KR20220102357A - Method for producing neural stem cells via 3d culture - Google Patents

Abstract

The present invention relates to a method for differentiating stem cells into neural stem cells in a short time by a specific culture method without conventional suspension culture, retinoic acid, co-culture (with stromal cells), a dual SMAD inhibitor (noggin & SB431542) treatment, or nervous system cell inducer treatment. The present invention provides a method for preparing neural stem cells. According to the present invention, early neural tissue is easily distinguished by transforming a marker gene into embryonic stem cells and differentiated into early nervous system cell aggregates without a neural lineage differentiation inducing factor by culturing the same in a three-dimensional form inside a drop of hydrogel. Also, by culturing the same in two dimensions, neural stem cells can be produced in a significantly shorter period (within about 3 weeks) compared to the conventional art, and thus a differentiation method is simplified and cost is reduced.

Description

Method for producing neural stem cells through three-dimensional culture {METHOD FOR PRODUCING NEURAL STEM CELLS VIA 3D CULTURE}

The present invention relates to pluripotent stem cells from neural stem cells in a short time by a specific culture method without conventional suspension culture, retinoic acid, co-culture, dual SMAD inhibitor (noggin & SB431542) treatment or treatment with a nervous system cell inducer. How to differentiate into

A stem cell refers to a cell that can proliferate indefinitely while maintaining an undifferentiated state, and can differentiate to have a specific function and form when given a certain environment and condition. Stem cells can be classified according to the method and source of their establishment, including "embryonic stem cells (ESC)" established from embryos and "adult stem cells" isolated from adult tissues. )", and "induced stem cells" artificially established through the process of somatic cell reprogramming. In addition, they can be classified according to their differentiation ability, and can be classified into “unipotency” capable of differentiating into one type of cell, “multipotency” capable of differentiating into several types of cells, and all tissue cells. It can be divided into "pluripotency" stem cells that can differentiate. Pluripotent stem cells include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Because it has the ability to differentiate into cells, its value is highly recognized in regenerative medicine. Embryonic stem cells are stem cells isolated from the inner cell mass (ICM) that will develop into the fetus during the blastocyst embryo, which is a very early stage after the formation of a fertilized egg and before implantation in the endometrium. It is a pluripotent cell that can differentiate into cells of any tissue produced by the embryonic germ layer of Human embryonic stem cells can continuously self-renewal under suitable in vitro culture conditions, and have pluripotency to differentiate into all types of cells constituting the human body. Therefore, in order to understand the basic knowledge about the development, differentiation and growth of the human body, as well as the development of cell therapy, which is a fundamental treatment method for damage to the human body or various diseases, search for drug efficacy of various new drug candidates, identification of the cause of disease, development of treatment, etc. In various aspects, the scope of application of research results on this subject is expanding. So far, pluripotent stem cells have been mainly generated by nuclear transfer and cell fusion (Philos Trans R Soc Lond B Biol Sci. 363(1500): 2079-2087). "Induced pluripotent stem cells (iPSCs)" are cells that exhibit characteristics similar to embryonic stem cells (ESCs). Induced pluripotent stem cells were derived from mouse fibroblasts in 2006 (Cell 126: 663-676 (2006)) and human fibroblasts in 2007 (Science 318: 1917-1920 (2007), ) Expression of defined factors was first created by increasing

Nervous system cells can be divided into two main types, the central nervous system cells constituting the brain and spinal cord, and the peripheral nervous system cells constituting the motor, sensory, and autonomic nerves. Neurons, astrocytes, and oligodendrocytes that make up the central nervous system (brain and spinal cord) are made by differentiating neural stem cells or neural progenitor cells differentiated from pluripotent stem cells. On the other hand, peripheral nerve cells (motor, autonomic, and sensory nerves) and Schwann cells that make up the peripheral nervous system are derived from neural crest stem cells (NCSCs) differentiated from pluripotent stem cells. is derived Therefore, central nervous system cells and peripheral nervous system cells follow different differentiation pathways from pluripotent stem cells, respectively, through neural progenitor cells and neural crest cells, and these different pathways depend on the surrounding environment and intracellular signaling system. is known to be Neural stem cells (NSCs) are self-renewing pluripotent stem cells of the nervous system, which have the ability to differentiate into neurons, astrocytes and oligodendrocytes. Neural stem cells can be obtained either directly from the fetal and adult central nervous system or by neural differentiation derived from pluripotent stem cells. NSC is also considered as a perspective treatment for the recovery treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. as well as spinal cord injuries that cause immobility.

