Method for producing skin-derived pluripotent precursor cells

Abstract

In the differentiation induction of the present invention from stem cells into skin-derived pluripotent precursor cells, the yield of skin-derived pluripotent precursor cells is increased. A method for producing skin-derived pluripotent precursor cells, which comprises a step of culturing neural crest stem cells in the presence of at least one selected from laminin and its fragments to differentiate them into skin-derived pluripotent precursor cells. The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and mutants thereof.

Description

Technical Field

[0001] The present invention relates to a method for producing skin-derived pluripotent precursor cells. Background Art

[0002] In recent years, regenerative medicine using artificially cultured cells or tissues has attracted attention. For example, treating hair loss by regenerating hair follicles is very important for improving the quality of life (QOL) of people in terms of appearance (social aspect) and health.

[0003] As one of the cell sources for artificially culturing cells or tissues, there are skin-derived pluripotent precursor cells (skin-derived precursor cells: hereinafter, also referred to as "SKPs" in this specification). SKPs are cells present in the dermal papilla and can differentiate into nerve cells, glial cells (glial cells), smooth muscle cells, adipocytes, osteocytes, dermal fibroblasts, dermal papilla cells, etc. In addition, SKPs play important functions in maintaining the dermal environment, tissue repair, and hair follicle formation (see Non-Patent Documents 1 and 2).

[0004] Currently, various clinical and non-clinical studies are being conducted in the field of regenerative medicine. For these studies, it is necessary to develop an effective method for obtaining a large amount of SKPs. As the methods for obtaining SKPs reported so far, there are methods of collecting and culturing as floating cell masses from human or non-human animal tissues (for example, see Non-Patent Document 1), and methods of producing using adherent cells cultured from human or non-human animal tissues (for example, see Non-Patent Document 3). Patent Document 1 discloses a method for producing SKPs, which includes a step of culturing and differentiating neural crest stem cells derived from human pluripotent stem cells in a differentiation induction medium containing an agonist of the Wnt signal into SKPs.

[0005] The culture of pluripotent cells such as ES cells or iPS cells is usually carried out in the co - existence of feeder cells. On the other hand, artificial cultured cells for human regenerative medicine are preferably cultured under feeder - free conditions without using feeder cells and without containing xenogeneic components (no xenobiotics). Methods for culturing pluripotent cells under feeder - free conditions using cell adhesion molecules instead of feeder cells have been developed. Patent Document 2 discloses a method for culturing human pluripotent stem cells using a culture substrate coated with an E8 fragment of laminin 511 or an E8 fragment of laminin 332. Patent Document 3 discloses a method for culturing mammalian cells, which includes the step of culturing stem cells such as ES cells or iPS cells in the presence of a modified laminin, which is composed of a laminin or a laminin fragment to which a fragment of a proliferation factor - binding site containing heparan sulfate proteoglycan, etc. is bound. Patent Document 4 discloses a method for culturing pluripotent stem cells, which includes the step of contacting laminin 421, laminin 121, or a fragment thereof with pluripotent stem cells. Patent Document 5 discloses a method for inducing the differentiation of pluripotent stem cells, which includes the step of contacting a conjugate of an E8 fragment of laminin and a fragment of a proliferation factor - binding site containing heparan sulfate proteoglycan with human pluripotent stem cells.

[0006] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2015 - 213495

[0007] Patent Document 2: Japanese Unexamined Patent Application Publication No. 2011 - 078370

[0008] Patent Document 3: International Publication No. 2012 / 137970

[0009] Patent Document 4: International Publication No. 2018 / 038242

[0010] Patent Document 5: International Publication No. 2018 / 088501

[0011] Non - Patent Document 1: Nature Cell Biology, 2001, 3: 778 - 784

[0012] Non - Patent Document 2: Cell Stem Cell, 2009, 5: 610 - 623

[0013] Non - Patent Document 3: PLoS One, 2012, 7(11): e50742 Summary of the Invention

[0014] In one aspect, the present invention provides a method for producing skin-derived pluripotent precursor cells, which includes a step of culturing neural crest stem cells in the presence of at least one selected from laminin and its fragments, and differentiating them into skin-derived pluripotent precursor cells. The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and mutants thereof.

[0015] In other aspects, the present invention provides an anchoring material for inducing and culturing the differentiation of neural crest stem cells into skin-derived pluripotent precursor cells, which contains at least one selected from laminin and its fragments as an active ingredient. The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and mutants thereof.

[0016] In another aspect, the present invention provides a method for producing neural crest stem cells, which includes a step of culturing pluripotent stem cells in the presence of at least one selected from laminin and its fragments that are bound to perlecan or fragments containing its proliferation factor binding site, and differentiating them into neural crest stem cells. The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and mutants thereof.

[0017] Furthermore, in other aspects, the present invention provides an anchoring material for inducing and culturing the differentiation of pluripotent stem cells into neural crest stem cells, which contains at least one selected from laminin and its fragments that are bound to perlecan or fragments containing its proliferation factor binding site as an active ingredient. The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and mutants thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the induction of differentiation from NCS cells to SKPs in the presence of laminin fragments. iPS-NCS: cells before the start of differentiation induction; iPS-SKPs P0: cells 4 days after differentiation induction; iPS-SKPs P1: cells after passage culture (all magnifications are 40 times). The lowermost part shows the fluorescence staining images of nestin and fibronectin in iPS-SKPs P1 (magnification: 50 times).

[0019] Figure 2It shows the induction of differentiation from iPS cells into SKPs in the presence of laminin fragments. The photographs show SKPs after subculture (all magnifications are 40-fold). Upper: culture in the presence of unmodified laminin fragments; Lower: culture in the presence of basement membrane proteoglycan-modified laminin fragments.

[0020] Figure 3 It shows the expression of marker genes in SKPs differentiated in the presence of basement membrane proteoglycan-modified laminin fragments. iPS-SKPs P0: cells after differentiation induction; iPS-SKPs P1: cells after subculture.

[0021] Figure 4 It shows the expression of marker proteins in SKPs differentiated in the presence of basement membrane proteoglycan-modified laminin fragments. Fluorescence microscope photographs of immunostained cells. A: expression of nestin; B: expression of fibronectin; C: expression of αSMA.

[0022] Figure 5 It shows the differentiation of SKPs differentiated in the presence of basement membrane proteoglycan-modified laminin fragments into tissue cells. Microscope photographs of stained cells. A: adipocytes; B: osteocytes; C: Schwann cells.

[0023] Figure 6 It shows the induction of differentiation from iPS cells into SKPs in the presence of laminin fragments under xenobiotic-free conditions. iPS-NCS (upper): cells before the start of differentiation induction; iPS-SKPs P0 (middle): cells 4 days after differentiation induction; iPS-SKPs P1 (lower): cells after subculture (all magnifications are 40-fold).

[0024] Figure 7 It shows the expression of marker genes in NCS cells differentiated in the presence of laminin fragments.

[0025] Figure 8 It shows the number of SKPs induced to differentiate in the presence of laminin fragments. LM: unmodified laminin fragments; P-LM: basement membrane proteoglycan-modified laminin fragments. The labels under each bar indicate the laminin types. Error bar = SD, *: P < 0.0005, **: P < 0.0001 (t-test). Detailed implementation mode

[0026] All patent documents, non-patent documents and other publications cited in this specification are hereby incorporated by reference in their entirety in this specification.

[0027] In this specification, "pluripotent stem cells" refer to undifferentiated cells that have the pluripotency to differentiate into various tissues that make up an adult and the ability of self-renewal. The pluripotent stem cells used in the present invention can be appropriately selected. Specific examples of pluripotent stem cells include: embryonic stem cells (hereinafter also referred to as "ES cells"), embryonic carcinoma cells (hereinafter also referred to as "EC cells"), embryonic germ stem cells (hereinafter also referred to as "EG cells"), induced pluripotent stem cells (iPS cells), and the like. These cells can be prepared by conventional methods or commercially available cells can be used. The pluripotent stem cells used in the present invention are preferably ES cells or iPS cells, and more preferably iPS cells. In addition, the pluripotent stem cells used in the present invention only need to be pluripotent stem cells derived from mammals, preferably pluripotent stem cells derived from humans, mice, rats, cows, pigs, and non-human primates, and more preferably human-derived pluripotent stem cells. Among them, human-derived ES cells or iPS cells are preferred, and human-derived iPS cells are more preferred.

[0028] "Skin-derived precursor cells (SKPs)" in this specification refer to: undifferentiated cells with the ability of self-renewal, which have the ability to differentiate into nerve cells, glial cells (such as microglia, astrocytes, oligodendrocytes, ependymal cells, Schwann cells, satellite cells, etc.), smooth muscle cells, adipocytes, osteocytes, dermal fibroblasts, dermal papilla cells, and the like. SKPs can be identified based on the expression of marker proteins such as nestin, fibronectin, αSMA, etc., and preferably can be identified based on the co-expression of nestin and fibronectin. Alternatively, SKPs can be identified based on the expression of marker genes such as Nestin gene, Snail gene, Slug gene, Sox9 gene, Dermo-1 gene, BMP-4 gene, Wnt-5a gene, etc.

[0029] "Neural crest stem cells" (hereinafter also referred to as "NCS cells") in this specification refer to: pluripotent stem cells with the ability of self-renewal and multi-differentiation, which migrate from the dorsal side of the neural tube into the body during vertebrate development and contribute to the formation of various tissues. Neural crest stem cells can be identified based on the expression of well-known markers such as paired box 6 (PAX6), nerve growth factor receptor (p75), etc.

[0030] The expression of a marker protein or a marker gene in a cell can be detected by conventional methods. For example, the expression of a marker protein in a cell can be detected by immunohistochemical staining, Western blotting, ELISA, etc. using an antibody against the marker protein. In addition, for example, the expression of a marker gene in a cell can be detected by PCR, microarray, sequencing, etc.

[0031] In this specification, "Wnt signal" refers to a series of pathways activated by the binding of a Wnt protein to its receptor, including the β-catenin pathway, the PCP pathway, and the Ca 2+ pathway. The activation of the Wnt signal controls various cellular functions such as cell proliferation or differentiation, organ formation, or cell movement during early development. Preferably, "Wnt signal" in this specification refers to the β-catenin pathway (Canonical cascade). In this pathway, the binding of a Wnt protein to its receptor stabilizes β-catenin, which migrates into the nucleus and functions as a transcription factor, thereby activating gene transcription. In addition, β-catenin is also thought to be involved in cell adhesion. On the other hand, when the β-catenin pathway is turned off, β-catenin forms a degradation complex composed of multiple proteins and is degraded after phosphorylation by the serine / threonine kinases Glycogensynthase 3 (GSK-3) or Casein kinase 1α (CK1α) in this complex, thereby maintaining β-catenin at a low level in the cell.

[0032] In this specification, a "Wnt signal agonist" refers to a factor that activates the above-mentioned Wnt signal, preferably a factor that activates the above-mentioned β-catenin pathway (Canonical cascade). As an example of a factor that activates the β-catenin pathway, factors that promote the nuclear migration of β-catenin by inhibiting β-catenin phosphorylases such as GSK-3 and thereby activate gene transcription factors can be cited. Therefore, a GSK-3 inhibitor, which is an inhibitor of β-catenin phosphorylase, can be cited as an example of a factor that activates the β-catenin pathway.

[0033] Laminin is one of the main extracellular matrices and is a protein involved in cell adhesion, metastasis, proliferation, etc. Laminin is a heterotrimeric molecule with three different subunits (α chain, β chain, γ chain). To date, five α chains (α1, α2, α3, α4, α5), three β chains (β1, β2, β3), and three γ chains (γ1, γ2, γ3) have been discovered, and many isomers exist in laminin depending on their combinations. Members of the laminin family are named according to the types of subunits. For example, laminin composed of α5 chain, β1 chain, and γ1 chain is called laminin 511.

