참조). 또한 원시 신경줄기세포를 LSC 배양액에서 계대 배양하였고, 이후 마트리겔(Matrigel)이 코팅된 세포 배양디쉬에 저밀도로 원시 신경줄기세포를 계대시키고 복측 로제트 타입 신경줄기세포(Ventral rosette type NSCs, vrNSCs)로 분화하기 위해 FEP 배지[BM, 20 ng/ml bFGF, 20 ng/ml EGF 및 0.5 μM Purmorphamine 함유] 조건에서 배양하였다. 배양 8 일 후, 로제트 모양의 콜로니가 나타났다. 또한 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre-OPCs)의 확립을 위해, FEP 배양액에서 FPP 배양액[BM, 20 ng/ml bFGF 및 20 ng/ml PDGF 함유]으로 교체하여 배양하였다(도 1D 참조).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).
그 결과, 상기 방법으로 확립된 희소돌기아교세포로의 분화가 특화된 신경줄기세포(pre-OPCs)에서 희소돌기아교 전구세포의 대표적인 마커인 OLIG2 및 NKX2.2의 발현이 상당수 증가한 것을 확인하였다(도 1E 참조).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).
또한, PDGF 배양액[BM, 20 ng/ml PDGF, 10 ng/ml NT-3, 10 ng/ml IGF-1, 10 uM Forskolin 및 60 ng/ml T3 함유]으로 교체하여 2주간의 공배양 후(도 1F 참조), 8주간의 분화를 살펴본 결과, 신경교세포의 모양이 나타나는 것을 확인하였고, 해당 세포들은 성상교세포의 대표적인 마커인 GFAP 및 S100β를 발현하는 것으로 나타났다(도 1G 참조). 또한, 희소돌기아교세포에서는 O4, MBP, MAG 등 성숙한 형태의 마커를 발현하는데, 희소돌기아교세포로의 분화가 특화된 신경줄기세포(pre-OPCs)에서 분화된 희소돌기아교세포(oligodendrocytes)에서 O4, MBP, MAG가 발현되고 있음을 확인함에 따라, 상기 방법에 의해 희소돌기아교세포로의 분화가 이루어진 것을 확인하였다(도 1H 참조).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 ).
<실시예 2><Example 2>
희소돌기아교세포로의 분화가 특화된 신경줄기세포로부터 오가노이드 확립Establishment of organoids from neural stem cells specialized for differentiation into oligodendrocytes
상기 실시예 1에서 확립된 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre-OPCs)로부터 오가노이드를 하기의 방법에 따라 형성시켰고, pre-OPCs로부터 형성된 오가노이드 및 원시 신경줄기세포(pNSCs)로부터 형성된 오가노이드에 대한 비교 분석을 다음과 같은 방법을 통해 수행하였다.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.
먼저, 상기 실시예 1에서 확립된 PAX6 및 SOX-1을 발현하는 원시 신경줄기세포(Primitive NSCs, pNSCs)와 OLIG2 및 NKX2.2를 발현하는 희소돌기아교세포로 특화된 신경줄기세포(pre-OPCs)를 4일간 공배양을 통하여 구(sphere)를 형성하게 한 다음, 마트리겔에 임베딩(Embedding) 후 RA 배양액(BM 및 0.5uM Retinoic Acids(RA) 함유)에서 7일 동안 배양하였다. 이후, PDGF 배양액에서 14일 동안 추가 배양 후, 실험에 따라 5주~9주 동안 교세포(glial) 분화배양액(BM, 10 uM Forskolin, 60 ng/ml T3, 20 ug/ml 아스코르빅산 함유)에서 배양하였다. 이러한 배양 순서에 대한 모식도는 도 2A에 나타내었다. 4주 동안의 배양을 완료한 후, 원시 신경줄기세포와 희소돌기아교세포로의 특화된 신경줄기세포로부터 얻어진 오가노이드들에 대한 특징을 분석하였다.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.