Techniques for inducing neural stem cells from human embryonic stem cells are being continuously studied. Conventionally, suspension culture was performed for differentiation from pluripotent stem cells into neural stem cells or neurons, and during the culture process, retinoic acid, co-culture (cultured with stromal cells), dual SMAD inhibitors ( noggin & SB431542) have been used previously. Differentiation-induced neural progenitor cells are differentiated into specific neurons, or transplanted into an animal model of cranial nervous system disease at the stage of neural progenitor cells or more differentiated neurons, and are used to confirm their clinical applicability. However, the conventional method has a disadvantage in that it takes a long time (eg, 1 month or more) through several stages of differentiation from undifferentiated human embryonic stem cells to specific neurons.

Diseases of the central nervous system are largely divided into brain diseases and spinal cord diseases. Spinal cord injury is caused by a sudden injury, and when the function of the spinal cord is damaged, problems such as loss of motor control, loss of sensation, and loss of bladder control occur, and people live in pain for the rest of their lives. In addition, the number of people suffering from many central nervous system diseases is rapidly increasing with the aging of the world. It is urgent.

Therefore, in order to develop cell replacement therapy, which is considered the most fundamental treatment for central nervous system diseases, it is necessary to establish a method for efficiently differentiating pluripotent stem cells into neural stem cells, which are the parental cells of central nervous system cells. do.

An object of the present invention is to provide a method for efficiently producing neural stem cells (NSC) from pluripotent stem cells.

Another object of the present invention is to provide neural stem cells.

Another object of the present invention is to provide a composition for treating spinal cord injury.

In addition, it is an object of the present invention to provide a composition for treating neurodegenerative diseases.

In order to solve the above problems, the present invention provides a method for producing neural stem cells.

In addition, the present invention provides a neural stem cell prepared by the above method.

In addition, the present invention provides a composition for treating spinal cord injury comprising the neural stem cells.

In addition, the present invention provides a composition for treating neurodegenerative diseases comprising the neural stem cells.

According to the method of the present invention, early neural tissue was easily distinguished by transforming the marker gene into embryonic stem cells, and by culturing it in a drop of hydrogel in a three-dimensional form, the initial nervous system cell aggregate without a neural system differentiation inducing factor Since neural stem cells can be produced in a significantly shorter period (within about 3 weeks) compared to the prior art by two-dimensional culture, the differentiation method is simple and the cost is reduced.

1 is a diagram confirming the differentiation of Sox1-GFP ESCs into neural stem cells (NSCs):
A: Schematic of the NSC induction protocol;
B: single cells of Sox1-GFP ESCs embedded in matrigel droplets;
C: Aggregate formation of Sox1-GFP ESCs embedded in Matrigel (day 3);
D: Sox1-GFP expressing aggregates (day 10); and
Scale bar: 200 μm.
2 is a diagram showing neural stem cells (NSCs) prepared using Sox1-GFP-expressing ESCs aggregates:
A: GFP-expressing aggregates attached to gelatin-coated dishes (day 1);
B: GFP-expressing aggregates attached to gelatin-coated dishes (day 3);
C to E: NSCs prepared from aggregates at passages 3, 4 and 5 showing typical NSC morphology.
3 is a diagram confirming the characteristics of the prepared NSCs:
A: Results of immunocytochemical analysis using NSC-specific markers; and
B: Real-time RT-PCR analysis result using NSC marker gene.
4 is a diagram confirming the differentiation capacity of the prepared NSCs:
A: Results of immunocytochemical analysis using markers of neuronal subtypes (neurons, astrocytes and oligodendrocytes) of cells differentiated from NSCs; and
B: Real-time RT-PCR analysis results using neuron, astrocyte and oligodendrocyte-specific marker genes.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples for the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express a preferred embodiment of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

In addition, the term "step for" or "step of" as used herein does not mean "step for".