[0034] In this specification, the "E8 fragment of laminin" (hereinafter, also simply referred to as "laminin E8") refers to a fragment composed of a trimer of a fragment obtained by removing globular domains 4 and 5 from the C-terminal fragment of the laminin α chain (hereinafter referred to as "α-chain E8"), the C-terminal fragment of the β chain (hereinafter referred to as "β-chain E8"), and the C-terminal fragment of the γ chain (hereinafter referred to as "γ-chain E8"). The molecular weight of this trimer is about 150 to about 170 kDa. α-chain E8 is usually composed of about 770 amino acids, and about 230 amino acids on the N-terminal side are involved in the formation of the trimer. β-chain E8 is usually composed of about 220 to about 230 amino acids. γ-chain E8 is usually composed of about 240 to about 250 amino acids, and the glutamic acid residue from the C-terminal part to the 3rd position is essential for the integrin-binding activity of laminin E8 (see The Journal of Biological Chemistry, 2007, 282: 11144-11154).

[0035] The amino acid sequences of the α-chain, β-chain, and γ-chain of mammalian laminin and the nucleotide sequences of the genes encoding them can be obtained from publicly known databases (such as GenBank [www.ncbi.nlm.nih.gov / genbank / ]). Table 1 shows the GenBank accession numbers of each chain constituting human laminin. Laminin can be produced by publicly known methods such as a method of purifying from laminin-high-expressing cells and a method of producing as a recombinant protein. In addition, laminin fragments can be produced by publicly known methods such as a method of digesting full-length laminin with a protease and a method of directly expressing laminin fragments as recombinants. Alternatively, laminin or its fragments can be purchased as commercial products.

[0036] [Table 1]

[0037] Amino acid sequence Nucleotide sequence Human laminin α1 chain NP_005550 NM_005559 Human laminin α2 chain NP_000417 NM_000426 Human laminin α3 chain NP_000218 NM_000227 Human laminin α4 chain NP_002281 NM_002290 Human laminin α5 chain NP_005551 NM_005560 Human laminin β1 chain NP_002282 NM_002291 Human laminin β2 chain NP_002283 NM_002292 Human laminin β3 chain NP_000219 NM_000228 Human laminin γ1 chain NP_002284 NM_002293 Human laminin γ2 chain NP_005553 NM_005562 Human laminin γ3 chain NP_006050 NM_006059

[0038] It is desired to increase the yield of SKPs during the differentiation induction of neural crest stem cells into SKPs as described in Patent Document 1. In addition, SKPs cultured under feeder-free conditions, which are suitable as a cell source for regenerative medicine, are desired.

[0039] The inventors of the present invention found that in the differentiation induction culture of neural crest stem cells into SKPs, cell culture can be carried out under feeder-free conditions by using a specific laminin species as a culture attachment material. In addition, the inventors of the present invention found that under this culture condition, the differentiation from neural crest stem cells into SKPs is promoted, and the yield of SKPs is increased.

[0040] According to the present invention, skin-derived precursors (SKPs) that can be effectively differentiated into nerve cells, glial cells, smooth muscle cells, adipocytes, osteocytes, dermal papilla cells, etc. can be produced. In addition, according to the present invention, it is possible to induce the differentiation of pluripotent stem cells into neural crest stem cells and the differentiation of neural crest stem cells into SKPs under feeder-free conditions. Therefore, according to the present invention, SKPs suitable as a cell source for regenerative medicine can be provided.

[0041] In one aspect, the present invention provides a method for producing skin-derived pluripotent precursor cells (SKPs). In this method, neural crest stem cells (NCS cells) are induced to differentiate into SKPs. More specifically, the method includes a step of culturing NCS cells in the presence of laminin and / or its fragments to differentiate them into SKPs.

[0042] As the laminin that can be used for the differentiation induction culture of these SKPs, at least one selected from laminin 111 (α1β1γ1), laminin 121 (α1β2γ1), laminin 332 (α3β3γ2), laminin 421 (α4β2γ1), laminin 511 (α5β1γ1), and laminin 521 (α5β2γ1) can be mentioned, and preferably at least one selected from laminin 111, laminin 332, laminin 421, and laminin 511 is selected.

[0043] Any one of the laminin and its fragments can be used, or the two can be used in combination. Therefore, the laminin and / or its fragments that can be used for the differentiation induction culture of these SKPs are preferably at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their fragments, and more preferably at least one selected from laminin 111, laminin 332, laminin 421, and laminin 511, and their fragments.

[0044] As an example of the laminin fragment, any fragment of laminin that has the same cell adhesion activity as the full-length protein of the laminin species mentioned above can be used, and its molecular weight and structure are not particularly limited. As a preferred example of the laminin fragment that can be used for the differentiation induction culture of these SKPs, fragments of the laminin species mentioned above having integrin-binding activity can be mentioned. As a more preferred example, fragments containing the E8 fragment of the laminin species mentioned above can be mentioned. As a further preferred example, the E8 fragment of the laminin species mentioned above can be mentioned.

[0045] The laminin and / or its fragment capable of being used for the differentiation induction culture of the SKPs can be human-derived laminin and / or its fragment, or can be non-human mammalian-derived laminin and / or its fragment. Preferably, the laminin and / or its fragment is the laminin and / or its fragment of the same origin as the produced SKPs, and more preferably is human laminin and / or its fragment.

[0046] The laminin and / or its fragment capable of being used for the differentiation induction culture of the SKPs can be wild-type laminin and / or its fragment, or can be its mutant, or can be a combination thereof. As an example of the mutant of the laminin and / or its fragment, a polypeptide can be cited which is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added in a wild-type laminin or its fragment as a parent (such as laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, or these fragments) and has integrin-binding activity. Here, several means, for example, can be 2 to 10, can be 2 to 8, can be 2 to 6, and can also be 2 to 4. Therefore, in the present specification, laminin includes wild-type laminin and its mutant. The same applies to the laminin fragment.

[0047] Furthermore, the laminin and / or its fragment capable of being used for the differentiation induction culture of the SKPs can be a modified laminin and / or its fragment obtained by further modifying the above-mentioned laminin and / or its fragment. As an example of the modified laminin and / or its fragment, the laminin and / or its fragment bound to perlecan or a fragment containing its growth factor-binding site (also referred to as "perlecan-modified laminin / fragment" in the present specification) can be cited (refer to Patent Documents 3 and 5). From the viewpoint of the efficiency of differentiation induction into SKPs, the laminin and / or its fragment used in the differentiation induction culture of SKPs in the present invention is preferably perlecan-modified laminin / fragment. The perlecan or the fragment containing its growth factor-binding site used for this modification is preferably a fragment of the same origin as the laminin or its fragment, and more preferably is a fragment containing human perlecan (GenBank: NP_005520, NM_005529) or its growth factor-binding site. As the growth factor-binding site of perlecan, domains I to III of perlecan can be cited (for example, the region of the amino acid sequence of human perlecan from valine at the 22nd position from the N-terminus to proline at the 1676th position) (The Journal of Biological Chemistry, 2003, 278: 30106 - 30114), and among them, domain I (Gly25 -Pro 196 )。The basement membrane proteoglycan or a fragment containing its proliferation factor binding site can be produced, for example, by a known method such as a method of purifying it from cells highly expressing it or a method of producing it as a recombinant protein.

[0048] In the basement membrane proteoglycan-modified laminin / fragment, the basement membrane proteoglycan or a fragment containing its proliferation factor binding site only needs to bind to at least one of the N-terminal of the α-chain of laminin or its fragment, the C-terminal of the α-chain, the N-terminal of the β-chain, and the N-terminal of the γ-chain. Therefore, the basement membrane proteoglycan-modified laminin / fragment can have one, two, three, or four of the basement membrane proteoglycans or fragments containing its proliferation factor binding site, and each of them can all be basement membrane proteoglycans, or all be basement membrane proteoglycan fragments containing the proliferation factor binding site, or a part be basement membrane proteoglycans and a part be basement membrane proteoglycan fragments containing the proliferation factor binding site. The basement membrane proteoglycan-modified laminin / fragment used in the present invention preferably has one basement membrane proteoglycan or its proliferation factor binding site bound to the C-terminal of the α-chain of laminin or its fragment, and more preferably has one proliferation factor binding site of the basement membrane proteoglycan bound to the C-terminal of the α-chain of the laminin fragment. Preferred examples of the laminin, laminin fragment, and basement membrane proteoglycan and its proliferation factor binding site contained in the basement membrane proteoglycan-modified laminin / fragment are as described above.

[0049] The basement membrane proteoglycan-modified laminin / fragment can be prepared using known genetic recombination techniques, for example, according to the steps described in Patent Document 3. More specifically, the DNA encoding laminin or its fragment and the DNA encoding the basement membrane proteoglycan or a fragment containing its proliferation factor binding site are ligated to construct a DNA encoding a fusion protein of the basement membrane proteoglycan or a fragment containing its proliferation factor binding site and laminin or its fragment. By introducing a vector containing the DNA encoding the fusion protein into a host cell and expressing it, the target basement membrane proteoglycan-modified laminin / fragment can be prepared. Alternatively, by chemically binding the basement membrane proteoglycan or a fragment containing its proliferation factor binding site to an appropriate position on laminin or its fragment, the target basement membrane proteoglycan-modified laminin / fragment can be synthesized.

[0050] The laminin and / or its fragment available in the above-mentioned present invention may contain sequences for their construction, isolation, purification, or binding to the aforementioned basement membrane proteoglycan or its fragment, such as tag sequences, linker sequences, and the like.

[0051] In the method for producing SKPs of the present invention, NCS cells are cultured in the presence of the laminin and / or its fragment to differentiate into SKPs. In this step, the laminin and / or its fragment is used as a culture anchoring material for inducing the differentiation of NCS cells into SKPs. Therefore, in the method of the present invention, it is not necessary to use feeder cells as in the conventional method for inducing the differentiation of SKPs, and culture under feeder-free conditions becomes possible.

[0052] The NCS cells used in the method for producing SKPs of the present invention can be prepared by known methods. For example, NCS cells can be prepared by collecting NCS cells from early embryos, proliferating them as needed, and inducing differentiation from pluripotent stem cells. The NCS cells used in the method of the present invention are preferably NCS cells derived from pluripotent stem cells obtained by inducing the differentiation of pluripotent stem cells, and more preferably NCS cells derived from induced pluripotent stem cells (iPS cells) obtained by inducing the differentiation of induced pluripotent stem cells (iPS cells).

[0053] Therefore, in one embodiment, the method for producing SKPs of the present invention may further include the following step: inducing the differentiation of NCS cells from pluripotent stem cells to produce NCS cells derived from pluripotent stem cells. In a preferred embodiment, the step of producing NCS cells derived from the pluripotent stem cells includes culturing the pluripotent stem cells in the presence of laminin and / or its fragment to differentiate them into NCS cells.

[0054] Furthermore, if necessary, before the above-mentioned induction of differentiation into NCS cells, the pluripotent stem cells can be pre-cultured. This pre-culture is also preferably carried out in the presence of laminin and / or its fragment. After this pre-culture, the pluripotent stem cells maintained can be used for the induction of differentiation into the NCS cells.

[0055] The type of laminin and / or its fragment used in the production process of the NCS cells is not particularly limited, and examples thereof include: laminin and / or its fragment that can be used for the above-mentioned differentiation induction culture of SKPs; laminin, laminin fragment, and modified laminin used for the culture of pluripotent stem cells as described in Patent Documents 2 to 5. Preferably, the laminin and / or its fragment used in this process can be at least one selected from laminin and its fragments that can be used for the above-mentioned differentiation induction culture of SKPs. The laminin and / or its fragment used in this process can be of the same type as the laminin and / or its fragment used for the differentiation induction of the above-mentioned SKPs, or can be of different types. In a preferred embodiment, the laminin and / or its fragment used in the production process of the NCS cells is a basement membrane proteoglycan-modified laminin / fragment that can be used for the above-mentioned differentiation induction culture of SKPs. By using the basement membrane proteoglycan-modified laminin / fragment, the efficiency of differentiation induction into NCS cells can be improved. In a preferred embodiment of the present invention, after culturing pluripotent stem cells in the presence of a basement membrane proteoglycan-modified laminin / fragment that can be used for the above-mentioned differentiation induction culture of SKPs to differentiate them into NCS cells, the NCS cells are continuously cultured to differentiate them into SKPs.

