Composition for inducing differentiation of cranial neural crest stem cells into pericytes, method for producing perivascular cells from cranial neural crest stem cells, and cell therapeutic agent composition for preventing or treating brain nervous system diseases using same perivascular cells or bone diseases using same cranial neural crest stem cells

Abstract

The present invention relates to a composition for inducing differentiation of cranial neural crest stem cells into perivascular cells, a method for producing perivascular cells from cranial neural crest stem cells, and a cell therapeutic agent composition for preventing or treating brain nervous system diseases using the perivascular cells or bone diseases using the cranial neural crest stem cells. The composition and method for inducing differentiation of cranial neural crest stem cells into perivascular cells according to an aspect of the present invention enable the specific production of from cranial neural crest stem cells. In addition, the perivascular cells can be used as a cell therapeutic agent for brain nervous system diseases and using the cranial neural crest stem cells can be used as a cell therapeutic agent for bone diseases.

Description

A composition for inducing differentiation of cranial neural crest stem cells into perivascular cells, a method for producing perivascular cells from cranial neural crest stem cells, and a cell therapy composition for preventing or treating brain and nervous system diseases using the perivascular cells or bone diseases using the cranial neural crest stem cells

[0001] The present invention relates to a composition for inducing differentiation of two neural crest stem cells into perivascular cells and a method for producing perivascular cells from two neural crest stem cells.

[0002] The present invention relates to a cell therapy composition for preventing or treating a brain nervous system disease, comprising pericytes produced from two neural crest stem cells.

[0003] The present invention relates to a cell therapy composition for preventing or treating bone diseases, including two neural crest stem cells.

[0004] Nervous system cells can be broadly divided into two types: central nervous system and motor nervous system cells, which constitute the brain and spinal cord; and peripheral nervous system cells, which constitute sensory nerves, autonomic nerves, etc. Neurons, astrocytes, and oligodendrocytes, which constitute the central nervous system (brain and spinal cord) and motor nerves, can be differentiated from neural stem cells (neural progenitor cells: NPCs) differentiated from pluripotent stem cells, whereas peripheral nervous system cells (autonomic nerves and sensory nerves) and Schwann cells, which constitute the peripheral nervous system, are derived from neural crest stem cells (NCSCs) differentiated from pluripotent stem cells. Therefore, it is known that central nervous system cells and peripheral nervous system cells are created from pluripotent stem cells along different differentiation pathways, passing through neural progenitor cells and neural crest cells, respectively, and that these different pathways are influenced by the surrounding environment and intracellular signaling systems.

[0005] Neural crest stem cells, which exist in the human body during embryonic or fetal development, are widely located along the rostro-caudal axis, from the head and neck to the coccyx. Depending on the location along the rostro-caudal axis, neural crest stem cells are further subdivided into cranial, trunk, cardiac, and sacral neural crest stem cells. Depending on their type, neural crest stem cells differentiate into different cells, tissues, and organs.

[0006] Early neural crest stem cells in embryonic development do not have information about the spinal axis, and during the embryonic elongation period along with the formation of the neural tube, the neural crest also naturally acquires information about the spinal axis location while moving along the elongating spinal axis, and accordingly, the differentiation of cranial, trunk, cardiac, and sacral neural crest cells appears.

[0007] Neural crest stem cells migrate from the posterior portion of the neural folds during the migratory stage of development, and are distributed to a wide range of tissues and organs throughout the body. After the migratory stage, neural crest stem cells are known to differentiate into neurons and glial cells of the peripheral nervous system, melanocytes of the skin, endocrine cells, and various mesenchymal cells, and are distributed to nervous and non-neural tissues and organs in adults (Dev Dyn 2007;236:3242).

[0008] Meanwhile, pericytes, along with endothelial cells and smooth muscle cells, are a type of component cell of the vasculature. They are vascular mural cells embedded within the basement membrane of the microvasculature, forming unique local contacts with the vascular endothelium. Although the presence and role of pericytes have been underestimated for a long time, they are thought to play a significant role in the blood-brain barrier. In recent years, pericytes have been gaining attention as an essential element in the formation of the microvasculature, and are emerging as important regulators of vascular development, stabilization, maturation, and remodeling.

[0009] Although various methods are being explored to produce perivascular cells with such potential value, there is currently no information on processing factors for specific differentiation of cranial neural crest stem cells into perivascular cells, and the discovery of such processing factors is necessary.

[0010] Furthermore, various cells are involved in maintaining and repairing brain and nervous system functions, as well as in the regeneration and maintenance of bone tissue. However, specific methods for using these cells to prevent or treat brain and nervous system and bone diseases have not yet been established. Consequently, the need for cell therapy for the prevention or treatment of brain and nervous system and bone diseases is growing, and research and development in this area is urgently needed.

[0011] [Prior Art Literature]

[0012] [Non-patent literature]

[0013] (Non-patent Document 0001) Delfino-Machin, M., Chipperfield, TR, Rodrigues, FS, & Kelsh, RN (2007). The proliferating field of neural crest stem cells. Developmental dynamics: an official publication of the American Association of Anatomists, 236(12), 3242-3254.

[0014] One aspect of the present invention provides a composition for inducing differentiation of cranial neural crest stem cells into pericytes, comprising PDGF-BB (Platelet-Derived Growth Factor-BB), BMP4 (Bone Morphogenetic Protein 4), or a combination thereof.

[0015] Another aspect is to provide a medium for inducing differentiation of two neural crest stem cells into perivascular cells, including the composition for inducing differentiation.

[0016] Another aspect provides a method for producing pericytes, comprising the step of culturing two neural crest stem cells in the differentiation induction medium.

[0017] Another aspect is to provide perivascular cells produced by the above method for producing perivascular cells.

[0018] Another aspect is to provide a cell therapy composition for preventing or treating a brain nervous system disease, which comprises perivascular cells produced by the above method for producing perivascular cells.

[0019] Another aspect provides a method for preventing or treating a neurological disease, comprising a step of administering perivascular cells produced by the method for producing perivascular cells to an individual in need thereof.

[0020] Another aspect provides a use of perivascular cells produced by the method for producing perivascular cells for use in the manufacture of a cell therapy composition for preventing or treating a brain and nervous system disease.

[0021] Another aspect provides a use of perivascular cells produced by the method for producing perivascular cells for use in the prevention or treatment of brain and nervous system diseases.

[0022] Another aspect is to provide a cell therapy composition for preventing or treating bone diseases comprising two neural crest stem cells.

[0023] Another aspect provides a method of preventing or treating a bone disease comprising administering two neural crest stem cells to a subject in need thereof.

[0024] Another aspect provides the use of cranial neural crest stem cells for use in the manufacture of a cell therapy composition for the prevention or treatment of bone diseases.

[0025] Another aspect provides the use of cranial neural crest stem cells for the prevention or treatment of bone diseases.

[0026] One aspect of the present invention provides a composition for inducing differentiation of cranial neural crest stem cells into pericytes, comprising PDGF-BB (Platelet-Derived Growth Factor-BB), BMP4 (Bone Morphogenetic Protein 4), or a combination thereof.

[0027] The above PDGF-BB is one of the types of Platelet-Derived Growth Factor (PDGF) and is a dimeric glycoprotein composed of two B subunits. PDGF can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB). For the desired effect of the present invention, the composition for inducing differentiation can include PDGF-AA, PDGF-AB, or PDGF-BB, and preferably can include PDGF-BB.