All technical terms used in the present invention, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In one aspect, the present invention comprises the steps of (A) transforming an undifferentiated stem cell into a marker gene specifically expressed in neural lineage cells; (B) forming a cell aggregate by three-dimensional culture; (C) selecting a cell aggregate expressing the marker gene; And (D) relates to a method for producing neural stem cells (NSC) comprising the step of culturing the selected cell aggregate in two dimensions.

In one embodiment, the stem cells can be selected from the group consisting of embryonic stem cells (Embryonic Stem Cells, ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells, embryonic tumor cells and adult stem cells. and may be pluripotent stem cells (PSCs), more preferably embryonic stem cells.

In one embodiment, the marker gene may be any one selected from the group consisting of Sox1 ( Sry-related-HMG box 1 ), Olig2 and Sox2 , and Sox1 ( Sry-related- HMG box 1 ).

In one embodiment, the marker gene may further include a detectable label, wherein the detectable label is a fluorescent label, a probe, a luminescent material, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radiolabel. , tag and hapten, and the fluorescent label is Atto dye, Alexafluor dye, quantum dot, hydroxycoumarin, aminocoumarin, methoxycoumarin, cascade blue, pacific blue, pacific orange, lucifer yellow , NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, True Red, FluorX, Fluorescein, Body P-FL, Cy2, Cy3, Cy3B, Cy3. 5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lysamine Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 Conjugate, Indo-1, Fluo-3, Fluo-4 , DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation) ), mKeima-red, Tagged CFP, Suggestive 1, mTFP1, GFP (S65A mutation), Midorish cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, Tagged GFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusavira Orange, mOrange, Allophycocyanin (APC) , mKO, TurboRFP, tdTomato, TaggedRFP, Dsred monomer, Dsred2 ("RFP"), mStrawberry, TurboFP602, Asred2, mRFP1, J-red, R-phycoerythrin (RPE) , B-phycoerythrin (BPE), m cherry, Hc red 1, KATUSA, P3, peridinine chlorophyll ( PerCP), mKate (tag FP635), TurboFP635, mPlum, and mRaspberry.

In one embodiment, the stem cells transformed with the marker gene may be cultured in the absence of a feeder before three-dimensional culture.

In one embodiment, the three-dimensional culture may be one in which cells are cultured inside a hydrogel, comprising the steps of (a) isolating undifferentiated stem cells transformed with a marker gene into single cells; (b) mixing the separated single cells with the hydrogel and curing the mixture; and (c) culturing in a pluripotent stem cell medium.

In one embodiment, the three-dimensional cultured embryonic stem cells may be differentiated into early nervous system cells.

In one embodiment, the hydrogel may include an extracellular matrix material or an extracellular matrix-like material, wherein the extracellular matrix-like material is gelatin, polyethylene glycol, collagen, alginate, chitosan, hyaluronic acid, dextran, fibrin , cellulose, fibroin, fibronectin, laminin, cordroitin sulfate, matrigel, and mixtures thereof.

In one embodiment, the hydrogel mixed with the isolated single cells may form a three-dimensional cell culture structure while curing, and may be in the form of drops, drops or droplets.

In one embodiment, the hydrogel can be cured through a sol-gel phase transformation.

In one embodiment, the cell aggregate may be an aggregate of cells differentiated into early nervous system cells, may be in the form of a spheroid, or may be a two-layered aggregate.

In one embodiment, the three-dimensional culture may be performed for 8 to 14 days.

In one embodiment, the two-dimensional culture may be culturing the cell aggregate on the hydrogel surface, and may be performed for 3 to 7 days.

In one embodiment, the two-dimensional culture neural stem cell proliferation (expand) medium by culturing the cell aggregate on the surface of the hydrogel can be proliferated.

In one embodiment, the step (D) may further include the step of separating the expanded (expanded) cell aggregates into single cells by two-dimensional culture in step (D) and subculture.

