WO2015064661A1 - ポリペプチド組成物およびこれを用いた多能性幹細胞の培養方法 - Google Patents
ポリペプチド組成物およびこれを用いた多能性幹細胞の培養方法 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/0018—Culture media for cell or tissue culture
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- C—CHEMISTRY; METALLURGY
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Definitions
- the present invention relates to a polypeptide composition and a method for culturing pluripotent stem cells using the same.
- pluripotent stem cells are derived from somatic cells, unlike embryonic stem cells, and thus have the advantage of fewer ethical problems.
- pluripotent stem cells are in an undifferentiated state. It is required to be maintained over a long period of time.
- feeder cells such as mouse fibroblasts are used to culture undifferentiated pluripotent stem cells for a long period of time.
- a feeder cell derived from a heterologous animal such as mouse fibroblast
- a foreign substance such as an antigenic substance derived from the heterologous animal may be mixed into the culture solution.
- totipotent or pluripotent stem cells for medical use or similar uses, it is required to culture these cells in the absence of feeder cells.
- Japanese Patent Application Laid-Open No. 2001-17183 discloses a cellular composition that does not contain feeder cells and contains proliferating primate progenitor cells.
- the cellular composition further comprises an extracellular matrix. It is disclosed.
- Japanese Patent Laid-Open No. 2010-29186 discloses a cell culture substrate obtained by coating a plasma polymerized cell culture surface with a coating solution containing an extracellular matrix protein and an aqueous solvent at a predetermined concentration. The quality is described as having good adhesion that helps to avoid the differentiation of embryonic stem cells.
- Biomaterials, 2010, Nov; Vol.31 (32), pp.8281-8288 and Nature Biotechnology, 2010, Vol.28, No.6, pp.606-610 can contribute to long-term culture of embryonic stem cells
- a recombinant peptide or a synthetic peptide consisting of a vitronectin partial sequence specifically, a polypeptide which is the amino acid sequence from the first to the 52nd in the amino acid sequence of natural vitronectin (Biomaterials, 2010, Nov; Vol.31 (32) , pp.8281-8288), and polypeptides having amino acid sequences 41 to 52 including RGD sequences (see Nature Biotechnology, 2010, Vol.28, No.6, pp.606-610), respectively. It is disclosed. Since these polypeptides are non-biological samples, they are known to be excellent in that they can avoid the possibility of contamination with antigenic substances and can be industrially produced.
- heterologous feeder cells such as fibroblasts derived from mice, etc.
- the use of animal-derived ingredients should be eliminated as much as possible.
- materials derived from living bodies, whether of the same type or different types are not preferable from an industrial point of view, such as a very small amount of extraction or performance variations depending on donors.
- studies on culturing stem cells under chemically identified conditions that do not contain foreign animal-derived components or antigenic substances have been actively conducted. However, no material has been found that exhibits practical cell culture performance and can replace feeder cells.
- Biomaterials, 2010, Nov; Vol. 31 (32), pp. 8281-8288 and Nature Biotechnology, 2010, Vol. 28, No. 6, pp. 606-610 discloses an example of carrying out long-term culture of embryonic stem cells using a recombinant peptide consisting of a partial sequence of human vitronectin or a synthetic peptide.
- a recombinant peptide consisting of a partial sequence of human vitronectin or a synthetic peptide.
- these recombinant peptides are not sufficient for cell culture performance of pluripotent stem cells.
- the present invention can provide a polypeptide composition capable of inducing cell culture performance of pluripotent stem cells, particularly excellent cell proliferation ability, and a method for culturing pluripotent stem cells using the same.
- polypeptides (a) to (c) (a) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, (b) 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 A polypeptide having the following amino acid sequence and having the ability to culture pluripotent stem cells, and (c) having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1, And at least one selected from the group consisting of polypeptides capable of culturing pluripotent stem cells, and polypeptides (a) to (c) are represented by the following (1-i) to (1-iii): Any one amino acid sequence: (1-i) an amino acid sequence consisting of amino acid residues 1 to 44 in the amino acid sequence represented by SEQ ID NO: 1, (1-ii) from amino acid sequence (1-i) Amino acid An amino acid sequence comprising an amino acid sequence having 80% or more identity with a sequence and having cell adhe
- polypeptide composition which is 20 mass% or less of the total mass of the contained polypeptide.