[0056] In the production process of the NCS cells, the laminin and / or its fragment is used as a culture attachment material during the differentiation induction culture of pluripotent stem cells into NCS cells. Therefore, in this process, it is not necessary to use feeder layer cells as in the conventional culture method of pluripotent stem cells, and culture under feeder-free conditions becomes possible.

[0057] The pre-culture of pluripotent stem cells, the differentiation induction culture of pluripotent stem cells into NCS cells, and the differentiation induction culture of NCS cells into SKPs in the present invention are preferably carried out ex vivo and do not include culture on or within a living human or animal individual. Therefore, the laminin and / or its fragment used as a culture attachment material in the present invention is preferably used ex vivo and not for culture on or within a living human or animal individual.

[0058] In the present invention, as long as the laminin and / or its fragment exerts the function of culture attachment, it can be used in any method. For example, cells can be cultured on a culture substrate containing the laminin and / or its fragment. Examples of the culture substrate containing the laminin and / or its fragment include a culture substrate having a coating containing the laminin and / or its fragment (such as a culture plate, a culture mesh, a culture dish, etc.). As an example thereof, a culture substrate on which the laminin and / or its fragment is adsorbed by coating with a coating agent containing the laminin and / or its fragment can be mentioned. The content of the laminin and / or its fragment in such a culture substrate is preferably: per 1 cm of the area of the culture substrate in contact with the culture 2 is 0.05 to 50 μg, more preferably 0.1 to 10 μg. For example, per 1 cm of the coated area 2 a coating agent containing preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg of the laminin and / or its fragment is used to coat the culture substrate so that the laminin and / or its fragment is adsorbed onto the culture substrate.

[0059] Alternatively, in the present invention, cell culture can be carried out in a culture medium supplemented with the laminin and / or its fragment. When adding the laminin and / or its fragment to the culture medium, the laminin and / or its fragment can be added to the culture medium before inoculating cells, or the laminin and / or its fragment can also be added together with cells to the culture medium. It is only necessary to adjust the content of the laminin and / or its fragment in the culture medium so that the final concentration per 1 mL of the culture medium becomes preferably 0.1 to 100 μg, more preferably 0.2 to 20 μg.

[0060] In the culture substrate or coating agent or culture medium containing the laminin and / or its fragment, an attachment material or a cell adhesion molecule other than the laminin and / or its fragment can be contained. Examples of the attachment material or cell adhesion molecule include: gelatin, collagen, Matrigel, fibronectin, poly-L-lysine, etc.

[0061] In the culture medium for inducing the differentiation of NCS cells into SKPs and the culture medium for inducing the differentiation of pluripotent stem cells into NCS cells, a differentiation medium commonly used for the induction of stem cell differentiation can be used. In addition, a maintenance medium commonly used for the maintenance culture of stem cells can be used in the pre-culture of pluripotent stem cells. The basal medium of these culture media can be appropriately selected from the basal media commonly used for stem cell culture, and commercially available products can also be used. Examples of such basal media include: MEM medium (Minimum Essential Medium), BME medium (Basal Medium Eagle), IMDM medium (Iscove’s Modified Dulbecco’s Medium), DMEM medium (Dulbecco’s Modified Eagle’s Medium), Ham medium, RPMI medium (Roswell Park Memorial Institute medium), Fischer’s medium, and their mixed media. Among them, DMEM / Ham’s F12 medium (hereinafter, also simply referred to as “DMEM / F12 medium”) is preferred. When culturing under feeder-free conditions, it can be appropriately selected from the basal media prepared for feeder-free culture of stem cells, and commercially available products can also be used. Examples thereof include StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) and the like.

[0062] The culture medium can be a serum-containing medium, a serum-free medium, or a medium containing a serum substitute, and a medium containing a serum-free medium or a serum substitute is preferred. The serum substitute is a composition containing components such as albumin, transferrin, fatty acids, collagen precursors, trace elements (such as lead, selenium, etc.), and growth factors (EGF (epidermal growth factor), bFGF (basic fibroblast growth factor), etc.) contained in serum. As long as it has the ability to promote cell proliferation, its composition is not limited. Specific examples include B-27 (trademark) additive, N2 additive, KnockOut Serum Replacement, etc. Furthermore, components commonly used in stem cell culture media such as vitamins, buffers, inorganic salts, antibiotics (such as penicillin, kanamycin, streptomycin), and 2-mercaptoethanol can be contained in the culture medium as needed. It is preferred that the culture medium is a xeno-free human pluripotent induced stem cell medium that does not contain components of xenobiotic origin relative to the cultured cells. Among them, as long as feeder-free culture, preferably further xeno-free culture, can be achieved, commercially available products of this culture medium can also be used.

[0063] The differentiation medium for inducing the differentiation of the NCS cells into SKPs preferably contains at least one growth factor selected from serum substitutes, EGF, and bFGF, and more preferably contains a serum substitute, EGF, and bFGF. As the serum substitute, preferably the above-mentioned B-27 (trademark) additive, N2 additive, or KnockOut Serum Replacement, and more preferably the B-27 (trademark) additive. These growth factors can be prepared by conventional methods or commercially available products can also be used. The content of the growth factor in the differentiation medium can be appropriately set according to culture conditions, the type of pluripotent stem cells used, the type of inhibitor, etc. For example, the concentration of the serum substitute in the medium is preferably 0.5% by mass or more, more preferably 1% by mass or more, and preferably 20% by mass or less, more preferably 5% by mass or less, or the concentration range is preferably 0.5 to 20% by mass, more preferably 1 to 5% by mass. In addition, for example, the concentrations of EGF and bFGF in the medium are each preferably 1 ng / mL or more, more preferably 10 ng / mL or more, and preferably 100 ng / mL or less, more preferably 50 ng / mL or less, or the concentration range is preferably 1 to 100 ng / mL, more preferably 10 to 50 ng / mL.

[0064] Furthermore, by containing a Wnt signal agonist in the differentiation medium, the induction of the differentiation of NCS cells into SKPs can be promoted, and the yield of SKPs can be increased. The Wnt signal agonist used in the present invention is as defined above. As preferred examples, factors that activate the β-catenin pathway and factors that inhibit the degradation of intracellular β-catenin such as GSK-3 inhibitors can be cited. A GSK-3 inhibitor is preferred.

[0065] Examples of GSK-3 inhibitors include: aminopyrimidine compounds (e.g., GSK-3 Inhibitor XVI, trade name CHIR99021, etc.; CAS 252917-06-9), bis-indole (indirubin) compounds (hereinafter also referred to as "BIO") (e.g., (2’Z,3’E)-6-bromoindirubin-3’-oxime; CAS 667463-62-9), acetone oxime compounds of BIO (hereinafter also referred to as "BIO-acetone oxime") (e.g., (2’Z,3’E)-6-bromoindirubin-3’-acetone oxime), thiazolidine (TDZD) compounds (e.g., 4-benzyl-2-methyl-1,2,4-thiazolidine-3,5-dione), oxothiazolidine-3-thione compounds (e.g., 2,4-dibenzyl-5-oxothiazolidine-3-thione), thiophenyl α-chloromethyl ketone compounds (e.g., 2-chloro-1-(4,4-dibromo-thiophen-2-yl)-ethanone), phenyl α-bromomethyl ketone compounds (e.g., α-4-dibromoacetophenone), thiazole-containing urea compounds (e.g., N-(4-methoxybenzyl)-N’-(5-nitro-1,3-thiazol-2-yl)urea), 1H-pyrazolo[3,4-b]quinoxalin-3-amine: trade name NSC693868 (CAS 40254-90-8), (2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione: trade name SB216763 (CAS 280744-09-4), 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione: trade name SB415286 (CAS 264218-23-7), GSK-3β inhibitor XII: trade name TWS119 (CAS 601514-19-6), GSK-3β peptide inhibitors (e.g., H-KEAPPAPPQSpP-NH 2 ) and the like. Among them, at least one selected from GSK-3 inhibitor XVI (CAS 252917-06-9), (2’Z,3’E)-6-bromoindirubin-3’-oxime (CAS 667463-62-9), 1H-pyrazolo[3,4-b]quinoxalin-3-amine (CAS 40254-90-8), (2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (CAS 280744-09-4), 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione (CAS 264218-23-7) and GSK-3β inhibitor XII (CAS 601514-19-6) is preferred, and GSK-3 inhibitor XVI is more preferred.

[0066] The Wnt signal agonists exemplified above can be prepared by conventional methods or commercially available products can also be used. The content of the Wnt signal agonist contained in the differentiation medium can be appropriately set within the range where the Wnt signal is activated and cell proliferation does not stop according to culture conditions, the type of Wnt signal agonist used, and the like. For example, when GSK-3 inhibitor XVI is used as the Wnt signal agonist, the concentration of this Wnt signal agonist in the medium is preferably 0.5 μM or more, more preferably 2 μM or more, and preferably 5 μM or less, more preferably 4 μM or less. Alternatively, the concentration range of this Wnt signal agonist in the medium is preferably 0.5 to 5 μM, more preferably 2 to 4 μM. Further preferably, the concentration of this Wnt signal agonist in the medium is 3 μM.

[0067] On the other hand, the differentiation medium for inducing differentiation culture from pluripotent stem cells into NCS cells preferably contains a TGFβ signal inhibitor and / or a BMP signal inhibitor. Thereby, the induction of differentiation into NCS cells is promoted, and as a result, the yield of the target SKPs is increased. Examples of this TGFβ signal inhibitor include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide (for example, SB431542 (trade name); CAS 301836-41-9), etc. Examples of this BMP signal inhibitor include noggin, LDN193189 (CAS 1062368-24-4), etc. These TGFβ signal inhibitors and BMP signal inhibitors can be prepared by conventional methods or commercially available products can also be used. The content of this TGFβ signal inhibitor and BMP signal inhibitor in the differentiation medium for producing this NCS cell can be appropriately set according to culture conditions, the type of pluripotent stem cell used, the type of inhibitor, and the like. For example, the concentration of SB431542 in this medium is preferably 1 μM or more, more preferably 5 μM or more, and preferably 30 μM or less, more preferably 20 μM or less, or the concentration range is preferably 1 to 30 μM, more preferably 5 to 20 μM. In addition, for example, the concentration of noggin in this medium is preferably 10 ng / mL or more, more preferably 100 ng / mL or more, and preferably 1000 ng / mL or less, more preferably 700 ng / mL or less, or the concentration range is preferably 10 to 1000 ng / mL, more preferably 100 to 700 ng / mL.

[0068] The culture conditions for inducing differentiation from pluripotent stem cells into NCS cells can be appropriately set according to the type of cells used, etc. For example, when using iPS cells as pluripotent stem cells, the culture period for inducing differentiation into NCS cells is preferably 1 to 20 days. The culture conditions for inducing differentiation from NCS cells into SKPs can also be appropriately set separately. For example, by preferably culturing NCS cells in a differentiation induction medium containing a Wnt signal agonist for 3 to 5 days, the cells can be effectively differentiated into SKPs. In the present invention, the cell culture method can be either adherent culture or suspension culture, but adherent culture is preferably selected.

