Fibronectin fragment used in the production of stem cells

Abstract

The present invention aims to solve the problems associated with conventional methods for producing stem cells, and provides a method for producing a large amount of stem cells in a short period of time using fibronectin fragments. Stem cells are efficiently proliferated by culturing them in the presence of a novel recombinant fibronectin fragment.

Description

Technical Field

[0001] The present invention relates to a method for producing stem cells having the ability to differentiate into various cells.

Background Art

[0002] Stem cells are defined as cells that have the ability to divide and produce the same cells as themselves (self-renewal ability) and the ability to differentiate into different types of cells, and can proliferate without limit. Among the two daughter cells generated from stem cells, at least one can continue to be the same stem cell to supply differentiated cells.

[0003] Currently, extensive research is being conducted on methods for producing stem cells. For example, as a substrate for culturing stem cells, feeder cells derived from mice, Matrigel, extracellular matrices such as laminin and fibronectin, etc. can be used.

[0004] As a study on a method for producing stem cells using fibronectin or fibronectin fragments, for example, Non-Patent Document 1 can be cited. Non-Patent Document 1 discloses a method for culturing human embryonic stem cells (ES cells) on a 120 kDa fibronectin fragment (hereinafter referred to as 120k-fr) to proliferate human ES cells while maintaining pluripotency. However, the growth rate of cells on 120k-fr was slower than that on full-length fibronectin. In addition, for stem cells to be used in regenerative medicine, quality and safety need to be ensured. However, since full-length fibronectin and commercially available 120k-fr are derived from natural fibronectin, there is a high risk of bringing in viruses and the like carried by the origin organisms.

[0005] Thus, a technique for producing a sufficient amount of stem cells in a short period using fibronectin or fibronectin fragments has not yet been established.

Prior Art Documents

Non-Patent Literature

[0006]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention aims to solve the problems of conventional stem cell production methods and provides a method for producing a large amount of stem cells in a short period using a fibronectin fragment.

Means for Solving the Problems

[0008] As a result of intensive research to solve the above problems, the present inventors have found that stem cells can proliferate efficiently by culturing them in the presence of a novel recombinant fibronectin fragment, and have completed the present invention.

[0009] That is, the present invention provides: [1] A stem cell, (a) a recombinant polypeptide comprising a repeat selected from the group consisting of III-1 to 7 of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the repeat selected from the group consisting of III-1 to 7, (b) a recombinant polypeptide comprising the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the III-8 to 10 repeats, and (c) A recombinant polypeptide comprising the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the above III-12 to 14 repeats, A method for producing stem cells, which includes the step of culturing in the presence of [2] The production method according to [1], which includes the step of culturing stem cells in the presence of a recombinant polypeptide containing the recombinant polypeptides of (a), (b) and (c) in the same molecule. [3] The production method according to [1] or [2], wherein the recombinant polypeptide of (a) is a recombinant polypeptide containing the III-1 to 3 repeats or III-4 to 6 repeats of human fibronectin, or a recombinant polypeptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the above III-1 to 3 repeats or III-4 to 6 repeats. [4] The production method according to [3], wherein the recombinant polypeptide is a recombinant polypeptide containing the amino acid sequence described in SEQ ID NO: 19 or 20, or a recombinant polypeptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence described in SEQ ID NO: 19 or 20. [5] The production method according to any one of [1] to [4], wherein the step of culturing stem cells in the presence of a recombinant polypeptide is carried out in a state where the solid phase coated with the recombinant polypeptide is in contact with the stem cells. [6] The production method according to [5], wherein the solid phase is a cell culture device or a cell culture carrier. [7] The production method according to [5], wherein the solid phase is a dish, plate, flask, bag, bead, membrane or slide glass. [8] The production method according to any one of [1] to [7], wherein the stem cells are human-derived pluripotent stem cells or neural stem cells. [9] The production method according to [8], wherein the stem cells are induced pluripotent stem cells.

[10] A recombinant polypeptide comprising a repeat selected from the group consisting of III-1 to III-7 of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the repeat selected from the group consisting of III-1 to III-7, (b) A recombinant polypeptide comprising the III-8 to III-10 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the III-8 to III-10 repeats, and (c) A recombinant polypeptide comprising the III-12 to III-14 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the III-12 to III-14 repeats, A recombinant polypeptide containing them in the same molecule,

[11] The polypeptide according to

[10] , wherein the recombinant polypeptide of (a) is a recombinant polypeptide comprising the III-1 to III-3 repeats or III-4 to III-6 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the III-1 to III-3 repeats or III-4 to III-6 repeats,

[12] The recombinant polypeptide according to

[10] , which is a recombinant polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19 or 20, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 19 or 20,

[13] A solid phase coated with the recombinant polypeptide according to any one of

[10] to

[12] ,

[14] The solid phase according to

[13] , which is an equipment or carrier for cell culture coated with the recombinant polypeptide,

[15] The solid phase according to

[13] , which is a dish, plate, flask, bag, bead, membrane or slide glass coated with the recombinant polypeptide, relates to.

Advantages of the Invention

[0010] The present invention provides a method for producing stem cells. According to the method of the present invention, stem cells can be efficiently proliferated, the undifferentiated state of stem cells can be maintained, and stem cells can be efficiently induced. Since the method has a high cell proliferation rate and the stem cells obtained by the present invention have the ability to differentiate into desired cells, it is suitably used, for example, in regenerative medicine. Therefore, the method of the present invention is expected to make a great contribution to the medical field. Further, the present invention provides a novel recombinant fibronectin fragment.

Brief Description of the Drawings

[0011]

Figure 1

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Embodiments for Carrying Out the Invention

[0012] The present invention will be described in detail below. <Fibronectin> Human-derived and mammalian-derived fibronectins have been well studied, and the following are mainly findings on human-derived plasma fibronectin.

[0013] Fibronectin is a large glycoprotein with a molecular weight of approximately 250 kDa (monomer) that exists in blood, on the cell surface, in the extracellular matrix, etc., and is known to have various functions such as cell adhesion. Fibronectin consists of a domain structure (see Figure 1 below), and its amino acid sequence contains three types of similar sequences. The three types of similar sequences are called type I repeat, type II repeat, and type III repeat, respectively. Among them, the type III repeat is composed of 87 - 96 amino acid residues, and the homology of the amino acid sequence between each repeat is 17 - 40%. There are 15 type III repeats in fibronectin. Among them, the first, second, and third (hereinafter referred to as III - 1, III - 2, and III - 3, respectively) are in the self - association domain, the fourth, fifth, and sixth (hereinafter referred to as III - 4, III - 5, and III - 6, respectively) are in the DNA - binding domain, the eighth, ninth, and tenth (hereinafter referred to as III - 8, III - 9, and III - 10, respectively) are in the cell - binding domain, and the twelfth, thirteenth, and fourteenth (hereinafter referred to as III - 12, III - 13, and III - 14, respectively) are contained in the heparin - binding domain. III - 10 contains a region having binding activity to integrin α5β1 (also called VLA - 5), and the core sequence is RGD. In addition, a region called IIICS exists in the part closer to the C - terminal side of fibronectin. IIICS contains a sequence consisting of 25 amino acids called CS - 1, and this sequence shows binding activity to integrin α4β1 (also called VLA - 4).

[0014] The amino acid sequences of III - 1 to 14 and CS - 1 of human fibronectin are shown in the sequence listing of this specification as SEQ ID NO: 1 to 14 and 15, respectively.

[0015] 1. Method for producing stem cells of the present invention The method for producing stem cells of the present invention is characterized by including the step of culturing stem cells in the presence of a polypeptide which is a recombinant fibronectin fragment.

