Methods for inducing differentiation from pluripotent stem cells to ectodermal cells and methods for making such cells

Abstract

To provide methods for inducing differentiation to ectodermal cells excellent in differentiation-inducing efficiency from pluripotent stem cells to ectodermal cells and excellent in culture operability as well as methods for making such cells.SOLUTION: Methods for inducing differentiation from pluripotent stem cells to ectodermal cells comprise the steps of: (1) coating a cell culture substrate with one or more substances selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin and collagen where the substrate comprises a region having cell adhesiveness and cell-proliferating property and a region having neither of them and then seeding pluripotent stem cell 1; (2) conducting adhesion culture of the seeded pluripotent cells in the presence of an undifferentiation-maintaining medium in feeder-free conditions to form agglutination of pluripotent stem cells 2 adhered to the cell culture substrate; (3) culturing the formed agglutination of pluripotent stem cells in the presence of a culture medium containing a differentiation-inducing agent so that the cells adhered to the cell culture substrate form germ layer 3; and (4) culturing the formed germ layer in the presence of a culture medium containing the differentiation-inducing agent in the adhered state to the cell culture substrate to form ectodermal cell aggregates 4.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for inducing differentiation from pluripotent stem cells into ectodermal cells, and a production method thereof, which is excellent in differentiation induction efficiency and culture operability. [Background technology]

[0002] There is growing interest in drug discovery using neural cells induced to differentiate from induced pluripotent stem cells (iPS cells) for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). By utilizing iPS cells derived from patients with the disease, it is expected that the mechanism of disease, which was previously difficult to elucidate, and drug discovery research and regenerative medicine will progress. In general, differentiation into neural cells is induced through the formation of pluripotent stem cell aggregates called embryoid bodies and neural stem cell aggregates called neurospheres, and then differentiation into each peripheral nerve cell is induced. In addition, it is known that the state of the embryoid body during the differentiation induction process into the target cell greatly contributes to the cell fate, such as the differentiation pathway, and how to efficiently produce homogeneous embryoid bodies is an issue.

[0003] In conventional methods, embryoid bodies and neurospheres have been prepared (e.g., Patent Document 3) by suspension culture methods such as hanging drop culture (e.g., Patent Document 1) and non-adhesive surface culture (e.g., Patent Document 2). However, the above culture methods have the problem that the size of the aggregates formed is non-uniform, which tends to cause differences in differentiation induction efficiency between individuals.

[0004] In addition, since cell aggregates of different sizes are arranged three-dimensionally, there is a risk of localized nutritional deficiency. Another method using the suspension culture method is to seed cells on a cell-non-adhesive substrate having a fine uneven surface, and then allow the cells to settle to form cell aggregates according to the uneven surface size (for example, Patent Document 4). However, in the method of Patent Document 4, since the cell aggregates are not fixed to the substrate surface, there is a problem that the culture operation requires skill, such as a part of the cell aggregates being removed together with the medium when the medium is replaced. In addition, due to the nature of the substrate, it is difficult to replace the entire amount of the medium, so nutritional deficiency of differentiation induction factors and the like is likely to occur, and there is a concern that the differentiation induction efficiency is poor. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2008 / 001938 [Patent Document 2] International Publication No. 2017 / 123791 [Patent Document 3] JP 2002-291469 A [Patent Document 4] International Publication No. 2018 / 123663 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the invention is to provide a method for inducing differentiation from pluripotent stem cells to ectodermal cells and a production method thereof, which are excellent in differentiation induction efficiency and culture operability. [Means for solving the problem]

[0007] The inventors discovered that the above-mentioned problems can be solved by seeding pluripotent stem cells onto a cell culture substrate having island-shaped cell adhesion regions to form embryoid bodies adhered to the cell culture substrate, and inducing differentiation of the adhered embryoid bodies, thereby completing the present invention.

[0008] That is, the present invention provides a method for inducing differentiation of pluripotent stem cells into ectodermal cells and a method for producing ectodermal cells, which comprises the following steps (1) to (4). <1> A method for inducing differentiation of pluripotent stem cells into ectodermal cells, comprising the following steps (1) to (4): (1) A process for coating a cell culture substrate having the following two regions (A) and (B) with one or more substances selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin, and collagen, and then seeding pluripotent stem cells on the substrate. (A) Area with cell adhesion and cell proliferation: 0.001 to 5 mm 2 An island-like area. (B) A region adjacent to the region (A), which does not have cell adhesiveness or cell proliferation properties. (2) A step of subjecting the pluripotent stem cells seeded in the step (1) to feeder-free adherent culture in the presence of an undifferentiated state maintenance medium to form pluripotent stem cell aggregates adhered onto a cell culture substrate. (3) A step of culturing the pluripotent stem cell aggregates formed in the step (2) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form embryoid bodies. (4) A step of culturing the embryoid bodies formed in the step (3) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form ectodermal cell aggregates.

[0009] <2> The cell culture substrate contains a hydrophilic polymer layer on its surface, and the (A) region is a region in which a part of the hydrophilic polymer layer is decomposed or modified by any one of plasma treatment, ultraviolet light treatment, and corona discharge treatment, or a combination thereof. <1> The differentiation induction method described in the above. <3> The differentiation induction factor in the step (3) contains an ectodermal induction factor, a mesodermal induction factor, and an endodermal induction factor. <1> or <2> The differentiation induction method described in the above. <4> The ectodermal cell aggregate formed in the step (4) is characterized in that it has PAX6 and SOX1. <1> ~ <3> The differentiation induction method according to any one of the preceding claims. <5> The ectodermal cell is a neural stem cell or a neural cell. <1> ~ <4> The differentiation induction method according to any one of the preceding claims. <6> The ectodermal cells are motor neurons. <1> ~ <5> The differentiation induction method according to any one of the preceding claims.

