Method for differentiation of neural crest stem cells with body axis information

Abstract

The present invention relates to a method for selectively differentiating and isolating neural crest cells having human body axis information from pluripotent stem cells. According to the method of the present invention, it is possible to produce early neural crest cells that correspond to the embryonic neurulation stage and have not yet formed human body axis information, and to produce neural crest cells having specific human body axis information, which can be specifically isolated and cultured in vitro. This makes it possible to obtain various neural crest cells at once, and by studying the biological process of neural crest cells acquiring body axis information and elucidating the developmental mechanisms of tissues and organs, it is possible to develop a variety of cell therapeutic agents optimized for the location characteristics of the human body.

Description

[Technical Field]

[0001] The present invention relates to a method for selectively differentiating and isolating neural crest cells bearing human axial information from pluripotent stem cells. [Background technology]

[0002] Nervous system cells can be broadly divided into two types: central nervous system cells, which make up the brain and spinal cord, and peripheral nervous system cells, which make up motor neurons, sensory neurons, autonomic neurons, etc. The neurons, astrocytes, and oligodendrocytes that make up the central nervous system (brain and spinal cord) and motor neurons can be generated by differentiation of neural stem cells or neural progenitor cells (NPCs), which are differentiated from pluripotent stem cells. In contrast, peripheral neurons (autonomic and sensory neurons) and Schwann cells that make up the peripheral nervous system are derived from neural crest stem cells (NCSCs), which are differentiated from multipotent stem cells. Therefore, central nervous system cells and peripheral nervous system cells are generated from pluripotent stem cells via neural progenitor cells and neural crest cells, respectively, along different differentiation pathways. These different pathways are known to be influenced by the surrounding environment and intracellular signaling systems.

[0003] Human neural crest stem cells present during embryonic or fetal development are distributed widely along the rostrocaudal axis, which extends from the head to the tailbone. They are further subdivided into cranial, trunk, cardiac, and sacral neural crest stem cells according to their position along the spinal axis, and differentiate into different cells, tissues, and organs depending on their type. During early embryonic development, neural crest stem cells lack spinal axis information. During embryonic elongation, along with neural tube formation, they naturally acquire spinal axis information as they migrate along the spinal axis, where the neural crest also elongates. This results in the emergence of cranial, trunk, cardiac, and sacral neural crest cell subdivisions. Neural crest stem cells migrate from the posterior neural fold during the migratory stage of development and then distribute to a wide range of tissues and organs in the human body. After migration, neural crest stem cells differentiate into neurons and glial cells in the peripheral nervous system, melanocytes in the skin, endocrine cells, and various mesenchymal cells, which then distribute to adult nervous and non-nervous tissues and organs (Dev Dyn 2007;236:3242).

[0004] In recent years, neural crest stem cell-like cells have been found to exist postnatally in peripheral nerves, dorsal root ganglia, gut, hair follicles, dermal papillae, cornea, and dental pulp. Among these, adult human neural crest stem cell-like cells have been isolated exclusively from hair follicles, dermal papillae, and dental pulp (J Cell Biochem 2009;107:1046). Previous reports have identified neural crest stem cells isolated from dermal papillae or hair follicles in adult humans. However, because dermal papillae and hair follicles contain numerous appendages derived from epidermal cells, the cells that compose neurospheres from these tissues are likely to contain not only neural crest cells but also heterogeneous cells derived from skin appendages, posing challenges for their application as cell therapy (PNAS 2005;102:5530). Although previous studies have demonstrated the existence of neural crest stem cells or neural crest stem-like cells in adult humans, conventional techniques have had the drawback of requiring cell culture for as long as three months to isolate and culture neural crest stem cells, and the possibility of various cellular transformations, such as transformation and dedifferentiation, cannot be ruled out. Furthermore, given the nature of neural crest stem cells, which differentiate into various cells that reside and function in various human organs and tissues, adult neural crest stem cells or neural crest stem-like cells cultured using conventional techniques are likely to have undergone significant differentiation and lost their initial cellular properties. Furthermore, while conventional techniques have used specific markers to isolate neural crest stem cells, a problem has arisen in that no markers specifically expressing neural crest stem cells at different developmental stages have been identified. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide a method for producing early neural crest stem cells.

[0006] Another object of the present invention is to provide a method for producing neural crest stem cells.

[0007] Another object of the present invention is to provide a composition for detecting early neural crest stem cells.

[0008] Another object of the present invention is to provide a method for detecting early neural crest stem cells.

[0009] It is also an object of the present invention to provide an application for detecting early neural crest stem cells.

[0010] Another object of the present invention is to provide a use for producing a composition for detecting early neural crest stem cells.

[0011] Another object of the present invention is to provide a composition for detecting neural crest stem cells.