[0016] The stem cells used in the present invention are not limited as long as they have the ability to divide and produce the same cells as themselves (self-renewal ability) and the ability to differentiate into different types of cells. Stem cells are classified as follows according to the difference in their differentiation ability, and any of the stem cells can be used in the present invention.

[0017] (1) Totipotent stem cell: Refers to stem cells that can differentiate into all cell types that form an individual, including in vitro tissues such as the placenta. Examples include fertilized eggs (and up to 4 - 8 cell divisions). (2) Pluripotent stem cell: Refers to stem cells that do not form an individual but can differentiate into all cell lineages belonging to the three germ layers (endoderm, mesoderm, and ectoderm). Although not particularly limited, examples include the inner cell mass of the blastocyst stage, embryonic stem cells (ES cells) established therefrom, induced pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), embryonic germ stem cells (EG cells), nuclear transfer ES cells (ntES cells), etc. Pluripotent stem cells are sometimes referred to as omnipotent cells. (3) Multipotent stem cell: Refers to stem cells that can differentiate into a limited number of cell lineages but can differentiate into diverse cell types. Although not particularly limited, examples include hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, etc. Generally, differentiation beyond germ layers cannot be achieved, but there are exceptions. (4) Oligopotent stem cell: Refers to stem cells that can differentiate into only several cell types. Although not particularly limited, examples include neural stem cells, etc. (5) Unipotent stem cell: Refers to stem cells in which the cell types that can differentiate are limited to one type. They can divide and proliferate as stem cells or differentiate into other cell types other than stem cells. Although not particularly limited, examples include muscle stem cells, germ stem cells (oogonial stem cells, spermatogonial stem cells), etc. Unipotent stem cells are sometimes referred to as progenitor cells.

[0018] The origin of the stem cells used in the present invention is not particularly limited, and stem cells derived from any organism, preferably a mammal, can be used. The age and sex of the organism are not particularly limited. In one embodiment, cells derived from primates (e.g., chimpanzee, Japanese macaque, human) are used. Most preferably, human-derived cells are used, but the present invention is not limited thereto.

[0019] In a preferred embodiment of the present invention, the stem cells are preferably pluripotent stem cells, more preferably iPS cells, and even more preferably human iPS cells. Although various methods are known for the production of iPS cells, the method of the present invention is not applicable only to iPS cells produced by a specific method. It can also be applied to established iPS cells, for example, established human iPS cell lines (such as the 253G1 line).

[0020] When producing stem cells by the method of the present invention for the purpose of administration to humans, preferably cells collected from a donor whose type of histocompatibility antigen matches or is similar to that of the recipient are used for the production of the stem cells. For example, cells collected from the recipient himself / herself are used for the production of the stem cells.

[0021] The method for producing stem cells of the present invention is a method for producing stem cells, which is characterized by including a culturing step in the presence of a polypeptide described below (hereinafter sometimes referred to as the culturing step of the present invention).

[0022] In the method for producing stem cells of the present invention, stem cells are cultured in the presence of polypeptide (a), polypeptide (b), and polypeptide (c). The method of the present invention can be carried out in the presence of polypeptide (a), polypeptide (b), and polypeptide (c). Further, in the presence of a mixture of two polypeptides, namely a polypeptide containing (a) and (b) in the same molecule and polypeptide (c), in the presence of a mixture of two polypeptides, namely a polypeptide containing (b) and (c) in the same molecule and polypeptide (a), in the presence of a mixture of two polypeptides, namely a polypeptide containing (a) and (b) in the same molecule and a polypeptide containing (b) and (c) in the same molecule, or in the presence of one polypeptide containing (a), (b), and (c) in the same molecule, the stem cells may be cultured. However, the one polypeptide containing (a), (b), and (c) in the same molecule is different from full-length fibronectin.

[0023] Polypeptide (a) is a recombinant polypeptide containing a repeat selected from the group consisting of III-1 to III-7 of human fibronectin, or a recombinant polypeptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence of the repeat selected from the group consisting of III-1 to III-7. In the polypeptide (a), the "repeat selected from the group consisting of III-1 to III-7 of human fibronectin" may be at least one repeat, preferably three repeats, or even all seven repeats. The polypeptide (a) is particularly preferably a polypeptide containing III-1, III-2, and III-3 repeats, or a polypeptide containing III-4, III-5, and III-6 repeats, or a polypeptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence of III-1 to III-3 repeats or III-4 to III-6 repeats.

[0024] Polypeptide (b) is a recombinant polypeptide containing the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the above III-8 to 10 repeats. That is, the polypeptide (b) is a polypeptide containing all of III-8, III-9, and III-10.

[0025] Polypeptide (c) is a recombinant polypeptide containing the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the above III-12 to 14 repeats. That is, the polypeptide (c) is a polypeptide containing all of III-12, III-13, and III-14.

[0026] As a polypeptide containing (a) and (b) within the same molecule, a 120 kDa fibronectin fragment (120k-fr) is exemplified. 120k-fr is a protein with a molecular weight of approximately 120 kDa, containing III-1, III-2, III-3, III-4, III-5, III-6, III-7, III-8, III-9, III-10 in order from the N-terminal side. The predicted amino acid sequence of 120k-fr (932 amino acid residues) is shown in the sequence listing of this specification as SEQ ID NO: 16. By preparing DNA encoding the amino acid sequence of 120k-fr and combining it with an appropriate host-vector system, 120k-fr can be produced as a recombinant polypeptide. Also, commercially available 120k-fr may be used.

[0027] As polypeptides containing (b) and (c) within the same molecule, CH-271 and CH-296 are exemplified.

[0028] CH-271 is a recombinant protein with a molecular weight of approximately 60 kDa (549 amino acid residues), containing III-8, III-9, III-10, III-12, III-13, and III-14 in order from the N-terminal side. The amino acid sequence of CH-271 is shown in the Sequence Listing of this specification as SEQ ID NO: 17.

[0029] CH-296 is a recombinant protein with a molecular weight of approximately 63 kDa (574 amino acid residues), containing III-8, III-9, III-10, III-12, III-13, III-14, and CS-1 in order from the N-terminal side. The amino acid sequence of CH-296 is shown in the Sequence Listing of this specification as SEQ ID NO: 18. CH-296 is commercially available as Retronectin (registered trademark, manufactured by Takara Bio Inc.).

[0030] For example, the method for producing the stem cells of the present invention can be carried out by using the above-mentioned 120k-fr in combination with CH-271 or CH-296.

[0031] A polypeptide having (a), (b), and (c) in the same molecule can also be used in the method for producing the stem cells of the present invention. Although the present invention is not particularly limited, examples of the polypeptide containing (a), (b), and (c) in the same molecule include the following FCH-296 and DCH-296.

[0032] FCH-296 is a recombinant polypeptide with a molecular weight of approximately 96 kDa (881 amino acid residues), which contains III-1, III-2, III-3, III-8, III-9, III-10, III-12, III-13, III-14, and CS-1 in order from the N-terminal side. The amino acid sequence of FCH-296 is shown in the Sequence Listing of this specification as SEQ ID NO: 19. Among SEQ ID NO: 19, amino acid numbers 1 to 298 correspond to (a), amino acid numbers 299 to 307 correspond to the GS linker, amino acid numbers 308 to 585 correspond to (b), amino acid numbers 586 to 856 correspond to (c), and amino acid numbers 857 to 881 correspond to CS-1. Note that amino acid numbers 94 to 111 of SEQ ID NO: 19 are regions other than type III repeats that exist between III-1 and III-2. FCH-296 is a novel polypeptide produced for the first time in the present invention.