[0069] In a preferred embodiment, the differentiated SKPs are subcultured one or more times. By performing subculture, SKPs can be obtained as cell aggregates with high purity. The subculture method and the number of subculture times of SKPs can be appropriately selected from common subculture methods according to culture conditions, etc. For example, for adherent culture cells, after detaching the cells with an enzyme, etc., subculture is carried out by dilution culture, and for suspension culture cells, subculture is carried out by dilution culture.

[0070] The differentiated NCS cells or SKPs can be identified based on the expression of the respective cell marker proteins or marker genes described above. Or the differentiation of the cells into NCS cells or into SKPs can be confirmed based on cell morphology under microscopic observation, etc.

[0071] In the method for producing SKPs of the present invention, the differentiation from NCS cells into SKPs occurs with a high probability, so it is not necessarily necessary to isolate and recover SKPs from the cultured cells. On the other hand, in order to further improve the purity of SKPs, it is also possible to isolate and recover SKPs from the culture. The isolation and recovery of SKPs can be carried out using conventional methods. For example, it can be carried out by a method using a cell sorter, a method using magnetic beads, etc.

[0072] As described above, in the present invention, the laminin and / or its fragment is used as a culture attachment material in the induction culture of differentiation from pluripotent stem cells into NCS cells and in the induction culture of differentiation from NCS cells into SKPs. Therefore, in another embodiment, the present invention provides an attachment material for the induction culture of differentiation from NCS cells into SKPs, which contains the laminin and / or its fragment as an active ingredient. Furthermore, the present invention provides an attachment material for the induction culture of differentiation from pluripotent stem cells into NCS cells, which contains the laminin and / or its fragment as an active ingredient. In a preferred embodiment, the laminin and / or its fragment contained in the attachment material for the induction culture of differentiation from pluripotent stem cells into NCS cells is the above-mentioned basement membrane proteoglycan-modified laminin / fragment.

[0073] In one embodiment, the anchoring material of the present invention is provided in the following manner: a culture substrate containing the laminin and / or its fragment, such as a culture substrate having a coating containing the laminin and / or its fragment (such as a culture plate, a culture mesh, a culture dish, etc.), more specifically, a culture substrate adsorbed with the laminin and / or its fragment by coating with a coating agent containing the laminin and / or its fragment; a coating agent containing the laminin and / or its fragment; a culture medium containing the laminin and / or its fragment, etc. The culture substrate, the coating agent, and the culture medium containing the laminin and / or its fragment may contain an anchoring material or a cell adhesion molecule other than the above-mentioned laminin and / or its fragment. In addition, the coating agent may also contain other components contained in a coating agent for a normal culture substrate (for example, a buffer as a solvent). In addition, the culture medium may also contain other components contained in a normal culture medium (for example, the basal medium, serum, serum substitute, growth factor, etc. as described above). When using this culture medium in the differentiation induction culture from NCS cells to SKPs, this culture medium preferably contains the above-mentioned Wnt signal agonist, and more preferably contains the Wnt signal agonist, a serum substitute, EGF, and bFGF. On the other hand, when using this culture medium in the differentiation induction culture from pluripotent stem cells to NCS cells, this culture medium preferably contains the above-mentioned TGFβ signal inhibitor and / or BMP signal inhibitor.

[0074] In one embodiment, the anchoring material of the present invention may be composed of the laminin and / or its fragment. In this case, the anchoring material of the present invention is preferably used by adding it to a coating agent for a culture substrate or adding it to a culture medium.

[0075] When the anchoring material of the present invention is a culture substrate containing the laminin and / or its fragment, for every 1 cm of the area of contact between the culture substrate and the culture 2 , the content of the laminin and / or its fragment in this culture substrate is preferably 0.05 - 50 μg, more preferably 0.1 - 10 μg. When the anchoring material of the present invention is a coating agent containing the laminin and / or its fragment, for every 1 cm of the area coated with the coating agent 2, the content of the laminin and / or its fragment in the coating agent is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg. When the fixing material of the present invention is a culture medium containing the laminin and / or its fragment, as the final concentration per 1 mL of the culture medium, the content of the laminin and / or its fragment in the culture medium is preferably 0.1 to 100 μg, more preferably 0.2 to 20 μg. When the fixing material of the present invention is added to the coating agent of the culture medium material and used, its usage amount can be adjusted so that the content of the laminin and / or its fragment in the coating agent falls within the above range. Or, when the fixing material of the present invention is added to the culture medium and used, its usage amount can be adjusted so that the final concentration of the laminin and / or its fragment per 1 mL of the culture medium falls within the above range.

[0076] Furthermore, in another embodiment, the present invention provides a culture medium material for inducing differentiation culture from NCS cells to SKPs, which contains the fixing material of the present invention. In addition, the present invention provides a cell culture kit for inducing differentiation culture from NCS cells to SKPs, which contains the fixing material of the present invention.

[0077] Furthermore, in another embodiment, the present invention provides a culture medium material for inducing differentiation culture from pluripotent stem cells to NCS cells, which contains the fixing material of the present invention. In addition, the present invention provides a cell culture kit for inducing differentiation culture from pluripotent stem cells to NCS cells, which contains the fixing material of the present invention. In a preferred embodiment, the laminin and / or its fragment contained in the fixing material of the present invention in the culture medium material or cell culture kit for inducing differentiation culture from the pluripotent stem cells to NCS cells is the above-mentioned basement membrane proteoglycan-modified laminin / fragment.

[0078] In a preferred embodiment, the culture medium material of the present invention is a culture medium material (such as a culture plate, a culture mesh, a culture dish, etc.) having a coating containing the laminin and / or its fragment, and more preferably a culture medium material that adsorbs the laminin and / or its fragment by coating with a coating agent containing the laminin and / or its fragment. In a preferred embodiment, in addition to the laminin and / or its fragment, the cell culture kit of the present invention may also contain, as needed, a culture medium material, a differentiation medium or its additive for inducing differentiation from pluripotent stem cells to NCS cells or from NCS cells to SKPs, a culture medium or its additive for maintaining the culture of pluripotent stem cells, etc. The laminin and / or its fragment contained in the cell culture kit of the present invention can be coated on the culture medium material, or can be contained in the coating agent or culture medium for coating the culture medium material.

[0079] In addition, as an exemplary embodiment, the present invention includes the following substances, manufacturing methods, uses, methods, etc. However, the present invention is not limited to these embodiments.

[0080] [1] A method for producing skin-derived pluripotent progenitor cells, wherein:

[0081] It includes a step of culturing neural crest stem cells in the presence of at least one selected from laminin and its fragments to differentiate them into skin-derived pluripotent progenitor cells,

[0082] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their mutants.

[0083] [2] The method according to [1], wherein preferably it further includes a step of producing the neural crest stem cells,

[0084] This step includes a step of culturing pluripotent stem cells in the presence of at least one selected from laminin and its fragments to differentiate them into neural crest stem cells,

[0085] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their mutants.

[0086] [3] The method according to [2], wherein preferably it further includes a step of pre-culturing the pluripotent stem cells in the presence of at least one selected from laminin and its fragments.

[0087] [4] The method according to any one of [1] to [3], wherein the laminin fragment is preferably a laminin fragment having integrin-binding activity,

[0088] More preferably, it is a laminin fragment containing the laminin E8 fragment.

[0089] [5] The method according to any one of [1] to [4], wherein at least one selected from the laminin and its fragments preferably includes the laminin or its fragment bound to perlecan or a fragment containing its proliferation factor-binding site,

[0090] More preferably, it is the laminin or its fragment bound to perlecan or a fragment containing its proliferation factor-binding site.

[0091] [6] According to the method described in [5], wherein the basement membrane proteoglycan or a fragment containing its proliferative factor binding site is preferably a fragment containing any one of domains I to III of the basement membrane proteoglycan,

[0092] more preferably a fragment containing domain I of the basement membrane proteoglycan.

[0093] [7] According to the method described in any one of [1] to [6], wherein the culturing of neural crest stem cells preferably in the presence of at least one selected from the laminin and its fragments includes: culturing the cells on a culture substrate containing at least one selected from the laminin and its fragments, or culturing the cells in a culture medium containing at least one selected from the laminin and its fragments.

[0094] [8] According to the method described in any one of [2] to [7], wherein the culturing of pluripotent stem cells preferably in the presence of at least one selected from the laminin and its fragments includes: culturing the cells on a culture substrate containing at least one selected from the laminin and its fragments, or culturing the cells in a culture medium containing at least one selected from the laminin and its fragments.

[0095] [9] According to the method described in [7] or [8], preferably, when the area of contact between the culture substrate and the culture is 1 cm 2 , the usage amount of at least one selected from the laminin and its fragments is preferably 0.05 - 50 μg, more preferably 0.1 - 10 μg, or as the final concentration per 1 mL of the culture medium is preferably 0.1 - 100 μg, more preferably 0.2 - 20 μg, or,

[0096] when at least one selected from the laminin and its fragments is added and used in the coating agent of the culture substrate, the content of at least one selected from the laminin and its fragments in the coating agent is preferably 0.05 - 50 μg, more preferably 0.1 - 10 μg per 1 cm 2 of the coating area.

[0097]

[10] According to the method described in any one of [1] to [9], wherein the culturing of the neural crest stem cells preferably includes the step of culturing the cells in a differentiation medium containing a Wnt signal agonist.

[0098]

[11] According to the method described in

[10] , wherein the Wnt signal agonist is preferably a factor that activates the β-catenin pathway,

[0099] more preferably a phosphorylase inhibitor of β-catenin,

[0100] More preferably, it is a GSK-3 inhibitor.

[0101] More preferably, it is at least one selected from the group consisting of aminopyrimidine compounds, bis-indole (indirubin) compounds (BIO) or their acetone oxime compounds, thiazolidine (TDZD) compounds, oxothiazolidine-3-thione compounds, thiophenyl α-chloromethyl ketone compounds, phenyl α-bromomethyl ketone compounds, thiazole-containing urea compounds, 1H-pyrazolo[3,4-b]quinoxalin-3-amine, (2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione, 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione, GSK-3β inhibitor XII, and GSK-3β peptide inhibitors.

[0102] More preferably, it is at least one selected from the group consisting of GSK-3 Inhibitor XVI, (2’Z,3’E)-6-bromoindirubin-3’-oxime (CAS 667463-62-9), 1H-pyrazolo[3,4-b]quinoxalin-3-amine, (2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione, 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione, and GSK-3β inhibitor XII.

[0103] More preferably, it is GSK-3 Inhibitor XVI.

[0104]

[12] According to the method described in

[10] or

[11] , wherein the concentration of the Wnt signal agonist in the differentiation medium is preferably 0.5 - 5 μM, more preferably 2 - 4 μM.

[0105]

[13] According to the method described in any one of [1] -

[12] , preferably, the culturing of the neural crest stem cells includes the step of culturing the cells in a differentiation medium containing a growth factor.

[0106] The growth factor is preferably at least one selected from the group consisting of serum substitute, EGF, and bFGF.

[0107] More preferably, it is serum substitute, EGF, and bFGF.

[0108]

[14] According to the method described in

[13] , wherein the concentration of the serum substitute in the differentiation medium is preferably 0.5 - 20% by mass, more preferably 1 - 5% by mass.

[0109] The concentrations of EGF and bFGF in the differentiation medium are preferably 1 to 100 ng / mL, more preferably 10 to 50 ng / mL.

[0110]

[15] The method according to any one of [2] to

[14] , wherein the step of preferably culturing the pluripotent stem cells to differentiate them preferably includes culturing the cells in a differentiation medium containing at least one selected from a TGFβ signal inhibitor and a BMP signal inhibitor.

[0111]

[16] The method according to

[15] , wherein preferably the TGFβ signal inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide, and the BMP signal inhibitor is noggin.

[0112]

[17] The method according to any one of [1] to

[16] , wherein preferably the skin-derived pluripotent progenitor cells co-express nestin and fibronectin.