[0012] Another object of the present invention is to provide a method for detecting neural crest stem cells.

[0013] Another object of the present invention is to provide a use for detecting neural crest stem cells.

[0014] A further object of the present invention is to provide a use for producing a composition for detecting neural crest stem cells. [Means for solving the problem]

[0015] To achieve the above object, the present invention provides a composition for detecting early neural crest stem cells, which comprises a preparation for measuring the expression level of the P75 NGFR or HNK1 gene.

[0016] The present invention also provides a method for detecting early neural crest stem cells, comprising the step of measuring the expression level of the P75 NGFR or HNK1 gene in an isolated sample.

[0017] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0018] In one embodiment, the method for detecting early neural crest stem cells may further include a step of identifying the cells as early neural crest stem cells when the expression level of the p75 NGFR or HNK1 gene is increased or positive compared to a control group.

[0019] The present invention also provides use of a preparation that measures the expression level of the P75 NGFR or HNK1 gene for detecting early neural crest stem cells.

[0020] The present invention also provides use of a preparation for measuring the expression level of the P75 NGFR or HNK1 gene for the manufacture of a composition for detecting early neural crest stem cells.

[0021] The present invention also provides the use of p75 NGFR or HNK1 for detecting early neural crest stem cells.

[0022] The present invention also provides a composition for detecting neural crest stem cells that contains information about the body axis, the composition comprising a preparation for measuring the expression level of the ETS1 gene or ZIC1 gene.

[0023] The present invention also provides a method for detecting neural crest stem cells that have axis information, comprising the step of measuring the expression level of the ETS1 gene or ZIC1 gene in an isolated sample.

[0024] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0025] In one embodiment, the method for detecting neural crest stem cells with body axis information may further include a step of identifying the cells as neural crest stem cells with body axis information if the expression level of the ETS1 gene or ZIC1 gene is increased or positive compared to a control group.

[0026] The present invention also provides use of a preparation that measures the expression level of the ETS1 gene or ZIC1 gene for detecting neural crest stem cells that provide information on the body axis.

[0027] The present invention also provides use of a preparation for measuring the expression level of the ETS1 gene or ZIC1 gene for producing a composition for detecting neural crest stem cells that has information on the body axis.

[0028] The present invention also provides uses for detecting neural crest stem cells that have body axis information from the ETS1 gene or ZIC1 gene.

[0029] The present invention also provides a method for producing early neural crest stem cells.

[0030] Furthermore, the present invention provides a method for producing neural crest stem cells. [Effects of the Invention]

[0031] According to the method of the present invention, it is possible to produce early neural crest cells, which correspond to the embryonic neurulation stage and have not yet formed human body axis information, and to produce neural crest cells with specific human body axis information, and these can be specifically isolated and cultured in vitro. This makes it possible to obtain various neural crest cells at once, and by studying the biological process of neural crest cells to acquire body axis information and elucidating the developmental mechanisms of tissues and organs, it is possible to develop various cell therapeutic agents optimized for the location characteristics of the human body. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 shows the stages of germ cell development during human embryogenesis. [Figure 2] Figure 2 shows cell surface markers for the isolation of early neural crest stem cells. ((a) Chicken embryo; and (b) Human pluripotent stem cells.) [Figure 3] FIG. 3 shows the expression of early neural crest stem cell markers (p75 NGFR and HNK1) and axial markers (HOX) in early neural crest stem cells that were differentiated from pluripotent stem cells for 5 days. [Figure 4] FIG. 4 shows the process of neural crest stem cell generation. [Figure 5] FIG. 5 shows a comparison of gene expression levels between early neural crest stem cells on day 5 of differentiation and neural crest stem cells having an axis on day 10 of differentiation. [Figure 6] FIG. 6 shows the expression levels of ZIC1 and ETS-1 when early neural crest stem cells are present alone without treatment with an axis formation stimulating factor. [Figure 7] Figure 7 shows the analysis of gene expression in neural crest stem cells differentiated to secondary differentiation after addition of axis-forming stimulating factors. ((a): ETS1 / ZIC1 expression rate in a group treated with cell signaling proteins and cultured without isolating neural crest stem cells; (b): ETS1 / ZIC1 expression rate in a group treated with cell signaling proteins during the process of isolating and culturing early neural crest stem cells alone; (c): Expression levels of TWIST1 (a marker gene for cranial neural crest cells) and HOX genes in early neural crest stem cells treated with FGF2 during secondary differentiation; (d): Expression levels of ZIC1 and ETS1 genes in single cells after FGF2 treatment during secondary differentiation; and (e): Expression levels of ETS1 / ZIC1 in single cells after FGF2 treatment during secondary differentiation.) [Figure 8] FIG. 8 shows an analysis of the mechanism by which signal transduction factors control the body axis of early neural crest cells. [Figure 9] and [Figure 10] Figures 9 and 10 show the simultaneous generation of multiple axially specified neural crest cell populations within a single batch by treating early neural crest stem cells isolated by primary differentiation with various stimulating factors for body axis formation. (a): mRNA expression levels of marker genes for cranial neural crest stem cells; (b) and (c): Verification of the differentiation ability of cranial neural crest stem cells into target cells; (d): mRNA expression levels of marker genes for cardiac neural crest stem cells; and (e) and (f): Verification of the differentiation ability of cardiac neural crest stem cells into target cells. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be described in detail below by way of examples with reference to the accompanying drawings. However, the following examples are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, such detailed description may be omitted and the present invention is not limited thereby. The present invention is susceptible to various modifications and applications within the scope of equivalents as interpreted from the claims below.

