Method for differentiating neural crest stem cells containing axial specialization information

Abstract

The present invention relates to a method for selectively differentiating and separating neural crest cells containing human axial specialization information from pluripotent stem cells, and according to the method of the present invention, early neural crest cells corresponding to the embryonic neural tube formation stage and not having formed human axial information can be prepared. The neural crest cells with specific human body axial information can be prepared and subjected to specific separation and in-vitro culture, so that various neural crest cells can be obtained at a time, and the development process of tissues and organs can be revealed by researching the biological process of the neural crest cells with the axial information; thus, it is possible to develop various cell therapeutic agents optimized for body part characteristics.

Description

Technical Field

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

[0002] Nervous system cells can be roughly divided into two categories: the central nervous system and motor nerve cells that make up the brain and spinal cord, and the peripheral nervous system cells that make up sensory nerves, autonomic nerves, etc. The nerve cells (neurons), astrocytes, and oligodendrocytes that make up the central nervous system (brain and spinal cord) and motor nerves can be further differentiated from neural progenitor cells (neural stem cells or neural progenitor cells; NPCs) differentiated from totipotent stem cells, while the peripheral nerve cells (autonomic nerves and sensory nerves) and Schwann cells that make up the peripheral nervous system are derived from neural crest stem cells (NCSC) differentiated from totipotent stem cells. Therefore, central nervous system cells and peripheral nervous system cells are produced from totipotent stem cells along different differentiation pathways via neural progenitor cells and neural crest cells, respectively, and these different pathways are known to depend on the surrounding environment and intracellular signaling systems.

[0003] During embryonic or fetal development, neural crest stem cells in the human body are widely distributed on the rostro-caudal axis extending from the head and neck to the coccyx. According to the position of the rostro-caudal axis, they are subdivided into cranial neural crest stem cells, trunk neural crest stem cells, cardiac neural crest stem cells, sacral neural crest stem cells, etc., and there are differences in the target cells, tissues, and organs differentiated according to their types. In the early stages of embryonic development, neural crest stem cells do not have rostro-caudal axis information. With the formation of the neural tube and the elongation of the embryo, the neural crest also moves along the elongated rostro-caudal axis and naturally obtains the rostro-caudal axis position information, thus generating the regional division of cranial neural crest cells, trunk neural crest cells, cardiac neural crest cells, and sacral neural crest cells. Neural crest stem cells begin to migrate from the posterior part of the neural fold during the migratory stage of development, and will migrate and distribute to a wide range of tissues and organs in the body. It is known that after the migratory stage, neural crest stem cells differentiate into neurons, glial cells, melanocytes, endocrine system cells, and various mesenchymal cells of the peripheral nervous system, and are distributed to the nervous system or non-nervous system tissues and organs of adults (Dev Dyn 2007; 236: 3242).

[0004] Recently, it has been found that neural crest stem cell-like cells still exist in the peripheral nerves, dorsal root ganglia, gut, hair follicle, dermal papillae, cornea, and dental pulp after birth. Among them, adult neural crest stem cell-like cells have been isolated only from the hair follicle, dermal papillae, and dental pulp (J Cell Biochem 2009; 107: 1046). Previously, it has been reported that neural crest stem cells have been isolated from adult dermal papillae or hair follicles. However, since there are many appendage organs derived from epidermal cells in the dermal papillae or hair follicles, when forming neurospheres from this tissue, in addition to neural crest stem cells, it is very likely that heterogeneous cells derived from skin appendage organs are also mixed, which causes problems when it is used as a cell therapy agent (PNAS 2005; 102: 5530). In addition, although existing research results have proven the fact that neural crest stem cells or neural crest stem cell-like cells also exist in adult humans, existing techniques require up to 3 months of cell culture to isolate and culture neural crest stem cells, and there is a problem that various cell transformations such as cell transformation and dedifferentiation cannot be excluded due to long-term culture. In addition, considering the characteristic that neural crest stem cells can differentiate into various cell types with different functions in various organs or tissues of the human body, adult neural crest stem cells or neural crest stem cell-like cells cultured using existing techniques are very likely to have undergone significant differentiation and lost their early multi-directional differentiation potential. In addition, although existing techniques use specific markers to isolate neural crest stem cells, the problem is that there is currently no marker that can specifically represent the developmental stage of neural crest stem cells. Summary of the Invention

[0005] Technical Problem

[0006] An object of the present invention is to provide a method for preparing early neural crest stem cells.

[0007] In addition, an object of the present invention is to provide a method for preparing neural crest stem cells.

[0008] In addition, an object of the present invention is to provide a composition for detecting early neural crest stem cells.

[0009] In addition, an object of the present invention is to provide a method for detecting early neural crest stem cells.

[0010] In addition, an object of the present invention is to provide a use for detecting early neural crest stem cells.

[0011] In addition, an object of the present invention is to provide a use for preparing a composition for detecting early neural crest stem cells.

[0012] In addition, an object of the present invention is to provide a composition for detecting neural crest stem cells.