[0113]

[18] The method according to any one of [1] to

[17] , wherein preferably the neural crest stem cells are human-derived neural crest stem cells.

[0114]

[19] The method according to any one of [1] to

[18] , wherein preferably the neural crest stem cells are neural crest stem cells derived from pluripotent stem cells.

[0115]

[20] The method according to any one of [1] to

[19] , wherein preferably the neural crest stem cells are cultured without using feeder cells.

[0116]

[21] The method according to any one of [1] to

[20] , wherein preferably the neural crest stem cells are cultured in the absence of xenogeneic components.

[0117]

[22] A method for producing neural crest stem cells, wherein

[0118] it includes a step of culturing pluripotent stem cells in the presence of at least one selected from laminin and its fragments, which are bound to perlecan or fragments containing its proliferation factor binding site, so that the pluripotent stem cells differentiate into neural crest stem cells.

[0119] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their mutants.

[0120]

[23] The method according to

[22] , wherein preferably, the method further comprises a step of preliminarily maintaining and culturing the pluripotent stem cells in the presence of at least one selected from laminin and its fragments, which are conjugated with basement membrane proteoglycan or fragments containing its proliferative factor-binding site.

[0121]

[24] The method according to

[22] or

[23] , wherein the laminin fragment is preferably a laminin fragment having integrin-binding activity,

[0122] and more preferably a laminin fragment containing a laminin E8 fragment.

[0123]

[25] The method according to any one of

[22] to

[24] , wherein the basement membrane proteoglycan or the fragment containing its proliferative factor-binding site is preferably a fragment containing any one of domains I to III of basement membrane proteoglycan,

[0124] and more preferably a fragment containing domain I of basement membrane proteoglycan.

[0125]

[26] The method according to any one of

[22] to

[25] , wherein the culturing of the cells in the presence of at least one selected from laminin and its fragments, which are conjugated with the basement membrane proteoglycan or fragments containing its proliferative factor-binding site, preferably includes: culturing the cells on a culture substrate containing at least one selected from laminin and its fragments, which are conjugated with the basement membrane proteoglycan or fragments containing its proliferative factor-binding site; or culturing the cells in a culture medium containing at least one selected from laminin and its fragments, which are conjugated with the basement membrane proteoglycan or fragments containing its proliferative factor-binding site.

[0126]

[27] The method according to

[26] , wherein preferably, when the culture substrate is in contact with the culture, the usage amount of at least one selected from laminin and its fragments, which are conjugated with the basement membrane proteoglycan or fragments containing its proliferative factor-binding site, is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg per 1 cm 2 of the contact area, or the final concentration in the culture medium is preferably 0.1 to 100 μg, more preferably 0.2 to 20 μg per 1 mL.

[0127] Alternatively,

[0128] when at least one selected from laminin and its fragments, which are conjugated with the basement membrane proteoglycan or fragments containing its proliferative factor-binding site, is added to the coating agent of the culture substrate for use, the coating area per 1 cm 2, the content of at least one selected from laminin and its fragments, which is selected from those conjugated with the basement membrane proteoglycan or fragments containing its proliferation factor binding site, in the coating agent is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg.

[0129]

[28] According to the method described in any one of

[22] to

[27] , wherein the step of preferably culturing the pluripotent stem cells to differentiate preferably includes culturing the cells in a differentiation medium containing at least one selected from a TGFβ signal inhibitor and a BMP signal inhibitor.

[0130] More preferably, the TGFβ signal inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide, and the BMP signal inhibitor is noggin.

[0131]

[29] According to the method described in any one of

[22] to

[28] , preferably the pluripotent stem cells are human-derived pluripotent stem cells.

[0132]

[30] According to the method described in any one of

[22] to

[29] , wherein preferably the pluripotent stem cells are cultured without using feeder cells.

[0133]

[31] According to the method described in any one of

[22] to

[30] , wherein preferably the pluripotent stem cells are cultured in the absence of xenogeneic components.

[0134]

[32] An adhesion material, which uses at least one selected from laminin and its fragments as an active ingredient, for the differentiation induction culture of neural crest stem cells into skin-derived pluripotent progenitor cells, wherein

[0135] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511 and laminin 521, and their mutants.

[0136]

[33] An adhesion material, which uses at least one selected from laminin and its fragments conjugated with the basement membrane proteoglycan or fragments containing its proliferation factor binding site as an active ingredient, for the differentiation induction culture of pluripotent stem cells into neural crest stem cells, wherein

[0137] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511 and laminin 521, and their mutants.

[0138]

[34] The anchoring material according to

[32] or

[33] , wherein the laminin fragment is preferably a laminin fragment having integrin-binding activity,

[0139] more preferably a laminin fragment containing a laminin E8 fragment.

[0140]

[35] The anchoring material according to any one of

[32] to

[34] , wherein at least one selected from the laminin and its fragments preferably contains the laminin or its fragment bound to a basement membrane proteoglycan or a fragment containing its growth factor binding site,

[0141] more preferably the laminin or its fragment bound to a basement membrane proteoglycan or a fragment containing its growth factor binding site.

[0142]

[36] The anchoring material according to any one of

[33] to

[35] , wherein the basement membrane proteoglycan or the fragment containing its growth factor binding site is preferably a fragment containing any one of domains I to III of the basement membrane proteoglycan,

[0143] more preferably a fragment containing domain I of the basement membrane proteoglycan.

[0144]

[37] The anchoring material according to any one of

[32] to

[36] , which is preferably a culture substrate containing at least one selected from the laminin and its fragments, or a culture substrate having a coating containing at least one selected from the laminin and its fragments, or a coating agent containing at least one selected from the laminin and its fragments, or a culture medium containing at least one selected from the laminin and its fragments.

[0145]

[38] The anchoring material according to

[37] , wherein for every 1 cm 2 of the area of contact between the culture substrate and the culture, the content of at least one selected from the laminin and its fragments in the culture substrate is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg,

[0146] or as the final concentration per 1 mL of the culture medium, the content of at least one selected from the laminin and its fragments in the culture medium is preferably 0.1 to 100 μg, more preferably 0.2 to 20 μg, or,

[0147] for every 1 cm 2 of the area coated with the coating agent, the content of at least one selected from the laminin and its fragments in the coating agent is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg.

[0148]

[39] A culture medium material for inducing the differentiation of neural crest stem cells into skin-derived pluripotent precursor cells, which contains the anchoring material described in any one of the above

[32] ,

[34] to

[38] .

[0149]

[40] A cell culture kit for inducing the differentiation of neural crest stem cells into skin-derived pluripotent precursor cells, which contains the anchoring material described in any one of the above

[32] ,

[34] to

[38] .

[0150]

[41] A culture medium material for inducing the differentiation of pluripotent stem cells into neural crest stem cells, which contains the anchoring material described in any one of the above

[33] to

[38] .

[0151]

[42] A cell culture kit for inducing the differentiation of pluripotent stem cells into neural crest stem cells, which contains the anchoring material described in any one of the above

[33] to

[38] .

[0152]

[43] Use of at least one selected from laminin and its fragments in the manufacture of an anchoring material for inducing the differentiation of neural crest stem cells into skin-derived pluripotent precursor cells, wherein,

[0153] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511 and laminin 521, and their mutants.

[0154]

[44] Use of at least one selected from laminin and its fragments conjugated with perlecan or a fragment containing its growth factor binding site in the manufacture of an anchoring material for inducing the differentiation of pluripotent stem cells into neural crest stem cells, wherein,

[0155] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511 and laminin 521, and their mutants.

[0156]

[45] Use of at least one selected from laminin and its fragments as an anchoring material for inducing the differentiation of neural crest stem cells into skin-derived pluripotent precursor cells, wherein,

[0157] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511 and laminin 521, and their mutants.

[0158]

[46] Use of at least one selected from laminin and its fragments, which is conjugated with a basement membrane proteoglycan or a fragment containing its proliferative factor binding site, as an anchoring material for inducing differentiation culture of pluripotent stem cells into neural crest stem cells, wherein,

[0159] The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their mutants.

[0160]

[47] The use according to any one of

[43] to

[46] , wherein the laminin fragment is preferably a laminin fragment having integrin binding activity,

[0161] More preferably, it is a laminin fragment containing a laminin E8 fragment.

[0162]

[48] The use according to any one of

[43] to

[47] , wherein at least one selected from the laminin and its fragments preferably contains the laminin or its fragment conjugated with a basement membrane proteoglycan or a fragment containing its proliferative factor binding site,

[0163] More preferably, it is the laminin or its fragment conjugated with a basement membrane proteoglycan or a fragment containing its proliferative factor binding site.

[0164]

[49] The use according to any one of

[44] ,

[46] to

[48] , wherein the basement membrane proteoglycan or the fragment containing its proliferative factor binding site is preferably a fragment containing any one of domains I to III of the basement membrane proteoglycan,

[0165] More preferably, it is a fragment containing domain I of the basement membrane proteoglycan.

[0166]

[50] The use according to any one of

[43] to

[49] , wherein preferably the anchoring material is a culture medium material containing at least one selected from the laminin and its fragments, or a culture medium material having a coating containing at least one selected from the laminin and its fragments, or a coating agent containing at least one selected from the laminin and its fragments, or a culture medium containing at least one selected from the laminin and its fragments.

[0167]

[51] The use according to

[50] , wherein for every 1 cm of the area of contact between the culture medium material and the culture, 2 the content of at least one selected from the laminin and its fragments in the culture medium material is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg, or

[0168] As the final concentration per 1 mL of the medium, the content of at least one selected from laminin and its fragments in the medium is preferably 0.1 to 100 μg, more preferably 0.2 to 20 μg, or

[0169] per 1 cm of the area coated with the coating agent 2 , the content of at least one selected from laminin and its fragments in the coating agent is preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg.

[0170]

[52] At least one selected from laminin and its fragments, which is used as an anchoring material for inducing the differentiation culture of neural crest stem cells into skin-derived pluripotent progenitor cells. Here, the laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their mutants.

[0171]

[53] At least one selected from laminin and its fragments, which is bound to basement membrane proteoglycan or a fragment containing its proliferation factor binding site and is used as an anchoring material for inducing the differentiation culture of pluripotent stem cells into neural crest stem cells. Among them, the laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and their mutants.

[0172]

[54] The method according to any one of [1] to

[31] , which is an ex vivo method.

[0173]

[55] The method according to any one of [1] to

[31] , wherein the culture does not include culturing on or within a living human or animal individual.

[0174]

[56] The application according to any one of

[43] to

[51] , which is an application in an ex vivo method.

[0175]

[57] The application according to any one of

[43] to

[51] , wherein the application does not include application on or within a living human or animal individual.

[0176] Examples

[0177] Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

[0178] Reference Example 1 Preparation of Laminin

[0179] As a laminin fragment for use as a fixing material, the E8 fragment of laminin 111 (LM111E8), the E8 fragment of laminin 121 (LM121E8), the E8 fragment of laminin 332 (LM332E8), the E8 fragment of laminin 421 (LM421E8), the E8 fragment of laminin 511 (LM511E8), and the E8 fragment of laminin 521 (LM521E8) were prepared according to the method described in International Publication No. 2014 / 103534.

[0180] Preparation of basement membrane proteoglycan-modified laminin in Reference Example 2

[0181] (1) Preparation of basement membrane proteoglycan-modified LM511E8 (P-LM511E8)

[0182] According to the method described in International Publication No. 2014 / 199754, the domain I (Gly 25 -Pro 196 ) of human basement membrane proteoglycan (hereinafter also referred to as Pln-D1) was fused to the C-terminal of the E8 fragment of the human laminin α5 chain to obtain a laminin 511E8 fragment (hereinafter also referred to as basement membrane proteoglycan-modified LM511E8, or P-LM511E8).