그 결과, 상기 실험에 사용한 원시 신경줄기세포(Primitive NSCs, pNSCs)와 희소돌기아교세포로 특화된 신경줄기세포(pre-OPCs)의 특징을 면역화학염색법을 통해 확인하였는데, 원시 신경줄기세포는 PAX6 및 SOX-1을 발현하는 것을 확인할 수 있었고, 희소돌기아교세포로 특화된 신경줄기세포는 OLIG2 및 NKX2.2를 발현하는 것을 확인할 수 있었다(도 2B 및 2C). 또한 원시 신경줄기세포와 희소돌기아교세포로의 특화된 신경줄기세포로부터 얻어진 오가노이드들의 모양을 현미경으로 관찰한 결과, 이들 두 가지의 세포가 신경구를 형성하는 것을 확인할 수 있었으나 그 형태는 다른 것으로 확인되었다(도 2D 및 2E 참조). 또한 RA 처리 7일 후의 관찰 결과에서, 원시 신경줄기세포로부터 형성된 오가노이드는 뇌 오가노이드에서 관찰되는 큰 신경 상피 버드의 형성이 확인되는 것으로 나타난 반면, 본 발명의 희소돌기아교세포로의 특화된 신경줄기세포로부터 형성된 오가노이드는 직접 신경분화와 일치하는 방사형의 돌출된 프로세스를 관찰할 수 있었다(도 2F 및 2G 참조).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).
면역염색화학 분석결과, 원시 신경줄기세포로부터 얻어진 오가노이드는 VZ(Ventricular zone)이 크게 형성되는 것으로 나타났고 TUJ1+ 미성숙 뉴런을 포함하는 것으로 나타난 반면, 희소돌기아교세포로의 특화된 신경줄기세포로부터 얻어진 오가노이드에서는 배양 2주째에 작은 VZ의 형성을 확인하였다(도 2H 참조).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).
또한, 다양한 신경세포의 마커 분석결과, 희소돌기아교세포로의 특화된 신경줄기세포로부터 얻어진 오가노이드에서는 성숙한 신경세포의 마커인 TH와 GABA가 나타나는 신경세포로 분화가 촉진된 것을 확인할 수 있었고(도 2I 참조), 나아가 운동뉴런의 마커인 OLIG2가 여전히 발현되는 것으로 확인되었고, 반면 운동뉴런 마커인 HB9+는 관찰되지 않았다(도 2J 참조).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 ).
이러한 결과를 토대로, 본 발명자들은 본 발명에 따른 희소돌기아세포로 분화가 특화된 신경줄기세포로부터 형성된 오가노이드는 원시 신경줄기세포로부터 형성된 오가노이드보다 분화가 빠르게 진행되는 것을 확인할 수 있었다.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.
<실시예 3> <Example 3>
신경줄기세포로부터 형성된 오가노이드에서 신경교세포로의 분화 검증Verification of differentiation from organoids formed from neural stem cells to glial cells
희소돌기아교세포로 특화된 신경줄기세포(pre-OPCs)로부터 얻어진 오가노이드에서 신경교세포로(Glia)의 분화가 빠르게 진행되었는지를 면역염색화학법을 통해 분석하였다.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.
그 결과, 신경교세포로의 분화 2주차에 신경교 전구세포(Glial progenitor cells)의 마커인 BLBP가 pre-OPCs로부터 형성된 오가노이드(스페로이드)에서 발현되는 것을 확인하였고(도 3A), 3주차에는 성상교세포(astrocytes)의 전형적인 마커인 S100β 와 GFAP의 발현을 면역염색화학법(도 3B) 및 유세포 분석법(도 3C)을 통해 확인할 수 있었다. 반면, 원시 신경줄기세포(pNSCs)로부터 형성된 오가노이드에서는 분화 4주차에서도 이러한 마커의 발현을 확인할 수 없었으며, 분화 5주차의 경우, S100β 와 GFAP의 발현도 매우 미비한 것으로 나타났다.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.
오가노이드의 특장점으로 알려진 형태학적으로 다양한 성상교세포가 존재하는 것을 면역염색화학법을 통해 확인할 수 있었고(도 3D), 유전자 발현 또한 다양한 성상교세포가 혼합되어 있는 것을 확인할 수 있었다(도 3E).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).
또한, 본 발명에 따른 희소돌기아교세포로 특화된 신경줄기세포로부터 오가노이드를 형성하는 배양 방법은 기존 2D 분화 방법에 비해 더 짧은 시간에 오가노이드로부터 성상교세포로의 분화 효율을 월등히 높일 수 있는 것으로 나타났다(도 3F 참조).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).