[0013] In addition, an object of the present invention is to provide a method for detecting neural crest stem cells.

[0014] In addition, an object of the present invention is to provide a use for detecting neural crest stem cells.

[0015] Furthermore, an object of the present invention is to provide a use for preparing a composition for detecting neural crest stem cells.

[0016] Technical solution

[0017] To achieve the above object, the present invention provides a composition for detecting early neural crest stem cells, the composition comprising an agent for measuring the expression level of the p75NGFR or HNK1 gene.

[0018] In addition, the present invention provides a method for detecting early neural crest stem cells, the method comprising the step of measuring the expression level of the p75 NGFR or HNK1 gene in an isolated sample.

[0019] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0020] In one embodiment, the method for detecting early neural crest stem cells may further comprise the following steps: when the expression level of the p75 NGFR or HNK1 gene is further increased or positive compared with a control group, it is determined as early neural crest stem cells.

[0021] In addition, the present invention provides a use of an agent for measuring the expression level of the p75 NGFR or HNK1 gene in detecting early neural crest stem cells.

[0022] In addition, the present invention provides a use of an agent for measuring the expression level of the p75 NGFR or HNK1 gene in preparing a composition for detecting early neural crest stem cells.

[0023] In addition, the present invention provides a use of early neural crest stem cells for detecting p75 NGFR or HNK1.

[0024] In addition, the present invention provides a composition for detecting neural crest stem cells having axial specialization information, the composition comprising an agent for measuring the expression level of the ETS1 gene or the ZIC1 gene.

[0025] In addition, the present invention provides a method for detecting neural crest stem cells with axial specialization information, the method comprising the step of measuring the expression level of the ETS1 gene or the ZIC1 gene in an isolated sample.

[0026] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0027] In one embodiment, the method for detecting neural crest stem cells with axial specialization information may further comprise the following steps: when the expression level of the ETS1 gene or the ZIC1 gene is further increased or positive compared with a control group, it is determined as neural crest stem cells with axial specialization information.

[0028] In addition, the present invention provides the use of a preparation for measuring the expression level of the ETS1 gene or the ZIC1 gene in detecting neural crest stem cells with axial specialization information.

[0029] In addition, the present invention provides the use of a preparation for measuring the expression level of the ETS1 gene or the ZIC1 gene in preparing a composition for detecting neural crest stem cells with axial specialization information.

[0030] In addition, the present invention provides the use of a preparation for detecting neural crest stem cells with axial specialization information of the ETS1 gene or the ZIC1 gene.

[0031] In addition, the present invention provides a method for preparing early neural crest stem cells.

[0032] In addition, the present invention provides a method for preparing neural crest stem cells.

[0033] Advantages of the Invention

[0034] According to the method of the present invention, early neural crest cells corresponding to the stage before the formation of human axial information during embryonic neurulation can be prepared, neural crest cells with specific human axial information can be prepared, and they can be specifically isolated and cultured in vitro, so that various types of neural crest cells can be obtained at one time. By studying the biological processes of neural crest cells with axial information, the development processes of tissues and organs can be revealed, thus having the effect of being able to develop various cell therapeutics optimized for the characteristics of human body parts. Brief Description of the Drawings

[0035] Figure 1 is a diagram depicting the developmental stages of embryonic cells during human embryonic development.

[0036] Figure 2 is a diagram for confirming cell surface markers for the isolation of early neural crest stem cells:

[0037] (a): Chicken embryo; and

[0038] (b): Human pluripotent stem cells.

[0039] Figure 3 It is a figure for confirming the expression of early neural crest stem cell markers (P75 NGFR and HNK1) and axial specialization markers (HOX) in early neural crest stem cells differentiated from pluripotent stem cells for 5 days.

[0040] Figure 4 It is a figure showing the preparation process of neural crest stem cells.

[0041] Figure 5 It is a figure comparing the gene expression levels of early neural crest stem cells on the 5th day of differentiation with neural crest stem cells with axial specialization on the 10th day of differentiation.

[0042] Figure 6 It is a figure for confirming the gene expression levels of ZIC1 and ETS1 genes in the case of not treating with axial specialization stimulating factors and only the presence of early neural crest stem cells alone.

[0043] Figure 7 It is a figure for analyzing the gene expression of neural crest stem cells undergoing secondary differentiation by adding axial specialization stimulating factors:

[0044] (a): The expression ratio of ETS1 / ZIC1 in the group of non-isolated neural crest stem cells treated and cultured with cell signaling proteins;

[0045] (b): The expression ratio of ETS1 / ZIC1 in the group of only isolating early neural crest stem cells and further culturing alone, treated and cultured with cell signaling proteins;

[0046] (c): The expression levels of TWIST1 gene (cranial neural crest stem cell marker gene) and HOX gene in early neural crest stem cells treated with FGF2 during secondary differentiation;

[0047] (d): The expression levels of ZIC1 and ETS1 genes in single cells after treatment with FGF2 during secondary differentiation; and

[0048] (e): The expression ratio of ETS1 / ZIC1 genes in single cells after treatment with FGF2 during secondary differentiation.