[0183] (1-1) Preparation of expression vectors for each E8 fragment of human laminin α5 chain, β1 chain, and γ1 chain

[0184] First, using the cloning plasmid pBluescript KS(+) (Stratagene) as a template, PCR was performed using the following 3 primer sets to prepare 3 types of pBluescript KS(+) into which a DNA sequence encoding a 6×His tag (HHHHHH), a DNA sequence encoding an HA (hemagglutinin) tag (VPVDVPDVA), or a DNA sequence encoding a FLAG tag (DVKDDDDK) was inserted on the 5' side of EcoRV within the multiple cloning site of the plasmid.

[0185] (i) Primer for introducing 6×His tag

[0186] 5'-ATGATGATGAAGCTTATCGATACCGT-3' (forward, SEQ ID NO: 1)

[0187] 5'-CATCATCATGATATCGAATTCCTGCA-3' (reverse, SEQ ID NO: 2)

[0188] (ii) Primer for introducing HA tag

[0189] 5’-ATCATATGGATAAAGCTTATCGATACCGT-3’ (Forward, Sequence No. 3)

[0190] 5’-GTGCCAGATTATGCAGATATCGAATTCCT-3’ (Reverse, Sequence No. 4)

[0191] (iii) Primers for FLAG tag introduction

[0192] 5’-ATCCTTGTAATCAAGCTTATCGATACCGT-3’ (Forward, Sequence No. 5)

[0193] 5’-GATGATGATAAGGATATCGAATTCCT-3’ (Reverse, Sequence No. 6)

[0194] Next, using the plasmid containing the full-length nucleotide sequences of human laminin α5 chain, β1 chain, and γ1 chain (The Journal of Biological Chemistry, 2004, 279: 10946 - 10954) as a template, PCR was performed using the following primers to amplify the regions corresponding to the E8 fragments of the α5 chain (Ala 2534 -Ala 3327 ), β1 chain E8 fragment (Leu 1561 -Leu 1786 ), and γ1 chain E8 fragment (Asn 1362 -Pro 1609 ).

[0195] (iv) Primers for amplifying the E8 fragment of the α5 chain

[0196] 5’-GCTGCCGAGGATGCTGCTGGCCAGG-3’ (Forward, Sequence No. 7)

[0197] 5’-CTAGGCAGGATGCCGGGCGGGCTGA-3’ (Reverse, Sequence No. 8) (v) Primers for amplifying the E8 fragment of the β1 chain

[0198] 5’-CTTCAGCATAGTGCTGCTGACATTG-3’ (Forward, Sequence No. 9)

[0199] 5’-TTACAAGCATGTGCTATACACAGCAAC-3’ (Reverse, Sequence No. 10)

[0200] (vi) Primers for amplifying the E8 fragment of the γ1 chain

[0201] 5’-AATGACATTCTCAACAACCTGAAAG-3’ (Forward, Sequence No. 11)

[0202] 5’-CTAGGGCTTTTCAATGGACGGGGTG-3’ (reverse, SEQ ID NO: 12)

[0203] After inserting the amplified DNA fragment into the EcoRV site of the multiple cloning site of pBluescript KS(+) appended with a tag sequence, the amplified DNA fragment containing the coding sequence for the 5'-side tag sequence was excised with restriction enzymes EcoRI and HindIII and inserted into the mammalian cell expression vector pSecTag2B (Invitrogen, containing the DNA sequence encoding the signal peptide of mouse Ig-κ chain V-J2-C), and expression vectors for human α5 chain E8 fragment (containing a 6×His tag on the N-terminal side), human β1 chain E8 fragment (containing an HA tag on the N-terminal side), and human γ1 chain E8 fragment (containing a FLAG tag on the N-terminal side) were respectively prepared.

[0204] (1-2) Preparation of expression vector for basement membrane proteoglycan domain-fused laminin α5 chain E8 fragment

[0205] Using the human laminin α5 chain E8 fragment expression vector as a template, PCR was performed with the following primers to amplify a DNA fragment encoding the C-terminal part (Leu 570 -Pro 772 ) of the human laminin α5 chain E8 fragment and the DNA fragment of the linker sequence between human laminin α1 chain G3-G4 domains (DAEDSKLLPEPRAFP, SEQ ID NO: 13).

[0206] (vii) Amplification primers for introducing linker sequence

[0207] 5’-CCTCAAGCGGCTGAACACGACAGGCG-3’ (forward, SEQ ID NO: 14)

[0208] 5’-ATATGGATCCTGGAAAAGCCCGGGGCTCTGGCAAGAGCTTGCTGTCCTCTGCATCAGGCCCCAGGCCCGG-3’ (reverse, SEQ ID NO: 15, containing the recognition sequence of restriction enzyme BamHI)

[0209] The obtained DNA fragment was digested with restriction enzymes AscI (the recognition sequence of this restriction enzyme exists within the DNA sequence encoding the C-terminal part of the human laminin α5 chain E8 fragment) and BamHI to prepare DNA fragment 1.

[0210] Using the human basement membrane proteoglycan expression vector (Journal of Biological Chemistry, 2010, 285(47):36645-36655) as a template, PCR was performed using the following primers to amplify a DNA fragment encoding the C-terminal of domain I (Pln-D1) of human basement membrane proteoglycan (Gly 25 -Pro 196 ) with a His-tag sequence attached.

[0211] (viii) Primers for amplifying Pln-D1 sequence

[0212] 5’-ATATATATGGATCCGGGCTGAGGGCATACGATGGCTTGTCTCTG-3’ (forward, sequence number 16, containing the restriction enzyme BamHI recognition sequence)

[0213] 5’-ATATATATGCGGCCGCCTAATGATGATGATGATGATGTGGGAACTGGGGCACTGTGCCCAG-3’ (reverse, sequence number 17, containing the restriction enzyme NotI recognition sequence)

[0214] The resulting DNA fragment was digested with restriction enzymes BamHI and NotI to prepare DNA fragment 2.

[0215] In the expression vector fragment containing the N-terminal part (Met 1 -Asp 610 ) of the human laminin α5 chain E8 fragment obtained by digesting the human laminin α5 chain E8 fragment expression vector with restriction enzymes AscI and NotI, the above DNA fragments 1 and 2 were inserted to construct a Pln-D1 fusion human laminin α5 chain E8 fragment expression vector.

[0216] (1-3) Expression and purification of Pln-D1 fusion laminin fragment

[0217] The Pln-D1 fusion human laminin α5 chain E8 fragment expression vector was mixed with the human β1 chain E8 fragment (containing an HA tag on the N-terminal side) expression vector and the human γ1 chain E8 fragment (containing a FLAG tag on the N-terminal side) expression vector, transfected into 293F cells derived from human kidney, cultured for 72 hours, and then the culture medium was recovered. The E8 fragment of Pln-D1 fusion laminin 511 (P-LM511E8) was purified by affinity chromatography using Ni-NTA agarose and ANTI-FLAG M2 affinity Gel (Sigama).

[0218] (2) Preparation of basement membrane proteoglycan-modified LM111E8 (P-LM111E8)

[0219] Except for changing the α5 chain E8 to α1 chain E8, the E8 fragment of laminin 111 fused with Pln-D1 (P-LM111E8) was prepared according to the same steps as in (1).

[0220] (3) Preparation of basement membrane proteoglycan-modified LM121E8 (P-LM121E8)

[0221] The α5 chain E8 was changed to α1 chain E8, and the β1 chain E8 was changed to β2 chain E8. Except for this, the E8 fragment of laminin 121 fused with Pln-D1 (P-LM121E8) was prepared according to the same steps as in (1).

[0222] (4) Preparation of basement membrane proteoglycan-modified LM332E8 (P-LM332E8)

[0223] Except that the α chain, β chain and γ chain used α3 chain, β3 chain and γ2 chain, the E8 fragment of laminin 332 fused with Pln-D1 (P-LM332E8) was prepared according to the same steps as in (1).

[0224] (5) Preparation of basement membrane proteoglycan-modified LM421E8 (P-LM421E8)

[0225] Except that the α chain, β chain and γ chain used α4 chain, β2 chain and γ1 chain, the E8 fragment of laminin 421 fused with Pln-D1 (P-LM421E8) was prepared according to the same steps as in (1).

[0226] (6) Preparation of basement membrane proteoglycan-modified LM521E8 (P-LM521E8)

[0227] Except for changing the β1 chain E8 to β2 chain E8, the E8 fragment of laminin 521 fused with Pln-D1 (P-LM521E8) was prepared by the same steps as in (1).

[0228] Example 1 Construction of laminin anchoring material

[0229] (1) Construction of laminin anchoring material

[0230] Coat various laminins prepared in Reference Example 1 or laminin modified with basement membrane proteoglycan prepared in Reference Example 2 on the culture substrate in advance, or add them to the culture medium simultaneously with the cells. In the case of pre-coating, dissolve various laminins in Dulbecco’s phosphate-buffered saline (DPBS) (manufactured by Life Technologies, catalog number: 14190-250), and spread the resulting laminin solution (containing 2.5 μg / mL of laminin) on the surface of the culture substrate (35 mm diameter culture dish) (final concentration of laminin: about 0.5 μg / cm of the coating area) 2 ). After standing at 37 °C for 1 hour, wash with DPBS to form a coating on the culture substrate. In the case of adding to the culture medium, adjust the amount of various laminins added to the culture medium so that the final concentration in the culture medium is in the range of 1.25 μg / mL. In the culture environment containing these laminin anchoring materials, perform cell culture in the following examples.

[0231] Example 2 Production of skin-derived pluripotent progenitor cells (SKPs)

[0232] (1) Culture of iPS cells

[0233] As pluripotent stem cells, human-derived iPS cells (clone name: 1231A3, purchased from CiRA, Kyoto University iPS Research Institute) were used. Among them, the 1231A3 strain is an iPS cell obtained by introducing pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1 into peripheral blood mononuclear cells of an African American female using an episomal vector. The iPS cells were maintained and cultured according to the culture method recommended by the obtaining institution. That is, as a culture medium for human iPS cells, StemFit (registered trademark) (AK02N, Ajinomoto Co., Inc.) was used, and the cells were cultured in an incubator at 37 °C with 5% CO 2 2 according to the method described in Sci.Rep, 4, 2014, 3594; DOI: 10.1038 / srep03594. As laminin fragments for the anchoring material, LM111E8, LM121E8, LM332E8, LM421E8, LM511E8, and LM521E8 (pre-coated on the culture dish in advance according to the steps described in Example 1) were used.

[0234] (2) Preparation of neural crest stem (NCS) cells derived from human iPS cells

[0235] Differentiate the iPS cells cultured in (1) into NCS cells based on the methods described in Nature Protocols, 2010, 5: 688-701 or Cell Reports, 2013, 3: 1140-1152. Induce the differentiation into NCS cells by culturing the iPS cells in StemFit (registered trademark) AK02N (without additive C) medium containing noggin (manufactured by R&D Systems, catalog number: 6057-NG-100 / CF, 500 ng / mL) and / or SB431542 (manufactured by TOCRIS, catalog number: 1614, 10 μM) for 5 days to 2 weeks. Among them, as the attachment material in the differentiation induction culture, no passage is performed during the differentiation induction from iPS cells to NCS cells. Therefore, the coated laminin fragment still exists during the iPS cell culture.