As used herein, the term “stem cells” refers to undifferentiated cells having self-renewal and differentiation proliferative capacity. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation capacity. The pluripotent stem cells refer to cells having the ability to differentiate into all tissues or cells constituting a living body, and the pluripotent stem cells are not all types, but have the ability to differentiate into multiple types of tissues or cells. cells that have A unipotent stem cell refers to a cell having the ability to differentiate into a specific tissue or cell. Examples of pluripotent stem cells include embryonic stem cells (ES Cells), undifferentiated germline cells (EG Cells), iPS cells, and the like. Examples of pluripotent stem cells include mesenchymal stem cells (adipose-derived, Bone marrow-derived, umbilical cord blood or umbilical cord-derived), hematopoietic stem cells (derived from bone marrow or peripheral blood, etc.), nervous system stem cells, adult stem cells such as reproductive stem cells, etc. Committed stem cells, which exist in a low state and produce only hepatocytes through vigorous division after activation, are examples.

As used herein, the term "pluripotency or pluripotent" refers to three germ layers: endoderm (eg, gastrointestinal inner layer, gastrointestinal tract, lung), mesoderm (eg, muscle, bone, blood, urogenital) , or ectoderm (eg, epidermal tissue and nervous system). As used herein, “pluripotent cell” is used interchangeably with “pluripotent stem cell”. On the other hand, a pluripotent stem cell can be one of several types of cells within a particular organ. For example, pluripotent blood stem cells can develop into red blood cell progenitor cells, white blood cells, or platelet-producing cells. Adult stem cells are pluripotent stem cells.

As used herein, the term "induced pluripotent stem cells" (iPSCs) is a type of pluripotent stem cells artificially induced from mast cells. CiPSCs are iPSCs, but they differ in some ways from iPSCs in that they are not genetically engineered to confer pluripotency.

As used herein, the term "hydrogel" refers to a material in which a liquid having water as a dispersion medium solidifies through a sol-gel phase change to lose fluidity and form a porous structure, and if it is a hydrogel suitable for cell culture, it is not particularly limited. No, in one embodiment of the present invention was used Matrigel.

As used herein, the term “differentiation” refers to a phenomenon in which the structure or function of a cell is specialized while the cell divides and proliferates and grows.

As used herein, the term "dedifferentiation" refers to a phenomenon in which differentiated cells acquire a 'stem cell'-like or multipotent state before differentiating into one or more different tissue types.

As used herein, the term “drop or droplet” refers to any binding volume of a hydrogel material (which may be a liquid or a gel). The volume of a droplet of water is generally less than 1.0 mL, but may be greater than this for viscous substances, and the single bound volume of the medium is referred to as a droplet.

As used herein, the term "spheroid" refers to a three-dimensional structure formed to such an extent that cells are aggregated so that the cross section can be generally seen as circular or oval.

As used herein, the term "medium" refers to a medium capable of supporting the growth and survival of cells in vitro , and the pluripotent stem cell medium is a medium capable of differentiating into early nervous system cells in the art. All conventional and appropriate media used are included. Such pluripotent stem cell medium includes, for example, Epiblast stem cell medium, but is not limited thereto.

As used herein, the term "gelatin" is an induced protein obtained by hydrolyzing collagen (collagen), which is a natural high molecular protein constituting animal bones, hides, tendons, cartilage, and the like. The general composition of gelatin may be about protein: 84 to 90%, water: 8 to 12%, and mineral salts: 2 to 4%. In the present invention, gelatin may have various gel strength and viscosity. In the present invention, gelatin can be separated or synthesized from natural materials, and the type of raw material is not limited, but preferably, it can be produced by hydrolyzing collagen separated from skins, bone shells, etc. of mammals or fish.

In one aspect, the present invention relates to a neural stem cell prepared by the method of the present invention.

In one embodiment, the neural stem cells express Nestin, Sox2, N-Cadherin and Musashi, and can be differentiated into neurons, astrocytes and oligodendrocytes.

In one aspect, the present invention relates to a composition for treating spinal cord injury comprising the neural stem cells of the present invention as an active ingredient.