[0049] Figure 8 It is a figure for analyzing the mechanism of axial specialization of early neural crest stem cells based on signaling factors.

[0050] Figure 9 and Figure 10It is a figure showing that early neural crest stem cells separated by primary differentiation are treated with various axial specialization stimulating factors, and it is confirmed that multiple axially specified neural crest populations are generated simultaneously within the same batch:

[0051] (a): mRNA expression levels of cranial neural crest stem cell marker genes;

[0052] (b) and (c): Verifying the ability of cranial neural crest stem cells to differentiate into target cells;

[0053] (d): mRNA expression levels of cardiac neural crest stem cell marker genes; and

[0054] (e) and (f): Verifying the ability of cardiac neural crest stem cells to differentiate into target cells. Detailed implementation manners

[0055] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings in terms of the embodiments of the present invention. However, the following embodiments are only presented as examples of the present invention. If it is determined that the detailed description of the technologies or configurations well-known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description thereof may be omitted, and the present invention is not limited thereto. The present invention can be variously modified and applied within the scope of description of the following claims and their equivalents.

[0056] In addition, the terminology used in this specification is for appropriately expressing the preferred embodiments of the present invention, and these may vary according to the intention of the user, operator, or the convention in the field of the present invention. Therefore, the definition of these terms should be based on the content of the entire specification. Throughout the specification, when a certain part "includes" a certain component, unless there is a particularly contrary description, this does not mean excluding other components, but rather means that other components may also be included.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the present invention. Although any methods and materials similar or equivalent to those described herein can be used in testing the practice of the present invention, the preferred materials and methods are described herein.

[0058] On the one hand, the present invention relates to a composition for detecting early neural crest stem cells, and the composition contains a preparation for measuring the expression level of the p75 NGFR or HNK1 gene.

[0059] On the one hand, the present invention relates to a method for detecting early neural crest stem cells, and the method includes the step of measuring the expression level of the p75 NGFR or HNK1 gene in a separated sample.

[0060] In one embodiment, the sample can be any substance, biological fluid, tissue or cell derived from a subject.

[0061] In one embodiment, the method for detecting early neural crest stem cells may further include the following steps: compared with a control group, when the expression level of the p75 NGFR or HNK1 gene is further increased or positive, it is determined as an early neural crest stem cell.

[0062] On the one hand, the present invention relates to the use of a preparation for measuring the expression level of the p75 NGFR or HNK1 gene in detecting early neural crest stem cells.

[0063] On the one hand, the present invention relates to the use of a preparation for measuring the expression level of the p75 NGFR or HNK1 gene in preparing a composition for detecting early neural crest stem cells.

[0064] On the one hand, the present invention relates to the use of a preparation for detecting early neural crest stem cells of p75 NGFR or HNK1.

[0065] In one embodiment, the early neural crest stem cells can be neural crest stem cells at the neural plate border-stage during the neurulation stage of the embryo.

[0066] In one embodiment, the early neural crest stem cells can be neural crest stem cells that express the PAX7 gene and protein.

[0067] In one embodiment, the early neural crest stem cells can be present in the neural plate.

[0068] In one embodiment, the early neural crest stem cells can be PAX7, MSX1, AP2alpha, p75 NGFR or HNK1 positive cells, more preferably p75 NGFR or HNK1 positive cells.

[0069] In one embodiment, the preparation for measuring the gene expression level can be a preparation for measuring the mRNA or protein expression level of the gene.

[0070] In one embodiment, the preparation for measuring the mRNA expression level of a gene may include the nucleic acid sequence of the marker, the nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize the fragments of the nucleic acid sequence and the complementary sequence, and the measurement can be carried out 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 reverse transcription polymerase chain reaction (competitive RT-PCR), ribonuclease and S1 nuclease protection assay, in situ hybridization technique, nucleic acid microarray, northern blotting, or DNA chip.

[0071] In one embodiment, the preparation for measuring the protein expression level of a marker may include an antibody, an antibody fragment, an aptamer, an avidity multimer, or peptidomimetics that specifically recognize the full-length protein of the gene or its fragment, and the measurement can be carried out by a method selected from the group consisting of: western blot, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (FACS), mass spectrometry, or protein microarray.

[0072] On the one hand, the present invention relates to a composition for detecting neural crest stem cells with axial specification information, and the composition contains a preparation for measuring the expression level of the ETS1 gene or the ZIC1 gene.

[0073] On the one hand, the present invention relates to a method for detecting neural crest stem cells with axial specification information, and the method includes the step of measuring the expression level of the ETS1 gene or the ZIC1 gene in an isolated sample.

[0074] In one embodiment, the sample can be any substance, biological fluid, tissue, or cell derived from a subject.

[0075] In one embodiment, the method for detecting neural crest stem cells with axial specialization information may further include the following steps: when the expression level of the ETS1 gene or the ZIC1 gene is further increased or positive compared to the control group, it is determined as neural crest stem cells with axial specialization information.