- An amino acid sequence comprising 80% or more amino acid sequence and having cell adhesion ability to pluripotent stem cells, and (1-iii) amino acid sequence (1-i), wherein one or several amino acids are deleted, substituted or A first region comprising an added amino acid sequence and represented by an amino acid sequence having cell adhesion ability to pluripotent stem cells, and any one of the following amino acids (2-i) to (2-iii):
- Array ( -I) An amino acid sequence represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID NO: 3), (2-ii) an amino acid sequence represented by SEQ ID NO: 3 having an identity of 80% or more, and the ability of the support to adsorb to the cell culture surface
- a polypeptide composition wherein the multimeric polypeptide of the body or more is 20% by mass or less of the total mass of the polypeptide contained in the composition.
- the dimer or higher multimeric polypeptide cross-linked via a cysteine residue at any position in the polypeptide is 20% by mass or less of the total mass of the polypeptide contained in the composition.
- the polypeptide composition according to any one of [1] to [3], comprising at least one polypeptide cross-linked between the residue and a cysteine residue corresponding to the 31st amino acid residue object.
- the polypeptide (a) to (c) or (d) in the composition corresponds to the fifth amino acid residue in the amino acid sequence contained in the first region and represented by SEQ ID NO: 1.
- the polypeptide composition according to any one of [1] to [3], which contains a polypeptide that is intramolecularly crosslinked. [6]
- the binding constant between the polypeptide contained in the composition and plasminogen activator inhibitor-1 is greater than 0.06, and any one of [1] to [3] Polypeptide composition.
- the polypeptide (a) to (c) or (d) in the composition further includes a third region comprising any one of the following amino acid sequences of (3a-i) to (3a-iii):
- a fourth region comprising any one amino acid sequence of the following (4a-i) to (4a-iii) is further added:
- polypeptides (a) to (c): (a) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, (b) 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 A polypeptide having the following amino acid sequence and having the ability to culture pluripotent stem cells, and (c) having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1, And at least one selected from the group consisting of a polypeptide having the ability to culture pluripotent stem cells, and a large amount of a dimer or more that is intermolecularly cross-linked via a cysteine residue contained in the polypeptide
- the polypeptide composition wherein the body polypeptide is 20% by mass or less of the total mass of the polypeptide contained in the composition.
- a method for culturing pluripotent stem cells comprising culturing pluripotent stem cells in the presence of the polypeptide composition according to any one of [1] to [9].
- a support having a cell culture surface and a polypeptide contained in the polypeptide composition according to any one of [1] to [9] disposed on the cell culture surface of the support Incubator.
- the present invention it is possible to provide a polypeptide composition capable of inducing cell culture performance of pluripotent stem cells, particularly excellent cell proliferation ability, and a method for culturing pluripotent stem cells using the same.