As used herein, the term "Spinal Cord Injury (SCI)" refers to damage to the spinal cord due to an acquired accident, so that the performance of sensory signals and motor signals passing through the damaged site is affected, so sensory neurons (sensory neurons) ) or motor neuron transmission disorder, resulting in partial or complete paralysis of human body functions. Specifically, the spinal cord injury may be caused by trauma such as a traffic accident or a fall. The spinal cord injury may be divided into a complete injury and an incomplete injury according to the extent of the injury. Spinal cord injury may be accompanied by symptoms of loss of function mainly due to paralysis of motor nerves below the injured site. Specifically, complete damage may result in complete loss of motor and sensory functions of the spinal cord due to complete transverse amputation of the spinal cord. Incomplete damage, unlike complete damage, may appear in a state in which some sensory and motor functions below the damaged area are preserved. Symptoms may vary depending on the injured part, and for example, quadriplegia, blood pressure, pulse, body temperature, and respiratory rate may all decrease due to hard water injury. Paraplegia of the lower extremities, loss of sensory function, and loss of large intestine, bladder, and sexual function due to subpleural injury may be seen.

In one aspect, the present invention relates to a composition for treating neurodegenerative diseases comprising the neural stem cells of the present invention as an active ingredient.

As used herein, the term "neurological degenerative disease" refers to abnormalities in motor control ability, cognitive function, perceptual function, sensory function and autonomic nerve function due to reduction or loss of nerve cell function, and progressive cognitive function according to the main symptoms. It can be divided into disability, progressive ataxia, muscle weakness and muscle atrophy. The neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic brain disease (stroke), dehydration It may be selected from the group consisting of demyelinating disease, multiple sclerosis, epilepsy and neurodegenerative diseases, but is not particularly limited thereto.

As used herein, the term “prevention” refers to any action of inhibiting the progression or delaying the onset of neurodegenerative diseases including spinal cord injury by the composition of the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of neurodegenerative diseases, including spinal cord injury, are improved or beneficially changed by the composition of the present invention.

A composition is indicated to be "pharmaceutically or physiologically acceptable" if the recipient animal can tolerate administration of the composition, or if administration of the composition to that animal is suitable. When the amount administered is physiologically important, the agent can be said to have been administered in a "therapeutically effective amount". An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, administration method, target site, patient's condition, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively.

As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition.

The composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose. , mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal or topical administration according to a desired method) ) or oral administration, and the dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the subject's age, weight, sex, physical condition, etc. is selected taking into account. It is self-evident that the concentration of the active ingredient included in the composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg/ml, it may be toxic to the human body.

The compositions of the present invention may be formulated into a variety of oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, coloring, flavoring and sweetening agents. The formulation may be prepared by conventional mixing, granulating or coating methods. In addition, a representative formulation for parenteral administration is an injection formulation, and examples of the solvent for the injection formulation include water, Ringer's solution, isotonic saline or suspension. The sterile, fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil including mono- and di-glycerides can be used for this purpose. In addition, the injection preparation may use a fatty acid such as oleic acid.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Induction of differentiation into early nervous system cells using a 3D culture system