[0076] In one embodiment, the present invention relates to the use of a preparation for measuring the expression level of the ETS1 gene or the ZIC1 gene in detecting neural crest stem cells with axial specialization information.

[0077] In one embodiment, the present invention relates to the use of a preparation for measuring the expression level of the ETS1 gene or the ZIC1 gene in preparing a composition for detecting neural crest stem cells with axial specialization information.

[0078] In one embodiment, the present invention relates to the use of the ETS1 gene or the ZIC1 gene in detecting neural crest stem cells with axial specialization information.

[0079] In one embodiment, neural crest stem cells with axial specialization 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, more preferably cranial neural crest stem cells.

[0080] In one embodiment, the preparation for measuring the gene expression level can be a preparation for measuring the mRNA or protein expression level of the gene.

[0081] In one embodiment, the preparation for measuring the mRNA expression level of the gene may include the nucleic acid sequence of the marker, the nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize the fragments of the nucleic acid sequence and the complementary sequence, and its measurement can be carried out by a method selected from the group consisting of: polymerase chain reaction, real-time polymerase chain reaction, reverse transcription polymerase chain reaction, competitive reverse transcription polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip.

[0082] In one embodiment, the preparation for measuring the expression level of the protein of the biomarker may include an antibody, an antibody fragment, an aptamer, an avidity multimer, or peptidomimetics that specifically recognize the full length of the protein of the gene or a fragment thereof, and the measurement can be performed by a method selected from the group consisting of: Western blot, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (FACS), mass spectrometry, or protein microarray.

[0083] As used herein, the terms "detect" or "measure" refer to quantifying the concentration of the object to be detected or measured.

[0084] In the present invention, the term "primer" refers to a short nucleic acid sequence having a short free 3'-hydroxyl group, which can form base pairs with a complementary template and serve as a starting point for replicating the template strand. In an appropriate buffer solution and temperature, in the presence of a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) reagent and four different nucleoside triphosphates, the primer can initiate DNA synthesis.

[0085] In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which can specifically bind to mRNA, ranging from as short as a few bases to as long as several hundred bases, and is labeled so that the presence or absence of a specific mRNA can be confirmed. The probe can be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is carried out using a probe complementary to the gene, and the gene expression level can be diagnosed based on whether hybridization occurs. The selection of a suitable probe and the hybridization conditions can be modified based on what is well known in the art, and thus the present invention does not make specific limitations thereon.

[0086] The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many methods known in the art. Non-limiting examples of such modifications include methylation, capping, replacement of natural nucleotides with one or more homologs, or modifications between nucleotides, such as modification to a neutral linker (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or a charged linker (e.g., phosphorothioate, dithiophosphate, etc.).

[0087] In the present invention, suitable conditions for the hybridization of the probe with the cDNA molecule can be determined through a series of processes by optimizing the procedures. These procedures are carried out by those skilled in the art through a series of processes to establish a protocol for the laboratory. For example, conditions such as temperature, component concentration, hybridization and washing time, buffer components and their pH value and ionic strength depend on various factors such as the length of the probe, the GC content, and the target nucleotide sequence. The detailed conditions for hybridization can be confirmed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y (1999). For example, among the stringent conditions, high-stringency conditions refer to hybridization at 65 °C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), 1 mM EDTA, and washing at 68 °C in 0.1x SSC (standard saline citrate) / 0.1% SDS. Alternatively, high-stringency conditions refer to washing at 48 °C in 6x SSC / 0.05% sodium pyrophosphate. These stringent conditions refer to, for example, washing at 42 °C in 0.2x SSC / 0.1% SDS.

[0088] In the present invention, the term "antibody", which is well-known in the art, refers to a specific protein molecule against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the protein expressed by the gene, and the preparation method of the antibody can be prepared using well-known methods. It also includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, as long as it is a polyclonal antibody, monoclonal antibody, or has antigen-binding properties, its partial is also included in the antibody of the present invention, and includes all immunoglobulin antibodies. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

[0089] On the one hand, the present invention relates to a kit, which includes a composition for detecting the early neural crest stem cells or neural crest stem cells of the present invention.

[0090] On the one hand, the present invention relates to a method for preparing early neural crest stem cells, the method comprising: the step of culturing pluripotent stem cells; the step of differentiating the cultured cells into early neural crest stem cells; and the step of isolating the early neural crest stem cells.

[0091] In one embodiment, the early neural crest stem cells can be neural crest stem cells at the neural plate border-stage during the neurulation stage of the embryo.

[0092] In one embodiment, the early neural crest stem cells can be neural crest stem cells that express the PAX7 gene and protein.

[0093] In one embodiment, the early neural crest stem cells can be present in the neural plate.

[0094] In one embodiment, the early neural crest stem cells can be PAX7, MSX1, AP2alpha, p75 NGFR or HNK1 positive cells, more preferably p75 NGFR or HNK1 positive cells.