- FIG. 2 is a gel image of SDS-PAGE of polypeptide compositions I-1 (lane (B)) and I-2 (lane (A)) according to an example of the present invention. It is a graph which shows the relationship of the coupling
- the polypeptide composition of the present invention comprises the following polypeptides (a) to (c): (A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, (B) a polypeptide having an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 and having the ability to culture pluripotent stem cells; (C) a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1 and having a culture ability for pluripotent stem cells, Wherein the polypeptides (a) to (c) are any one of the following amino acid sequences (1-i) to (1-iii): (1-i) an amino acid sequence consisting of amino acid residues 1 to 44 in the amino acid sequence represented by SEQ ID NO: 1, (1-ii) an amino acid sequence comprising an amino acid sequence having 80% or more identity with the amino acid sequence consisting of amino acid sequence (1-i) and having cell adhesion ability to pluripot
- polypeptide composition of the present invention comprises a polypeptide (d) consisting of 40 to 450 amino acid residues
- the polypeptide (d) comprises the following (1-i) to (1- iii) any one amino acid sequence: (1-i) an amino acid sequence consisting of amino acid residues 1 to 44 in the amino acid sequence represented by SEQ ID NO: 1, (1-ii) an amino acid sequence comprising an amino acid sequence having 80% or more identity with the amino acid sequence consisting of amino acid sequence (1-i) and having cell adhesion ability to pluripotent stem cells, and (1-iii) A first region represented by an amino acid sequence comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (1-i) and having cell adhesion ability to pluripotent stem cells
- the present inventors have intensively studied to develop a recombinant protein that allows pluripotent stem cells to proliferate, and include a predetermined human vitronectin N-terminal partial sequence and have cell adhesion ability to pluripotent stem cells. It contains a large amount of polypeptides having amino acid sequences not in the form of multimers that are intermolecularly cross-linked via a cysteine residue on the N-terminal side of human vitronectin but in the form of monomers that are not cross-linked. It has been found that the proliferation ability of pluripotent stem cells is enhanced by culturing pluripotent stem cells in the presence of the polypeptide composition. The present invention is based on this finding. Hereinafter, embodiments of the present invention will be described in detail.
- the polypeptide composition of the present invention comprises at least one selected from the group consisting of the above-mentioned polypeptides (a) to (c), and is selected from the group consisting of polypeptides (a) to (c).
- a dimer or higher multimeric polypeptide which is at least one kind and is intermolecularly cross-linked via a cysteine residue contained in the first region is 20% of the total mass of the polypeptide contained in the composition. It is a composition of mass% or less. This polypeptide composition can enhance the proliferation ability of pluripotent stem cells.
- Another polypeptide composition of the present invention includes the above-described polypeptide (d), and is a polypeptide (d), which is an intermolecular crosslink via a cysteine residue contained in the first region.
- the dimer or higher multimeric polypeptide is a composition of 20% by mass or less of the total mass of the polypeptide contained in the composition.
- the polypeptide composition can enhance the proliferation ability of pluripotent stem cells while maintaining an undifferentiated state.
- the term “process” is not limited to an independent process, and is included in the term if the intended purpose of this process is achieved even when it cannot be clearly distinguished from other processes. .
- a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- the amount of each component in the composition is such that when there are a plurality of substances corresponding to each component in the composition, the plurality of substances present in the composition unless otherwise specified. Means the total amount.
- amino acid residues in amino acid sequences are represented by one-letter code (for example, “G” for glycine residue) or three-letter code (for example, “Gly” for glycine residue) well known in the art. There is a case.
- “%” relating to the amino acid sequence of a polypeptide is based on the number of amino acid (or imino acid) residues unless otherwise specified.
- amino acid residues used for a particular amino acid residue in an amino acid sequence insert two or more amino acid sequences to be compared in a manner well known in the art, Taking into account deletions and substitutions, when aligned so that the number of identical amino acid residues is the largest, it matches the position of a specific amino acid residue in the reference amino acid sequence , Is meant to denote an amino acid residue in the other amino acid sequence.
- identity with respect to amino acid sequence can refer to a value calculated using the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4thEd—Chapter 18). For example, 80% or more identity to SEQ ID NO: 1 means that Max.
- amino acid sequence with amino acid deletion, substitution or addition in an amino acid sequence excludes combinations of two or more amino acid deletions, substitutions and additions contained in the amino acid sequence. is not.
- polypeptides (a) to (c) are as follows: (A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, (B) a polypeptide having an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 and having the ability to culture pluripotent stem cells; (C) A polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1, and having the ability to culture pluripotent stem cells.
- SEQ ID NO: 1 DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDKYYRVNL
- pluripotent stem cells in a polypeptide means having the proliferation activity of pluripotent stem cells.
- pluripotent stem cells are seeded at a cell density of 250 cells / well on the surface of the cell culture adsorbed with the polypeptide and cultured for 8 days. When removed by washing, evaluation can be made based on whether or not there are 2500 cells / well (10 times the number of seeded cells) or more of adherent cells.
- the number of adherent cells can be quantified by a method for quantifying the activity of alkaline phosphatase expressed by pluripotent stem cells, the MTT test, or the like.