[0010] <7> A method for producing cells obtained by inducing differentiation of pluripotent stem cells into ectodermal cells, comprising the following steps (1) to (4): (1) A process for coating a cell culture substrate having the following two regions (A) and (B) with one or more substances selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin, and collagen, and then seeding pluripotent stem cells on the substrate. (A) Area with cell adhesion and cell proliferation: 0.001 to 5 mm 2 An island-like area. (B) A region adjacent to the region (A), which does not have cell adhesiveness or cell proliferation properties. (2) A step of subjecting the pluripotent stem cells seeded in the step (1) to feeder-free adherent culture in the presence of an undifferentiated state maintenance medium to form pluripotent stem cell aggregates adhered onto a cell culture substrate. (3) A step of culturing the pluripotent stem cell aggregates formed in the step (2) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form embryoid bodies. (4) A step of culturing the embryoid bodies formed in the step (3) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form ectodermal cell aggregates. Effect of the Invention

[0011] The present invention provides a method for inducing differentiation from pluripotent stem cells to ectodermal cells, which is excellent in differentiation induction efficiency and culture operability. Embryoid bodies induced to differentiate in a culture system immobilized on a cell culture substrate having the above-mentioned regions (A) and (B) can maintain any shape (diameter of the embryoid body, aspect ratio of the embryoid body), and are less affected by the concentration gradient of differentiation induction factors, making it possible to synchronize the differentiation progress of each cell. Furthermore, by inducing differentiation in a culture system in which cell aggregates are feeder-free and immobilized on a cell culture substrate, the inclusion of impurities can be reduced. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of the differentiation induction method of the present invention. [Diagram 2] Schematic diagram of the cross section of a cell culture substrate. [Diagram 3] 1 is a phase-contrast micrograph of an embryoid body on a cell culture substrate in Example 1. [Figure 4] Graph showing the change over time in diameter and aspect ratio of embryoid bodies in Example 1. [Diagram 5] 1 is a phase-contrast micrograph showing pseudopod extension at the outer edge of embryoid bodies in Example 1 and Comparative Example 1. [Figure 6] 4 is a phase-contrast micrograph of embryoid bodies in Comparative Examples 2 to 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment for carrying out the present invention (hereinafter, simply referred to as "the present embodiment") will be described in detail. The present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be carried out by appropriately modifying it within the scope of its purpose.

[0014] As used herein, "pluripotent stem cells" refer to cells that have the property of being able to differentiate into various cells (undifferentiated or pluripotent). In addition, as used herein, "ectodermal cells" refers to a general term for cells contained in the ectoderm formed by early embryos (ectodermal cells) or cells contained in tissues derived from the ectoderm (including neural stem cells and neural cells).

[0015] Furthermore, the term "neural stem cells" refers to cells in a state where differentiation induction into neuroectoderm has progressed and they can be induced to differentiate into glial cells (astrocytes, oligodendrocytes), central nerves (dopamine nerves, GABA nerves), and peripheral nerves (motor nerves, sensory nerves). In the present specification, "maintenance of undifferentiated state" refers to a state in which cultured cells have pluripotency. Methods for evaluating the maintenance of undifferentiated state are not particularly limited, and examples include analysis of cell surface markers by alkaline phosphatase staining, analysis of cell surface / cell nuclear markers by immunostaining and flow cytometry, analysis of gene expression levels by real-time RT-PCR, confirmation of formation of germinal bodies, which are unique structures formed by pluripotent stem cells, and teratoma assays to determine in vivo differentiation into three germ layers from pathological sections.

[0016] In addition, in this specification, "differentiation" refers to a state in which the expression of membrane proteins, transcription factors, etc. specific to a cell type located downstream of pluripotent stem cells in the differentiation induction pathway of pluripotent stem cells is confirmed. Furthermore, "differentiation induction" refers to progressing cell differentiation by culturing cells in the presence of specific proteins or genes, natural products, synthetic chemicals, etc. In addition, as used herein, the term "cell aggregate" refers to a three-dimensional structure formed by the aggregation of multiple cells. Furthermore, the term "embryoid body" refers to a spherical cell aggregate observed in the early stage of embryo development, and exhibits properties of ectoderm, mesoderm, and endoderm similar to those of early embryos.

[0017] In addition, in this specification, the term "marker" refers to a protein or gene specific to a particular cell. A marker possessed by a pluripotent stem cell is referred to as an "undifferentiated marker", a marker possessed by an ectodermal cell is referred to as an "ectodermal marker", a marker possessed by a mesodermal cell is referred to as a "mesodermal marker", a marker possessed by an endoderm cell is referred to as an "endodermal marker", a marker possessed by a neural stem cell is referred to as a "neural stem cell marker", and a marker possessed by a neuronal cell is referred to as a "neuronal cell marker". Pluripotent stem cells do not possess ectodermal, mesodermal, or endodermal markers. Furthermore, an embryoid body possesses ectodermal, mesodermal, and endodermal markers. In this specification, "cell adhesiveness" refers to the ease with which cells adhere to a cell culture substrate at culture temperature, "having cell adhesiveness" refers to the ability of cells to adhere to a cell culture substrate at culture temperature, and "not having cell adhesiveness" refers to the inability of cells to adhere to a cell culture substrate at culture temperature.

[0018] In addition, in this specification, "cell proliferation ability" refers to the ease with which cells proliferate at the culture temperature, "having cell proliferation ability" refers to the ability of cells to proliferate at the culture temperature, and "not having cell proliferation ability" refers to the inability of cells to proliferate at the culture temperature.