[0236] (3) Induce the differentiation from NCS cells into SKPs

[0237] Using DMEM / F12 medium (catalogue number: 10565-018, manufactured by Life Technologies) containing B-27 (trademark) supplement (manufactured by Life Technologies, catalogue number: 17504-044, 2% by mass), EGF (manufactured by R&D Systems, catalogue number: 336-EG-200, 20 ng / mL), bFGF (manufactured by Wako, catalogue number: 064-04541, 40 ng / mL), penicillin / streptomycin (manufactured by Life Technologies, catalogue number: 15140-122, 50 U, 50 μg / mL) and CHIR99021 (manufactured by Cayman, catalogue number: 13122, 3 μM), the NCS cells prepared in (2) were cultured for 3 to 5 days to induce the differentiation of NCS cells into SKPs. Among them, as the adherent material in the differentiation induction culture, no passage was carried out during the differentiation induction from NCS cells to SKPs. Therefore, during the culture of iPS cells, the coated laminin fragment still remained. Then, using a cell culture isolation / dispersion solution (trade name: Accutase, manufactured by BD Biosciences, catalogue number: 561527), the cells differentiated into SKPs were subcultured without the laminin fragment adherent material. Then, they were further cultured in DMEM / F12 medium containing B-27 (trademark) supplement (2% by mass), EGF (20 ng / mL), bFGF (40 ng / mL), and penicillin / streptomycin (manufactured by Life Technologies, catalogue number: 15140-122, 50 U, 50 μg / mL).

[0238] (4) Expression of labeled protein

[0239] The expression of marker proteins in the cells induced to differentiate in (3) was studied. For the subcultured cells (iPS-SKPs P1) induced to differentiate in the presence of laminin, the expression of marker proteins (nestin and fibronectin) specific to SKPs was studied by immunohistofluorescence staining. The cells were washed with D-PBS(-), fixed with 4% paraformaldehyde for 15 minutes. After washing the fixed cells with D-PBS(-), they were treated with a PBS solution of Triton X-100 (0.5% by mass) for 5 minutes, washed again with D-PBS(-), and blocked with 10% goat serum (manufactured by Nichirei Corporation, catalog number: 426041) at room temperature for 1 hour. Thereafter, the cells were treated with the primary antibody shown in Table 2 (room temperature, 2 hours) and the secondary antibody (room temperature, 1 hour), stained with Hoechst 33258 (manufactured by Dojindo Laboratories, catalog number: H341), embedded in Fluoromount G (manufactured by Southern Biotech, catalog number: 0100-01), and the expression of the marker protein was observed under a fluorescence microscope.

[0240] [Table 2]

[0241]

[0242] (5) Results

[0243] Micrographs showing the induction of differentiation from NCS cells to SKPs are shown in Figure 1 . iPS-NCS ( Figure 1 , paragraph 1) shows the micrograph of the cells before the start of differentiation induction, iPS-SKPs P0 ( Figure 1 , paragraph 2) shows the micrograph of the cells 4 days after differentiation induction (before passage), and iPS-SKPs P1 ( Figure 1 , paragraph 3) shows the micrograph of the cells after subculture (all magnifications are 40 times). In addition, the expression of the marker proteins (nestin and fibronectin) of SKPs in the obtained cells was observed under a fluorescence microscope. Among them, through the simultaneous Hoechst staining, it was confirmed that viable cells were present at the positions where marker expression was observed ( Figure 1 , paragraph 4) (all magnifications are 50 times). As Figure 1 shown, differentiation from NSC to SKPs was observed in the culture environment containing fragments of laminin 111, 121, 332, 421, 511, and 521. In particular, in the presence of fragments of laminin 111, 332, 421, and 511, differentiation into SKPs was induced with high efficiency.

[0244] Preparation of SKPs in the presence of modified laminin fragments in Example 3

[0245] (1) Preparation of SKPs

[0246] According to the same steps as (1) to (3) in Example 2, NCS cells were prepared from iPS cells, and SKPs were induced to differentiate from the obtained NCS cells. The concentration of CHIR99021 in the culture medium for inducing differentiation of SKPs was set to 3 μM. The culture period for inducing differentiation of SKPs was set to 4 days. As the laminin fragments used as the attachment materials in the maintenance culture of iPS cells, the induction of differentiation into NCS cells, and the induction of differentiation into SKPs, LM111E8, LM121E8, LM332E8, LM421E8, LM511E8, and LM521E8, as well as the basement membrane proteoglycan-modified laminin fragments P-LM111E8, P-LM121E8, P-LM332E8, P-LM421E8, P-LM511E8, and P-LM521E8 (all pre-coated on the culture dish according to the steps described in Example 1) were used. The differentiated SKPs were passaged in the absence of the laminin fragment attachment material. The total number of days of culture from iPS cells to obtaining SKPs was 9 days.

[0247] The microscopic photographs of the SKPs obtained in the step shown in (1) are shown in Figure 2 . As Figure 2 shown, in the presence of the basement membrane proteoglycan-modified laminin fragment, an increase in the differentiation induction efficiency from NSC to SKPs was observed compared with the culture in the presence of the unmodified laminin fragment.

[0248] (2) Expression of marker genes

[0249] The expression of marker genes in the cells induced to differentiate in (1) was studied. For the cells induced to differentiate in the presence of the basement membrane proteoglycan-modified laminin fragment (P-LM111E8), the expression of the marker genes shown in Table 3 was studied by PCR method before passage culture (iPS-SKPs P0) and after passage culture (iPS-SKPs P1) after differentiation induction. Among the markers shown in Table 3, the Nestin gene, Slug gene, Sox9 gene, Dermo-1 gene, BMP-4 gene, and Wnt-5a gene are SKPs markers (Nat Cell Biol, 2004, 6: 1082-1093, Stem Cell, 2005, 23: 727-737), and the Versican gene and CD133 gene are dermal papilla cell markers (J Dermatol Sci, 39, 2005, 147-154). The GAPDH gene was used as a control.

[0250] Total RNA was extracted from the cell samples using the RNeasy Mini kit (manufactured by QIAGEN, catalog number: 74104). The concentration of the extracted total RNA was measured, and using a certain amount of total RNA, a reverse transcription reaction was carried out using the High capacity RNA-to-cDNA Kit (manufactured by Applied Biosystems, catalog number: 4387406). Using 1 μL of the obtained cDNA sample as a template, PCR reaction was carried out in a 50 μL system using the primer sets shown in Table 3. The enzyme used was KOD-Plus-Ver.2 (manufactured by TOVOBO, catalog number: KOD-211). The PCR was carried out according to the reaction program of [94°C, 2 min → (98°C, 10 s; 63°C, 30 s; 68°C, 30 s) × 25 - 35 cycles]. Using 1.5% agarose gel (manufactured by Takara Bio, catalog number: 50071) / TBE buffer (manufactured by Kanto Chemical Co., Inc., catalog number: 46510-78), 5 μL of the reaction solution was electrophoresed at 100 V.

[0251] [Table 3]

[0252]

[0253] The results of the electrophoresis are shown in Figure 3 . As Figure 3 shown, the expression of SKPs marker genes was detected in the cells after differentiation induction. Most of these SKPs markers showed no change in expression between P0 and P1, or increased expression in P1. Therefore, it was confirmed that the cells induced to differentiate in (1) were SKPs.

[0254] (3) Expression of marker proteins

[0255] The expression of marker proteins in the cells induced to differentiate in (1) was studied. For the cells after subculture (iPS-SKPs P1) induced to differentiate in the presence of proteoglycan-modified laminin fragment (P-LM111E8), according to the same steps as in Example 2

[0256] (4) Through immunohistofluorescence staining, the expression of marker proteins (nestin, fibronectin, and αSMA) specific to SKPs was studied. The primary and secondary antibodies used are shown in Table 4.

[0257] [Table 4]

[0258]

[0259] The results are shown in Figure 4 . Among them, Figure 4 (A), (B), and (C) are fluorescence microscope photos showing the expression of nestin, fibronectin, and αSMA, respectively. As Figure 4 shown, the expression of marker proteins specific to SKPs can be seen in almost all cells after subculture. Therefore, it was confirmed that the cells induced to differentiate in (1) are SKPs.

[0260] Example 4 Differentiation induction of SKPs into tissue cells

[0261] For the SKPs after subculture induced to differentiate in the presence of proteoglycan-modified laminin fragment (P-LM111E8) obtained in Example 3, the ability to differentiate into tissue cells was studied.

[0262] (1) Differentiation induction into adipocytes

[0263] The subcultured SKPs were seeded at 3×10 5Cells were seeded in 35-mm diameter culture dishes. After culturing for 24 hours, the medium was replaced with MEM medium (manufactured by Life Technologies, catalog number: 42360-032) containing 3-isobutyl-1-methylxanthine (manufactured by Sigma Aldrich, catalog number: I7018, 0.45 nM), insulin (manufactured by Sigma Aldrich, catalog number: I3536, 2.07 μM), dexamethasone (manufactured by Sigma Aldrich, catalog number: D4902, 100 nM), rabbit serum (manufactured by Sigma Aldrich, catalog number: R4505, 15%), and penicillin / streptomycin (manufactured by Life Technologies, catalog number: 15140-122, 100 U, 100 μg / mL), and the cells were further cultured for 2 weeks. Thereafter, an Oil Red O staining Kit (manufactured by ScienCell Research Laboratories, catalog number: 0843) was used to perform Oil Red O staining of the cells according to the attached instructions.

[0264] (2) Induction of differentiation into osteocytes

[0265] The passaged SKPs were seeded at 3×10 5 cells in 35-mm diameter culture dishes. After culturing for 24 hours, the medium was replaced with MEM medium (manufactured by Life Technologies, catalog number: 42360-032) containing dexamethasone (manufactured by Sigma Aldrich, catalog number: D4902, 100 nM), β-glycerophosphate (manufactured by Sigma Aldrich, catalog number: G9422, 10 mM), L-ascorbic acid-2-phosphate (manufactured by Sigma Aldrich, catalog number: A8960, 50 μM), bovine serum (manufactured by Hyclone, catalog number: SH30070.03, 10% by mass), and penicillin / streptomycin (manufactured by Life Technologies, catalog number: 15140-122, 100 U, 100 μg / mL), and the cells were further cultured for 2 weeks. Thereafter, an Alkaline Phosphatase Substrate Kit (manufactured by Vector Laboratories, catalog number: SK-5300) was used to perform alkaline phosphatase staining of the cells according to the attached instructions.

[0266] (3) Induction of differentiation into Schwann cells

[0267] After passage culture, SKPs were used with a culture medium for SKPs [DMEM / F12 medium (manufactured by Life Technologies, catalog number: 10565-018) containing 2% B-27 (trademark) additive (manufactured by Life Technologies, catalog number: 17504-044), 20 ng / mL EGF (manufactured by R&D Systems, catalog number: 336-EG-200), 40 ng / mL bFGF (manufactured by Wako, catalog number: 064-04541), 50 U penicillin, 50 μg / mL streptomycin (manufactured by Life Technologies, catalog number: 15140-122)], at 4.8×10 4Cells were seeded in a culture dish (35 mm diameter culture dish) coated with laminin 111 (manufactured by Sigma Aldrich, catalog number: L4544) diluted 25-fold in advance and 0.1 mg / mL poly-L-lysine (manufactured by Sigma Aldrich, catalog number: P4707). After culturing for 24 hours, the medium was changed to DMEM / F12 medium (manufactured by Life Technologies, catalog number: 10565-018) containing 5 μM Forskoline (manufactured by Sigma Aldrich, catalog number: F3917), 50 ng / mL Heregulin-1β (manufactured by Peprotech, catalog number: 100-03), 2% N2 supplement (manufactured by Life Technologies, catalog number: 17502-048), and 1% bovine serum (manufactured by Hyclone, catalog number: SH30070.03E), and further cultured for 2 to 3 weeks. During the culture, the medium was changed every 2 to 3 days. The obtained cells were washed with D-PBS(-) and fixed with 4% paraformaldehyde for 15 minutes. After washing the fixed cells with D-PBS(-), they were treated with a PBS solution containing 0.5% Triton X-100 for 5 minutes, washed again with D-PBS(-), and blocked with 10% goat serum (manufactured by Nichirei, catalog number: 426041) at room temperature for 1 hour. Then, the cells were treated with a primary antibody (anti-S100β antibody, manufactured by Sigma Aldrich, catalog number: S2532, at room temperature for 2 hours) and a secondary antibody (Alexa Fluor 488 goat anti-mouse IgG(H+L), manufactured by Life Technologies, catalog number: A11029, at room temperature for 1 hour). Thereafter, nuclear staining was performed with DAPI (manufactured by DOJINDO, catalog number: FK045), and the cells were embedded in Fluoromount G (manufactured by Southern Biotech, catalog number: 0100-01), and the expression of the marker protein (S100β) specific to Schwann cells was observed under a fluorescence microscope.