Even with the direct differentiation of pluripotent stem cells (PSCs), it is almost impossible to obtain a pure population of desired cells, and various types of unwanted cells are induced together with the desired cells and form a heterogeneous cell population. To efficiently identify differentiation into , lineage-specific markers were used. That is, to select early nervous system cell types during the differentiation of PSCs, Sox1 ( Sry-related-HMG box), which is important for neurodevelopmental differentiation and specific for the early formation of neuroepithelial cells in the neural tube, as a marker for identifying early stages of neuronal development (Sry-related-HMG box ) (Sox1-GFP) transgenic ESCs expressing green fluorescence protein (GFP) under the control of 1 ) were used. Specifically, Sox1-GFP ESCs were cultured in a 37° C. 5% CO ₂ incubator under feeder-free conditions. At this time, the medium used was serum-free N2B27 medium (N2 supplement (Gibco), B27 supplement (Gibco), 100x penicillin/streptomycin/glutamine (Gibco), LIF (Leukemia inhibitory factor) (ESGRO; Chemicon), 1:1 mixture of DMEM/F12 medium (Gibco) and Neurobasal medium (Gibco) with 3 μM GSK inhibitor (Sigma) and 1 μM MEK inhibitor (Sigma)). One day before subculture, Matrigel was placed in a liquid state at 4°C, and then undifferentiated Sox1-GFP ESCs were made into single cells using trypsin-EDTA (Gibco), and the number of cells was counted with a hematocyte. Thus, 3,000 single cells were aliquoted into 50 μl Matrigel (Corning) drop of a 4-well dish. Thereafter, the matrigel droplets were solidified in a 5% CO ₂ incubator at 37° C. for 1 hour, and the solidified matrigel droplets were treated with DMEM/F12 (Gibco), 100x penicillin/streptomycin/glutamine (Gibco), 5% Knockout serum. (Gibco), 100x NEAA (Non-essential amino acid) (Gibco), 20 ng/ml Activin A supplement (PeproTech), and 10 ng/ml bFGF supplement (R&D system) incubated in Epi medium to Sox1-GFP ESCs When the differentiation was induced (FIG. 1B), Sox1-GFP ESCs proliferated to form spherical aggregates like ESC colonies in a 3D environment about 3 days after culture (FIG. 1C). A fraction (about 30%) of Sox1-GFP ⁺ aggregates began to form bilayer aggregates expressing Sox1-GFP (Sox1-GFP ⁺ ) after 7 days of incubation in matrigel droplets (Fig. 1D). That is, aggregates composed of cells uniformly expressing Sox1-GFP appeared on the 10th day of culture ( FIG. 1E ), confirming that Sox1-GFP ESCs were differentiated into early neural cells or neuroepithelial cells within 7 days of 3D culture. Thereafter, this aggregate was used for induction of neural stem cells (NSC) in Example 2 ( FIG. 2A ).

Example 2. Preparation of neural stem cells using a 2D culture system

Neural stem cells (NSCs) were induced in the 2D culture system by selecting aggregates in which almost all cells expressed Sox1-GFP on the 10th day after culturing by the 3D culture method in Example 1 above. Specifically, after 3D culture of Sox1-GFP ESCs in Matrigel, Matrigel was removed using Cell Recovery Solution (Corning) on days 11 to 12. Then, using a fluorescence microscope, colonies (aggregates) in which almost all cells of the neurospheres express Sox1-GFP were selected and transferred to a gelatin-coated 24-well dish in NSC expansion medium (DMEM). /F12 (Gibco), 100x penicillin/streptomycin/glutamine (Gibco), B27 supplement (Gibco), 10ng/ml epidermal growth factor (EGF) (Gibco) and 10ng/ml basic fibroblast growth factor (bFGF) (R&D system) ) cells were cultured in 2D for 3 to 4 days ( FIGS. 2A and B). The proliferated neurospheres were treated with 0.25% trypsin (Gibco) to separate single cells, and then subcultured in gelatin-coated dishes with NSC growth medium every 2 to 3 days, polar-shaped cells, typical of NSCs, were formed. (Fig. 2C). Thereafter, it was confirmed that NSCs that actively self-renew through subsequent culture were produced ( FIGS. 2D and E). Through this, it was confirmed that ESCs effectively differentiated into 2D NSCs within 17 days through 3D culture (about 10 days) and 2D culture (about 7 days).

Please indicate the number of incubation days as a range or unify it.