[0034] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intention of a user or operator, or on the practices in the field to which the present invention pertains. Therefore, definitions of these terms should be based on the overall content of this specification. Throughout the specification, when a part is said to "include" a certain element, this does not mean that other elements are excluded, but that other elements may also be included, unless otherwise specified.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the testing and practice of the present invention, the preferred methods and materials are described herein.

[0036] In one aspect, the present invention relates to a composition for detecting early neural crest stem cells, comprising a preparation for measuring the expression level of the p75 NGFR or HNK1 gene.

[0037] In one aspect, the present invention relates to a method for detecting early neural crest stem cells, comprising the step of measuring the expression level of the P75 NGFR or HNK1 gene in an isolated sample.

[0038] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0039] In one embodiment, the method for detecting early neural crest stem cells may further include a step of identifying the cells as early neural crest stem cells if the expression level of the P75 NGFR or HNK1 gene is increased or positive compared to a control group.

[0040] In one embodiment, the present invention relates to the use of a preparation for measuring the expression level of the P75 NGFR or HNK1 gene for detecting early neural crest stem cells.

[0041] In one aspect, the present invention relates to the use of a preparation for measuring the expression level of the P75 NGFR or HNK1 gene for the manufacture of a composition for detecting early neural crest stem cells.

[0042] In one aspect, the present invention relates to the use of P75 NGFR or HNK1 for detecting early neural crest stem cells.

[0043] In one embodiment, the early neural crest stem cells may be neural crest stem cells corresponding to the neural plate border stage of embryonic neurulation.

[0044] In one embodiment, the early neural crest stem cells may be neural crest stem cells that express the PAX7 gene and protein.

[0045] In one embodiment, early neural crest stem cells can be present in the neural plate.

[0046] In one embodiment, the early neural crest stem cells can be PAX7-, MSX1-, AP2α-, P75 NGFR-, or HNK1-positive cells, and more preferably P75 NGFR- or HNK1-positive cells.

[0047] In one embodiment, the preparation for measuring the expression level of a gene may be a preparation for measuring the expression level of the mRNA or protein of the gene.

[0048] In one embodiment, a preparation for measuring the expression level of mRNA of a gene may comprise a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair that specifically recognizes a fragment of the nucleic acid sequence and the complementary sequence, a probe, or a primer pair and a probe, and the measurement may be performed by a method selected from the group consisting of 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.

[0049] In one embodiment, the preparation for measuring the expression level of a marker protein may comprise an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full-length protein of the gene or a fragment thereof, and the measurement may be performed by a method selected from the group consisting of 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.

[0050] In one aspect, the present invention relates to a composition for detecting neural crest stem cells having information on axial specification, comprising a preparation for measuring the expression level of the ETS1 gene or ZIC1 gene.

[0051] In one aspect, the present invention relates to a method for detecting neural crest stem cells having axial information, comprising the step of measuring the expression level of the ETS1 gene or the ZIC1 gene in an isolated sample.

[0052] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0053] In one embodiment, the method for detecting neural crest stem cells with body axis information may further include a step of identifying the cells as neural crest stem cells with body axis information if the expression level of the ETS1 gene or ZIC1 gene is increased or positive compared to a control group.

[0054] In one embodiment, the present invention relates to the use of a preparation that measures the expression level of the ETS1 gene or ZIC1 gene for detecting neural crest stem cells that have information on the body axis.

[0055] In one embodiment, the present invention relates to the use of a preparation for measuring the expression level of the ETS1 gene or ZIC1 gene for the manufacture of a composition for detecting neural crest stem cells that has information on the body axis.

[0056] In one embodiment, the present invention relates to use for detecting neural crest stem cells having axial information of the ETS1 gene or ZIC1 gene.