- vitronectin means human vitronectin. It has been confirmed that natural vitronectin is a glycoprotein having a sugar chain as part of its sequence.
- the polypeptide (b) preferably has an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and has culturing ability of pluripotent stem cells, more preferably , Having an amino acid sequence having an identity of 95% or more with the amino acid sequence represented by SEQ ID NO: 1, and having the ability to culture pluripotent stem cells.
- the polypeptide (c) is preferably a polypeptide having an amino acid sequence in which 1 to 5 amino acids are deleted, substituted or added in SEQ ID NO: 1 and having the ability to culture pluripotent stem cells. .
- the polypeptide according to the present invention is preferably (a1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, and (b1) an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1.
- the polypeptide of (b1) preferably consists of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and has the ability to culture pluripotent stem cells.
- the polypeptide (c1) is preferably composed of an amino acid sequence in which one or several, preferably 1 to 5 amino acids are deleted, substituted or added in SEQ ID NO: 1, and pluripotent stem cells
- the culture performance is as follows.
- Polypeptides (a) to (c) include a first region represented by any one of the following amino acid sequences (1-i) to (1-iii).
- (1-iii) An amino acid sequence consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (1-i) and having cell adhesion ability to pluripotent stem cells.
- the polypeptide site consisting of the 1st to 44th amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 is a domain known as the somatomedin B (SMB) domain of human vitronectin.
- SMB domain is known to interact with plasminogen activator inhibitor 1 (PAI-1).
- PAI-1 plasminogen activator inhibitor 1
- the SMB domain includes an amino acid sequence represented by CSYYQSC (SEQ ID NO: 2) corresponding to the 25th to 31st 7 amino acid residues in the vitronectin amino acid sequence, and the 45th to 47th 3 in the amino acid sequence of vitronectin. Both the RGD sequence, which is a cell adhesion motif corresponding to an amino acid residue, is included.
- the first region in the polypeptides (a) to (c) only needs to have any one selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 2 and the RGD sequence. From the viewpoint of proliferation, the first region in the polypeptides (a) to (c) preferably includes both of these sequences.
- the first region of (1-i) to (1-iii) is a sequence located relatively to the N-terminal side of natural vitronectin and exhibits adhesion to undifferentiated pluripotent stem cells, As a result, it is presumed that pluripotent stem cells can be proliferated. For this reason, the polypeptide including the first region is superior in cell adhesiveness and more excellent in cell proliferation ability than the polypeptide not including the first region.
- the first region containing a predetermined amino acid sequence has excellent cell adhesion. Therefore, the polypeptide of the present invention can favorably proliferate cells, particularly pluripotent stem cells.
- the polypeptides (a) to (c) according to the present invention having such an amino acid sequence can proliferate pluripotent stem cells over a long period of time.
- Polypeptide has“ cell adhesion ability to pluripotent stem cells ” means that the pluripotent stem cells exhibit adhesion to the polypeptide. Whether or not pluripotent stem cells are adherent to a polypeptide can be evaluated by a known evaluation method. For example, 24 hours after seeding pluripotent stem cells on the culture surface coated with the polypeptide, the cells are rinsed lightly with a phosphate buffer (PBS) or the like, and the pluripotent stem cells remaining on the culture surface after rinsing are removed. Evaluation can be based on quantity.
- PBS phosphate buffer
- the amino acid sequence represented by (1-ii) is preferably composed of an amino acid sequence having 90% or more identity with the amino acid sequence consisting of the amino acid sequence (1-i), and has cell adhesion ability to pluripotent stem cells. More preferably, the amino acid sequence is composed of an amino acid sequence having 95% or more identity with the amino acid sequence consisting of the amino acid sequence (1-i), and has an ability to adhere to cells against pluripotent stem cells. .
- the amino acid sequence represented by (1-iii) preferably comprises an amino acid sequence in which 1 to 5 amino acids are deleted, substituted or added in amino acid sequence (1-i), and is a pluripotent stem cell Is an amino acid sequence having the ability to adhere to cells.