[0019] The differentiation induction method of the present invention uses a cell culture substrate having the following two regions: (A) Area with cell adhesion and cell proliferation: 0.001 to 5 mm 2 An island-like area. (B) A region adjacent to the region (A) and not having cell adhesive properties. By using a cell culture substrate having the (A) region as the cell culture substrate, pluripotent stem cells can be adherently cultured on the cell culture substrate in step (1) described below, and an embryoid body adhered to the cell culture substrate can be formed in step (2) described below. If the (A) region is not present, pluripotent stem cells cannot be adherently cultured on the cell culture substrate, and an embryoid body adhered to the cell culture substrate cannot be formed. Furthermore, by using a cell culture substrate having the (B) region as the cell culture substrate, pluripotent stem cells can be grown only in the prophase (A) region, and an embryoid body of uniform size can be formed. By forming an embryoid body of uniform size, differentiation induction efficiency can be increased. If the (B) region is not present, an embryoid body of uniform size cannot be formed.

[0020] The area of ​​the region (A) is preferably 0.005 to 3 mm in order to form an embryoid body of a size suitable for differentiation into ectodermal cells. 2 More preferably, the area is 0.01 to 1.0 mm 2 is particularly preferred, and the area is 0.03 to 0.8 mm 2 is most preferred.

[0021] The shape of the region (A) is not particularly limited, but is preferably a circle, ellipse, or regular polygon, and more preferably a circle, as these are suitable for increasing the efficiency of differentiation induction.

[0022] The cell culture substrate used in the differentiation induction method of the present invention is suitable for forming uniform-sized embryoid bodies and increasing differentiation induction efficiency, and therefore preferably contains a hydrophilic polymer layer on its surface, and region (A) is a region in which a part of the hydrophilic polymer layer has been decomposed or modified by any one of plasma treatment, ultraviolet light treatment, corona discharge treatment, or a combination of these. By having a hydrophilic polymer layer on the surface of the cell culture substrate, adsorption of proteins that contribute to substrate-cell adhesion in region (B) can be suppressed, making it a region that does not have cell adhesiveness or cell proliferation properties.

[0023] In addition, by decomposing or modifying a part of the hydrophilic polymer layer, cell adhesiveness or cell proliferation properties can be imparted to region (A). Furthermore, it is more preferable that region (A) is a plasma-treated region, since this enhances the cell adhesiveness and cell proliferation properties of region (A) and is suitable for forming an embryoid body in a short period of time.

[0024] The thickness of the hydrophilic polymer layer is preferably 10 nm or more, more preferably 50 nm or more, particularly preferably 100 nm or more, and most preferably 500 nm or more, since it is suitable for making the (B) region a region having no cell adhesiveness or cell proliferation properties. Also, the thickness is preferably 1000 nm or less, more preferably 500 nm or less, particularly preferably 100 nm or less, and most preferably 50 nm or less, since it is suitable for making the (A) region a region having cell adhesiveness and cell proliferation properties.

[0025] The method for forming a layer using the hydrophilic polymer can be at least one of a method for forming a chemical bond and a method using physical interaction. Examples of the method for forming a chemical bond include a method for forming a reactive functional group, such as ultraviolet irradiation, electron beam irradiation, gamma ray irradiation, plasma treatment, and corona treatment. In addition, a crosslinking reaction on the substrate surface by an organic reaction using ions or radicals as a reaction source is also possible. Examples of the method using physical interaction include coating, brush coating, dip coating, spin coating, bar coding, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating, which use a matrix having excellent compatibility with the target hydrophilic polymer as a coating material. It is possible to use the following technique.

[0026] The type of the hydrophilic polymer is not particularly limited, but examples include those having a polar group such as a hydroxy group, an amino group, or a polyethylene glycol group, and those having an amphoteric structure such as a betaine structure or a phosphorylcholine group. Since these are suitable for making the (B) region a region that does not have cell adhesiveness and cell proliferation properties, a hydroxy group, a phosphorylcholine group, or a polyethylene glycol group is preferred, a hydroxy group or a phosphorylcholine group is more preferred, and a phosphorylcholine group is particularly preferred.

[0027] The hydrophilic polymer is preferably a random copolymer or block copolymer having both hydrophilic and hydrophobic monomer units, since it is suitable for suppressing the elution of the hydrophilic polymer from the cell culture substrate and suppressing the influence on quality caused by the polymer being mixed into cell aggregates or embryoid bodies, and more preferably a random copolymer having both hydrophilic and hydrophobic monomer units. In addition, as the composition ratio of the copolymer, since it is suitable for making the (B) region a region having no cell adhesiveness or cell proliferation properties, the hydrophilic monomer units are preferably 30 wt% or more, more preferably 40 wt% or more, particularly preferably 50 wt% or more, and most preferably 60 wt% or more. Furthermore, since it is suitable for suppressing the elution of the hydrophilic polymer, the hydrophobic monomer units are preferably 20 wt% or more, more preferably 30 wt% or more, particularly preferably 40 wt% or more, and most preferably 50 wt% or more.

[0028] The hydrophilic monomer unit is not particularly limited other than being hydrophilic, and examples thereof include those having an amino group such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, and the like. acrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate, and other polyethylene glycol groups and methoxyethyl groups; methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide, and other acrylate groups;2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexylphosphat; Examples of the phosphorylcholine group include those having a phosphorylcholine group, such as phosphorylcholine, 10-acrylamidodecylphosphorylcholine, and ω-(meth)acrylamide(poly)oxyethylenephosphorylcholine.

[0029] The hydrophobic monomer unit is not particularly limited other than being hydrophobic, and examples thereof include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate.

[0030] The hydrophilic polymer also preferably contains a reactive monomer unit, which is suitable for suppressing the elution of the hydrophilic polymer. As the reactive monomer unit, a UV-reactive monomer unit is preferable, which is capable of immobilizing the hydrophilic polymer on the substrate by a short-term treatment, and examples of the reactive monomer unit include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate.

[0031] The hydrophilic polymer may also contain a temperature-responsive monomer unit, for example, a (meth)acrylamide compound such as acrylamide or methacrylamide; N,N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, Nt-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfuryl Examples of the compound include N-alkyl-substituted (meth)acrylamide derivatives such as acrylamide and N-tetrahydrofurfuryl methacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide and N,N-diethylacrylamide; (meth)acrylamide derivatives having a cyclic group such as 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide.