[0057] In one embodiment, the neural crest stem cells having body axis information can be cranial neural crest stem cells, vagal neural crest stem cells, cardiac neural crest stem cells, trunk neural crest stem cells, or sacral neural crest stem cells, and are more preferably cranial neural crest stem cells.

[0058] In one embodiment, the preparation for measuring the expression level of a gene may be a preparation for measuring the expression level of the mRNA or protein of the gene.

[0059] In one embodiment, a preparation for measuring the expression level of mRNA of a gene may comprise a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair that specifically recognizes the nucleic acid sequence and a fragment of the complementary sequence, a probe, or a primer pair and a probe, and the measurement may be performed by a method selected from the group consisting of polymerase chain reaction, real-time polymerase chain reaction, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, Northern blotting, or DNA chip.

[0060] In one embodiment, the preparation for measuring the expression level of a marker protein may comprise an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full-length protein of the gene or a fragment thereof, and the measurement may be performed by a method selected from the group consisting of Western blot, ELISA, radioimmunoassay (RIA), radial immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation, complement fixation assay, FACS, mass spectrometry, or protein microarray.

[0061] The terms "detecting" or "measuring" as used herein mean quantifying the concentration of the object detected or measured.

[0062] In the present invention, the term "primer" refers to a nucleic acid sequence having a short free hydroxyl group at the 3' end, which can form a base pair with a complementary template and act as a replication origin for the template strand. A primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature.

[0063] In the present invention, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, which can specifically bind to mRNA and is as short as a few bases or as long as several hundred bases. It is labeled and can be used to confirm the presence or absence of a specific mRNA. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to the gene, and the expression level of the gene can be diagnosed based on the presence or absence of hybridization. The selection of a suitable probe and hybridization conditions can be varied based on those known in the art, and are not particularly limited in the present invention.

[0064] The primers or probes of the present invention can be chemically synthesized using phosphoramidite solid-phase synthesis or other widely known methods. Furthermore, such nucleic acid sequences can be modified using many techniques known in the art. Non-limiting examples of these modifications include methylation, capping, substitution of one or more natural nucleotides with their analogs, and internucleotide modifications, such as uncharged linkers (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphothioates, phosphorodithioates, etc.).

[0065] In the present invention, suitable conditions for hybridizing a probe to a cDNA molecule can be determined through a series of optimization procedures. Such procedures are carried out by those skilled in the art to establish protocols for use in laboratories. For example, conditions such as temperature, component concentrations, 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 stringent conditions, high stringency conditions refer to hybridization in 0.5 M NaHPO4, 7% SDS (sodium dodecylsulfate), and 1 mM EDTA at 65°C, followed by washing in 0.1×SSC (standard saline citrate) / 0.1% SDS at 68°C. Alternatively, high stringency conditions refer to washing in 6×SSC / 0.05% tetrasodium pyrophosphate at 48°C. Low stringency conditions refer to washing in 0.2×SSC / 0.1% SDS at 42°C, for example.

[0066] In the present invention, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed by the gene, and the antibody can be produced using widely known methods. This also includes partial peptides that can be produced from the protein. The form of the antibody of the present invention is not particularly limited, and the antibody of the present invention includes polyclonal antibodies, monoclonal antibodies, and any part thereof that has antigen-binding ability, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes specialized antibodies such as humanized antibodies.

[0067] In one aspect, the present invention relates to a kit comprising the early neural crest stem cells or the composition for detecting neural crest stem cells of the present invention.

[0068] In one aspect, the present invention relates to a method for producing early neural crest stem cells, comprising the steps of culturing pluripotent stem cells, differentiating the cultured cells into early neural crest stem cells, and isolating the early neural crest stem cells.

[0069] In one embodiment, the early neural crest stem cells may be neural crest stem cells corresponding to the neural plate border stage of embryonic neurulation.

[0070] In one embodiment, the early neural crest stem cells may be neural crest stem cells that express the PAX7 gene and protein.

[0071] In one embodiment, early neural crest stem cells can be present in the neural plate.

[0072] In one embodiment, the early neural crest stem cells can be PAX7-, MSX1-, AP2α-, P75 NGFR-, or HNK1-positive cells, and more preferably P75 NGFR- or HNK1-positive cells.

[0073] In one embodiment, the early neural crest stem cells may be neural crest stem cells that do not form axial information.

[0074] In one embodiment, differentiation may be carried out for 4 to 7 days.

[0075] In one aspect, the present invention relates to a method for producing neural crest stem cells, comprising the steps of culturing pluripotent stem cells, allowing the cultured cells to undergo primary differentiation into early neural crest stem cells, isolating the early neural crest stem cells, and allowing the early neural crest stem cells to undergo secondary differentiation into neural crest stem cells having body axis information.