[0028] The above BMP4 is a member of the bone morphogenetic protein family. For the desired effect of the present invention, the composition for inducing differentiation may include BMP, preferably BMP4.

[0029] In one specific example, the composition for inducing differentiation may contain PDGF-BB at a concentration of 10 to 100 ng / ml. Specifically, the composition for inducing differentiation may contain PDGF-BB at a concentration of 10 to 100 ng / ml, 10 to 80 ng / ml, 10 to 60 ng / ml, 10 to 55 ng / ml, 10 to 50 ng / ml, 20 to 100 ng / ml, 20 to 80 ng / ml, 20 to 60 ng / ml, 30 to 90 ng / ml, 30 to 70 ng / ml, 30 to 50 ng / ml, 40 to 100 ng / ml, 40 to 80 ng / ml, 40 to 60 ng / ml, 40 to 55 ng / ml, 45 to 100 ng / ml, 45 to 70 ng / ml, 45 to 55 ng / ml or 50 ng / ml.

[0030] In one specific example, the composition for inducing differentiation may contain BMP4 at a concentration of 1 to 20 ng / ml. Specifically, the composition for inducing differentiation may contain BMP4 at a concentration of 1 to 20 ng / ml, 1 to 15 ng / ml, 1 to 10 ng / ml, 1 to 8 ng / ml, 1 to 6 ng / ml, 1 to 5 ng / ml, 3 to 20 ng / ml, 3 to 15 ng / ml, 3 to 10 ng / ml, 3 to 8 ng / ml, 3 to 6 ng / ml, 3 to 5 ng / ml, 4 to 20 ng / ml, 4 to 15 ng / ml, 4 to 10 ng / ml, 4 to 8 ng / ml, 4 to 6 ng / ml, 4 to 5 ng / ml or 5 ng / ml.

[0031] Another aspect provides a medium for inducing differentiation of neural crest stem cells into perivascular cells, comprising the composition for inducing differentiation.

[0032] The above differentiation-inducing medium includes all media commonly used for culturing cells, and may be a basic medium such as DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal essential Medium), improved MEM, BME (Basal Medium Eagle), RPMI1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI1640, F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), or a medium containing a component capable of inducing differentiation, but is not limited thereto.

[0033] The component capable of inducing the above differentiation may include at least one of PDGF-BB, BMP4, which can induce differentiation of two neural crest stem cells into pericytes, and bFGF, BMP4, which can induce differentiation of early neural crest stem cells into two neural crest stem cells.

[0034] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to self-replicate and differentiate into two or more different types of cells. The stem cell may be a totipotent stem cell, a pluripotent stem cell, or a multipotent stem cell.

[0035] In this specification, the term "differentiation" means a phenomenon in which structures or functions become specialized while cells divide and proliferate and grow, that is, cells, tissues, etc. of a living organism change in form or function to perform their respective assigned tasks.

[0036] As used herein, the term "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell being considered.

[0037] Another aspect provides a method for producing pericytes, comprising the step of culturing two neural crest stem cells in the differentiation induction medium.

[0038] The production method according to one specific example may further include a step of isolating or obtaining the produced perivascular cells.

[0039] In a production method according to one specific example, the perivascular cells may have one or more of the following characteristics:

[0040] (a) Decreased expression of p75 NGFR, SOX10, or both compared to pre-culture neural crest stem cells;

[0041] (b) increased expression of one or more of PDGFR-beta, NG2, and CD146 compared to pre-culture neural crest stem cells; and

[0042] (c) At least 90% of the cells express at least one of PDGFR-beta, NG2, and CD146.

[0043] In relation to (c) above, specifically, at least 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the cells may express one or more of PDGFR-beta, NG2, and CD146.

[0044] In a production method according to one specific example, the culturing may be performed for 5 to 20 days. Specifically, it may be performed for 5 to 20 days, 5 to 15 days, 7 to 14 days, or 10 to 15 days.

[0045] In this specification, the measurement of the expression level of a marker protein or marker gene can be measured using an agent that measures the level of a protein or an agent that measures the level of a gene.

[0046] As used herein, the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.

[0047] In one specific example, the agent for measuring the level of the protein may include one or more selected from the group consisting of antibodies, antibody fragments, interacting proteins, oligopeptides, ligands, nanoparticles, aptamers, avidity multimers, and peptidomimetics that specifically bind to the protein or a peptide fragment thereof.

[0048] The measurement thereof may be performed by a method selected from the group consisting of, but not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (FACS), mass spectrometry, or protein microarray.

[0049] The above protein may include not only the protein itself, but also protein isoforms or protein variants that may be produced by splicing and variable promoters, or may be produced due to genetic changes such as mutations or polymorphisms.

[0050] The antibody may refer to a substance that specifically binds to the protein and causes an antigen-antibody reaction. The antibody may include a polyclonal antibody, a monoclonal antibody, or a recombinant antibody. The antibody may be readily produced using techniques well known in the art. The antibody may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibody may include a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of the antibody molecule.

[0051] The above-mentioned interacting protein refers to a protein having a protein-protein interaction (PPI) with the above-mentioned protein, and may refer to a protein having a highly specific physical contact as a result of a biochemical action regulated by interactions including electrostatic force, hydrogen bonding, and hydrophobic effect.

[0052] The above oligopeptides are peptides composed of 2 to 20 amino acids and may include, but are not limited to, dipeptides, tripeptides, tetrapeptides and pentapeptides.

[0053] The above aptamer refers to a single-stranded oligonucleotide, and may refer to a nucleic acid molecule having binding activity to the protein. The aptamer may have various secondary or tertiary structures depending on its base sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be linear or cyclic in shape.

[0054] In one specific example, the agent for measuring the level of the gene may be an agent for measuring the level of mRNA of the gene, and may include one or more selected from the group consisting of a primer, a probe, a peptide nucleic acid (PNA), and an antisense nucleotide that specifically binds to the gene.

[0055] The measurement thereof may be performed by a method selected from the group consisting of, but not limited to, polymerase chain reaction (PCR), real-time polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, RNase and S1 nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting, or DNA chip.

[0056] The above primer is a fragment that recognizes a target gene sequence, and may include a pair of forward and reverse primers. Preferably, the primer may be a pair of primers that provide analysis results with specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, and thus the primer only amplifies the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be imparted.

[0057] The above probe refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a target nucleic acid, for example, mRNA, and may be labeled so as to confirm the presence or absence, content, and expression level of a specific mRNA. The probe may be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions may be appropriately selected according to techniques known in the art.

[0058] The above primers or probes can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modifications between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0059] Suitable conditions for hybridizing probes with cDNA molecules can be determined through a series of optimization procedures. These procedures are implemented in a series of steps by those skilled in the art to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength depend on various factors such as the length and GC content of the probe and the target nucleotide sequence. Detailed hybridization conditions can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and M. L. M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., NY (1999). For example, among the above stringency conditions, high stringency conditions mean hybridization at 65°C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), 1 mM EDTA, and washing at 68°C in 0.1 x SSC (standard saline citrate) / 0.1% SDS. Alternatively, high stringency conditions mean washing at 48°C in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean washing at 42°C in 0.2 x SSC / 0.1% SDS, for example.