[0032] Other methods for producing the cell culture substrate used in the differentiation induction method of the present invention include a method in which a substance that promotes or inhibits cell adhesion is coated on a portion of the cell culture substrate by photolithography or inkjet printing, and a method in which the surface of the cell culture substrate is treated with plasma, ultraviolet light, corona discharge, or a combination of these, and then coated with a temperature-responsive polymer.

[0033] The cell culture substrate used in the differentiation induction method of the present invention may be sterilized. There is no particular limitation on the sterilization method, but high-pressure steam sterilization, UV sterilization, gamma ray sterilization, ethylene oxide gas sterilization, etc. can be used. High-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred because they are suitable for suppressing denaturation of the block copolymer, UV sterilization and ethylene oxide gas sterilization are more preferred because they are suitable for suppressing deformation of the substrate, and ethylene oxide gas sterilization is particularly preferred because it is excellent in mass productivity of the cell culture substrate.

[0034] In the present invention, the substrate used for preparing the cell culture substrate is not particularly limited, but commonly used polymer compounds such as glass, polystyrene, polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polyethylene, polypropylene, and polyethylene methacrylate, ceramics, and metals can be used. Polystyrene is most preferred because of its excellent transparency and ease of molding and surface modification.

[0035] The differentiation induction method and production method of the present invention are characterized in that differentiation is induced through the following steps (1) to (4). (1) A step of coating a cell culture substrate with one or more substances selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin, and collagen, and seeding pluripotent stem cells thereon. (2) A step of subjecting the pluripotent stem cells seeded in the step (1) to feeder-free adherent culture in the presence of an undifferentiated state maintenance medium to form pluripotent stem cell aggregates adhered onto a cell culture substrate. (3) A step of culturing the pluripotent stem cell aggregates formed in the step (2) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form embryoid bodies. (4) A step of culturing the embryoid bodies formed in the step (3) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form ectodermal cell aggregates. Step (1) in the differentiation induction method of the present invention is a step of coating the cell culture substrate with one or more substances selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin, and collagen, and seeding pluripotent stem cells. By coating the region (A) with one or more substances selected from the group consisting of Matrigel, laminin, vitronectin, fibronectin, and collagen, cell adhesiveness and cell proliferation properties can be imparted to the region (A). If none of Matrigel, laminin, vitronectin, fibronectin, and collagen is coated, cell adhesiveness and cell proliferation properties cannot be imparted. Since these are suitable for imparting cell adhesiveness and cell proliferation properties, a combination of at least four types including laminin is more preferable, and a combination of laminin and Matrigel, laminin and fibronectin, or laminin and collagen is particularly preferable, and laminin alone is most preferable.

[0036] The matrigel, laminin, vitronectin, fibronectin and collagen may be natural products, or may be artificially synthesized using recombinant gene technology or the like, or may be fragments obtained by cleavage with restriction enzymes or the like, or synthetic proteins or synthetic peptides based on these biological substances.

[0037] In the present invention, as the Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Gibco) can be suitably used due to their ease of availability.

[0038] The type of laminin is not particularly limited, and for example, laminin 511, laminin 521, or laminin 511-E8 fragment, which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells, can be used. The laminin may be a natural product, may be artificially synthesized by recombinant gene technology, or may be a synthetic protein or peptide based on the laminin. In view of ease of availability, for example, iMatrix-511 (manufactured by Nippi Corporation) can be suitably used as a commercially available product.

[0039] The vitronectin may be a natural product, may be artificially synthesized by gene recombination technology, or may be a synthetic protein or peptide based on the vitronectin. Examples of suitable antibodies that can be used include human plasma-derived tyrosine (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Gibco).

[0040] The fibronectin may be a natural product, may be artificially synthesized by gene recombination technology, or may be a synthetic protein or peptide based on the fibronectin. In view of availability, commercially available products such as fibronectin solution, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.) and Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

[0041] The type of collagen is not particularly limited, and for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, may be artificially synthesized by gene recombination technology, or may be a synthetic peptide based on the collagen. In view of availability, for example, collagen I, human (manufactured by Corning Incorporated) or collagen IV, human (manufactured by Corning Incorporated) can be preferably used as a commercially available product.

[0042] The type of cells in the step (1) can be appropriately selected from stem cells having differentiation pluripotency such as ES cells and iPS cells, but iPS cells are preferred because they are suitable for applying the differentiation induction method of the present invention to regenerative medicine and drug discovery. The cell seeding method in the step (1) is not particularly limited, but includes a method in which a cell suspension in which cells are monodispersed is added to a cell culture substrate, or a method in which cell aggregates are added to a cell culture substrate. Since cell aggregates are uniformly formed in the (A) region, it is preferable to monodisperse and seed the cells. When human-derived iPS cells are monodispersed and cultured, it is preferable to add a Rho-associated kinase (ROCK) inhibitor at the time of seeding in order to suppress apoptosis due to low cell density. As the ROCK inhibitor, for example, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·H2O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name: Y-27632) and the like can be used. The concentration of the ROCK inhibitor added to the medium is within a range that is effective for maintaining the survival of human cells and does not affect the undifferentiated state of human cells, and is preferably 1 μM to 50 μM, more preferably 3 μM to 20 μM, even more preferably 5 μM to 15 μM, and most preferably 8 μM to 12 μM. In addition, the ROCK inhibitor is preferably removed 24 hours after cell seeding by a method such as replacing the medium with one not containing a ROCK inhibitor.