[0033] DCH-296 is a recombinant polypeptide with a molecular weight of approximately 93 kDa (851 amino acid residues), which contains III-4, III-5, III-6, III-8, III-9, III-10, III-12, III-13, III-14, and CS-1 in order from the N-terminal side. The amino acid sequence of DCH-296 is shown in the Sequence Listing of this specification as SEQ ID NO: 20. Among SEQ ID NO: 20, amino acid numbers 1 to 268 correspond to (a), amino acid numbers 269 to 277 correspond to the GS linker, amino acid numbers 278 to 555 correspond to (b), amino acid numbers 556 to 826 correspond to (c), and amino acid numbers 827 to 851 correspond to CS-1. DCH-296 is a novel polypeptide produced for the first time in the present invention.

[0034] The polypeptides (a) to (c) used in the present invention may include an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the repeat selected from the group consisting of III-1 to 7, the III-8 to 10 repeat, or the III-12 to 14 repeat, as long as they are functionally equivalent or retain the function of proliferating stem cells, maintaining the undifferentiated state of stem cells, or inducing stem cells. In the present specification, "one or several" is not particularly limited, but is in the range of 1 to 15, preferably in the range of 1 to 10, more preferably in the range of 1 to 5, and particularly preferably in the range of 1 to 3. Although not particularly limited, for example, instead of including III-1 (SEQ ID NO: 1), a polypeptide containing an amino acid sequence in which the N-terminal 9 amino acids of III-1 are deleted (SEQ ID NO: 23), an amino acid sequence in which the N-terminal 5 amino acids of III-1 are deleted (SEQ ID NO: 24), or an amino acid sequence in which the N-terminal 3 amino acids of III-1 are deleted (SEQ ID NO: 25) is also included in the polypeptide.Furthermore, examples of the polypeptide comprising (a) to (c) include a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of FCH-296 (SEQ ID NO: 19) or the amino acid sequence of DCH-296 (SEQ ID NO: 20). More specifically, but not limited thereto, FCH-296 lacking 9 amino acids at the N-terminus (SEQ ID NO: 29), FCH-296 lacking 6 amino acids at the N-terminus (SEQ ID NO: 30), FCH-296 lacking 5 amino acids at the N-terminus (SEQ ID NO: 31), FCH-296 lacking 3 amino acids at the N-terminus (SEQ ID NO: 32), FCH-296 with 3 amino acids inserted at the N-terminus (SEQ ID NO: 33), FCH-296 with 6 amino acids inserted at the N-terminus (SEQ ID NO: 34), FCH-296 with 9 amino acids inserted at the N-terminus (SEQ ID NO: 35), FCH-296 with 11 amino acids inserted at the N-terminus (SEQ ID NO: 36), FCH-296 with 12 amino acids inserted at the N-terminus (SEQ ID NO: 37), FCH-296 with 14 amino acids inserted at the N-terminus (SEQ ID NO: 38), FCH-296 with 15 amino acids inserted at the N-terminus (SEQ ID NO: 39), FCH-296 with HKRHEEGH inserted at the N-terminus (SEQ ID NO: 40), FCH-296 with HKRH inserted at the N-terminus (SEQ ID NO: 41), FCH-296 with HH inserted at the N-terminus (SEQ ID NO: 42), FCH-296 with HHH inserted at the N-terminus (SEQ ID NO: 43), FCH-296 having a His-tag at the N-terminus (SEQ ID NO: 21), and DCH-296 having a His-tag at the N-terminus (SEQ ID NO: 22) are exemplified.

[0035] In addition, as long as the polypeptides (a) to (c) used in the present invention are functionally equivalent or retain the function of proliferating stem cells, the function of maintaining the undifferentiated state of stem cells, or the function of inducing stem cells, they may include an amino acid sequence having identity with the amino acid sequence of the repeat selected from the group consisting of III-1 to 7, the III-8 to 10 repeat, or the III-12 to 14 repeat. Although not particularly limited, examples include polypeptides having an amino acid sequence having 80% or more, preferably 90% or more, particularly preferably 95% or more identity with the amino acid sequence of the repeat selected from the group consisting of III-1 to 7, the III-8 to 10 repeat, or the III-12 to 14 repeat.

[0036] Substitutions, deletions, insertions, or additions of amino acids (hereinafter sometimes referred to as "amino acid substitutions, etc.") preferably change the physicochemical properties, etc. of the polypeptide to such an extent that the function of the original polypeptide can be maintained. For example, amino acid substitutions, etc. are preferably conservative ones that do not substantially change the properties (e.g., hydrophobicity, hydrophilicity, charge, pK, etc.) of the original polypeptide. For example, amino acid substitutions are substitutions within each of the following groups: 1. glycine, alanine; 2. valine, isoleucine, leucine; 3. aspartic acid, glutamic acid, asparagine, glutamine; 4. serine, threonine; 5. lysine, arginine; 6. phenylalanine, tyrosine. Deletions, additions, and insertions of amino acids are preferably deletions, additions, and insertions of amino acids having properties similar to those around the target site in the polypeptide, within a range that does not substantially change the properties around the target site.

[0037] Amino acid substitutions, etc. may occur naturally due to species differences or individual differences, or may be artificially introduced. Artificial introduction may be carried out by known methods, and there is no particular limitation. For example, by using a nucleic acid in which a substitution, deletion, addition, or insertion of a base has been introduced into the nucleic acid encoding the aforementioned polypeptide by a known technique, a polypeptide containing an amino acid sequence having one or several amino acid substitutions, etc. in the amino acid sequence of the polypeptide can be produced.

[0038] As used herein, "functionally equivalent" or "equivalent function" means being functionally equivalent or having an equivalent function to the corresponding polypeptide into which no amino acid substitution, etc. has been introduced. That is, when producing the stem cells described below using the polypeptide as the comparison target, it means that the same cell proliferation rate of the stem cells can be obtained as when using the corresponding polypeptide into which no amino acid substitution, etc. has been introduced, the undifferentiated state of the same stem cells is maintained, or the same induction rate of the stem cells can be obtained. That is, the function of the polypeptide can be appropriately confirmed by evaluating its properties in accordance with the method described in the examples below.

[0039] The polypeptide used in the present invention may contain peptides and amino acid residues other than the above type III repeats, and / or regions present in fibronectin other than the above type III repeats, such as CS-1, etc., as long as the usefulness in culturing stem cells is not lost. For example, arbitrary peptides and amino acid residues can be introduced into the regions other than the above type III repeats, and examples include the polypeptide of the present invention in which amino acid residues or peptides are inserted as a linker between each repeat, and the polypeptide of the present invention to which a peptide (tag) useful for purification of a recombinant polypeptide is added. As the linker, a glycine-serine linker (GS linker) is exemplified, but is not limited thereto. As the tag, a polyhistidine-tag (His-tag), a Flag-tag, or a Glutathione S-Transferase tag (GST-tag) is exemplified, but is not limited thereto. Although not particularly limited, for example, the FCH-296 polypeptide (SEQ ID NO: 21) having a His-tag at the N-terminus and the DCH-296 polypeptide (SEQ ID NO: 22) having a His-tag at the N-terminus are included in the polypeptide used in the present invention.

[0040] In the culturing step of the present invention, stem cells are cultured while maintaining an undifferentiated state and at a high cell growth rate. As described also in the examples below, the method for producing stem cells of the present invention is clearly more useful because the cell growth rate is significantly higher and the undifferentiated state can be maintained at a high level as compared with the method of using the known fibronectin fragments 120k-fr, CH-271, or CH-296 alone. Furthermore, by using the above method for the expansion culture of stem cells, there is a great advantage that a high cell growth rate and maintenance of the undifferentiated state can be achieved without using feeder cells.