따라서 본 발명에 따른 희소돌기아교세포로 특화된 신경줄기세포로부터 오가노이드를 형성할 수 있는 배양방법 및 상기 방법으로 형성된 오가노이드는 성상교세포로의 분화가 빠른 특징이 있고, 실제 인간의 뇌와 유사하게 다양한 성상교세포가 혼합하여 존재하는 특징이 있어, 뇌질환의 치료를 위한 세포치료제로서 더욱 효과적으로 사용될 수 있음을 알 수 있었다.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.
<실시예 4> <Example 4>
신경줄기세포로부터 형성된 오가노이드에서 희소돌기아교세포로의 분화 및 수초 형성능 검증Validation of differentiation and myelination ability from organoids formed from neural stem cells to oligodendrocytes
나아가 본 발명자들은 본 발명에 따른 희소돌기아교세포로 특화된 신경줄기세포로부터 얻어진 오가노이드 안에서 희소돌기아교세포로의 분화가 빠르게 진행되었는지를 면역염색화학법으로 분석하였다.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.
그 결과, 희소돌기아교세포로 특화된 신경줄기세포로부터 얻어진 오가노이드 안에서 분화 4주차에 희소돌기아교 전구세포의 마커인 SOX10의 발현을 확인하였고(도 4A), SOX10 양성 세포에서 OLIG2, PDGFRa 및 O4를 발현하는 것을 확인할 수 있었으며(도 4B), 6주차에는 희소돌기아교세포 마커인 MBP의 발현도 확인할 수 있었다(도 4C). 또한, 8주차의 오가노이드는 4주차의 오가노이드에 비해 희소돌기아교세포 관련 마커들의 발현량이 증가한 것으로 나타났고(도 4D 참조), 특히 8주차에는 오가노이드 안에서 시험관 내 수초(In vitro myelination) 형성이 이루어지는 것을 확인하였다(도 4E 및 4F).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 ).
나아가 본 발명자들은 본 발명에 따른 오가노이드 배양법을 이용하여 수초 형성능 평가를 통한 약물 스크리닝이 가능한지를 확인하기 위해, 기존에 재수초 형성(remyelination) 유도약물로 알려진 T3 (60 ng/ml), Miconazole (1uM) 및 Benztropine (1uM)을 상기 오가노이드에 처리한 결과, 대조군인 DMSO를 처리한 군에 비해 상기 약물을 처리한 군에서, 수초 형성이 더 많이 진행된 것을 확인할 수 있었고(도 4G), 구체적으로 상기 약물 중에서 특히 Miconazole을 처리한 오가노이드에서 가장 많은 양의 수초가 형성된 것을 확인할 수 있었다(도 4H 및 4I). 또한 본 발명자들은 Miconazole을 처리한 오가노이드에서 희소돌기아교세포를 분리한 후, 수초가 없는 Shiverer 쥐에 이식한 결과 MBP+ 희소돌기아교세포, 즉 수초가 형성되는 것을 확인하였고(도 4J 참조), 이식 후 12주 후에 전자현미경을 통해 수초 집단이 형성된 것을 확인할 수 있었다.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.
따라서, 상기 결과를 토대로 본 발명자들은 본 발명에 의한 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 오가노이드를 형성할 수 있는 배양 방법은 원시 신경줄기세포를 사용하는 것에 비하여 더 짧은 시간 안에 희소돌기아교세포 및 성상교세포로의 분화가 가능하고 또한 재수초 형성능을 평가할 수 있어, 탈수초성 질환의 치료를 위한 약물 스크리닝에 유용하게 사용할 수 있음을 알 수 있었고, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 형성된 오가노이드는 탈수초성 질환의 치료를 위한 세포치료제로 유용하게 사용 가능함을 알 수 있었다.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.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.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) 인간 만능성 줄기세포(Human pluripotent stem cell; hPSC)를 LIF(Leukemia Inhibitory Factor), SB431542 화합물 및 CHIR 99021 화합물을 포함하는 LSC 배지로 배양하여 원시 신경줄기세포(Primitive NSC; pNSC)로 분화시키는 단계;(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) 상기 원시 신경줄기세포를 bFGF, EGF 및 퍼모프아민(Purmorphamine)을 포함하는 FEP 배지로 배양하여 복측 로제트 타입 신경줄기세포(Ventral rosette type NSC; vrNSC)로 분화시키는 단계;(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) 상기 복측 로제트 타입 신경줄기세포를 bFGF, PDGF 및 퍼모프아민(Purmorphamine)를 포함하는 FPP 배지로 배양하여 희소돌기아교세포로 분화가 특화된 신경줄기세포(pre Oligodendrocyte Precursor Cell; pre-OPC)로 분화시키는 단계; 및(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) 상기 희소돌기아교세포로 분화가 특화된 신경줄기세포를 RA(retinoic acid) 함유 배지; PDGF(Platelet-derived growth factor), IGF1(Insulin like growth factor), HGF(Hepatocyte growth factor) 및 포스콜린(forskolin) 함유 배지; 및 포스콜린(forskolin), 아스코르빅산 및 T3(3,3′,5-triiodo-L-thyronine) 함유 배지;로 순차적으로 배지를 교체하면서 배양하여 희소돌기아교세포를 포함하는 오가노이드를 형성하는 단계를 포함하는,(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,
    희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법.A method for producing an organoid comprising oligodendrocytes from neural stem cells specialized for differentiation into oligodendrocytes.