[0043] The cell recovery method is not particularly limited, but for example, enzymatic treatment with trypsin or collagenase, chelating treatment with ethylenediamineacetic acid (EDTA), physical treatment with a scraper, etc., can be used alone or in combination. When using enzymatic treatment, it is preferable that the treatment is free of animal-derived components. From the viewpoint of easy availability, commercially available products such as TrypLE Select (Gibco), TrypLE Express (Gibco), Accutase (Nacalai Tesque), and Accumax (Nacalai Tesque) can be preferably used.

[0044] The cell seeding density in the step (1) is set to 1.0×102 cells / cm, which is suitable for forming uniform cell aggregates in a short time. 2 More than 1.2×10 is preferable. 3 cells / cm 2 More preferably, 2.0×10 3 cells / cm 2 More than 3.0×10 is particularly preferable. 3 cells / cm 2 More than 1.0×10 is most preferable. In addition, it is preferable to suppress cell death due to nutrient deficiency in the medium. 5 cells / cm 2 Less than 5.0×10 is preferable. 4 cells / cm 2 Less than or equal to 2.5×10 is even better. 4 cells / cm 2 The following is particularly preferred: 5.0×10 3 cells / cm 2 The following are most preferred:

[0045] In the step (2) of the differentiation induction method of the present invention, the pluripotent stem cells seeded in the step (1) are cultured in a feeder-free manner in the presence of an undifferentiated state maintenance medium to form pluripotent stem cell aggregates adhered to the cell culture substrate. In the present invention, "feeder-free" refers to a method of directly seeding and culturing pluripotent stem cells on a cell culture substrate without using cells (feeder cells) inactivated by gamma-ray irradiation or the like. By culturing the pluripotent stem cells seeded in the step (1) in a feeder-free manner in the presence of an undifferentiated state maintenance medium in the presence of an undifferentiated state maintenance medium, the pluripotent stem cells can proliferate in the (A) region and form cell aggregates layered in the outward direction of the substrate surface. If an undifferentiated state maintenance medium is not used, the cell aggregates lose their differentiation pluripotency, and the efficiency of inducing differentiation into ectodermal cells decreases. In addition, by culturing in a feeder-free manner, it is easier to maintain the nutrients of the medium during culture, and the cell survival rate and maintenance of undifferentiation can be increased. In addition, because the method is an adherent culture, it is easier to change the medium compared to a suspension culture, making it easier to culture, and because the entire medium can be changed, the concentration of waste products excreted from the cells can be kept low. In addition, the effect of concentration gradients in the culture system is low, making it excellent in differentiation induction efficiency.

[0046] The undifferentiated state maintenance medium preferably contains at least one of factors that act to maintain the undifferentiated state of pluripotent stem cells, such as basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), insulin-like growth factor (IGF), activin A, Wnt, insulin, transferrin, ethanolamine, 2-mercaptoethanol, selenic acid, oleic acid, and sodium bicarbonate, and most preferably contains bFGF, which has a high effect of maintaining the undifferentiated state. The type of medium is not particularly limited, and examples thereof include culture media obtained by adding culture supplements such as factors that maintain undifferentiated state and non-essential amino acids to a basal medium such as DMEM, Ham's F12, or D-MEM / Ham's F12; Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.), StemFit AK03 (manufactured by Ajinomoto Co., Inc.), mTeSR1 (manufactured by STEMCELL TECHNOLOGIES), TeSR-E8 (manufactured by STEMCELL Examples of commercially available undifferentiated state maintenance media include those available from TECHNOLOGIES, ReproNaive (REPROCELL), ReproXF (REPROCELL), ReproFF (REPROCELL), ReproFF2 (REPROCELL), NutriStem (Biological Industries), iSTEM (Takara Bio), GS2-M (Takara Bio), and hPSC Growth Medium DXF (PromoCell). Primate ES Cell Medium, StemFit AK02N, and StemFit AK03 are preferred, StemFit AK02N and StemFit AK03 are more preferred, and StemFit AK02N (Ajinomoto Co., Inc.) is the most preferred, as they are suitable for stably maintaining the undifferentiated state of cells. Furthermore, since serum that is generally used in cell culture media also contains differentiation-inducing factors, it is preferable that the medium for maintaining the undifferentiated state of pluripotent stem cells is a serum-free medium.

[0047] In step (2), a ROCK inhibitor similar to that in step (1) may be added to increase the survival rate of the pluripotent stem cells. The suitable type and concentration of the ROCK inhibitor are the same as those described above.

[0048] In addition, in the step (2), the number of cells per unit area of ​​the (A) region is set to 1.0 × 10 because this is suitable for increasing the efficiency of differentiation induction in the steps (3) and (4) described below. 4 cells / cm 2 It is preferable to culture until the cell density reaches 2.0 × 10 or more. 4 cells / cm 2 More preferably, 5.0×10 4 cells / cm 2 More than 7.5×10 is particularly preferable. 4 cells / cm 2 More than this is most preferable. In addition, since this is suitable for maintaining the nutrients in the medium and increasing the cell survival rate in steps (3) and (4), the number of cells per unit area of ​​the (A) region is preferably 5.0 × 10 6 cells / cm 2 It is preferable to proceed to step (3) if the 6 cells / cm 2 Less than 5.0×10 is more preferable. 5 cells / cm 2 The following is particularly preferred: 1.0×10 5 cells / cm 2 The following are most preferred: In order to achieve the number of cells per unit area of ​​the (A) region, the culture in step (2) is preferably carried out for 1 to 48 hours, more preferably 6 to 36 hours, particularly preferably 12 to 36 hours, and most preferably 18 to 30 hours.

[0049] In addition, since this is suitable for increasing the efficiency of differentiation induction in steps (3) and (4) described below, it is preferable that the shape of the embryoid body formed in step (2) is hemispherical. In step (3) of the differentiation induction method of the present invention, the pluripotent stem cell aggregates formed in step (2) are cultured in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation induction factor to form embryoid bodies.