[0041] Regarding the preparation of polypeptides, information on fibronectin can be referred to, for example, by Kimiduka F., et al. [Kimiduka F., et al., Journal of Biochemistry (J. Biochem.), Vol. 110, pp. 284 - 291 (1991)], Kornbrihtt A. R., et al. [Kornbrihtt A. R., et al., EMBO Journal (EMBO J.), Vol. 4, No. 7, 1755 - 1759 (1985)], and Sekiguchi K., et al. [Sekiguchi K., et al., Biochemistry, Vol. 25, No. 17, 4936 - 4941 (1986)]. Also, for the nucleic acid sequence encoding fibronectin or the amino acid sequence of fibronectin, it is disclosed in Genbank Accession No. NM_002026, NP_002017.

[0042] The polypeptide used in the present invention is produced by recombinant DNA technology. In terms of the production or handling of the recombinant, it is desirable that the molecular weight of the polypeptide used in the present invention is 100 kDa or less. The polypeptides herein also include chemically modified forms such as acetylation.

[0043] In a preferred embodiment of the present invention, the culturing of stem cells is carried out in a state where the solid phase coated with the above-mentioned polypeptide is in contact with the stem cells. Examples of the solid phase include containers or carriers (such as microbeads) used for cell culture. The solid phase coated with the above-mentioned polypeptide has the ability to stably hold stem cells and is useful for culturing such cells. The culture container can be made of any material and shape as long as it does not inhibit the maintenance, survival, differentiation, maturation, and self-replication of cells. Examples of the material of the culture container include glass, synthetic resins and natural resins including non-woven fabrics, or metals. Also, examples of the shape of the culture container include polyhedrons such as triangular prisms, cubes, and rectangular parallelepipeds, cylinders, polyhedral pyramids such as triangular pyramids and square pyramids, cones, any shape like a gourd, spheres, hemispheres, circles, ellipses, semi-circles, etc.

[0044] The cell culture equipment used for culturing stem cells is not particularly limited. For example, dishes, plates, flasks, bags, membranes, glass slides, large culture tanks, bioreactors, hollow fiber type culture devices, etc. can be used. Preferably, a plate is used, and more preferably, a Tissue culture treated plate is used.

[0045] As for the bag, for example, a CO2 gas permeable bag for cell culture can be used. Also, when industrially manufacturing a large amount of stem cells, a large culture tank can be used. In addition, the culture can be carried out in either an open system or a closed system, but preferably, from the viewpoint of the safety of the obtained stem cells, it is preferable to perform the culture in a closed system.

[0046] The solid-phase coating, that is, the immobilization of the polypeptide on the solid surface can be carried out by known methods. For example, it can be carried out in the same manner as the immobilization of the fibronectin fragment described in WO97 / 18318 pamphlet and WO00 / 09168 pamphlet. If the polypeptide is immobilized on the solid phase, after obtaining the stem cells by the method of the present invention, it is only necessary to separate the cells from the solid phase, and the cells and the polypeptide of the present invention can be easily separated, and the contamination of the polypeptide or the like into the stem cells can be prevented.

[0047] More specifically, a coating solution in which the polypeptide is dissolved in sterile distilled water, buffer solution or physiological saline can be prepared and used for immobilization. Preferably, phosphate buffered saline (PBS) is used, and particularly preferably, a coating solution in which the polypeptide is dissolved in Dulbecco's PBS (D-PBS) as a solvent is used.

[0048] The molar concentration of the polypeptide in the coating solution is not particularly limited, and examples thereof include 1 to 100,000 nM, preferably 10 to 2,000 nM, and more preferably 30 to 1,000 nM. When using FCH-296 as the polypeptide, the above molar concentration expressed as a weight concentration is 0.1 to 1,000 μg / mL, preferably 1 to 200 μg / mL, and more preferably 3 to 100 μg / mL.

[0049] The coating can be carried out by adding the above coating solution to a culture vessel and holding it for an appropriate time. The conditions for holding the coating solution can be set as appropriate, and examples thereof include holding at room temperature for 1 hour or overnight at 4°C.

[0050] The container coated with the fibronectin fragment can be used as it is or stored at a low temperature, for example, 0 to 10°C until use. Immediately before use, the coating solution is removed from these culture utensils, washed twice with, for example, D-PBS, and then once with a cell culture medium as necessary, and then used for cell culture.

[0051] The method for producing stem cells of the present invention is carried out by performing a culturing step in the presence of the above polypeptide during the entire period or any arbitrary partial period of culturing in stem cell production. That is, the present invention encompasses those that include the above culturing step in a part of the stem cell production process.

[0052] The culturing process of the present invention includes the induction, maintenance, and expansion culture of stem cells, or the maintenance and expansion culture of stem cells. Therefore, the present invention provides a method for producing stem cells, which includes inducing, maintaining, and expanding stem cells in the presence of the above recombinant polypeptides (a), (b), and (c), and a method for producing stem cells, which includes maintaining and expanding stem cells in the presence of the above recombinant polypeptides (a), (b), and (c). In the method for producing stem cells of the present invention, useful stem cells for regenerative medicine and the like can be produced by appropriately adjusting the type of cells to be used in the method, the culture conditions, etc. and culturing the stem cells. In the present specification, the stem cells mean a cell population containing stem cells.

[0053] When the purpose is the induction of stem cells, in the culturing process of the present invention, there are no particular limitations on the type of cells at the start of culture and the method for inducing stem cells. The cells at the start of culture may be differentiated cells (also referred to as somatic cells) such as fibroblasts, hepatocytes, adipocytes, cardiomyocytes, blood cell lineage cells (T cells, B cells, hematopoietic stem cells, etc.), or may be stem cells of a type different from the stem cells after induction. Also, the method for inducing stem cells is not particularly limited as long as it is a known method, and examples include a method of contacting or introducing a low molecular weight compound into cells, a method of introducing a reprogramming factor into cells, a method of transplanting a nucleus into cells, and the like. A reprogramming factor can be introduced into cells as a protein, or a nucleic acid (RNA, DNA) encoding a reprogramming factor can be introduced into cells directly or using a vector. Examples of the above vector include a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a Sendai virus vector, a measles virus vector, an episomal vector, and the like.

[0054] When the purpose is the maintenance and expansion culture of stem cells, in the culturing process of the present invention, the cell concentration at the start of culture is not particularly limited, but for example, it is 0.005 to 20×10 5 cells / mL, preferably 0.02 to 5×10 5 cells / mL, more preferably 0.05 to 2×105 cells / mL is exemplified.

[0055] In the culturing step of the present invention, various media used for culturing stem cells can be used. When culturing pluripotent stem cells, for example, Cellartis (registered trademark) DEF-CS medium (manufactured by Takara Bio Inc.) can be used. Preferably, media that do not contain components derived from heterologous sources such as fetal bovine serum (FBS or FCS) and sheep serum, serum-free media, and media that do not contain unknown components (defined medium), etc. can be mentioned. Such xeno-free media can be appropriately prepared, but known media or commercially available media can be used as they are or after modification. As commercially available xeno-free media, for example, Cellartis (registered trademark) DEF-CS xeno-free medium (manufactured by Takara Bio Inc.), DXF (manufactured by PromoCell), TeSR-E8 medium (manufactured by Stemcell Technologies) can be used. When culturing neural stem cells, for example, RHB-A medium (manufactured by Takara Bio Inc.) can be used.

[0056] The culturing conditions of the cells are not particularly limited, and normal cell culturing conditions can be adopted. As the culturing conditions, culturing at a temperature of 37°C, humidity of 95%, and CO2 concentration of 5% is exemplified, but the present invention is not limited to such conditions. For example, culturing at a temperature of 30 to 40°C, humidity of 90 to 98%, and CO2 concentration of 3 to 7% is exemplified, but culturing can be carried out at temperatures, humidities, and CO2 concentrations outside the above ranges as long as the desired cell growth can be achieved. During culturing, it is preferable to add and dilute fresh medium to the cell culture solution at appropriate time intervals, replace the medium with fresh medium, or replace the equipment for cell culture. The medium used and other components used simultaneously can be set as appropriate.