[0095] In one embodiment, the early neural crest stem cells can be neural crest stem cells that have not yet formed axial information.

[0096] In one embodiment, the differentiation can be carried out for 4 to 7 days.

[0097] On the one hand, the present invention relates to a method for preparing neural crest stem cells, the method comprising: a step of culturing pluripotent stem cells; a step of initially differentiating the cultured cells into early neural crest stem cells; a step of isolating the early neural crest stem cells; and a step of secondarily differentiating the early neural crest stem cells into neural crest stem cells with axial specialization information.

[0098] In one embodiment, during secondary differentiation, it can be treated with WNT, BMP, BMP4, SHH, FGF2, NOTCH or bFGF, and more preferably treated with FGF2 (fibroblast growth factor 2).

[0099] In one embodiment, during secondary differentiation, it can be treated with bFGF or FGF2 and BMP or BMP4 simultaneously.

[0100] In one embodiment, during secondary differentiation, it can be treated with 5 to 100 ng / mL of FGF2.

[0101] In one embodiment, the expression level of the ZIC1 gene during secondary differentiation can be decreased compared with that before secondary differentiation, and the expression ratio of the ETS1 gene / ZIC1 gene during secondary differentiation can be increased compared with that before secondary differentiation.

[0102] In one embodiment, FGF2 can inhibit the expression of the ZIC1 gene during secondary differentiation, and FGF2 can promote the expression of the ETS1 protein through BMP4.

[0103] In one embodiment, the neural crest stem cells with axial specialization 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.

[0104] In one embodiment, the neural crest stem cells with axial specialization information can be cells expressing the gene or protein of SOX9 or SOX10.

[0105] In one embodiment, the initial differentiation can be carried out for 4 to 7 days, and the secondary differentiation can be carried out for 3 to 10 days.

[0106] In one embodiment, by using the method of the present invention to exclude the influence of non-neural crest stem cells and precisely regulate and transmit cell signal transduction directly acting on neural crest stem cells, neural crest stem cells with axial specialization information can be effectively and precisely prepared. In the examples of the present invention, it was confirmed that when bFGF was applied to the two-step differentiation method, compared with the existing method, the differentiation efficiency of cranial neural crest stem cells using the SOX9 reporter hESC increased from about 30% to about 80% (refer to Figure 8 of (c)-(d)).

[0107] Existing neural crest stem cell differentiation techniques stimulate the activities of cell signals such as FGF and WNT to differentiate and obtain neural crest cells in various parts of the human body, thereby ensuring cranial neural crest cells, trunk neural crest cells, etc. However, there has always been a drawback that only one type of neural crest stem cell can be obtained in each batch. On the other hand, in order to solve the problem of various limitations caused by the fact that only one type of neural crest stem cell can be differentiated and obtained in the existing one-step neural crest stem cell differentiation method, the present invention develops a two-step neural crest stem cell differentiation technique, which has the advantage of being able to simultaneously differentiate and obtain neural crest stem cells with information on various parts such as cranial, trunk, cardiac, and sacral in the same differentiation batch.

[0108] For this reason, it is important to ensure early neural crest cells that have differentiated from pluripotent stem cells into neural crest stem cells but have not yet established axial specialization information, that is, early neural crest cells before the neurulation stage.

[0109] In this regard, in the present invention, by staining neural crest cells in developing chicken embryos, it was confirmed that it is possible to label early neural crest cells using p75NGFR, and when differentiating human pluripotent stem cells in a newly prepared differentiation culture medium (PIM), it was confirmed that the expression of the p75 NGFR protein precedes the expression of the SOX10 protein expressed by neural crest stem cells with axial specialization information and migration ability. Therefore, pluripotent stem cells differentiate into early neural crest cells that have not yet established axial specialization information through 5 to 7 days of primary differentiation, separate the cells using a p75 NGFR antibody, and then, in the secondary differentiation process of culturing these cells again, treat them with bFGF and differentiate them into cranial neural crest stem cells, treat them with WNT and differentiate them into trunk neural crest stem cells, and verify these cells through changes in the expression of ZIC1 or ETS1.

[0110] In addition, although existing neural crest stem cell differentiation methods can differentiate into specific neural crest cells, they have limitations in that they cannot differentiate, ensure, or confirm early neural crest cells (located within the neural tube during neurogenesis and expressing the PAX7 protein) that have not yet obtained axial specialization information. Due to the mixture of various cell types such as neural crest stem cells and non-neural crest stem cells during cell differentiation, there are limitations in understanding the correct mechanism of signal transduction proteins such as bFGF and WNT.

[0111] On the other hand, the present invention can isolate pure early neural crest cells. Therefore, by delivering signaling proteins such as bFGF and WNT thereto, it is possible to more effectively differentiate and ensure axially-specified neural crest stem cells. At the same time, it was also confirmed whether the actions of signaling proteins such as BMP and SHH, which are known to play a role in the axial specification of neural crest stem cells, directly act on neural crest stem cells or indirectly affect neural crest cells by acting on mixed non-neural crest stem cells.