[0060] The above PNA refers to an artificially synthesized polymer similar to DNA or RNA. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which can greatly increase binding affinity and stability to DNA or RNA of a target sequence.

[0061] The above antisense may refer to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize with a target sequence within RNA by Watson-Crick base pairing, typically forming an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or approximate sequence complementarity to the target sequence.

[0062] Since information on the protein according to the present invention or the gene encoding it is known in the art, a person skilled in the art can design a preparation for measuring the level of the protein or the gene encoding it based on this.

[0063] According to one specific example, the method for producing pericytes, which includes a step of culturing the two neural crest stem cells in the differentiation induction medium, may further include a step of producing the two neural crest stem cells by culturing the initial neural crest stem cells in a medium containing BMP4, bFGF or a combination thereof.

[0064] In a production method according to one specific example, the two neural crest stem cells may be produced by a method including a step of culturing initial neural crest stem cells in a medium containing BMP4, bFGF, or a combination thereof.

[0065] The above bFGF (basic Fibroblast Growth Factor) is also called FGF2 (Fibroblast Growth Factor 2).

[0066] In a production method according to one specific example, the two neural crest stem cells may be differentiated into two neural crest stem cells without influence by non-neural crest stem cells by culturing the initial neural crest stem cells in a medium containing BMP4, bFGF or a combination thereof.

[0067] The production method according to one specific example may further include a step of isolating or obtaining the produced neural crest stem cells.

[0068] The above culturing can be performed for 3 to 10 days, 4 to 8 days, or 5 to 10 days.

[0069] The BMP4, bFGF or a combination thereof may be included in the medium at 5 to 100 ng / ml. Specifically, the BMP4, bFGF or a combination thereof may be included in the medium at 5 to 100 ng / ml, 10 to 80 ng / ml, 20 to 70 ng / ml, or 30 to 60 ng / ml.

[0070] In a production method according to one specific example, the two neural crest stem cells may have an increased expression ratio of ETS1 / ZIC1 compared to the initial neural crest stem cells before culture.

[0071] Specifically, the amount of ZIC1 gene expression may be reduced compared to the initial neural crest stem cells before culture, and accordingly, the expression ratio of ETS1 / ZIC1 in the two neural crest stem cells may be increased compared to the initial neural crest stem cells before culture.

[0072] In a production method according to one specific example, bFGF can suppress ZIC1 gene expression during secondary differentiation, and bFGF can promote ETS1 protein expression through BMP4.

[0073] The above-mentioned early neural crest stem cells may correspond to the neural plate border-stage during the neurulation stage of the embryo.

[0074] The above early neural crest stem cells may not have established axial specification information.

[0075] The above early neural crest stem cells may be present in the neural plate.

[0076] According to one specific example, the method for producing pericytes, which comprises a step of culturing the two neural crest stem cells in the differentiation induction medium, may further comprise a step of culturing pluripotent stem cells to obtain cells expressing p75 NGFR, HNK1, or both, thereby producing early neural crest stem cells.

[0077] According to one specific example, the method for producing pericytes, comprising the step of culturing the two neural crest stem cells in the differentiation induction medium, may further comprise the step of (a) culturing the early neural crest stem cells in a medium containing BMP4, bFGF or a combination thereof to produce the two neural crest stem cells, and / or (b) culturing the pluripotent stem cells to obtain cells expressing p75 NGFR, HNK1 or both to produce the early neural crest stem cells.

[0078] In a production method according to one specific example, the initial neural crest stem cells may be produced by a method including a step of culturing pluripotent stem cells to obtain cells expressing p75 NGFR, HNK1, or both.

[0079] In a production method according to one specific example, the initial neural crest stem cell may be a cell expressing one or more of PAX7, MSX1, AP2alpha, p75 NGFR and HNK1, and preferably a cell expressing p75 NGFR, HNK1 or both.

[0080] The cells expressing the above p75 NGFR, HNK1 or both may be cells in which the expression of p75 NGFR, HNK1 or both is increased compared to the pluripotent stem cells before culture by culturing the pluripotent stem cells.

[0081] In a production method according to one specific example, the initial neural crest stem cell may be a cell expressing p75 NGFR, HNK1, or both, and may express PAX7 more than SOX10. In addition, the initial neural crest stem cell may express p75 NGFR or HNK1 more than SOX10.

[0082] In a production method according to one specific example, the initial neural crest stem cells may not express SOX10.

[0083] In a production method according to one specific example, early neural crest stem cells that do not possess axial specification information can be isolated using a p75 NGFR antibody.

[0084] The above culturing can be performed for 3 to 10 days, 4 to 7 days, or 5 to 7 days.

[0085] Another aspect provides perivascular cells produced by the method for producing perivascular cells.

[0086] Another aspect provides a cell therapy composition for preventing or treating a brain and nervous system disease, which comprises perivascular cells produced by the above method for producing perivascular cells.

[0087] Another aspect provides a method for preventing or treating a neurological disease, comprising administering to a subject in need thereof perivascular cells produced by the method for producing perivascular cells.

[0088] Another aspect provides a use of perivascular cells produced by the method for producing perivascular cells for use in the manufacture of a cell therapy composition for preventing or treating a brain and nervous system disease.

[0089] Another aspect provides the use of pericytes produced by the method for producing pericytes for use in the prevention or treatment of brain and nervous system diseases.

[0090] In this specification, the terms "subject" and "patient" are used interchangeably. A subject may be an animal.

[0091] In this specification, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0092] In one specific example, the cell therapy composition for preventing or treating a brain nervous system disease may include the perivascular cells as an active ingredient.

[0093] The term “comprising as an effective ingredient” in this specification means including an effective amount capable of exhibiting a preventive or therapeutic effect of the cell therapy composition.

[0094] As used herein, the term "therapeutically effective amount" or "prophylactically effective amount" refers to an amount of a drug, e.g., a cell therapy agent, effective to achieve a desired therapeutic or prophylactic result. In some cases, the desired result is treatment of a disease or disorder in a subject. The therapeutically or prophylactically effective amount level may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route and excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field. The compositions of the present disclosure may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. That is, the total effective amount of the compositions of the present disclosure may be administered to a patient as a single dose, or may be administered in a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into consideration, it is important to administer the amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0095] As used herein, terms such as "treating," "treatment," "to treat," "palliating," or "to palliate" refer to therapeutic measures aimed at curing, slowing, alleviating symptoms, and / or arresting the progression of a diagnosed pathological condition or disorder. Therefore, those requiring treatment include those who have already been diagnosed with or are suspected of having a disorder.

[0096] In one specific example, the above-mentioned brain nervous system disease may include all diseases in which an abnormality or disorder occurs in some or all of the brain, spinal cord, cranial nerves, spinal nerves, and autonomic nervous system that constitute the brain nervous system.

[0097] In one specific example, the neurological disease may be at least one selected from the group consisting of ischemic stroke, hemorrhagic stroke, ischemic brain injury, traumatic brain injury, cerebral palsy, mental illness, and degenerative brain disease.

[0098] The above mental illness may be one or more selected from the group consisting of autism spectrum disorder, schizophrenia, intellectual disability, and Down syndrome.

[0099] The above degenerative brain diseases include dementia, vascular dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia. It may be one or more selected from the group consisting of, but is not limited to, these.

[0100] Another aspect provides a cell therapy composition for preventing or treating bone diseases comprising two neural crest stem cells.