[0076] In one embodiment, during secondary differentiation, WNT, BMP, BMP4, SHH, FGF2, Notch, or bFGF can be treated, and treatment with FGF2 (fibroblast growth factor 2) is more preferable.

[0077] In one embodiment, during secondary differentiation, bFGF or FGF2 and BMP or BMP4 can be administered together.

[0078] In one embodiment, during secondary differentiation, treatment with FGF2 can be performed at 5 to 100 ng / ml.

[0079] In one embodiment, during secondary differentiation, the expression level of the ZIC1 gene may decrease compared to before secondary differentiation, and during secondary differentiation, the expression rate of the ETS1 gene / ZIC1 gene may increase compared to before secondary differentiation.

[0080] In one embodiment, during secondary differentiation, FGF2 can suppress ZIC1 gene expression, and FGF2 can promote ETS1 protein expression via BMP4.

[0081] In one embodiment, neural crest stem cells having axial information can be cranial neural crest stem cells, vagal neural crest stem cells, cardiac neural crest stem cells, trunk neural crest stem cells, or sacral neural crest stem cells.

[0082] In one embodiment, neural crest stem cells having axial information can be cells that express the SOX9 or SOX10 gene or protein.

[0083] In one embodiment, primary differentiation may be carried out for 4 to 7 days, and secondary differentiation may be carried out for 3 to 10 days.

[0084] In one embodiment, the method of the present invention can be used to eliminate the influence of non-neural crest stem cells and precisely regulate and transmit cell signaling that directly acts on neural crest stem cells, thereby effectively and accurately producing neural crest stem cells that possess body axis information. In an embodiment of the present invention, when bFGF was applied to the two-step differentiation method, the differentiation efficiency of cranial neural crest stem cells using SOX9 reporter HESCs was confirmed to be improved from approximately 30% to approximately 80% compared to conventional methods (see Figure 8 (c) to (d)).

[0085] Conventional neural crest stem cell differentiation techniques obtain cranial neural crest cells, trunk neural crest cells, etc. by stimulating cell signaling activity such as FGF and WNT to differentiate and obtain neural crest cells from various locations in the human body, but this technique has the disadvantage of only obtaining one type of neural crest stem cell per batch. In contrast, the present invention overcomes the various limitations of the conventional one-step neural crest stem cell differentiation method, which only obtains one type of neural crest stem cell, by developing a two-step neural crest stem cell differentiation technique, which has the advantage of simultaneously differentiating and obtaining neural crest stem cells with locational information for various locations, such as the cranial, trunk, cardiac, and sacral, in a single batch.

[0086] To achieve this, it is important to secure early neural crest cells that have differentiated from pluripotent stem cells into neural crest stem cells but have not yet established axial information, i.e., before the neurulation stage.

[0087] In this study, we confirmed that it is possible to label early neural crest cells using p75 NGFR by staining neural crest cells in developing chick embryos. We also confirmed that when human pluripotent stem cells were differentiated in a newly prepared differentiation medium (PIM), p75 NGFR protein was expressed prior to SOX10 protein, which is expressed by neural crest stem cells with axial information and the ability to migrate. Therefore, early neural crest cells without axial information were differentiated into starch-producing stem cells by primary differentiation for 5-7 days, and then isolated using a p75 NGFR antibody. These cells were then recultured in a secondary differentiation process. They were then treated with bFGF to differentiate into cranial neural crest stem cells, and treated with WNT to differentiate into trunk neural crest stem cells. These cells were then examined for changes in ZIC1 or ETS1 expression.

[0088] Furthermore, although conventional neural crest stem cell differentiation methods can differentiate into specific neural crest cells, they have the limitation that they cannot differentiate, secure, or identify early neural crest cells (which are located in the neural tube during the neural tube formation process and express the PAX7 protein) for which information on the body axis has not yet been obtained.Furthermore, because various cell types such as neural crest stem cells and non-neural crest stem cells are mixed during the cell differentiation process, there is a limitation that the exact mechanisms of signaling proteins such as bFGF and WNT cannot be understood.

[0089] In contrast, the present invention enables the isolation of pure early neural crest cells, which can be used to transduce signaling proteins such as bFGF and WNT, thereby enabling more efficient differentiation and acquisition of axially specified neural crest stem cells. Furthermore, the role of signaling proteins such as BMP and SHH, which are known to contribute to the axial specification of neural crest stem cells, was also examined, whether they act directly on neural crest stem cells or indirectly on mixed non-neural crest stem cells, thereby affecting neural crest cells.