[0057] In a preferred embodiment of the present invention, when aiming at the maintenance and expansion culture of stem cells, the stem cells are cultured for, for example, 5 days or more, preferably 10 days or more, while performing medium replacement and subculture, using an appropriate medium in a container coated with the polypeptide used in the present invention. By this culture, the stem cells can be proliferated. That is, 80% or more, preferably 90% or more of the cell population obtained by this culture is stem cells.

[0058] Also, in a preferred embodiment of the present invention, when aiming at single cell cloning of stem cells, the serially diluted stem cells are seeded in a container coated with the polypeptide used in the present invention. Then, using an appropriate medium and performing medium replacement, it is cultured for, for example, 5 days or more, preferably 10 days or more until colonies appear. By obtaining the colonies, single cell cloning of stem cells can be performed. That is, 80% or more, preferably 90% or more of the cells in the colonies obtained by this culture are stem cells.

[0059] The stem cells obtained by the culture step of the present invention can be distinguished from other cells based on their morphological characteristics. Also, in the case of pluripotent stem cells, it can also be confirmed based on the expression of marker molecules serving as indicators of the undifferentiated state, such as alkaline phosphatase, stage-specific embryonic antigen (SSEA, for example, SSEA-4, etc.), tumor rejection antigen (TRA)-1-60, TRA-1-81, OCT4 or NANOG. The expression of the above-mentioned molecules (positive markers) can be confirmed, for example, using an antibody that recognizes the above-mentioned molecules. Regarding alkaline phosphatase, the expression can also be confirmed based on its enzyme activity. On the other hand, in the case of neural stem cells, although not particularly limited, it can also be confirmed based on the expression of neural stem cell marker molecules such as Nestin.

[0060] 80% or more, preferably 90% or more, more preferably 95% or more of the stem cells obtained by the culture step of the present invention express the above positive markers.

[0061] Furthermore, stem cells can be isolated from the cell population obtained by the culturing step of the present invention, and stem cells separated from other cells can be obtained. Antibodies that recognize molecules characteristic of stem cells are useful for isolating and purifying the stem cells obtained by the present invention. The stem cells thus isolated can be established as a cell line by a known method. That is, as one aspect of the present invention, there is provided a method for producing stem cells, which includes the steps of the method for producing a cell population containing the stem cells of the present invention and the step of isolating stem cells from the obtained cell population. Furthermore, by differentiating the stem cells thus obtained by a known method, it is also possible to produce various differentiated cells.

[0062] The stem cells obtained in the present invention and the differentiated cells obtained from the cells can also be used, for example, in research on stem cell differentiation, pharmaceutical screening for various diseases, efficacy and safety evaluation of pharmaceutical candidate compounds, and the like. According to the present invention, since a large number of stem cells can be obtained by a single operation, it is possible to obtain reproducible research results without being affected by cell lot differences as in the past.

[0063] 2. The polypeptide of the present invention The present invention provides a novel recombinant polypeptide useful for the production of stem cells. The polypeptide is a recombinant polypeptide containing the polypeptides (a) to (c) described in "1. Method for producing stem cells of the present invention" in the same molecule and is useful for the method. The polypeptide of the present invention has a function of proliferating stem cells, a function of maintaining the undifferentiated state of stem cells, and / or a function of inducing stem cells. The polypeptide of the present invention has a function equivalent to that of full-length fibronectin or a higher function than existing fibronectin fragments.

[0064] The polypeptide of the present invention is a recombinant polypeptide containing the following polypeptides (a) to (c) in the same molecule: (a) A recombinant polypeptide comprising a repeat selected from the group consisting of III-1 to 7 of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the repeat selected from the group consisting of III-1 to 7 above, (b) A recombinant polypeptide comprising the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the III-8 to 10 repeats above, and (c) A recombinant polypeptide comprising the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the III-12 to 14 repeats above.

[0065] Regarding the recombinant polypeptides (a), (b) and (c) above, they are as described in "1. Method for producing stem cells of the present invention".

[0066] Although not particularly limiting the present invention, for example, polypeptides having (a), (b), and (c) from the N-terminal side can be mentioned. Further, (b) and (c) preferably have binding activity to integrin α5β1 (also referred to as VLA-5) and binding activity to heparin, respectively.

[0067] Particularly preferred embodiments of the polypeptide of the present invention are polypeptides comprising the amino acid sequences set forth in SEQ ID NO: 19 or 20 in the Sequence Listing. Further, a recombinant polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 19 or 20 in the Sequence Listing, and having a function equivalent to that of the recombinant polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19 or 20, or retaining the function of proliferating stem cells, maintaining the undifferentiated state of stem cells, and / or inducing stem cells is also encompassed by the present invention. Although not particularly limited, for example, instead of comprising III-1 (SEQ ID NO: 1), an amino acid sequence in which the N-terminal 9 amino acids of III-1 are deleted (SEQ ID NO: 23), an amino acid sequence in which the N-terminal 5 amino acids of III-1 are deleted (SEQ ID NO: 24), or an amino acid sequence in which the N-terminal 3 amino acids of III-1 are deleted (SEQ ID NO: 25) are also included in the polypeptide. More specifically, FCH-296 lacking the N-terminal 9 amino acids (SEQ ID NO: 29), FCH-296 lacking the N-terminal 6 amino acids (SEQ ID NO: 30), FCH-296 lacking the N-terminal 5 amino acids (SEQ ID NO: 31), FCH-296 lacking the N-terminal 3 amino acids (SEQ ID NO: 32), FCH-296 with an insertion of 3 amino acids at the N-terminus (SEQ ID NO: 33), FCH-296 with an insertion of 6 amino acids at the N-terminus (SEQ ID NO: 34), FCH-296 with an insertion of 9 amino acids at the N-terminus (SEQ ID NO: 35), FCH-296 with an insertion of 11 amino acids at the N-terminus (SEQ ID NO: 36), FCH-296 with an insertion of 12 amino acids at the N-terminus (SEQ ID NO: 37), FCH-296 with an insertion of 14 amino acids at the N-terminus (SEQ ID NO: 38), FCH-296 with an insertion of 15 amino acids at the N-terminus (SEQ ID NO: 39), FCH-296 with an insertion of HKRHEEGH at the N-terminus (SEQ ID NO: 40), FCH-296 with an insertion of HKRH at the N-terminus (SEQ ID NO: 41), FCH-296 with an insertion of HH at the N-terminus (SEQ ID NO: 42), FCH-296 with an insertion of HHH at the N-terminus (SEQ ID NO: 43), FCH-296 having a His-tag at the N-terminus (SEQ ID NO: 21), and DCH-296 having a His-tag at the N-terminus (SEQ ID NO: 22) are exemplified.

[0068] The polypeptide of the present invention can be produced using known recombinant DNA techniques. Known hosts and vectors can be used. For example, bacteria (such as Escherichia coli and Bacillus subtilis), yeast, filamentous fungi, insect cells, and animal cells (such as mammalian cells including human cells) can be used as hosts, and vectors compatible with each host can be used. The vector is loaded with a nucleic acid encoding the polypeptide of the present invention. The nucleic acid can be prepared by modifying a natural nucleic acid (such as DNA encoding human fibronectin) or by chemical synthesis. The polypeptide of the present invention expressed in a host into which a vector carrying the nucleic acid has been introduced or secreted into the culture supernatant of the host can be purified to the desired purity by known protein purification methods.

[0069] 3. Solid phase coated with the polypeptide of the present invention The present invention provides a solid phase coated with the polypeptide of the present invention. The solid phase is the solid phase described in "1. Method for producing stem cells of the present invention" and is useful for the method.