[0101] Another aspect provides a method of preventing or treating a bone disease comprising administering two neural crest stem cells to a subject in need thereof.

[0102] Another aspect provides the use of cranial neural crest stem cells for use in the manufacture of a cell therapy composition for the prevention or treatment of bone diseases.

[0103] Another aspect provides the use of cranial neural crest stem cells for the prevention or treatment of bone diseases.

[0104] In one specific example, the cell therapy composition for preventing or treating bone disease may include the cranial neural crest stem cells as an active ingredient.

[0105] In one specific example, the two neural crest stem cells may be produced by the method described above.

[0106] Specifically, another aspect provides a cell therapy composition for preventing or treating bone diseases comprising two neural crest stem cells produced by the following method:

[0107] (a) a step of culturing pluripotent stem cells;

[0108] (b) a step of primary differentiation of cultured cells into early neural crest stem cells;

[0109] (c) a step of isolating early neural crest stem cells; and

[0110] (d) A step of culturing the initial neural crest stem cells in a medium containing BMP4, bFGF or a combination thereof to secondary differentiate them into cranial neural crest stem cells.

[0111] Another aspect provides a method for preventing or treating a bone disease, comprising administering to a subject in need thereof two neural crest stem cells produced in steps (a) to (d).

[0112] Another aspect provides the use of cranial neural crest stem cells produced in steps (a) to (d) for use in the manufacture of a cell therapy composition for the prevention or treatment of bone diseases.

[0113] Another aspect provides the use of cranial neural crest stem cells produced in steps (a) to (d) for use in the prevention or treatment of bone diseases.

[0114] In one specific example, the cell therapy composition for preventing or treating bone disease may include two neural crest stem cells produced in steps (a) to (d) as an active ingredient.

[0115] In one specific example, the bone disease may include all diseases in which an abnormality or disorder occurs in part or all of the skull, facial bones, spine, thorax, pelvis, limb bones, etc. that constitute the skeleton.

[0116] In one specific example, the bone disease may be at least one selected from the group consisting of bone defect, bone defect caused by benign or malignant tumor, bone tumor, osteopenia, osteogenesis imperfecta, osteomalacia, osteosclerosis, bone fracture, compression fracture, nonunion fracture, osteoporosis, osteoarthritis, degenerative arthritis, rheumatoid arthritis, Paget's disease of bone, osteonecrosis, osteotrophy, and periodontal disease, but is not limited thereto.

[0117] In one specific example, the initial neural crest stem cells of the method comprising steps (a) to (d) may correspond to the neural plate border-stage among the neurulation stages of the embryo.

[0118] In one specific example, the initial neural crest stem cells of the method may be neural crest stem cells expressing the PAX7 gene and protein.

[0119] In one specific example, the initial neural crest stem cells of the method may be present in the neural plate.

[0120] In one specific example, the initial neural crest stem cells of the method may be neural crest stem cells that have not formed axial information.

[0121] In one specific example, the initial neural crest stem cells of step (c) may be cells expressing PAX7, MSX1, AP2alpha, p75 NGFR or HNK1, and are preferably cells expressing p75 NGFR, HNK1 or both.

[0122] In one specific example, the cell expressing p75 NGFR, HNK1 or both may be a cell in which the expression of p75 NGFR, HNK1 or both is increased compared to the pluripotent stem cell before culturing by culturing the pluripotent stem cell.

[0123] In one specific example, the initial neural crest stem cells of step (c) may be cells expressing p75 NGFR, HNK1, or both, and may express PAX7 more than SOX10. In addition, the initial neural crest stem cells may express p75 NGFR or HNK1 more than SOX10.

[0124] In one specific example, the initial neural crest stem cells of step (c) may not express SOX10.

[0125] In one specific example, the step (c) may be a step of isolating early neural crest stem cells that do not have axial specification information using a p75 NGFR antibody.

[0126] In one specific example, the step (d) may be a step of secondary differentiation into two neural crest stem cells without influence by non-neural crest stem cells by treating with BMP4, bFGF or a combination thereof.

[0127] In one specific example, during the secondary differentiation, BMP4, bFGF, or a combination thereof may be included in the medium at 5 to 100 ng / ml. Specifically, BMP4, bFGF, or a combination thereof may be included in the medium at 5 to 100 ng / ml, 10 to 80 ng / ml, 20 to 70 ng / ml, or 30 to 60 ng / ml.

[0128] In one specific example, the amount of ZIC1 gene expression during the secondary differentiation may decrease compared to the initial neural crest stem cells before the secondary differentiation, and accordingly, the expression ratio of the ETS1 gene / ZIC1 gene in the two neural crest stem cells may increase compared to the initial neural crest stem cells before the secondary differentiation.

[0129] In one specific example, during the secondary differentiation, bFGF can suppress ZIC1 gene expression, and bFGF can promote ETS1 protein expression through BMP4.

[0130] In one specific embodiment, the primary differentiation and / or the secondary differentiation may be performed for 3 to 10 days, 4 to 7 days, or 5 to 7 days.

[0131] The production method according to one specific example may further include a step of isolating or obtaining the produced neural crest stem cells.

[0132] The term "cell therapy" in this specification refers to a therapeutic agent that uses autologous, allogenic, or xenogenic cells to restore tissue function, and is used to treat a disease.

[0133] The above cell therapy agent may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be, for example, saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (human serum albumin), or a mixture of one or more of these components. Other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, a target organ-specific antibody or other ligand may be combined with the carrier to specifically act on the target organ.

[0134] The above cell therapy agent may additionally contain diluents, dispersants, surfactants, binders, and lubricants, and may be formulated into an injectable formulation, such as an aqueous solution, suspension, or emulsion.

[0135] The dose of cells administered according to one specific example is 1.0× 10 5 1.0× 10 8It can be cells / kg (body weight). However, the dosage can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage considering these factors. The number of administrations can be once or twice or more within the range of clinically acceptable side effects, and the administration site can be administered in one or more sites. For animals other than humans, the same dosage as for humans per kg can be administered, or the dosage converted from the above can be administered based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and human. Possible routes of administration include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or insertion of an implantable device or material.

[0136] As target animals for treatment according to one specific example, humans and other mammals for the purpose can be exemplified, and specifically include humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc.

[0137] According to the composition and method for inducing differentiation of cranial neural crest stem cells into perivascular cells according to an aspect of the present invention, perivascular cells can be specifically produced from cranial neural crest stem cells. In addition, the perivascular cells can be utilized as a cell therapy for brain and nervous system diseases, and the cranial neural crest stem cells can be utilized as a cell therapy for bone diseases.

[0138] Figure 1 is a diagram depicting the stages of cell development within an embryo during human embryonic development.

[0139] Figure 2 is a diagram confirming cell surface markers for isolation of early neural crest stem cells:

[0140] (a): chicken embryo; and

[0141] (b): Human pluripotent stem cells.

[0142] Figure 3 is a diagram confirming the expression of early neural crest stem cell markers (P75 NGFR and HNK1) and axial specification marker (HOX) in early neural crest stem cells differentiated from pluripotent stem cells for 5 days.

[0143] Figure 4 is a diagram showing the production process of neural crest stem cells.

[0144] Figure 5 is a diagram comparing the gene expression levels of early neural crest stem cells on the 5th day of differentiation and neural crest stem cells with axial specifications on the 10th day of differentiation.