Example 3. Confirmation of characteristics of neural stem cells

In order to confirm whether the NSCs prepared in Examples 1 and 2 exhibit typical NSC characteristics, immunocytochemistry (ICC) and real-time RT-PCR analysis were performed using major NSC markers. Specifically, for ICC analysis, the NSCs prepared by 2D culture in Example 2 were fixed with 4% paraformaldehyde (PFA) at 4° C. for 20 minutes, washed with PBS (welgene), and 3% BSA (Gibco) and After treatment with PBS containing 0.3% Triton X-100 (Sigma), it was incubated at room temperature (24° C., RT) for 45 minutes. Then, NSCs markers Nestin, Sox2, Pax6 and Musashi antibodies (anti-Nestin (Nestin; monoclonal, 1:500, Millipore), anti-Sox2 (Sox2; polyclonal, 1:500, Millipore), anti-Musashi) -1 (Musashi-1; polyclonal, 1:500, Millipore) and anti-Pax6 (Pax6; monoclonal, 1:500, Adcam)) were used as primary antibodies, and fluorescent substances (Alexa fluor 488 or 568; Molecular probes) , Eugene, OR, USA) labeled secondary antibody was used. In addition, immunocytochemical analysis was performed by staining with DAPI to indicate nuclei. In addition, for real-time RT-PCR analysis, RNA was extracted from NSCs prepared by 2D culture in Example 2 using TRIzol reagent (Invitrogen), and then SuperScript Ⅲ reverse transcriptase (Invitrogen, Grand Island, NY, USA) ) to synthesize cDNA from 1 mg of total RNA using the primer set in Table 1 and TOPreal™ qPCR 2x PreMIX (Enzynomics) in Roche LightCycler 480 in real-time PCR (Real-time polymerase chain reaction) at 95 ° C. It was repeated 3 times under the conditions of 45 cycles for 10 seconds, 10 seconds at 60°C, and 20 seconds at 72°C.

fowrard Reverse Nestin 5'-AAGTTCCCAGGCTTCTCTTG-3' 5'-GTCTCAAGGGTATTAGGCAAGG-3' Sox2 5'-GAACGCCTTCATGGTATGGTCC-3' 5'-CGGGTGCTCCTTCATGTGC-3' N-Cadherin 5'-GTACCGCAGCATTCCATTCAGG-3' 5'-GGCAATCAAGTGGAGAACCCC-3' Oct4 5'-GGCTTCAGACTTCGCCTTCT-3' 5'-TGGAAGCTTAGCCAGGTTCG-3' Olig2 5'-GCCTGCTCAAGTCACCGTCG-3' 5'-CACACGCCATCAACACTGGTCGC-3' S100b 5'-GGTTGCCCTCATTGATTGTCTTCC-3' 5'-CATCCCCATCTTCGTCCAGCC-3' GFAP 5'-CTGATGTCTACCAGGCGGAGC-3' 5'-CCAGGTTGTTCTCTGCCTCCAG-3' NeuN 5'-CACCTCCGCCTCATCCCAC-3' 5'-GTCTGTGCTGCTTCATCATCTGCC-3' TUBB3 5'-GCTCACGCAGCAGATGTTCG-3' 5'-GGATTGTCACACACGGCTACC-3' Actb 5'-CGCCATGGATGACGATATCG-3' 5'-CGAAGCCGGCTTTGCACATG-3'

As a result of ICC analysis, it was confirmed that the NSC-specific markers Musashi, Nestin, Sox2 and N-Cadherin were expressed in the NSCs prepared in Example 2 (FIG. 3A), and Nestin in the real-time RT-PCR analysis result , Sox2 and N-Cadherin were shown to be expressed in NSCs prepared by the method of the present invention, but Oct4 , a pluripotency marker, was not expressed ( FIG. 3B ). In addition, Sox2 , a marker for both pluripotent stem cells and NSCs, was highly expressed in both ESCs and NSCs.

Example 4. Confirmation of differentiation ability of neural stem cells

Since NSCs are multipotent stem cells capable of differentiating into various subtypes of neurons and glial cells, the Sox1-GFP ESCs-derived NSCs prepared in the above example are neurons, astrocytes ( The ability to differentiate into astrocytes) and oligodendrocytes was confirmed through ICC and real-time RT-PCR analysis. Specifically, neuronal differentiation medium (DMEM/F12 medium (Gibco) supplemented with 1% FBS (Hyclone), N2 (Gibco), B27 supplement (Gibco) and 1x penicillin/streptomycin/glutamine (Gibco)) and 1:1 mixed medium of neurobasal medium (Gibco) for 3 weeks to randomly differentiate into neurons and glial cells, and ICC and real-time RT using primers specific for each cell-specific marker and related genes -PCR analysis confirmed differentiation.