[0050] The differentiation induction factor in the step (3) preferably contains an ectodermal induction factor, a mesodermal induction factor and an endodermal induction factor, since it is suitable for increasing the efficiency of differentiation induction into an embryoid body. The ectodermal induction factor is preferably any one of Noggin (BMP inhibitor), dorsomorphin (BMP inhibitor), SB431542 (TGF-β inhibitor) and an activin inhibitor, since it is suitable for increasing the efficiency of differentiation induction into an embryoid body, and more preferably contains a BMP inhibitor and a TGF-β inhibitor. In addition, the mesodermal induction factor is preferably any one of activin A, CHIR99021 (GSK3β inhibitor) and bone morphogenetic protein (BMP) 4, since it is suitable for increasing the efficiency of differentiation induction into an embryoid body, and more preferably contains a GSK3β inhibitor. Furthermore, the endodermal induction factor may be a low molecular weight compound such as activin A or CHIR99021.

[0051] Furthermore, the concentration of the differentiation-inducing factor added to the culture medium is not particularly limited, but is preferably 0.1 to 10 μM, more preferably 1.0 to 7.5 μM, and most preferably 2.0 to 5.0 μM, which is suitable for increasing the efficiency of inducing differentiation into embryoid bodies. The medium containing the differentiation-inducing factor is preferably changed every 24 hours during culture in order to ensure that differentiation induction proceeds sufficiently. By changing the medium within 24 hours, it is possible to prevent a shortage of the differentiation-inducing factor in the medium and to allow all cells to differentiate uniformly.

[0052] As the ectodermal markers possessed by the embryoid body formed in the step (3), PAX6, Nestin, SOX1, SOX2, SOX10, Notch1, E Cadherin, and MAP2 are preferable, and PAX6, Nestin, SOX1, and SOX2 are more preferable. As the mesodermal markers, Tbx1, Brachyury, MSX1, and Flk-1 are preferable, and Tbx1 and Brachyury are more preferable. As the endoderm markers, FOXA2, SOX17, GATA4, GATA6, CXCR4, HNF3β, HNF4α, and αFP are preferable, and FOXA2 and SOX17 are more preferable. As the neural stem cell markers, PAX6, Nestin, Olig2, SOX1, SOX2, and DCX are preferable, and PAX6, Nestin, SOX1, and Olig2 are more preferable.

[0053] In the step (4) of the differentiation induction method of the present invention, the embryoid body formed in the step (3) is cultured in a state of being attached to a cell culture substrate in the presence of a medium containing a differentiation induction factor to form an ectodermal cell aggregate. By inducing differentiation of the embryoid body formed in the step (3) in a state of being attached to a cell culture substrate, it is expected that the differentiation will proceed uniformly since there is little influence due to the concentration gradient of the induction factor. The medium is not particularly limited, and examples thereof include a culture medium obtained by adding a factor acting for ectodermal induction and a culture supplement to a basal medium such as DMEM, Ham's F12, or D-MEM / Ham's F12.

[0054] The type of cells to be induced to differentiate from the embryoid body in the step (4) is not particularly limited except that they are ectodermal cells, but for example, epidermal ectoderm-derived cells and neuroectoderm-derived cells are preferred, and neuroectoderm-derived cells are more preferred because they are suitable for use in regenerative medicine and drug discovery. The neuroectoderm-derived cells are not particularly limited, but for example, Schwann precursor cells, myelin Schwann cells, non-myelin Schwann cells, radial glial cells, oligodendrocyte precursor cells, oligodendrocytes, astrocytes, and other glial cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, cholinergic neurons, motor neurons, sensory neurons, and other nerve cells are preferred. When the ectoderm-derived cells are nerve cells, it is preferable to culture them for 5 to 14 days in the presence of a medium containing B27 supplements, TGF-β inhibitors, GSK3β inhibitors, LIF, bFGF, retinoic acid, and Purmorphamine, and then culture them for 3 to 5 days in the presence of a medium containing B27 supplements, TGF-β inhibitors, GSK3β inhibitors, LIF, bFGF, retinoic acid, Purmorphamine, and DAPT. When differentiating them into motor nerve cells, it is preferable to culture them for 5 to 40 days in the presence of a medium containing B27 supplements, rhBDNF, rhGDNF, ascorbic acid, retinoic acid, and DAPT after the above-mentioned culture of the nerve cells. EXAMPLES

[0055] The present invention will be described in detail below with reference to the embodiments for carrying out the present invention. However, these are merely examples for explaining the present invention, and are not intended to limit the present invention to the following contents. Furthermore, the present invention can be practiced with appropriate modifications within the scope of the gist of the present invention. Unless otherwise specified, commercially available reagents were used. <Diameter and aspect ratio of embryoid body> The embryoid bodies were observed over time using an inverted phase-contrast microscope (Olympus Corporation, model number: IX73) and a digital camera (Olympus Corporation, model number: DP73) to measure the lengths of the long and short axes. The aspect ratio (long axis / short axis) was calculated from the determined long and short axis lengths and used to evaluate the morphology of the embryoid bodies. In the examples of the present invention, a circular pattern was formed, and the aspect ratio would theoretically be 1.0.