[0145] Figure 6 is a diagram showing the expression levels of ZIC1 and ETS1 when only early neural crest stem cells exist without treatment with axial specification stimulating factors.

[0146] Figure 7 is a diagram analyzing gene expression of secondary differentiated neural crest stem cells by adding axial specification stimulating factors:

[0147] (a): ETS1 / ZIC1 expression ratio of the group cultured by treating cell signaling proteins without isolating neural crest stem cells;

[0148] (b): ETS1 / ZIC1 expression ratio of the group cultured by treating cell signaling proteins during the process of isolating only early neural crest stem cells and further culturing them alone;

[0149] (c): Expression levels of TWIST1 gene (marker gene of cranial neural crest stem cells) and HOX genes in early neural crest stem cells treated with FGF2 during the secondary differentiation phase;

[0150] (d): Gene expression levels of ZIC1 and ETS1 in single cells after FGF2 treatment at the secondary differentiation stage; and

[0151] (e): Expression ratio of ETS1 / ZIC1 in single cells after FGF2 treatment at the secondary differentiation stage.

[0152] Figure 8 is a diagram analyzing the mechanism of axial specification retention of early neural crest stem cells by signal transduction factors.

[0153] Figures 9 and 10 are diagrams confirming the simultaneous creation of multiple axially specified neural crest populations within a single batch by treating various axial specification stimulating factors to early neural crest stem cells isolated through primary differentiation:

[0154] (a): mRNA expression levels of two neural crest stem cell marker genes;

[0155] (b) and (c): Verification of differentiation potential of two neural crest stem cells into target cells;

[0156] (d): mRNA expression level of cardiac neural crest stem cell marker genes; and

[0157] (e) and (f): Verification of differentiation potential of cardiac neural crest stem cells into target cells.

[0158] Figure 11 is a diagram analyzing the expression levels of PDGFR-beta and NG2, which are perivascular cell marker genes, according to treatment with PDGF-BB, PDGF-BB+BMP4, PDGF-BB+BMP4+LDN-193189, PDGF-BB+ActivinA, and PDGF-BB+ActivinA+SB-431542 in two neural crest stem cells.

[0159] Figure 12 is a diagram showing the production process of pericytes from two neural crest stem cells.

[0160] Figure 13 is a diagram showing morphological analysis of cells on day 14 of differentiation from two neural crest stem cells into pericytes, and analysis of the expression of pericyte marker proteins PDGFR-beta and NG2.

[0161] Figure 14 is a flow cytometric analysis of cells expressing PDGFR-beta and NG2, which are pericyte marker proteins, on the 14th day of differentiation from two neural crest stem cells into pericytes.

[0162] Figure 15 is a diagram analyzing the expression levels of mRNA of neural crest stem cell marker genes NGFR p75 and SOX10, and perivascular cell marker genes PDGFR-beta, NG2, and CD146 on days 0, 7, and 14 of differentiation from two neural crest stem cells into pericytes.

[0163] Figure 16 is a diagram analyzing the expression ratio of CD146, a pericyte protein marker, on days 0 and 14 of differentiation of two neural crest stem cells into pericytes.

[0164] Figure 17 is a diagram analyzing the blood vessel formation ability of HUVECs in the group of HUVECs alone, the group of HUVECs treated with bFGF, and the group of HUVECs co-cultured with differentiated pericytes.

[0165] Figure 18 is a diagram analyzing the point of disconnection in the group of HUVECs alone, the group of HUVECs treated with bFGF, and the group of HUVECs co-cultured with differentiated pericytes.

[0166] Figure 19 is a diagram analyzing the preservation or regeneration of neurons in the subventricular layer of brain tissue following treatment with pericytes on the 7th day of differentiation in an animal model of global ischemic injury.

[0167] Figure 20 is a diagram showing the results of observing changes in body weight and behavioral abnormalities in experimental animals during a new bone formation experiment using a skull defect model.

[0168] Figure 21 is a diagram showing the results of comparing the degree of new bone formation in the skull defect area analyzed by micro-CT at 8 weeks after surgery among the control group (Group 1; Vehicle), the group transplanted only with CollagenTape without cells (Group 2; Collagen sponge), and the group transplanted with CollagenTape loaded with cranial neural crest stem cells (Group 3; 1M+collagen sponge), in order to confirm the bone regeneration effect according to the transplantation of two neural crest stem cells.

[0169] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0170] Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0171] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0172] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0173]

[0174] Example 1. Marker selection of early neural crest stem cells (NCSCs)

[0175] To select cell surface markers specific for early neural crest stem cells (ECSs) that have not yet formed axial information (before committing to cranial / vagal / trunk / sacral KESCs) at the neurulation stage (Fig. 1) of human embryonic development, the expression of p75 NGFR was confirmed by immunofluorescence staining in the dorsal regional early neural crest of developing chick embryos after tissue clearing, and the expression of SOX10 and p75 NGFR was confirmed over time during differentiation of human pluripotent stem cells into KESCs.

[0176] As a result, it was confirmed that NGFR / HNK1 expression takes precedence over conventional SOX10 expression, which marks migrating neural crest stem cells, during the differentiation process of human pluripotent stem cells, and that pre-migrating early neural crest stem cells can be isolated using p75 NGFR or HNK1 antibodies (Fig. 2).

[0177]

[0178] Example 2. Primary differentiation into early neural crest stem cells at the neural plate border-stage.

[0179] Human pluripotent stem cell (hESC) clones were grown to single cells using an appropriate solution such as Accutase (Innovative Cell Technologies) or Versene (Thermo Fisher), and then seeded onto Geltrex ECM-coated plates at a density of 100,000 cells per well of a 24-well plate. When the hESC density reached 70%, the hESC maintenance medium was replaced with differentiation medium (PIM medium (DMEM / F-12; Thermo Fisher) containing 3 μM CHIR99021 (GSK-3 inhibitor, WNT activator), 0.5% KSR (Thermo Fisher), 2% B-27 supplement (Thermo Fisher), and 1% Glutamax (Thermo Fisher), pH 7.5) (day 0). Thereafter, the medium was replaced with PIM medium containing 3uM CHIR99021 on days 2 and 4, and added every other day to produce early neural crest stem cells that correspond to the neurulation stage of the embryo and have not yet formed the axial information of the human body (Table 1).

[0180]

[0181] DAY Badge CreationDay 0PIM + CHIR99021(3 uM)Day 1PIM + CHIR99021(3 uM)Day 2PIM + CHIR99021(3 uM)Day 3PIM + CHIR99021(3 uM)Day 4PIM + CHIR99021(3 uM)

[0182]

[0183] Example 3. Isolation and identification of early neural crest stem cells at the neural plate boundary stage.

[0184] 3-1. Isolation of early neural crest stem cells

[0185] To isolate early neural crest stem cells (NNSCs), which are early neural crest stem cells, the cells were collected on day 5 to day 7 of differentiation using Accutase, and the cell pellet was washed with PBS and resuspended in FACS buffer. Multi-cell clumps were removed by filtering twice using 35 um strainer snap-cap round bottom tubes, and NNSCs were isolated by labeling with antibodies to the NNSC surface antigen p75 NGFR or HNK1 using a FACS sorter (SONY SH-800).