As a result of ICC analysis, it was confirmed that NSCs prepared under the conditions of the present invention can differentiate into neurons (Tuj1 ⁺ and MAP2 ⁺ ), astrocytes (GFAP ⁺ and S100b ⁺ ) and oligodendrocytes (Sox10 ⁺ ) ( Figure 4A). In particular, it was confirmed that Sox1-GFP ESC-derived NSCs can be differentiated into late astrocyte stage through the expression of S100b, a late astrocyte marker. In addition, expression of neuronal ( Tuj1 and NeuN ), astrocyte ( GFAP and S100b ), and oligodendrocyte ( Olig2 ) marker genes was also observed in the results of quantitative RT-PCR analysis. It was confirmed that they can differentiate into glial cells and oligodendrocytes ( FIG. 4B ).

Claims (22)

(A) transforming the undifferentiated stem cells with a marker gene specifically expressed in neural lineage cells;
(B) forming a cell aggregate by three-dimensional culture;
(C) selecting a cell aggregate expressing the marker gene; and
(D) A method of producing neural stem cells (NSC) comprising the step of culturing the selected cell aggregate in two dimensions. The method of claim 1, wherein the stem cells are pluripotent stem cells (PSCs). According to claim 1, wherein the stem cells are embryonic stem cells (Embryonic Stem Cell, ESC), induced pluripotent stem cells (induced pluripotent stem cells, iPSCs), embryonic germ cells, embryonic tumor cells and selected from the group consisting of adult stem cells , a method for producing neural stem cells. The method of claim 1, wherein the marker gene is any one selected from the group consisting of Sox1 (Sry-related-HMG box 1) , Olig2 and Sox2 . The method of claim 1, further comprising a detectable label on the marker gene. 6. The method of claim 5, wherein the detectable label is selected from the group consisting of a fluorescent label, a probe, a luminescent material, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radiolabel, a tag, and a hapten. , a method for producing neural stem cells. The method of claim 1, wherein the three-dimensional culture is that the cells are cultured inside the hydrogel. The method of claim 7, wherein the hydrogel comprises an extracellular matrix material or an extracellular matrix-like material. The method of claim 8, wherein the extracellular matrix-like substance is gelatin, polyethylene glycol, collagen, alginate, chitosan, hyaluronic acid, dextran, fibrin, cellulose, fibroin, fibronectin, laminin, cordroitin sulfate, matrigel ) and a method for producing a neural stem cell selected from the group consisting of mixtures thereof. 8. The method of claim 7, wherein the three-dimensional culture comprises:
(a) separating the undifferentiated stem cells transformed with the marker gene into single cells;
(b) mixing the separated single cells with the hydrogel and curing the mixture; and
(c) a method for producing neural stem cells, comprising the step of culturing in a pluripotent stem cell medium. The method of claim 10, wherein the sol-cured through gel phase transformation, the method for producing neural stem cells. The method according to claim 1, wherein the cell aggregate is an aggregate of cells differentiated into early nervous system cells. The method of claim 1, wherein the three-dimensional culture is performed for 8 to 14 days. The method of claim 1, wherein the two-dimensional culture is to culture the cell aggregates on the hydrogel surface. The method of claim 1, wherein the two-dimensional culture is performed for 3 to 7 days. The method of claim 1, wherein the two-dimensional culture is cultured in a neural stem cell expansion medium. The method of claim 1, further comprising the step of sub-culturing by separating the cell aggregates expanded by two-dimensional culture in step (D) into single cells. A neural stem cell prepared by the method of claim 1. The neural stem cell according to claim 18, which expresses Nestin, Sox2, N-Cadherin or Musashi. The neural stem cell according to claim 18, which is capable of differentiating into neurons, astrocytes and oligodendrocytes. A composition for treating spinal cord injury comprising the neural stem cells of claim 18 as an active ingredient. A composition for treating neurodegenerative diseases comprising the neural stem cells of claim 18 as an active ingredient.

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