  2. 제1항에 있어서,According to claim 1,
    상기 배지는 DMEM/F12, 중탄산나트륨, 비타민 A가 없는 B-27 보충제, N-2 보충제, 페니실린-스트렙토마이신, L-글루타민 및 비필수아미노산을 포함하는 배지를 기초배지로 사용하는 것을 특징으로 하는, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법.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. 제1항에 있어서,According to claim 1,
    상기 희소돌기아교세포로 분화가 특화된 신경줄기세포는 OLIG2(Oligodendrocyte transcription factor 2) 및 NKX2.2(NK2 Homeobox 2)를 발현하는 것을 특징으로 하는, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법.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. 제1항에 있어서,According to claim 1,
    상기 오가노이드에는 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 분화된 신경세포 및 성상교세포가 더 포함되어 있는 것을 특징으로 하는, 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법.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. 제1항 내지 제4항 중 어느 한 항의 방법으로 제조된 희소돌기아교세포를 포함하는 오가노이드.An organoid comprising oligodendrocytes prepared by the method of any one of claims 1 to 4.
  6. 제5항의 희소돌기아교세포를 포함하는 오가노이드에 후보약물을 처리한 후, 수초 형성 여부를 분석하는 단계를 포함하는, 탈수초 질환의 치료제를 스크리닝하는 방법.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. 제6항에 있어서,7. The method of claim 6,
    상기 후보약물 처리에 의해 수초 형성이 촉진되는 경우, 상기 후보약물을 탈수초 질환의 치료제로 판단하는 단계를 더 포함하는 것을 특징으로 하는, 탈수초 질환의 치료제를 스크리닝하는 방법.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. 제5항의 희소돌기아교세포를 포함하는 오가노이드 또는 상기 오가노이드에서 분리된 희소돌기아교세포를 유효성분으로 포함하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물.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. 제8항에 있어서,9. The method of claim 8,
    상기 조성물은 수초화 재생을 촉진시키는 것을 특징으로 하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물.The composition is a cell therapy composition for the prevention or treatment of demyelination diseases, characterized in that it promotes myelination regeneration.
  10. 제8항에 있어서,9. The method of claim 8,
    상기 탈수초 질환은 급성 파종성 탈수초(acute disseminated demyelination), 레버 유전성 시각 신경병증(Leber hereditary optic neuropathy), 탈수초성 다발성 신경병증(demyelinating polyneuropathy) 및 샤르코 마리 투스병(Charcot Marie Tooth)으로 이루어진 군으로부터 선택되는 것을 특징으로 하는 탈수초 질환의 예방 또는 치료용 세포치료제 조성물.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. 제8항에 있어서,9. The method of claim 8,
    상기 오가노이드는 수초 형성 유도약물이 처리된 오가노이드인 것을 특징으로 하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물.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. 제11항에 있어서,12. The method of claim 11,
    상기 수초 형성 유도약물은 T3(3,3’,5-triiodo-L-thyronine), 미코나졸(Miconazole) 또는 벤즈트로핀(Benztropine)인 것을 특징으로 하는, 탈수초 질환의 예방 또는 치료용 세포치료제 조성물.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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