[0186]

[0187] 3-2. Identification of early neural crest stem cells

[0188] FACS analysis of cells differentiated from pluripotent stem cells for 5 days revealed that some of the cells were early neural crest stem cells expressing NGFR and / or HNK1, and many cells expressing p75 NGFR also co-expressed HNK1, with the average proportion of p75 NGFR+ / HNK1+ cells being 50% (Fig. 3). In addition, HOX gene expression patterns revealed that early neural crest stem cells on day 5 of differentiation had not yet undergone cranial specification (Fig. 3).

[0189] Accordingly, it was confirmed that early neural crest stem cells can be isolated by isolating only p75 NGFR positive cells.

[0190]

[0191] Example 4. Secondary differentiation into neural crest stem cells containing axial specification information.

[0192] In Example 3-1, the early neural crest stem cells (differentiated day 5 cells) expressing p75 NGFR or HNK1, isolated by FACS, were re-seeded at 300,000 cells per well in 24-well plates coated with Geltrex, Matrigel, or Laminin+Fibronectin. After 24 hours, the PIM medium added on day 5 for stabilization was gradually changed to Neurobasal medium containing 2% B-27 supplement, 1% N2 supplement, and 1% Glutamax together with bFGF (basic Fibroblast Growth Factor = FGF2 (Fibroblast Growth Factor 2)) (Table 2). bFGF, as an axial specification stimulating factor, was added to the culture medium on days 6 to 10 of differentiation, and the proliferated neural crest stem cells were purified by FACS using an antibody against the neural crest stem cell surface antigen p75 NGFR on days 11 to 14 of differentiation (Fig. 4).

[0193]

[0194] DAYMedia compositionDay 675% PIM + 25% NB (Neurobasal medium) + bFGF (20 ng / ml)Day 850% PIM + 50% NB + bFGF (20 ng / ml)Day 1025% PIM + 75% NB + bFGF (20 ng / ml)

[0195]

[0196] Example 5. Gene expression analysis of early neural crest stem cells

[0197] In Example 3, the expression of neural crest stem cell marker genes NGFR, HNK1, PAX7, MSX1, and AP2alpha, as well as the expression of neural crest stem cell differentiation promoting gene ZIC1 and cranial neural crest stem cell marker ETS1 in early neural crest stem cells at the neural plate boundary stage isolated using p75 NGFR antibody on the 5th day of differentiation, was analyzed by FACS, and the expression of PAX7 expressed at the neural plate boundary was confirmed at the protein level using immunofluorescence analysis.

[0198] As a result, the isolated early neural crest stem cells were found to express NGFR, HNK1, PAX7, MSX1, and AP2alpha (Fig. 5 (a) to (d)). In addition, the early neural crest stem cells at the neural plate boundary stage isolated on day 5 of differentiation were found to express ZIC1 significantly more than the cells before differentiation and the neural crest stem cells isolated on day 10 of differentiation, and ETS1 was found to increase as differentiation progressed (Fig. 5 (e)). Since the continuous expression of ZIC1 after developmental differentiation is a specification factor into trunk neural crest stem cells, not cranial neural crest stem cells, it was confirmed that the specification into cranial neural crest stem cells occurred between days 5 and 10 of differentiation.

[0199]

[0200] Example 6. Analysis of gene expression changes in axially specified neural crest stem cells.

[0201] 6-1. Gene expression analysis of secondary differentiated neural crest stem cells without axial specification stimuli

[0202] The early neural crest stem cells (expressing p75 NGFR or HNK1) on the 5th day of differentiation of Example 3-1 above were separated or cultured in a mixed cell state without separation for 5 days without adding FGF2, an axial specification stimulating factor. As a result of confirming the expression of the ZIC1 gene and ETS1 gene, it was found that the expression of the ZIC1 gene did not decrease, and the expression of the ETS1 gene increased with the additional number of differentiation days (Fig. 6 (a) and (b)). In addition, the expression ratio of the ZIC1 gene and ETS1 gene (ETS1 / ZIC1), which can estimate the specification of two neural crest stem cells, was found to be maintained without increasing (Fig. 6 (c)).

[0203] Through this, it was inferred that when early neural crest stem cells exist together with non-neural crest stem cells during the differentiation process, they can be destined to become two neural crest stem cells through cell-to-cell interaction, but when early neural crest stem cells exist alone, they cannot be destined to become two neural crest stem cells, and the main reason for this is that the expression of the ZIC1 gene does not decrease.

[0204]

[0205] 6-2. Gene expression analysis of secondary differentiated neural crest stem cells by adding axial specification stimulating factors.

[0206] The expression of ZIC1, ETS1, TWIST1, and HOX genes in the axial neural crest stem cells that were secondary cultured for 5 days after the 5th day of differentiation of the above Example 3-1 (NC only) or after treating the mixed cell state without separation with WNT (WNT activation by treating with GSK-3 inhibitor CHIR99021), BMP, SHH, or bFGF as an axial specification stimulating factor was confirmed. When bFGF was treated, the expression ratio of ETS1 / ZIC1 was found to increase in both the isolated early neural crest stem cell group and the unseparated mixed cell group (Fig. 7 (a)), and in particular, it was found to increase significantly in the isolated early neural crest stem cells (Fig. 7 (b)). Through this, it was found that the role of non-neural crest stem cells is to activate FGF signaling of early neural crest stem cells to induce cranial specification. In addition, neural crest stem cells treated with bFGF showed an increase in the expression of the TWIST1 gene, a marker gene of cranial neural crest stem cells, and a decrease in the HOX gene, which is known to not be expressed in cranial neural crest stem cells (Fig. 7 (c)). In addition, when the expression levels of the ZIC1 and ETS1 genes in single cells were compared, it was found that each single neural crest stem cell showed a significant decrease in the ZIC1 gene from day 4 after bFGF treatment (Fig. 7 (d) and (e)).

[0207]

[0208] Through this, it was confirmed that when only isolated neural crest stem cells exist, ZIC1 does not decrease due to the lack of interaction with cells other than neural crest stem cells, and that FGF2 can decrease this.

[0209]

[0210] Example 7. Analysis of the mechanism of axial specification retention of early neural crest stem cells by signal transduction factors.

[0211] After isolating early neural crest stem cells on the 5th day of differentiation, the protein expression of ETS1 was confirmed in neural crest stem cells treated with bFGF on the 5th to 10th day by immunochemical analysis, and the direct effects of various signaling factors on early neural crest stem cells were confirmed by FACS analysis.

[0212] As a result, it was shown that bFGF suppresses ZIC1 gene expression and affects the promotion of ETS1 protein expression through BMP4 (Fig. 8 (b)). When neural crest stem cells were differentiated without treating bFGF using SOX9: reporter hESC (GFP), known as a marker of cranial neural crest stem cells, the differentiation efficiency into cranial neural crest stem cells was approximately 25%, and when bFGF was treated, it was confirmed that the proportion of SOX9-expressing cells was over 80% (Fig. 8 (c) and (d)).

[0213]

[0214] Example 8. Confirmation of simultaneous generation of neural crest stem cell populations possessing multiple axial specification information.

[0215] 8-1. Confirmation of differentiation into neural crest stem cells possessing multiple axial specification information.