[0090] In other words, through the above method, the present invention confirmed that conditions such as the inhibition of ZIC1 by bFGF, the inhibition of WNT function by bFGF, and the promotion of ETS1 by BMP4 in pure early neural crest stem cells promote the cranial-axial specification of early neural crest stem cells, and that conditions such as the promotion of ZIC1 by WNT and the inhibition of bFGF function by SHH promote the trunk-axial specification of early neural crest stem cells.

[0091] The present invention will be described in more detail with reference to the following examples, which are merely for the purpose of illustrating the content of the present invention and are not intended to limit the scope of the present invention. [Example]

[0092] Marker selection of early neural crest stem cells (NCSCs) To identify cell surface markers specific to early neural crest stem cells, which correspond to the neurulation stage during human embryonic development (Figure 1) and have not yet formed human axial information (before specification into head, vagus, trunk, and sacral neural crest stem cells), we examined the expression of p75 NGFR in the dorsal regional early neural crest of developing chick embryos using tissue clearing and immunofluorescence staining. We also examined the expression of SOX10 and p75 NGFR over time during the differentiation of human pluripotent stem cells into neural crest cells.

[0093] As a result, we confirmed that NGFR / HNK1 expression prevails over SOX10 expression, which is a conventional labeling method for migrating neural crest stem cells, during the differentiation of human pluripotent stem cells. This indicates that pre-migrating early neural crest stem cells can be isolated using the P75 NGFR or HNK1 antibody (Figure 2). [Example]

[0094] Primary differentiation into early neural crest stem cells at the neural plate border stage Human pluripotent stem cell (hESC) clones were dissociated into single cells using an appropriate solution, such as Accutase (Innovative Cell Technologies) or Versene (Thermo Fisher Scientific), and then 100,000 cells were plated onto Geltrex ECM-coated 24-well plates at 100,000 cells per well. When hESCs reached 70% confluency, the maintenance medium was replaced with differentiation medium (PIM medium: DMEM / F-12; Thermo Fisher Scientific), 0.5% KSR (Thermo Fisher Scientific), 2% B-27 supplement (Thermo Fisher Scientific), and 1% Glutamax (Thermo Fisher Scientific), pH 7.5, containing 3 μM CHIR99021 (GSK-3 inhibitor, WNT activation) (day 0). Then, on days 2 and 4, the medium was replaced with PIM medium containing 3 μm CHIR 99021, and added every other day to generate early neural crest stem cells, which correspond to the embryonic neurulation stage and have not yet formed human body axis information (Table 1).

[0095] [Table 1] [Example]

[0096] Isolation and identification of early neural crest stem cells during neural plate border formation 3-1. Isolation of early neural crest stem cells

[0097] To isolate early neural crest stem cells (ESCs) at the neural plate boundary formation stage, cells were collected using Accutase on days 5 to 7 of differentiation. The cell pellets were washed with PBS and resuspended in FACS buffer. Cells were filtered twice using a 35 μm strainer snap-cap round-bottom tube to remove multiple cell clumps. ESCs were labeled with antibodies against the neural crest stem cell surface antigens p75 NGFR or HNK1 and isolated using a FACS sorter (SONY SH-800).

[0098] 3-2. Identification of early neural crest stem cells 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 that cells expressing p75 NGFR often co-expressed HNK1, with the average percentage of p75 NGFR+ / HNK1+ cells being 50% (Figure 3). Furthermore, analysis of the HOX gene expression patterns revealed that early neural crest stem cells on day 5 of differentiation had not yet undergone cranial specification (Figure 3).

[0099] This confirmed that early neural crest stem cells can be isolated by isolating only P75 NGFR-positive cells. [Example]

[0100] Secondary differentiation into axially-informed NCSCs Neural plate boundary-forming stage NCSCs (cells on day 5 of differentiation) expressing p75 NGFR or HNK1, isolated by FACS in Example 3-1, were aliquoted at 300,000 cells per well onto 24-well plates coated with Geltrex, Matrigel, or laminin plus fibronectin. After 24 hours, the PIM medium added on day 5 for stabilization was gradually replaced with Neurobasal medium containing 2% B-27 supplement, 1% N2 supplement, and 1% Glutamax, along with FGF2 (Fibroblast Growth Factor 2) (Table 2). FGF2 was added to the culture medium as an axis formation stimulator on days 6–10 of differentiation. Expanded neural crest stem cells were purified by FACS using an antibody against the neural crest stem cell surface antigen p75 NGFR on days 11–14 of differentiation (Figure 4).

[0101] [Table 2] [Example]

[0102] Analysis of gene expression in early neural crest stem cells In Example 3, early neural crest stem cells at the neural plate boundary formation stage, isolated using the P75 NGFR antibody on day 5 of differentiation, were analyzed by FACS for the expression of neural crest stem cell marker genes NGFR, HNK1, PAX7, MSX1, and AP2α, as well as the expression of neural crest stem cell differentiation-promoting gene ZIC1 and cranial neural crest stem cell marker ETS1. The expression of PAX7, which is expressed at the neural plate boundary, was confirmed at the protein level using immunofluorescence analysis.