[0070] The solid phase of the present invention is one in which the above polypeptide is immobilized on the surface of a suitable solid phase. Examples of the solid phase include cell culture equipment or cell culture carriers, specifically dishes, plates, flasks, bags, beads, membranes, and slide glasses. These are not particularly limited as long as they can be used in the method for producing stem cells of the present invention. Furthermore, for the immobilization of the polypeptide on the solid phase, the methods described for the method for producing stem cells of the present invention can be used.

[0071] Since the solid phase of the present invention can stably maintain stem cells on the solid phase surface, the efficiency of culture operations such as medium exchange can be improved. In addition, by preparing the solid phase of the present invention in advance, it becomes possible to immediately implement the method for producing stem cells of the present invention.

Examples

[0072] The present invention will be described more specifically with the following examples, but the scope of the present invention is not limited by these examples.

[0073] Preparation of Example 1: FCH-296 The FCH-296 polypeptide (SEQ ID NO: 21) having a His-tag consisting of a methionine residue and six histidine residues at the N-terminus was prepared by the following procedure.

[0074] The DNA encoding the polypeptide was artificially synthesized and incorporated into an expression plasmid. Escherichia coli was transformed with the plasmid, and the resulting transformant was cultured under conditions where the polypeptide was expressed. The cells collected from the culture were disrupted using an ultrasonic disruptor (manufactured by Kubota) to obtain a cell-free extract. Using this extract as a starting material, FCH-296 was purified by a series of column chromatographies on Ni-Chelating Sepharose (manufactured by GE Healthcare), Hydroxyapatite (40 μm, manufactured by Bio-Rad), and SP-Sepharose (manufactured by GE Healthcare). The confirmation of FCH-296 during the purification process was carried out by SDS-PAGE / CBB staining. The buffer of the obtained sample was replaced with [0.2 g / L KCl, 0.2 g / L KH2PO4, 8 g / L NaCl, 1.15 g / L Na2HPO4] to obtain 6 mL of an FCH-296 sample.

[0075] The FCH-296 sample showed a single band by SDS-PAGE / CBB staining. When the protein concentration of the FCH-296 sample was measured using a BCA protein quantification kit (manufactured by Pierce), it was 1.21 mg / mL (12.4 μM calculated from the molecular weight).

[0076] Preparation of Example 2: DCH-296 The DCH-296 polypeptide (SEQ ID NO: 22) having a His-tag consisting of a methionine residue and six histidine residues at the N-terminus was prepared by the following procedure.

[0077] DNA encoding the polypeptide was artificially synthesized and incorporated into an expression plasmid. Escherichia coli was transformed with the plasmid, and the resulting transformant was cultured under conditions in which the polypeptide was expressed. The cells collected from the culture were disrupted using an ultrasonic disruptor to obtain a cell-free extract. Using this extract as a starting material, DCH-296 was purified by a series of column chromatographies on Ni-Chelating Sepharose, Hydroxyapatite, and SP-Sepharose. Confirmation of DCH-296 during the purification process was carried out by SDS-PAGE / CBB staining. The buffer of the obtained sample was replaced with [0.2 g / L KCl, 0.2 g / L KH2PO4, 8 g / L NaCl, 1.15 g / L Na2HPO4] to obtain 3 mL of a DCH-296 sample.

[0078] The DCH-296 sample showed a single band by SDS-PAGE / CBB staining. When the protein concentration of the DCH-296 sample was measured using a BCA protein quantification kit, it was 1.03 mg / mL (11.0 μM calculated from the molecular weight).

[0079] Example 3 Evaluation of the adhesiveness of various fibronectin fragments to human iPS cells Full-length fibronectin (derived from human plasma; manufactured by SIGMA, F0895, final concentration 50 μg / ml), 120k-fr (manufactured by Millipore, F1904, final concentration 40 μg / ml), CH-271 (J. Biochem., Vol. 110, p284-291 (1991), final concentration 25 μg / ml), and CH-296 (Retronectin: manufactured by Takara Bio, final concentration 20 μg / ml) were each dissolved in D-PBS (manufactured by Promocell, C-40232) to prepare a coating solution. After adding 0.4 mL / well of the coating solution to a 12-well Tissue culture treated plate (manufactured by Corning, 3513), the plate was covered and left overnight at 4°C. The next day, the plate from which the coating solution had been removed was washed twice with 1 mL / well of D-PBS to obtain plates coated with each polypeptide. When coating with two fibronectin fragments, 0.4 mL / well of the second coating solution was added to the plate after the first coating, and left overnight at 4°C and washed in the same manner as the first coating. The prepared plates were covered and stored at 4°C until use.

[0080] Human iPS cells (253G1 strain) (manufactured by the Institute for Integrated Cell-Material Sciences, Kyoto University), passaged in Cellartis (registered trademark) DEF-CS medium (manufactured by Takara Bio, Y30010), were seeded onto a plate at 8×10 4 cells / well and cultured at 37°C and 5% CO2 in the same medium. From the next day, the medium was changed daily, and the cells were observed on the 6th day after the start of culture. The results are shown in Table 1.

[0081]

Table 1

[0082] No cell detachment was observed on the plates coated with 120k-fr / CH-271 and 120k-fr / CH-296. From these results, it was shown that by combining existing fibronectin fragments, cell adhesion to the plate can be enhanced.

[0083] Example 4 Evaluation of the Adhesion of FCH-296 and DCH-296 to Human iPS Cells Full-length fibronectin, 120k-fr, CH-271, CH-296, FCH-296, or DCH-296 was dissolved in D-PBS respectively to prepare coating solutions. Also, for CH-296, a high-concentration coating solution, and for FCH-296 and DCH-296, low-concentration coating solutions were prepared. Using these coating solutions, the 12-well Tissue culture treated plate was coated in the same manner as in Example 3.

[0084] Human iPS cells (253G1 strain) subcultured in DEF-CS medium were seeded onto the plate at 8×10 4 cells / well and cultured at 37 °C and 5% CO2. From the next day, the medium was changed daily, and the cells were observed on the 5th, 8th, and 11th days after the start of culture. The results are shown in Table 2.

[0085]

Table 2

[0086] On the 8th day, cell detachment was observed on the plates coated with the existing fibronectin fragments (120k-fr, CH-271, CH-296). On the other hand, no cell detachment was observed on the plates coated with DCH-296 and FCH-296 on the 8th day. In particular, in the case of FCH-296, no cell detachment was observed even in the wells coated with the low-concentration coating solution at the 11th day. From the above, it was shown that FCH-296 has cell adhesion equivalent to that of full-length fibronectin in the culture of human iPS cells.

[0087] Example 5 Long-Term Culture of Human iPS Cells (DEF-CS Medium) Full-length fibronectin or FCH-296 was dissolved in D-PBS respectively to prepare coating solutions of each concentration. Using these coating solutions, a 24-well Tissue culture treated plate (manufactured by Corning) was coated in the same manner as in Example 3.

[0088] Human iPS cells (253G1 strain) were suspended in DEF-CS medium and then seeded onto the plate. From the next day, the medium was changed daily and the cells were passaged every 3 - 4 days. The results of cell growth are shown in Figure 2. Also, on the 35th day (10th passage) after the start of culture, the TRA-1-60 positive cell rate and the SSEA4 positive cell rate were measured by flow cytometry. The results are shown in Table 3. TRA-1-60 and SSEA4 are pluripotent stem cell markers.

[0089]

Table 3

[0090] Even on the plate coated with FCH-296, human iPS cells grew while maintaining pluripotency, similar to the plate coated with fibronectin. From the above, it was shown that human iPS cells can be cultured long-term on the plate coated with FCH-296.

[0091] Example 6 Long-term culture of human iPS cells (DEF-CS xeno-free medium) It was investigated whether human iPS cells can be cultured long-term on a plate coated with FCH-296 even in a medium that does not contain components derived from animals or humans, that is, a xeno-free medium.