[0112] As a result, in the present invention, conditions such as ZIC1 inhibition by bFGF, WNT function inhibition by bFGF, and ETS1 promotion by BMP4 for pure early neural crest stem cells are confirmed by the above method to promote cranial-axial-specification of early neural crest stem cells, and conditions such as ZIC1 promotion by WNT and bFGF function inhibition by SHH promote trunk-axial-specification of early neural crest stem cells.

[0113] The present invention will be described in more detail by the following examples. However, the following examples are only used to concretize the content of the present invention, and the present invention is not limited thereto.

[0114] Example 1: Screening of Markers for Early Neural Crest Stem Cells (NCSCs)

[0115] In order to screen cell surface markers specific for early neural crest stem cells in the neurulation stage during human embryonic development ( Figure 1 ) and before the formation of human axial information (before specialization into cranial / vagal / trunk / sacral neural crest stem cells), the developing chicken embryo tissues were made transparent and the expression of p75 NGFR in the dorsal regional early neural crest was confirmed by immunofluorescence staining. During the differentiation of human pluripotent stem cells into neural crest cells, the expressions of SOX10 and p75 NGFR were detected over time.

[0116] The results showed that during the differentiation of human pluripotent stem cells, the expression of NGFR / HNK1 preceded the existing SOX10 expression that marks migrating neural crest stem cells, thereby confirming that pre-migrating early neural crest stem cells can be isolated using p75 NGFR or HNK1 antibodies ( Figure 2 ).

[0117] Example 2: Primary differentiation into early neural crest stem cells at the neural plate border stage

[0118] Human pluripotent stem cell (hESC) clones were made into single cells using an appropriate solution such as Accutase (Innovative Cell Technologies) or Versene (Thermo Fisher), and then seeded at 100,000 cells per well into a 24-well plate coated with Geltrex ECM. When the density of hESCs reached 70%, the maintenance medium of hESCs was changed to PIM medium (DMEM / F-12; ThermoFishser) containing 3 μM CHIR99021 (GSK-3 inhibitor, WNT activator), 0.5% KSR (Thermo Fishser), 2% B-27 supplement (Thermo Fishser), and 1% Glutamax (Thermo Fishser, pH 7.5) as the differentiation medium (day 0). Thereafter, the medium was changed to PIM medium containing 3 μM CHIR99021 on days 2 and 4 and supplemented every other day to prepare early neural crest stem cells at the neurulation stage of the embryo and without the formation of human axial information (Table 1).

[0119] Table 1

[0120] day Culture medium composition Day 0 PIM + CHIR99021 (3 μM) Day 1 PIM + CHIR99021 (3 μM) Day 2 PIM + CHIR99021 (3 μM) Day 3 PIM + CHIR99021 (3 μM) Day 4 PIM + CHIR99021 (3 μM)

[0121] Example 3: Isolation and identification of early neural crest stem cells at the neural plate border stage

[0122] 3-1. Isolation of early neural crest stem cells

[0123] To isolate early neural crest stem cells at the neural plate border stage, which are early neural crest stem cells, they were collected using Accutase from day 5 to day 7 of differentiation. The cell pellet was rinsed with PBS and resuspended in FACS buffer. Using 35μm filter snap-cap round bottom tubes, multiple cell clumps were removed by filtration twice. Antibodies against the neural crest stem cell surface antigens p75 NGFR or HNK1 were labeled, and early neural crest stem cells at the neural plate border stage were isolated using a FACS sorter (SONY SH-800).

[0124] 3-2. Identification of early neural crest stem cells

[0125] The results of FACS analysis of cells on day 5 of pluripotent stem cell differentiation showed that some of the cells were early neural crest stem cells expressing NGFR and / or HNK1. Cells expressing p75 NGFR often showed co-expression of HNK1. The proportion of p75NGFR+ / HNK1+ cells was on average 50% ( Figure 3 ). In addition, the results of identifying the HOX gene expression pattern showed that early neural crest stem cells on day 5 of differentiation had not formed cranial specification ( Figure 3 ).

[0126] Therefore, it was confirmed that early neural crest stem cells could be isolated by simply isolating p75 NGFR-positive cells.

[0127] Example 4: Secondary Differentiation of NCSCs Containing Axial Specification Information

[0128] The NCSCs at the neural plate border stage expressing p75 NGFR or HNK1 (cells on day 5 of differentiation) isolated by FACS in Example 3-1 above were re-seeded into a 24-well plate coated with Geltrex, Matrigel, or Laminin+Fibronectin at 300,000 cells per well. 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, 1% Glutamax, and FGF2 (fibroblast growth factor 2) (Table 2). From day 6 to day 10 of differentiation, FGF2 was added to the medium as an axial specification stimulator. From day 11 to day 14 of differentiation, the proliferated neural crest stem cells were purified by FACS using an antibody against the neural crest stem cell surface antigen p75 NGFR ( Figure 4 ).