[0056] <Composition of differentiation-inducing medium, etc.> Differentiation induction medium 1: 80% DMEM / F-12 (Fujifilm Wako Pure Chemical Industries, Ltd.), 20% KnockOut Serum Replacement XenoFree (Thermo Fisher Scientific), 0.1 mM non-essential amino acids (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 4 ng / mL basic fibroblast growth factor (bFGF), 3 μM SB431542 (Fujifilm Wako Pure Chemical Industries, Ltd.), 3 μM dorsomorphin (Sigma-Aldrich), 3 μM CHIR99021 (REPROCELL) Differentiation induction medium 2: 98% KBM serum-free medium for neural stem cell culture (Fujifilm Wako Pure Chemical Industries, Ltd.), 2% B27 supplement (Invitrogen), 20 ng / mL bFGF, 10 ng / mL recombinant human LIF (hLIF, Merck & Co., Ltd.), 2 μM SB431542, 3 μM CHIR99021, 2 μM retinoic acid (Sigma-Aldrich), 1 μM Purmorphamine (Merck & Co., Ltd.) Differentiation induction medium 3: 98% KBM serum-free medium for neural stem cell culture, 2% B27 supplement, 2ng / mL bFGF, 10ng / mL hLIF, 2μM SB431542, 3μM CHIR99021, 2μM retinoic acid, 1μM Purmorphamine, 5μM DAPT (added from "differentiation day 16", Sigma-Aldrich) Cell detachment solution: 1:1 mixture of TrypLE select (Gibco) and 0.5 mM EDTA solution (Invitrogen) Membrane permeabilization solution: 0.3% TritonX-100 (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% bovine serum albumin-containing PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) solution Blocking solution, antibody diluent, and flow cytometry analysis buffer: 1% BSA in PBS(-) [Example 1] Preparation of pluripotent stem cell aggregates A 35 mm diameter dish (manufactured by Sumitomo Bakelite Co., Ltd., product name: PrimeSurface (registered trademark)) coated on the surface with a hydrophilic polymer was covered with a metal mask (manufactured by Mitani Micronics Co., Ltd.) having multiple circular holes with a diameter of 0.2 mm. A plasma irradiation device (manufactured by Vacuum Device Co., Ltd., product name: Plasma Ion Bombarder PIB-20) was used to perform plasma treatment (gas pressure of 20 Pa, conduction current of 20 mA, irradiation time of 30 seconds) from above the metal mask to produce a cell culture substrate with areas of cell adhesiveness and cell proliferation.

[0057] StemFitAK02N (Ajinomoto Co., Inc.), an undifferentiated maintenance medium, was added to the patterned cell culture substrate at 2.0 mL / dish, and human iPS cells 201B7 strain were further cultured at 3900 cells / cm. 2 Then, iMatrix-511 solution (manufactured by Nippi Corporation) was added at a concentration of 2.5 μL / mL. The cells were cultured at 37°C in an environment with a CO2 concentration of 5%. In addition, Y-27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium until 24 hours after cell seeding. Multiple hemispherical cell aggregates adhered to the cell culture substrate were confirmed by observation under a phase contrast microscope.

[0058] Preparation of embryoid bodies 24 hours after cell seeding, 2.0 mL / dish of differentiation induction medium 1 was added to start differentiation induction. The time point when differentiation induction medium 1 was added was designated as "differentiation day 0". Thereafter, differentiation induction medium 1 was replaced every 24 hours of culture until "differentiation day 5". In addition, when the medium was replaced, the long and short axes of the embryoid bodies were measured using a phase contrast microscope according to the above procedure, and it was confirmed that embryoid bodies with a diameter of 0.2 mm, which is the hole size of the metal mask, were uniformly formed. In addition, the aspect ratio remained at 1.0 until differentiation day 5, and the shape of the embryoid bodies was also uniform, suggesting that the differentiation progress of the cells that make up the embryoid bodies was synchronized.

[0059] Preparation of ectodermal cell aggregates After inducing differentiation until "differentiation day 5", differentiation induction medium 1 was removed from the dish and differentiation induction medium 2 was added at 2.0 mL / dish. Thereafter, differentiation induction medium 2 was replaced every 48 hours of culture until "differentiation day 12". On "differentiation day 10", embryoid bodies were observed to be detached from the substrate surface. On "differentiation day 12", differentiation induction medium 2 in the dish was removed and differentiation induction medium 3 was added at 2.0 mL / dish. Thereafter, differentiation induction medium 3 was replaced every 48 hours of culture until "differentiation day 19".

[0060] <Measurement of cell number of embryoid body> On the "5th day of differentiation", differentiation induction medium 1 in the dish was removed, PBS(-) was added at 2.0 mL / dish, and the dish was washed. After washing, cell detachment solution was added at 1.0 mL / dish to the dish, and the dish was left to stand for 3 minutes at 37°C and CO2 concentration of 5%. After leaving the dish to stand for a predetermined time, the cell detachment solution was removed, differentiation induction medium 1 containing Y-27632 (concentration 10 μM) was added at 1.0 mL / dish, and the embryoid bodies were detached and dispersed from the substrate using a cell scraper (IWAKI). The obtained cell suspension was collected, and the number of cells was counted using a Luna automatic cell counter (Logos Biosystems).

[0061] <Cell counting of ectodermal cell aggregates> On the 19th day of differentiation, remove the differentiation-inducing medium 3 from the dish and add 2 portions of PBS(-). 1.0 mL / dish was added and washed. After washing, 1.0 mL / dish of cell detachment solution was added to the dishes and left to stand for 3 minutes at 37°C and 5% CO2 concentration. After leaving to stand for a specified time, the cell detachment solution was removed, 1.0 mL / dish of differentiation induction medium 1 containing Y-27632 (concentration 10 μM) was added, and the embryoid bodies were detached and dispersed from the substrate using a cell scraper (IWAKI). The obtained cell suspension was collected and the number of cells was counted using a Luna automatic cell counter (Logos Biosystems).

[0062] Flow cytometry analysis To evaluate the induction of differentiation into triploid bodies, neural stem cells, and nerve cells, ectoderm marker (PAX6), endoderm marker (FOXA2), mesoderm marker (TBX1), neural stem cell marker (SOX1), and nerve cell markers (NCAM1 and TUBB3) were evaluated. The cells on "differentiation day 5" and "differentiation day 19" collected by cell counting were separated into 1.5 mL sample tubes at 1.0 × 105 cells / tube. 0.5 mL / tube of 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, dispersed, and then left to stand at room temperature for 20 minutes for fixation. After fixation, centrifugation (room temperature, 800 × g, 5 minutes) was performed, the supernatant was removed, and 0.5 mL / tube of PBS (-) was added for washing. The washing operation was repeated three times. After washing, 0.5 mL / tube of membrane permeabilization solution was added, dispersed, and then left to stand at room temperature for 15 minutes for membrane permeabilization. After membrane permeabilization, centrifugation was performed, the supernatant was removed, and 0.5 mL of PBS(-) was added per tube for washing.