[0092] Full-length fibronectin or FCH-296 was dissolved in D-PBS respectively to prepare coating solutions of each concentration. Using these coating solutions, a 24-well Tissue culture treated plate was coated in the same manner as in Example 3. Also, coating was performed using Synthemax (registered trademark) II-SC Substrate (manufactured by Corning Inc.) recommended in DEF-CS xeno-free medium (manufactured by Takara Bio Inc.).

[0093] Human iPS cells (253G1 strain) were suspended in DEF-CS xeno-free medium and then seeded onto the plate. From the next day, the medium was changed daily, and the cells were passaged every 3 - 4 days. The results of cell growth are shown in Figure 3. On the 35th day (10th passage) after the start of culture, the TRA-1-60 positive cell rate and the SSEA4 positive cell rate were measured by flow cytometry. The results are shown in Table 4. TRA-1-60 and SSEA4 are pluripotent stem cell markers.

[0094]

Table 4

[0095] On the plate coated with FCH-296, human iPS cells grew while maintaining pluripotency, similar to the plates coated with fibronectin or Synthemax. From the above, it was shown that human iPS cells can be cultured long-term on plates coated with FCH-296 even in DEF-CS xeno-free medium.

[0096] Example 7 Long-term culture of human iPS cells (TeSR-E8 medium) TeSR-E8 medium is a xeno-free medium containing only the minimum 8 elements necessary for the maintenance of human iPS / ES cells. Whether human iPS cells can be cultured long-term on plates coated with FCH-296 was investigated even in TeSR-E8 medium.

[0097] Full-length fibronectin or FCH-296 was dissolved in D-PBS respectively to prepare coating solutions of each concentration. Using these coating solutions, a 24-well Tissue culture treated plate was coated in the same manner as in Example 3. Also, Vitronectin XF (manufactured by Corning), which is recommended in TeSR-E8 medium (manufactured by Stemcell Technologies), was used for coating.

[0098] Human iPS cells (253G1 strain) were suspended in TeSR-E8 medium and then seeded onto the plate. From the next day, the medium was changed daily, and the cells were passaged every 3 - 4 days. The results of cell growth are shown in Figure 4. On the 27th day after the start of culture (7th passage), the TRA-1-60 positive cell rate and the SSEA4 positive cell rate were measured by flow cytometry. The results are shown in Table 5. TRA-1-60 and SSEA4 are pluripotent stem cell markers.

[0099]

Table 5

[0100] On the plates coated with FCH-296, human iPS cells grew while maintaining pluripotency, similar to the plates coated with fibronectin or Vitronectin. From the above, it was shown that human iPS cells can be cultured long-term on plates coated with FCH-296 even in TeSR-E8 medium.

[0101] Example 8 Long-term culture of human neural stem cells It was examined whether human neural stem cells can be cultured long-term on plates coated with FCH-296.

[0102] FCH-296 was dissolved in D-PBS to prepare a coating solution at 30 μg / ml. Using this coating solution, a 12-well Tissue culture treated plate was coated in the same manner as in Example 3. Also, coating was performed using Laminin (manufactured by gibco, 10 μg / ml) recommended in RHB-A medium (manufactured by Takara Bio Inc.). Human neural stem cells were seeded at 1.5 - 2.0×10 5 cells / well on the above-coated plates and cultured at 37°C and 5% CO2. From the next day, the medium was changed every two days and the cells were passaged every 4 - 8 days. The results of cell growth are shown in Figure 5.

[0103] Also, on the 28th day (5th passage) after the start of culture, when the expression of Nestin, a neural stem cell marker, was confirmed by immunostaining, the cells retained the expression of Nestin on the plates coated with FCH-296 as well as on the plates coated with Laminin. From the above, it was shown that human neural stem cells can be cultured long-term on plates coated with FCH-296.

[0104] Example 9 Single-cell Cloning of Human iPS Cells Full-length fibronectin or FCH-296 was dissolved in D-PBS respectively to prepare coating solutions at each concentration. Using these coating solutions, a 96-well Half area Tissue culture treated plate (manufactured by Corning) was coated in the same manner as in Example 3.

[0105] Human iPS cells (253G1 strain) were suspended in DEF-CS medium and then seeded at 1 cell / well on the plates. From 2 days later, the medium was changed every two days. The appearance rate of colonies obtained on the 10th day is shown in Table 6.

[0106]

Table 6

[0107] Even on plates coated with FCH-296, single-cell-derived colonies of human iPS cells could be obtained in the same manner as on plates coated with fibronectin. In addition, some colonies were passaged and expanded in culture, and on day 16, the TRA-1-60 positive cell rate and the SSEA4 positive cell rate were measured by flow cytometry, and all were 90% or higher. From the above, it was shown that single-cell cloning of human iPS cells is possible on plates coated with FCH-296.

[0108] Example 10 Evaluation of adhesiveness to human iPS cells (F1CH-296, F2CH-296, F3CH-296) To examine which repeat among III-1, III-2, and III-3 is important, three kinds of polypeptides (F1CH-296, F2CH-296, and F3CH-296) as shown in FIG. 6 were prepared. That is, in the same manner as in Example 1, F1CH-296 (SEQ ID NO: 26), F2CH-296 (SEQ ID NO: 27), and F3CH-296 (SEQ ID NO: 28) having a His-tag consisting of a methionine residue and six histidine residues at the N-terminus were prepared.

[0109] Full-length fibronectin, CH-296, FCH-296, F1CH-296, F2CH-296, or F3CH-296 was each dissolved in D-PBS to prepare coating solutions of each concentration. Using these coating solutions, the 24 well Tissue culture treated plate was coated in the same manner as in Example 3. Note that 50 μg / ml of fibronectin is approximately 200 nM, and 20 μg / ml of CH-296, 30 μg / ml of FCH-296, and 24 μg / ml of F1CH-296 are approximately 320 nM.

[0110] Human iPS cells (253G1 strain) were suspended in DEF-CS medium and then seeded on plates. From the next day, the medium was changed daily, and the cells were observed on days 3, 6, 8, and 10 after the start of culture. The results are shown in Table 7 (-: no cell detachment, +: cell detachment).

[0111]

Table 7

[0112] The three polypeptides (F1CH-296, F2CH-296, and F3CH-296) showed similar trends. That is, for example, cell detachment was observed on the 8th day under the condition of 24 μg / ml. On the other hand, cell detachment was observed on the 3rd day with CH-296, and no cell detachment was observed with FCH-296. This indicates that the adhesion activities of the three polypeptides (F1CH-296, F2CH-296, and F3CH-296) are higher than that of CH-296 and lower than that of FCH-296. From the above, it was shown that the three type III repeats (III-1, III-2, III-3) have equivalent cell adhesiveness, and the cell adhesion activity is the highest when all three type III repeats are included.

[0113] Example 11 Evaluation of FCH-296 with Various N-Terminal Sequences FCH-296 with various N-terminal sequences was prepared. That is, FCH-296 lacking 9 amino acids at the N-terminus (SEQ ID NO: 29), FCH-296 lacking 6 amino acids at the N-terminus (SEQ ID NO: 30), FCH-296 lacking 5 amino acids at the N-terminus (SEQ ID NO: 31), FCH-296 lacking 3 amino acids at the N-terminus (SEQ ID NO: 32), FCH-296 (SEQ ID NO: 19), FCH-296 with 3 amino acids inserted at the N-terminus (SEQ ID NO: 33), FCH-296 with 6 amino acids inserted at the N-terminus (SEQ ID NO: 34), FCH-296 with 9 amino acids inserted at the N-terminus (SEQ ID NO: 35), FCH-296 with 11 amino acids inserted at the N-terminus (SEQ ID NO: 36), FCH-296 with 12 amino acids inserted at the N-terminus (SEQ ID NO: 37), FCH-296 with 14 amino acids inserted at the N-terminus (SEQ ID NO: 38), FCH-296 with 15 amino acids inserted at the N-terminus (SEQ ID NO: 39), FCH-296 with HKRHEEGH inserted at the N-terminus (SEQ ID NO: 40), FCH-296 with HKRH inserted at the N-terminus (SEQ ID NO: 41), FCH-296 with HH inserted at the N-terminus (SEQ ID NO: 42), and FCH-296 with HHH inserted at the N-terminus (SEQ ID NO: 43) were prepared.