[0268] (4) Microscopic observation

[0269] The microscopic images of the stained cells are shown in Figure 5 . Figure 5 (A), lipid stained in the cells was observed, indicating the differentiation of SKPs into adipocytes. Figure 5 (B), alkaline phosphatase-positive cells were seen, indicating the differentiation of SKPs into osteocytes.Figure 5 In (C), S100β-positive cells can be seen, indicating that SKPs differentiate into Schwann cells. Therefore, it was confirmed that the cells obtained in Example 3 are SKPs that can differentiate into glial cells such as adipocytes, osteocytes, and Schwann cells.

[0270] Preparation of SKPs under xeno-free conditions in Example 5

[0271] Under xeno-free conditions in the same steps as (1) to (3) of Example 2, NCS cells were prepared from iPS cells, and SKPs were induced to differentiate from the obtained NCS cells. As xeno-free reagents, noggin (manufactured by SIGMA, catalog number: H66416-10UG, 500 ng / mL), B-27 (trademark) additive XenoFree CTS (manufactured by Life Technologies, catalog number: A14867-01, 2% by mass), EGF (manufactured by HIGETA Shoyu Co., Ltd., catalog number: REG100UG, 20 ng / mL), and bFGF (manufactured by ReproCell, catalog number: RCHEOT005, 006, 40 ng / mL) were used. The concentration of CHIR99021 in the differentiation induction medium for SKPs was set to 3 μM. The culture period for the differentiation induction of SKPs was set to 4 days. As laminin fragments for use as attachment materials in the maintenance culture of iPS cells, the differentiation induction into NCS cells, and the differentiation induction into SKPs, LM111E8, LM511E8, and the basement membrane proteoglycan-modified laminin fragments P-LM111E8 and P-LM511E8 (all pre-coated on culture dishes according to the steps described in Example 1) were used.

[0272] Micrographs showing the differentiation induction of the obtained NCS cells into SKPs are shown in Figure 6 . iPS-NCS ( Figure 6 above) shows micrographs of the cells after the start of differentiation induction, and iPS-SKPs P0 ( Figure 6 middle) shows micrographs of the cells 4 days after differentiation induction (before passage), and iPS-SKPs P1 ( Figure 6 below) shows micrographs of the cells after passage culture (all magnifications are 40 times). As Figure 6 shown, in the presence of laminin fragments and basement membrane proteoglycan-modified laminin fragments, differentiation into SKPs can also be carried out under xeno-free conditions.

[0273] Example 6 Identification of neural crest stem (NCS) cells derived from human iPS cells

[0274] The properties of NCSs induced to differentiate in the presence of the laminin fragment and the basement membrane proteoglycan-modified laminin fragment (LM111E8 and P-LM111E8) obtained in the steps (1) to (2) of Example 2 were studied. According to the steps of Example 2(2), using cells from before the differentiation culture of NCS cells (0 days of differentiation, 0 d) to 8 days after culture (8 d), following the same steps as in Example 3(2), 2 μL of the cDNA sample was used as a template, and the primers and probes shown in Table 5 were used to perform a PCR reaction in a 20 μL system. Taqman Fast Universal PCR Master Mix (manufactured by Applied Biosystems, catalog number: 4352042) was used in the PCR. As an index of differentiation into NCS, the gene expression of the nerve growth factor receptor (p75) was detected. As an internal standard, RPLP0 was used. Regarding the change in gene expression, based on the expression level normalized by the internal standard, it was expressed as the relative expression level when the expression level of each gene at 0 days of differentiation (0 d) was set to 1.

[0275] [Table 5]

[0276] Gene name Primer ribosomal protein lateral stalk subunit P0(RPLP0) Hs99999902_m1 nerve growth factor receptor(p75) Hs00609976_m1

[0277] The results are shown in Figure 7 . As the differentiation induction proceeded, an increase in the expression of p75, a marker gene for NCS cells, was observed. Therefore, it was found that differentiation induction into NCS cells can be carried out on the laminin fragment and the modified laminin fragment.

[0278] Example 7 Differentiation induction rate of SKPs in the presence of laminin fragment

[0279] SKP differentiation induction was carried out in the same manner as in Example 3(1). As the laminin fragment used as the anchoring material, LM111E8, LM121E8, LM332E8, LM421E8, LM511E8, and LM521E8 as laminin fragments, and P-LM111E8, P-LM121E8, P-LM332E8, P-LM421E8, P-LM511E8, and P-LM521E8 as basement membrane proteoglycan-modified laminin fragments were used. The differentiated-induced SKPs (before passage: iPS-SKPs P0) were passaged at the same dilution rate in a 48-well plate (iPS-SKPs P1). The passage culture was carried out without the laminin fragment anchoring material. The cell number of the culture 3 days after passage was measured using Cell Counting Kit-8 (DOJINDO LABORATORIES, catalog number: 347-07621). After adding the reagent solution of the kit to the culture and culturing in an incubator with 5% CO 2 for a certain period of time (1 to 3 hours) at 37°C, the absorbance at 450 nm was measured with a microplate reader, and the respiratory activity (number of living cells) of the culture was evaluated therefrom.

[0280] The results are shown in Figure 8 . For all types of laminin fragments, the number of cells induced to differentiate using the basement membrane proteoglycan-modified laminin fragments was significantly more statistically than that of the non-modified fragments (t-test, P < 0.0005). Sequence Listing <110> Kao Corporation <110> The National University Corporation Osaka University <120> Method for Producing Skin-Derived Pluripotent Progenitor Cells <130> KS1675 <150> JP 2019-144899 <151> 2019 / 08 / 06 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 atgatgatga agcttatcga taccgt 26 <210> 2 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 2 catcatcatg atatcgaatt cctgca 26 <210> 3 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 3 atcatatgga taaagcttat cgataccgt 29 <210> 4 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 4 gtgccagatt atgcagatat cgaattcct 29 <210> 5 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 5 atccttgtaa tcaagcttat cgataccgt 29 <210> 6 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 6 gatgatgata aggatatcga attcct 26 <210> 7 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 gctgccgagg atgctgctgg ccagg 25 <210> 8 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 8 ctaggcagga tgccgggcgg gctga 25 <210> 9 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 9 cttcagcata gtgctgctga cattg 25 <210> 10 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 10 ttacaagcat gtgctataca cagcaac 27 <210> 11 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 11 aatgacattc tcaacaacct gaaag 25 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 12 ctagggcttt tcaatggacg gggtg 25 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 13 Asp Ala Glu Asp Ser Lys Leu Leu Pro Glu Pro Arg Ala Phe Pro 1 5 10 15 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 cctcaagcgg ctgaacacga caggcg 26 <210> 15 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 atatggatcc tggaaaagcc cggggctctg gcaagagctt gctgtcctct gcatcaggcc 60 ccaggcccgg 70 <210> 16 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 atatatatgg atccgggctg agggcatacg atggcttgtc tctg 44 <210> 17 <211> 61 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 atatatatgc ggccgcctaa tgatgatgat gatgatgtgg gaactggggc actgtgccca 60 g 61 <210> 18 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Forward primer of GAPDH <400> 18 cggagtcaac ggatttggtc g 21 <210> 19 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of GAPDH <400> 19 agccttctcc atggtggtga a 21 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer of nestin <400> 20 cagcgttgga acagaggttg 20 <210> 21 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of nestin <400> 21 gctggcacag gtgtctcaag 20 <210> 22 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Forward primer of Slug <400> 22 catctttggg gcgagtgagt cc 22 <210> 23 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of Slug <400> 23 cccgtgtgag ttctaatgtg tc 22 <210> 24 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer of Sox9 <400> 24 gtcagccagg tgctcaaagg 20 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of Sox9 <400> 25 acttgtaatc cgggtggtcc 20 <210> 26 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Forward primer of Dermo-1 <400> 26 gcaagaagtc gagcgaagat g 21 <210> 27 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of Dermo-1 <400> 27 ggcaatggca gcatcattca g 21 <210> 28 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Forward primer of BMP-4 <400> 28 ttctgcagat gtttgggctg c 21 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of BMP-4 <400> 29 agagccgaag ctctgcagag 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer of Wnt-5a <400> 30 ggatggctgg aagtgcaatg 20 <210> 31 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of Wnt-5a <400> 31 acacaaactg gtccacgatc 20 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer of versican <400> 32 acgatgccta ctttgccacc 20 <210> 33 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of versican <400> 33 tagtgaaaca caaccccatc c 21 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer of CD133 <400> 34 atggccctcg tactcggctc 20 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of CD133 <400> 35 cacgcggctg taccacatag 20

Claims

1. Method for producing skin-derived pluripotent precursor cells, wherein: It includes the step of culturing neural crest stem cells in the presence of at least one selected from laminin and its fragments to differentiate them into skin-derived pluripotent precursor cells, The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and the laminin fragment is a laminin fragment having integrin-binding activity and containing the laminin E8 fragment.

2. The method according to claim 1, wherein, It further includes the process of producing the neural crest stem cells, This process includes the step of culturing pluripotent stem cells in the presence of at least one selected from laminin and its fragments to differentiate them into neural crest stem cells, The laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, and laminin 521, and the laminin fragment is a laminin fragment having integrin-binding activity and containing the laminin E8 fragment.

3. The method according to claim 1 or 2, wherein, At least one selected from the laminin and its fragments is the laminin or its fragment conjugated with perlecan or a fragment containing its proliferative factor-binding site.

4. The method according to claim 1 or 2, wherein, The culturing of neural crest stem cells in the presence of at least one selected from the laminin and its fragments includes: culturing the cells on a culture substrate containing at least one selected from the laminin and its fragments, or culturing the cells in a culture medium containing at least one selected from the laminin and its fragments.

5. The method according to claim 2, wherein, The culturing of pluripotent stem cells in the presence of at least one selected from the laminin and its fragments includes: culturing the cells on a culture substrate containing at least one selected from the laminin and its fragments, or culturing the cells in a culture medium containing at least one selected from the laminin and its fragments.

6. The method according to claim 1 or 2, wherein, The culturing of the neural crest stem cells includes the step of culturing the cells in a differentiation medium containing a Wnt signal agonist.

7. The method according to claim 2, wherein, The culturing of the pluripotent stem cells includes the step of culturing the cells in a differentiation medium containing at least one selected from TGFβ signal inhibitors and BMP signal inhibitors.

8. The method according to claim 1 or 2, wherein, The skin-derived pluripotent precursor cells co-express nestin and fibronectin.

9. The method according to claim 1 or 2, wherein, The neural crest stem cells are human-derived neural crest stem cells.

10. The method according to claim 1 or 2, wherein, The cell culture is carried out without using feeder cells.

11. The method according to claim 1 or 2, wherein, The cell culture is carried out in the absence of xenogeneic components.

12. Use of at least one selected from laminin and its fragments in the preparation of an anchoring material for inducing the differentiation of neural crest stem cells into skin-derived pluripotent precursor cells, wherein, the laminin is at least one selected from laminin 111, laminin 121, laminin 332, laminin 421, laminin 511 and laminin 521, and the laminin fragment is a laminin fragment having integrin-binding activity and containing a laminin E8 fragment.

13. The use according to claim 12, wherein, the at least one selected from laminin and its fragments is the laminin or its fragment bound to perlecan or a fragment containing its growth factor-binding site.

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