[0216] The early neural crest stem cells (differentiated day 5 cells) at the neural plate boundary stage obtained through primary differentiation in Example 3-1 above were re-seeded and differentiated into cranial neural crest stem cells or cardiac neural crest stem cells by treatment with bFGF or CHIR99021+retinoic acid, respectively. Afterwards, the mRNA expression of cranial neural crest stem cell markers SOX9, TWIST1, and PRRX2, and the mRNA expression of cardiac neural crest stem cell markers cKIT, MEF2c, and MAFB were confirmed. As a result, the mRNA expression of cranial neural crest stem cell markers SOX9, TWIST1, and PRRX2 increased in the group treated with bFGF (Fig. 9 (a)), and the mRNA expression of cardiac neural crest stem cell markers cKIT, MEF2c, and MAFB increased in the group treated with CHIR99021+retinoic acid to induce WNT activation (Fig. 10 (d)).

[0217]

[0218] 8-2. Confirmation of the differentiation potential of neural crest stem cells possessing axial specification information into other cells.

[0219] In order to confirm whether each neural crest stem cell differentiated in the above Example 8-1 into a differentiation target cell at the corresponding location in the human body, the cranial neural crest stem cells differentiated by treating the initial neural crest stem cells in the above Example 8-1 with bFGF were differentiated into chondroblasts or osteoblasts, and then stained with Alcian blue and Alizarin red to confirm, and the expression of each related gene marker was confirmed. In addition, the cardiac neural crest stem cells differentiated by activating WNT from the initial neural crest stem cells in the above Example 8-1 were differentiated into SMCs (smooth muscle cells), and this was confirmed by immunofluorescence, and the expression of related marker genes was confirmed.

[0220] As a result, it was confirmed that the two neural crest stem cells differentiated into the target cells, chondroblasts or osteoblasts (Fig. 9 (b) and (c)), and that the cardiac neural crest stem cells differentiated into the target cells, SMCs (Fig. 10 (e) and (f)).

[0221]

[0222] Through this, it can be seen that multiple axially specified neural crest populations can be simultaneously generated within one batch through a two-step differentiation method according to one aspect of the present invention.

[0223]

[0224] Example 9. Identification of differentiation inducing factors from cranial neural crest stem cells into pericytes and establishment of differentiation inducing conditions.

[0225] To identify specific differentiation inducing factors from cranial neural crest stem cells into pericytes, which are neural crest stem cells possessing axial specification information, cranial neural crest stem cells were treated with PDGF-BB, PDGF-BB+BMP4, PDGF-BB+BMP4+LDN-193189, PDGF-BB+ActivinA, and PDGF-BB+ActivinA+SB-431542, respectively. Afterwards, the expression levels of PDGFR-beta and NG2, which are pericyte marker genes, were compared.

[0226] As a result, the expression levels of PDGFR-beta and NG2 were similar to those of primary human pericytes under conditions treated with PDGF-BB and BMP4 (Fig. 11).

[0227] Accordingly, PDGF-BB and BMP4 were selected as specific differentiation inducing factors from neural crest stem cells to pericytes.

[0228] Afterwards, the differentiation conditions and production method for pericytes or cerebral pericytes from cranial neural crest stem cells were established by culturing them in Neurobasal medium supplemented with B-27 supplement and N2 supplement and treating them with PDGF-BB and BMP4 for two weeks. Figure 12 is a schematic diagram illustrating this.

[0229]

[0230] Example 10. Confirmation of differentiation induction into pericytes from cranial neural crest stem cells - Morphological analysis and protein marker analysis

[0231] To confirm whether the two neural crest stem cells differentiated into pericytes by the production method of Example 9, morphological analysis and protein marker analysis were performed on cells differentiated for two weeks. PDGF-BB was treated at a concentration of 50 ng / ml, and BMP4 was treated at a concentration of 5 ng / ml.

[0232] Specifically, cells isolated on day 14 of differentiation were found to possess morphological characteristics similar to human primary pericytes, as determined by morphological analysis. Furthermore, immunohistochemical staining confirmed the expression of PDGFR-beta and NG2, marker proteins of pericytes (Fig. 13).

[0233] Additionally, flow cytometry analysis (FACS) of cells isolated on the 14th day of differentiation showed that more than 95% of cells expressed PDGFR-beta and NG2, which are perivascular cell marker proteins (Fig. 14).

[0234] The above results indicate that the neural crest stem cells differentiated into pericytes through the process of treating the two neural crest stem cells with PDGF-BB and BMP4.

[0235]

[0236] Example 11. Confirmation of differentiation of cranial neural crest stem cells into pericytes - Genetic marker analysis

[0237] To confirm whether the two neural crest stem cells were differentiated into pericytes by the production method of Example 9, genetic marker analysis was performed on cells on days 0, 7, and 14 of differentiation.

[0238] Specifically, cells on days 0, 7, and 14 of differentiation were isolated, and the mRNA expression levels of neural crest stem cell marker genes NGFR p75 and SOX10, and pericyte marker genes PDGFR-beta, NG2, and CD146 were compared and analyzed. As a result, it was shown that the expression levels of neural crest stem cell marker genes decreased and the expression trend of pericyte marker genes increased in the two neural crest stem cells treated with PDGF-BB and BMP4 on days 7 and 14 of differentiation (Fig. 15).

[0239] The above results indicate that the neural crest stem cells differentiated into pericytes through the process of treating the two neural crest stem cells with PDGF-BB and BMP4.

[0240]

[0241] Example 12. Isolation of pericytes derived from cranial neural crest stem cells

[0242] Perivascular cells produced by the production method of Example 9 were isolated.

[0243] Specifically, the expression rate of CD146, a pericyte protein marker, was 0.036% in cells on day 0 of differentiation, whereas the expression rate of CD146 in cells on day 14 of differentiation increased to 98.5% (Fig. 16). This indicates that cranial neural crest stem cells differentiated into pericytes after 14 days of differentiation.

[0244] Afterwards, perivascular cells expressing CD146 on the 14th day of differentiation were separated using a FACS sorter.

[0245]

[0246] Example 13. Analysis of angiogenesis following pericyte administration (in vitro)

[0247] In Example 12, the isolated perivascular cells were administered to HUVEC (Human umbilical vein endothelial cells) to perform a vasculogenesis assay.

[0248] As a result, it was confirmed that the vasculogenic ability of HUVECs increased when HUVECs were co-cultured with differentiated pericytes, compared to the group in which HUVECs were treated with bFGF alone or HUVECs (Fig. 17). In Fig. 17, the red arrow indicates the area where the tubular structure was not connected.

[0249] In addition, analysis of disconnected points revealed that HUVECs co-cultured with pericytes showed improved tubule structural connectivity and a decrease in disconnected points (Fig. 18).

[0250] The above results indicate that the perivascular cells obtained according to one aspect of the present invention have excellent blood vessel formation ability.

[0251]

[0252] Example 14. Confirmation of nerve cell preservation or regeneration following administration of pericytes (in vivo)

[0253] The perivascular cells obtained according to one aspect of the present invention were administered to a living body to confirm the nerve cell preservation or regenerative ability.

[0254] Specifically, the carotid artery of the Gerbillinae model was ligated for 10 minutes to induce ischemia, followed by reperfusion to induce global ischemic injury, and according to the method of Example 9, pericytes on the 7th day of differentiation were obtained by treating cranial neural crest stem cells with PDGF-BB and BMP4, and these were administered at a density of 0.4x10 6 / 100ul or 0.8x10 6 / ul was transplanted through the femoral vein.