[0103] The isolated early neural crest stem cells expressed NGFR, HNK1, PAX7, MSX1, and AP2α (Figure 5(a)-(d)). Furthermore, early neural crest stem cells at the neural plate boundary formation stage isolated on day 5 of differentiation expressed significantly more ZIC1 than predifferentiation cells or neural crest stem cells isolated on day 10 of differentiation, and ETS1 expression increased as differentiation progressed (Figure 5(e)). The sustained expression of ZIC1 after development and differentiation is a specification factor for trunk neural crest stem cells, not cranial neural crest stem cells, confirming that specification into cranial neural crest stem cells occurred between days 5 and 10 of differentiation. [Example]

[0104] Analysis of gene expression changes in axial NCSCs 6-1. Gene expression analysis of neural crest stem cells differentiated into secondary stages without axis formation stimuli The primary neural crest stem cells (expressing p75 NGFR or HNK1) from day 5 of differentiation in Example 3-1 were either isolated or in a mixed cell state without the addition of FGF2, an axis formation stimulating factor, and then secondary cultured for 5 days. The expression of the ZIC1 gene and the ETS1 gene was then examined. The results showed that the expression of the ZIC1 gene did not decrease, but increased with the number of days of differentiation (Figures 6(a) and (b)). Furthermore, the expression ratio of the ZIC1 gene and the ETS1 gene (ETS1 / ZIC1), which can be used to estimate the specification of cranial neural crest stem cells, was maintained without increasing (Figure 6(c)).

[0105] This led to the inference that when early neural crest stem cells exist together with non-neural crest stem cells during differentiation, they can be specialized into cranial neural crest stem cells through cell-cell interactions, but when early neural crest stem cells exist alone, they do not become specialized into cranial neural crest stem cells, and that this is mainly due to the lack of a decrease in ZIC1 gene expression.

[0106] 6-2. Analysis of gene expression in neural crest stem cells differentiated into secondary stages by adding axis formation stimulatory factors Early neural crest stem cells (expressing p75 NGFR or HNK1) from day 5 of differentiation in Example 3-1 were either isolated (NC only) or treated with WNT (WNT activation by treatment with the GSK-3 inhibitor CHIR99021), BMP, SHH, or FGF2 as axis formation stimuli in a non-isolated mixed cell state. After secondary culture for 5 days, the expression of ZIC1, ETS1, TWIST1, and HOX genes in the axial neural crest stem cells was examined. Treatment with FGF2 increased the expression of ETS1 / ZIC1 in both the isolated early neural crest stem cell group and the non-isolated mixed cell group ( Figure 7(a) ), with the expression level particularly notable in the isolated early neural crest stem cells ( Figure 7(b) ). This indicates that non-neural crest stem cells play a role in activating FGF signaling in early neural crest stem cells to induce cranial specification. Furthermore, FGF2-treated neural crest stem cells were shown to have increased expression of the TWIST1 gene, a marker gene for cranial neural crest stem cells, and decreased expression of HOX genes, which are known not to be expressed in cranial neural crest stem cells (Figure 7(c)). Furthermore, comparison of the expression levels of ZIC1 and ETS1 genes in single cells showed that the expression of ZIC1 gene was significantly reduced in each single neural crest stem cell from day 4 of FGF2 treatment (Figure 7(d) and (e)).

[0107] This confirmed that when isolated neural crest stem cells are present alone, there is a lack of interaction with other cells that are not neural crest stem cells, and ZIC1 does not decrease, but that FGF2 can decrease this. [Example]

[0108] Analysis of the mechanism by which signaling factors regulate the body axis of early neural crest stem cells After isolating early neural crest stem cells on day 5 of differentiation, we confirmed the expression of ETS1 serum protein in neural crest stem cells treated with FGF2 on days 5-10 by immunochemical analysis, and the direct effects of various signaling factors on early neural crest stem cells were confirmed by FACS analysis.