[0114] Instead of using FCH-296 (with His-tag, SEQ ID NO: 21) used in Examples 5 to 9, any of the FCH-296 with the various N-terminal sequences described above is used to carry out the content described in Examples 5 to 9. FCH-296 with various N-terminal sequences has the same effects as FCH-296.

Industrial Applicability

[0115] The present invention provides a method for producing a large amount of stem cells in a short period and a polypeptide used in the method.

Sequence Listing Free-Text

[0116] SEQ ID NO:1 ; Partial region of fibronectin named III-1 SEQ ID NO:2 ; Partial region of fibronectin named III-2 SEQ ID NO:3 ; Partial region of fibronectin named III-3 SEQ ID NO:4 ; Partial region of fibronectin named III-4 SEQ ID NO:5 ; Partial region of fibronectin named III-5 SEQ ID NO:6 ; Partial region of fibronectin named III-6 SEQ ID NO:7 ; Partial region of fibronectin named III-7 SEQ ID NO:8 ; Partial region of fibronectin named III-8 SEQ ID NO:9 ; Partial region of fibronectin named III-9 SEQ ID NO:10 ; Partial region of fibronectin named III-10 SEQ ID NO:11; Partial region of fibronectin named III-11 SEQ ID NO:12; Partial region of fibronectin named III-12 SEQ ID NO:13; Partial region of fibronectin named III-13 SEQ ID NO:14; Partial region of fibronectin named III-14 SEQ ID NO:15; Partial region of fibronectin named CS-1 SEQ ID NO:16; Fibronectin fragment named 120k-fr SEQ ID NO:17; Fibronectin fragment named CH-271 SEQ ID NO:18; Fibronectin fragment named CH-296 (RetroNectin) SEQ ID NO:19; Fibronectin fragment named FCH-296 SEQ ID NO:20; Fibronectin fragment named DCH-296 SEQ ID NO:21; His-tag FCH-296 SEQ ID NO:22; His-tag DCH-296 SEQ ID NO:23; N-terminal 9a.a. deletion of III-1 SEQ ID NO:24; N-terminal 5a.a. deletion of III-1 SEQ ID NO:25; N-terminal 3a.a. deletion of III-1 SEQ ID NO:26; His-tag F1CH-296 SEQ ID NO:27; His-tag F2CH-296 SEQ ID NO:28; His-tag F3CH-296 SEQ ID NO:29; N-terminal 9a.a. deletion of FCH-296 SEQ ID NO:30; N-terminal 6a.a. deletion of FCH-296 SEQ ID NO:31; N-terminal 5a.a. deletion of FCH-296 SEQ ID NO:32; N-terminal 3a.a. deletion of FCH-296 SEQ ID NO:33; N-terminal 3a.a. insertion of FCH-296 SEQ ID NO:34; N-terminal 6a.a. insertion of FCH-296 SEQ ID NO:35; N-terminal 9a.a. insertion of FCH-296 SEQ ID NO:36; N-terminal 11a.a. insertion of FCH-296 SEQ ID NO:37; N-terminal 12a.a. insertion of FCH-296 SEQ ID NO:38; N-terminal 14a.a. insertion of FCH-296 SEQ ID NO:39; N-terminal 15a.a. insertion of FCH-296 SEQ ID NO:40; N-terminal HKRHEEGH insertion of FCH-296 SEQ ID NO:41; N-terminal HKRH insertion of FCH-296 SEQ ID NO:42; N-terminal HH insertion of FCH-296 SEQ ID NO:43; N-terminal HHH insertion of FCH-296

Claims

**Claim 1** A method for producing stem cells, comprising culturing stem cells in the presence of three or two fibronectin fragments, wherein the three fibronectin fragments are recombinant polypeptides of the following (a), (b) and (c), respectively, and the two fibronectin fragments are each a single-molecule polypeptide composed of two recombinant polypeptides selected from the following (a) to (c) and the remaining one recombinant polypeptide, and the molar concentrations of the recombinant polypeptides of (a), (b) and (c) are 1 to 100,000 nM. (a) A recombinant polypeptide consisting of the III-1 to 3 repeats of human fibronectin, or a recombinant polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the III-1 to 3 repeats, which is functionally equivalent to the recombinant polypeptide consisting of the III-1 to 3 repeats of human fibronectin, or a recombinant polypeptide having the function of proliferating stem cells or maintaining the undifferentiated state of stem cells. (b) A recombinant polypeptide consisting of the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the III-8 to 10 repeats, which is functionally equivalent to the recombinant polypeptide consisting of the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide having the function of proliferating stem cells or maintaining the undifferentiated state of stem cells, and (c) A recombinant polypeptide consisting of the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the III-12 to 14 repeats, which is functionally equivalent to the recombinant polypeptide consisting of the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide having the function of proliferating stem cells or maintaining the undifferentiated state of stem cells. **Claim 2** The production method according to claim 1, wherein the step of culturing stem cells in the presence of the recombinant polypeptide is carried out in a state where the solid phase coated with the recombinant polypeptide is in contact with the stem cells. **Claim 3** The production method according to claim 1 or 2, wherein the solid phase is a cell culture device or a cell culture carrier. **Claim 4** The production method according to claim 3, wherein the solid phase is a dish, plate, flask, bag, bead, membrane or slide glass.

5. The production method according to any one of claims 1 to 4, wherein the stem cells are human-derived pluripotent stem cells or neural stem cells.

6. The production method according to claim 5, wherein the pluripotent stem cells are induced pluripotent stem cells.

7. A composition comprising three or two fibronectin fragments for use in the production method of stem cells, wherein the three fibronectin fragments are each a recombinant polypeptide of the following (a), (b) and (c), and the two fibronectin fragments are each a single-molecule polypeptide composed of two recombinant polypeptides selected from the following (a) to (c) and the remaining one recombinant polypeptide, and the molar concentrations of the recombinant polypeptides of (a), (b) and (c) are 1 to 100,000 nM. (a) A recombinant polypeptide consisting of the III-1 to 3 repeats of human fibronectin, or a recombinant polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the III-1 to 3 repeats, which is functionally equivalent to the recombinant polypeptide consisting of the III-1 to 3 repeats of human fibronectin, or a recombinant polypeptide having the function of proliferating stem cells or maintaining the undifferentiated state of stem cells. (b) A recombinant polypeptide consisting of the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the III-8 to 10 repeats, which is functionally equivalent to the recombinant polypeptide consisting of the III-8 to 10 repeats of human fibronectin, or a recombinant polypeptide having the function of proliferating stem cells or maintaining the undifferentiated state of stem cells, and (c) A recombinant polypeptide consisting of the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the III-12 to 14 repeats, which is functionally equivalent to the recombinant polypeptide consisting of the III-12 to 14 repeats of human fibronectin, or a recombinant polypeptide having the function of proliferating stem cells or maintaining the undifferentiated state of stem cells.

8. A solid phase coated with the composition according to claim 7.

9. The solid phase according to claim 8, which is an apparatus for cell culture or a carrier for cell culture coated with the composition.

10. The solid phase according to claim 8 or 9, which is a dish, plate, flask, bag, bead, membrane or slide glass coated with the composition.

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