[0129] Table 2

[0130] day Culture medium composition Day 6 75% PIM + 25% NB (Neurobasal medium) + FGF2 (20 ng / mL) Day 8 50% PIM + 50% NB + FGF2 (20 ng / mL) Day 10 25% PIM + 75% NB + FGF2 (20 ng / mL)

[0131] Example 5: Gene Expression Analysis of Early Neural Crest Stem Cells

[0132] In Example 3, the expressions of neural crest stem cell marker genes NGFR, HNK1, PAX7, MSX1, AP2alpha and 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 border stage isolated using the p75NGFR antibody on the 5th day of differentiation were analyzed by FACS, and the expression of PAX7 expressed at the neural plate border was verified at the protein level using immunofluorescence analysis.

[0133] The results showed that the isolated early neural crest stem cells expressed NGFR, HNK1, PAX7, MSX1 and AP2alpha ( Figure 5 (a) to (d) of). In addition, compared with pre-differentiated cells and neural crest stem cells isolated on the 10th day of differentiation, the expression of ZIC1 was significantly increased in early neural crest stem cells at the neural plate border stage isolated on the 5th day of differentiation, and ETS1 increased with the progress of differentiation ( Figure 5 (e) of). Since the continuous expression of ZIC1 after developmental differentiation is a specification factor for trunk neural crest stem cells rather than cranial neural crest stem cells, it was confirmed that the specification of cranial neural crest stem cells occurs between 5 and 10 days of differentiation.

[0134] Example 6: Analysis of Gene Expression Changes in Axially Specified NCSCs

[0135] 6-1. Gene expression analysis of neural crest stem cells undergoing secondary differentiation without axial specification stimulating factors

[0136] The early neural crest stem cells on the 5th day of differentiation (expressing p75 NGFR or HNK1) of Example 3-1 were isolated, or in the state of non-isolated mixed cells, and after 5 days of secondary culture without adding the axial specification stimulating factor FGF2, the expressions of ZIC1 gene and ETS1 gene were confirmed. The results showed that the expression of ZIC1 gene did not decrease, while the expression of ETS1 gene increased with the increase of differentiation days. Figure 6(a) and (b)). In addition, it can be estimated that the expression ratio of the ZIC1 gene and the ETS1 gene (ETS1 / ZIC1) for the specification of cranial neural crest stem cells did not increase but remained unchanged ( Figure 6 of (c)).

[0137] It is thus speculated that when early neural crest stem cells coexist with non-neural crest stem cells during the differentiation process, they can be specified into cranial neural crest stem cells through cell-cell interactions. However, when early neural crest stem cells exist alone, they cannot be specified into cranial neural crest stem cells. The main reason can be speculated to be the lack of a decrease in the expression of the ZIC1 gene.

[0138] 6-2. Gene expression analysis of neural crest stem cells undergoing secondary differentiation with axial specification stimulating factors added

[0139] Isolate (only NC) the early neural crest stem cells (expressing p75 NGFR or HNK1) on the 5th day of differentiation in Example 3-1, or in the state of non-isolated mixed cells. After treatment with WNT (activated with the GSK-3 inhibitor CHIR99021), BMP, SHH, or FGF2 as axial specification stimulating factors, followed by secondary culture for 5 days, confirm the expression of the ZIC1, ETS1, TWIST1, and HOX genes in the axially specified neural crest stem cells. The results show that when treated with FGF2, the expression ratio of ETS1 / ZIC1 increased in both the isolated early neural crest stem cell group and the non-isolated mixed cell group ( Figure 7 of (a)), especially, it was found to increase significantly in the isolated early neural crest stem cells ( Figure 7 of (b)). It can be seen from this that the role of non-neural crest stem cells is to activate the FGF signaling of early neural crest stem cells, thereby inducing cranial specification. In addition, the expression of the TWIST1 gene, which is a marker gene for cranial neural crest stem cells, increased in the neural crest stem cells treated with FGF2, and the HOX gene, which is known not to be expressed in cranial neural crest stem cells, decreased ( Figure 7 of (c)). In addition, when comparing the expression levels of the ZIC1 and ETS1 genes in single cells, the results show that starting from the 4th day of treatment with FGF2, the ZIC1 gene in each single neural crest stem cell decreased significantly ( Figure 7 of (d) and (e)).

[0140] It can thus be confirmed that when only isolated neural crest stem cells are present, ZIC1 does not decrease due to the lack of interaction with other cells of non-neural crest stem cells, while FGF2 can cause a decrease in ZIC1.

[0141] Example 7: Analysis of the mechanism for retaining axial specification of early neural crest stem cells based on signaling factors

[0142] Early neural crest stem cells were isolated on the 5th day of differentiation. Then, through immunochemical analysis, the expression of ETS1 protein in neural crest stem cells treated with FGF2 from the 5th day to the 10th day was confirmed. And through FACS analysis, the direct effects of various signaling factors on early neural crest stem cells were confirmed.