[0255] 120 hours after transplantation, neurons in the subventricular zone of brain tissue were observed. Observation results confirmed that neurons in the subventricular zone were preserved or regenerated (Fig. 19).

[0256] The above results indicate that perivascular cells obtained according to one aspect of the present invention can be used as a cell therapeutic agent for neurological diseases.

[0257]

[0258] Example 15. Confirmation of bone regeneration following administration of two neural crest stem cells (in vivo)

[0259] The bone regeneration ability was confirmed by administering the two neural crest stem cells obtained according to one aspect of the present invention to a living body.

[0260]

[0261] 1) Creation of a skull defect model and transplantation of cranial neural crest stem cells

[0262] Rats (SD rats; 8-week-old males) weighing approximately 250-300 g were anesthetized using an inhalation anesthesia device (Surgi-Vet, USA) with Isoflurane (Ifran solution, Hana Pharmaceutical) and O2 set at 4:4. Once anesthetized, the rats were placed on a 37℃ warm pad (Jeongdo B&P) and anesthesia was maintained by adjusting the Isoflurane and O2 to 2:2. After removing hair from the upper part of the skull, a midline incision was made and subperiosteal dissection was performed. An 8-mm circular bone defect was created in the midline of the exposed skull using an 8-mm trephine bur, taking care not to damage the dura mater. Then, CollagenTape (Zimmer) was cut into 8-mm circles and placed on the defect, and cells were transplanted. After 8 weeks, body weight was measured and behavioral abnormalities were checked through visual observation.

[0263] Group 1 (n=4, Vehicle): No cell transplantation - control group;

[0264] Group 2 (n=5, Collagen sponge): CollagenTape only, without cells;

[0265] Group 3 (n=5, 1M+collagen sponge): 1.0 × 10 6 CollagenTape treatment loaded with canine cranial neural crest stem cells.

[0266]

[0267] 2) micro-CT imaging

[0268] Eight weeks after surgery, the experimental animals were sacrificed for specimen collection, and the skull graft site, including the defect, was collected and stored in a 10% natural formalin solution for 3 days. Then, the graft site was photographed using Skyscan1276 high-resolution micro-CT (Bruker). The images of the graft site were separated from the bone using a CT analyzer (CTAn, Version 1.20.8, Bruker) program, and then 3D images were constructed using the CTVOX (Version 3.3.0.0, Bruker) program to obtain the final images.

[0269]

[0270] 3) Analysis of bone regeneration from the skull defect area

[0271] During the period of the new bone formation animal experiment using the rat skull defect model, no abnormalities in body weight or behavior were observed in any of the experimental animals (Fig. 20).

[0272] Eight weeks after surgery and transplantation, micro-CT analysis revealed that in the control group (Group 1), which underwent no transplantation and allowed for natural healing, new bone formation at the defect site remained extremely limited. Group 2, which received only CollagenTape without cells, showed a certain level of new bone formation compared to the control group, but the difference was not significant.

[0273] Meanwhile, in group 3, which was transplanted with CollagenTape loaded with the two neural crest stem cells of the present invention, it was confirmed that new bone formation was significantly increased compared to group 2, and it was confirmed that the transplantation of the two neural crest stem cells effectively promoted bone regeneration in the defect area (Fig. 21).

[0274]

[0275] The above results indicate that the two neural crest stem cells obtained according to one aspect of the present invention can be used as a cell therapeutic agent for bone diseases.

[0276]

[0277] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the experimental examples and embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for inducing differentiation of cranial neural crest stem cells into pericytes, comprising PDGF-BB (Platelet-Derived Growth Factor-BB), BMP4 (Bone Morphogenetic Protein 4), or a combination thereof.

2. A medium for inducing differentiation of two neural crest stem cells into perivascular cells, comprising the composition according to Article 1.

3. A method for producing perivascular cells, comprising a step of culturing two neural crest stem cells in a differentiation induction medium according to claim 2.

4. A method for producing perivascular cells in the third paragraph, wherein the perivascular cells have one or more of the following characteristics: (a) Decreased expression of p75 NGFR, SOX10, or both compared to pre-culture neural crest stem cells; (b) increased expression of one or more of PDGFR-beta, NG2, and CD146 compared to pre-culture neural crest stem cells; and (c) At least 90% of the cells express at least one of PDGFR-beta, NG2, and CD146.

5. In the third paragraph, the two neural crest stem cells, A method for producing pericytes, comprising the step of culturing early neural crest stem cells in a medium containing BMP4, bFGF or a combination thereof.

6. A method for producing perivascular cells, wherein the two neural crest stem cells in paragraph 5 have an increased expression ratio of ETS1 / ZIC1 compared to the initial neural crest stem cells before culture.

7. A method for producing perivascular cells in paragraph 5, wherein the early neural crest stem cells correspond to the neural plate border-stage among the neurulation stages of the embryo.

8. In paragraph 5, the initial neural crest stem cells are A method for producing perivascular cells, comprising a step of culturing pluripotent stem cells to obtain cells expressing p75 NGFR, HNK1, or both.

9. Perivascular cells produced by the method according to any one of claims 3 to 8.

10. A cell therapy composition for preventing or treating a brain nervous system disease, comprising perivascular cells produced by the method according to any one of claims 3 to 8.

11. A cell therapy composition for preventing or treating a brain disease, wherein the brain disease in paragraph 10 is at least one selected from the group consisting of ischemic stroke, hemorrhagic stroke, ischemic brain injury, traumatic brain injury, cerebral palsy, mental illness, and degenerative brain disease.

12. A cell therapy composition for preventing or treating bone disease containing cranial neural crest stem cells.

13. A cell therapy composition for preventing or treating bone disease according to claim 12, wherein the bone disease is at least one selected from the group consisting of bone defect, bone defect caused by benign or malignant tumor, bone tumor, osteopenia, osteogenesis imperfecta, osteomalacia, osteosclerosis, bone fracture, compression fracture, nonunion fracture, osteoporosis, osteoarthritis, degenerative arthritis, rheumatoid arthritis, Paget's disease of bone, osteonecrosis, osteotrophy, and periodontal disease.

14. Cell therapy composition for preventing or treating bone disease comprising two neural crest stem cells produced by the following method: (a) a step of culturing pluripotent stem cells; (b) A step for primary differentiation of cultured cells into early neural crest stem cells, The above early neural crest stem cells correspond to the neural plate boundary stage among the neural tube formation stages of the embryo; (c) A step for isolating early neural crest stem cells, The above-mentioned early neural crest stem cells are cells expressing p75 NGFR, HNK1, or both, and expressing PAX7 more than SOX10; and (d) A step of culturing the initial neural crest stem cells in a medium containing BMP4, bFGF or a combination thereof to secondary differentiate them into cranial neural crest stem cells.

15. A cell therapy composition for preventing or treating bone disease according to claim 14, wherein the bone disease is at least one selected from the group consisting of bone defect, bone defect caused by benign or malignant tumor, bone tumor, osteopenia, osteogenesis imperfecta, osteomalacia, osteosclerosis, bone fracture, compression fracture, nonunion fracture, osteoporosis, osteoarthritis, degenerative arthritis, rheumatoid arthritis, Paget's disease of bone, osteonecrosis, osteotrophy, and periodontal disease.

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