[0063] After washing, 0.5 mL / tube of PBS(-) containing 1% BSA was added, and the mixture was dispersed and left to stand at room temperature for 1 hour for blocking treatment. After blocking treatment, centrifugation was performed, the supernatant was removed, and 0.5 mL / tube of PBS(-) was added and washed. After washing, 0.1 mL / tube of diluted antibody solution was added, and the mixture was dispersed and reacted at room temperature and in the dark for 1 hour. After reaction, centrifugation was performed, the staining solution was removed, and 0.5 mL / tube of PBS(-) was added and washed. When an antibody not labeled with fluorescence was used in the antibody reaction, 0.1 mL / tube of diluted fluorescently labeled secondary antibody was added, dispersed, and reacted at room temperature and in the dark for 1 hour. After reaction, centrifugation was performed, the staining solution was removed, and 0.5 mL / tube of PBS(-) was added and washed. After washing, 0.5 mL / tube of antibody dilution was added, and the dispersed sample was analyzed by a flow cytometer (manufactured by Becton Dickinson Japan, product name: BD Accuri C6 Plus).

[0064] The results of flow cytometry analysis of cells on "day 5 of differentiation induction" are shown in Table 1, and the results of flow cytometry analysis of cells on "day 19 of differentiation induction" are shown in Table 2. On "day 5 of differentiation induction", 20.0% of cells had the ectoderm marker PAX6, 26.3% of cells had the mesoderm marker TBX1, 28.3% of cells had the endoderm marker FOXA2, and 20.8% of cells had the endoderm marker SOX1, confirming the creation of embryoid body-like structures. In addition, on "day 19 of differentiation induction", 11.4% of cells were confirmed to have the neuronal marker NCAM1 and 46.9% of cells were confirmed to have the neuronal marker TUBB3, confirming excellent efficiency of differentiation induction into neurons.

[0065] [Table 1]

[0066] [Table 2]

[0067] [Comparative Example 1: Undifferentiated maintenance culture on cell culture substrate] As the medium in Example 1, undifferentiated state maintenance medium StemFitAK02N (Ajinomoto Co., Inc.) was used instead of differentiation induction medium 1 to 3, and all other conditions were the same as those in Example 1. Culture was performed for 6 days (until "day 5 of differentiation induction" in Example 1). On the second day of culture ("day 1 of differentiation induction" in the example), pseudopod extension at the outer edge of the embryoid body confirmed in Example 1 was not confirmed, confirming that it worked to maintain undifferentiated state.

[0068] [Comparative Example 2: Differentiation induction on polystyrene substrate for cell culture] <Preparation of pluripotent stem cell aggregates and preparation of embryoid bodies> Embryoid bodies were prepared using a commercially available culture substrate (Tissue Culture treated polystyrene, manufactured by Eppemdorf) according to the method described in Example 1. The morphology of the formed structures showed an overall acute angle shape and were non-uniform in size.

[0069] Preparation of ectodermal cell aggregates The embryoid bodies were induced to differentiate into ectodermal cell aggregates according to the method described in Example 1. It was confirmed that the embryoid bodies reached 100% confluence on "days 7 to 10 of differentiation," and on "day 16 of differentiation," the phenomenon of the embryoid bodies peeling off in a sheet form upon medium replacement was confirmed. Flow cytometry analysis Flow cytometry analysis was carried out according to the method described in Example 1. The results of flow cytometry analysis of the cells on "day 5 of differentiation induction" are shown in Table 1, and the results of flow cytometry analysis of the cells on "day 19 of differentiation induction" are shown in Table 2. On "day 19 of differentiation induction", 10.6% of the cells had the neuronal marker NCAM1, and 26.1% had TUBB3, confirming poor efficiency of differentiation induction into neurons.

[0070] [Comparative Example 3: Undifferentiated maintenance culture on polystyrene substrate for cell culture] The human iPS cells used for seeding in Example 1 were directly subjected to flow cytometry analysis in the same manner as in Example 1, without carrying out any of the differentiation induction procedures in Example 1. [Explanation of symbols]

[0071] A: Cell adhesion and proliferation areas B: Area with no cell adhesiveness 1: Pluripotent stem cells 2: Pluripotent stem cell aggregate 3: Embryoid body 4: Ectodermal cell aggregate 5: Neuronal aggregates 10: Undifferentiated maintenance medium 11: Differentiation induction medium 1 12: Differentiation induction medium 2 13: Differentiation induction medium 3 20: Hydrophilic polymer 21: Base material

Claims

[Claim 1] A method for inducing differentiation of pluripotent stem cells into ectodermal cells, comprising the following steps (1) to (4): (1) A process for coating a cell culture substrate having two regions, (A) and (B), with one or more substances selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin, and collagen, and seeding pluripotent stem cells on the substrate. (A) Area having cell adhesion and cell proliferation properties of 0.001 to 5 mm ２ An island-like area. (B) a region adjacent to the (A) region, which does not have cell adhesiveness or cell proliferation properties. (2) A step of subjecting the pluripotent stem cells seeded in the step (1) to feeder-free adhesion culture in the presence of an undifferentiated state maintenance medium, thereby forming pluripotent stem cell aggregates adhered onto a cell culture substrate. (3) A step of culturing the pluripotent stem cell aggregates formed in the step (2) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form embryoid bodies. (4) A step of culturing the embryoid bodies formed in the step (3) in an adherent state on a cell culture substrate in the presence of a medium containing a differentiation-inducing factor to form ectodermal cell aggregates.

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