[0143] The results showed that FGF2 inhibited the expression of the ZIC1 gene and promoted the expression of ETS1 protein through the influence of BMP4 ( Figure 8 of (b)). When differentiating neural crest stem cells without FGF2 treatment using the known SOX9: reporter hESC as a cranial neural crest stem cell marker, the differentiation efficiency into cranial neural crest stem cells was approximately 25%, while when treated with bFGF, the proportion of SOX9-expressing cells was confirmed to be over 80% ( Figure 8 of (c), (d)).

[0144] Example 8: Confirmation of simultaneous generation of multiple axially specified neural crest stem cell populations

[0145] 8-1. Confirmation of the differentiation of multiple axially specified neural crest stem cells

[0146] The NCSCs (cells on the 5th day of differentiation) at the neural plate border stage obtained through primary differentiation in Example 3-1 were re-seeded and then treated with bFGF or CHIR99021 + retinoic acid respectively to differentiate them into cranial neural crest stem cells or cardiac neural crest stem cells. Subsequently, the mRNA expressions of SOX9, TWIST1, and PRRX2 as cranial neural crest stem cell markers and the mRNA expressions of cKIT, MEF2c, and MAFB as cardiac neural crest stem cell markers were confirmed. The results showed that in the bFGF treatment group, the mRNA expressions of SOX9, TWIST1, and PRRX2 as cranial neural crest stem cell markers increased ( Figure 9 of (a)), and in the group treated with CHIR99021 + retinoic acid to induce WNT activation, the mRNA expressions of cKIT, MEF2c, and MAFB as cardiac neural crest stem cell markers increased ( Figure 10 of (d)).

[0147] 8-2. Confirmation of the differentiation ability of axially specified neural crest stem cells into other cells

[0148] To confirm whether the neural crest cells in secondary differentiation in Example 8-1 differentiate into differentiation target cells at corresponding parts of the human body, the cranial neural crest stem cells obtained by treating early neural crest stem cells with bFGF for differentiation in Example 8-1 were differentiated into chondroblasts or osteoblasts, and then were confirmed by Alcian blue and alizarin red staining, and the expression of each related gene marker was confirmed. In addition, the cardiac neural crest stem cells obtained by differentiating early neural crest stem cells through WNT activation in Example 8-1 were differentiated into SMC (smooth muscle cells), and were confirmed by immunofluorescence method, and the expression of related marker genes was confirmed.

[0149] The results showed that the cranial neural crest stem cells differentiated into chondroblasts or osteoblasts as corresponding target cells ( Figure 9 (b) and (c) of Figure 10 ), and the cardiac neural crest stem cells differentiated into SMC as corresponding target cells (

[0150] (e) and (f) of ).

[0150] It can be seen therefrom that through the two-step differentiation method of the present invention, multiple axially specified neural crest populations can be generated simultaneously in the same batch.

Claims

1. A composition for detecting early neural crest stem cells, wherein, The composition comprises an agent for measuring the expression level of the p75 NGFR or HNK1 gene.

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 during the neural tube formation stage of the embryo.

3. A composition for detecting neural crest stem cells, the neural crest stem cells having axial specialization information, wherein, The composition comprises an agent for measuring the expression level of the ETS1 gene or ZIC1 gene.

4. The composition for detecting neural crest stem cells according to claim 3, wherein, The neural crest stem cells with axial specialization 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 method for preparing early neural crest stem cells, wherein, The method comprises: a) a step of culturing pluripotent stem cells; b) a step of differentiating the cultured cells into early neural crest stem cells; and c) a step of isolating the early neural crest stem cells.

6. The method for preparing early neural crest stem cells according to claim 5, wherein, The early neural crest stem cells correspond to the neural plate border stage during the neural tube formation stage of the embryo.

7. The method for preparing early neural crest stem cells according to claim 5, wherein, The early neural crest stem cells have not yet formed axial information.

8. The method for preparing early neural crest stem cells according to any one of claims 5 to 8, wherein, The early neural crest stem cells are PAX7, MSX1, AP2alpha, p75 NGFR or HNK1 positive cells.

9. A method for preparing neural crest stem cells, wherein, The method comprises: a) a step of culturing pluripotent stem cells; b) a step of primarily differentiating the cultured cells into early neural crest stem cells; c) a step of isolating the early neural crest stem cells; and d) a step of secondarily differentiating the early neural crest stem cells into neural crest stem cells with axial specialization information.

10. The method for preparing neural crest stem cells according to claim 9, wherein, The early neural crest stem cells are PAX7, MSX1, AP2alpha, p75 NGFR or HNK1 positive cells.

11. The method for preparing neural crest stem cells according to claim 9, wherein, Treat with WNT, BMP, BMP4, SHH, FGF2 (fibroblast growth factor 2), NOTCH or bFGF during secondary differentiation.

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

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

14. The method for preparing neural crest stem cells according to any one of claims 9 to 13, wherein, The neural crest stem cells with axial specialization 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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