[0109] The results showed that FGF2 suppressed ZIC1 gene expression and influenced the promotion of ETS1 protein expression by BMP4 (Figure 8(b)). When neural crest stem cells were differentiated using SOX9:Reporter HESC, a known marker for cranial neural crest stem cells, without FGF2 treatment, a differentiation efficiency of approximately 25% into cranial neural crest stem cells was observed, while when bFGF was treated, the percentage of SOX9-expressing cells was over 80% (Figure 8(c) and (d)). [Example]

[0110] Confirmation of simultaneous generation of multiple axially specified neural crest stem cell populations 8-1. Confirmation of neural crest stem cell differentiation along multiple axes NCSCs at the neural plate boundary formation stage (cells on day 5 of differentiation) obtained by primary differentiation in Example 3-1 were re-aliquoted and then treated with bFGF or CHIR99021 plus retinoic acid to differentiate into cranial neural crest stem cells or cardiac neural crest stem cells, respectively. The mRNA expression of cranial neural crest stem cell markers SOX9, TWIST1, and PRRX2, as well as cardiac neural crest stem cell markers cKIT, MEF2c, and MAFB, was then measured. The bFGF-treated group showed increased mRNA expression of cranial neural crest stem cell markers SOX9, TWIST1, and PRRX2 (Figure 9(a)), while the CHIR99021 plus retinoic acid-treated group showed increased mRNA expression of cardiac neural crest stem cell markers cKIT, MEF2c, and MAFB (Figure 10(d)).

[0111] 8-2. Confirmation of the differentiation potential of axially specified neural crest stem cells into other cells To confirm whether the neural crest cells differentiated in Example 8-1 into target cells in the corresponding locations in the human body, cranial neural crest stem cells, which were differentiated by treating early neural crest stem cells with bFGF in Example 8-1, were differentiated into chondroblasts or osteoblasts, which were then stained with Alcian Blue and Alizarin Red to confirm the expression of relevant gene markers. Additionally, cardiac neural crest stem cells, which were differentiated by WNT activation in Example 8-1, were differentiated into smooth muscle cells (SMCs), which were then confirmed by immunofluorescence to confirm the expression of relevant marker genes.

[0112] As a result, it was confirmed that cranial neural crest stem cells differentiated into the target cells, chondroblasts or osteoblasts (Figure 9(b) and (c)), and cardiac neural crest stem cells differentiated into the target cells, SMCs (Figure 10(e) and (f)).

[0113] This demonstrates that multiple axially specified neural crest cell populations can be simultaneously generated within a single batch through the two-step differentiation method of the present invention.

Claims

1. A composition for detecting early neural crest stem cells, comprising a preparation for measuring the expression level of p75NGFR or HNK1 gene.

2. 2. The composition for detecting early neural crest stem cells according to claim 1, wherein the early neural crest stem cells correspond to the neural plate border stage in the embryonic neurulation stage.

3. A composition for detecting neural crest stem cells, which has axial specification information, comprising a preparation for measuring the expression level of the ETS1 gene or the ZIC1 gene.

4. The composition for detecting neural crest stem cells described in claim 3, wherein the neural crest stem cells having body axis information are cranial neural crest stem cells, vagal neural crest stem cells, cardiac neural crest stem cells, trunk neural crest stem cells, or sacral neural crest stem cells.

5. a) culturing pluripotent stem cells; b) differentiating the cultured cells into early neural crest stem cells; c) isolating early neural crest stem cells; A method for producing early neural crest stem cells, comprising:

6. The method for producing early neural crest stem cells according to claim 5, wherein the early neural crest stem cells correspond to the neural plate boundary formation stage in the neural tube formation stage of an embryo.

7. The method for producing early neural crest stem cells according to claim 5, wherein the early neural crest stem cells do not form axial information.

8. The method for producing early neural crest stem cells according to any one of claims 5 to 8, wherein the early neural crest stem cells are PAX7-, MSX1-, AP2α-, p75 NGFR-, or HNK1-positive cells.

9. a) culturing pluripotent stem cells; b) allowing the cultured cells to differentiate into primary neural crest stem cells; c) isolating early neural crest stem cells; d) secondary differentiation of the early neural crest stem cells into neural crest stem cells with axial information; A method for producing neural crest stem cells, comprising:

10. The method for producing neural crest stem cells according to claim 9, wherein the early neural crest stem cells are PAX7-, MSX1-, AP2α-, p75 NGFR-, or HNK1-positive cells.

11. The method for producing neural crest stem cells according to claim 9, wherein WNT, BMP, BMP4, SHH, FGF2 (fibroblast growth factor 2), Notch, or bFGF is treated during secondary differentiation.

12. The method for producing neural crest stem cells according to claim 9, wherein the expression level of ZIC1 gene is reduced during secondary differentiation compared to before secondary differentiation.

13. The method for producing neural crest stem cells according to claim 9, wherein the expression rate of the ETS1 gene / ZIC1 gene is increased during secondary differentiation compared to before secondary differentiation.

14. The method for producing neural crest stem cells according to any one of claims 9 to 13, wherein the neural crest stem cells having body axis information are cranial neural crest stem cells, vagal neural crest stem cells, cardiac neural crest stem cells, trunk neural crest stem cells, or sacral neural crest stem cells.

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