- 1st to 44th amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 include the 5th amino acid residue, the 9th amino acid residue, the 19th amino acid residue, the 21st amino acid residue, A cysteine residue is included as the 25th amino acid residue, the 31st amino acid residue, the 32nd amino acid residue and the 39th amino acid residue.
- Polypeptides (a)-(c) may not contain cysteine residues corresponding to these eight cysteine residues, or may contain at least one.
- cysteine residues tend to crosslink with other cysteine residues under physiological conditions, when polypeptides (a) to (c) contain cysteine residues, cysteine residues at other sites in the polypeptide Between each other, intramolecular crosslinking may occur.
- Polypeptides (a) to (c) may contain two or more of the above-mentioned eight cysteine residues in the amino acid sequence shown in SEQ ID NO: 1 from the viewpoint of improving the proliferation promotion of pluripotent stem cells. More preferably, it is particularly preferable that two or more cysteine residues contained in the first region are intramolecularly crosslinked with each other.
- the combination of cysteine residues capable of forming an intramolecular bridge is a cysteine residue corresponding to the 5th amino acid residue and a cysteine residue corresponding to the 9th amino acid residue from the viewpoint of promoting the proliferation of pluripotent stem cells.
- Group cysteine residue corresponding to the 19th amino acid residue and cysteine residue corresponding to the 21st amino acid residue; cysteine residue corresponding to the 25th amino acid residue and corresponding to the 31st amino acid residue
- a combination of a cysteine residue corresponding to the 32nd amino acid residue and a cysteine residue corresponding to the 39th amino acid residue is a cysteine residue corresponding to the 5th amino acid residue and a cysteine residue corresponding to the 9th amino acid residue from the viewpoint of promoting the proliferation of pluripotent stem cells.
- Group cysteine residue corresponding to the 19th amino acid residue and cysteine residue corresponding to the 21st amino acid residue
- Polypeptides (a)-(d) preferably contain any one of the four preferred combinations of cysteine residues capable of forming intramolecular bridges, and more preferably contain all four.
- a specific cysteine residue may be displayed in combination with a number indicating the position of the amino acid sequence shown in SEQ ID NO: 1, for example, the 25th position in the amino acid sequence shown in SEQ ID NO: 1.
- Cys25 may be displayed.
- the number of amino acid residues in the first region can be 3 to 60 amino acid residues from the viewpoint of cell adhesion and proliferation of pluripotent stem cells, and is preferably 10 to 55 amino acid residues. preferable.
- the number of amino acid residues (amino acid length) contained in the polypeptides (a) to (c) is not particularly limited as long as it is in the range of 400 to 550, and is 450 to 520 in that it has high homology to human vitronectin. It is preferable that it is 450 to 459.
- the polypeptide (d) according to the present invention is as follows.
- a polypeptide comprising 40 to 450 amino acid residues comprising: (2-i) an amino acid sequence represented by PRPSLAKKQRFRHRNRKYRSQRGHSRGRNQN (SEQ ID NO: 3);
- the polypeptide (d) Since the polypeptide (d) has the ability to culture pluripotent stem cells, the polypeptide (d) preferably has “cell adhesion to pluripotent stem cells” and “adsorption ability of the support to the cell culture surface”.
- the support is a part of an incubator having a surface to which the polypeptide according to the present invention is applied when cell culture is performed using the culture method of the present invention. That is, since the first region represented by any one of the amino acid sequences (1-i) to (1-iii) has excellent cell adhesion, cells, particularly pluripotent stem cells are excellent. Can be grown.
- a polypeptide (d) containing such an amino acid sequence can proliferate pluripotent stem cells over a long period of time while maintaining the undifferentiated state.
- the second region represented by any one of the amino acid sequences (2-i) to (2-iii) contributes to the adsorptivity of the support to the cell culture surface.
- Polypeptide (d) containing such an amino acid sequence exhibits good adhesion of the support to the cell culture surface.
- the polypeptide (d) includes both the first region and the second region, thereby maintaining the pluripotent stem cells in an undifferentiated state without being detached from the cell culture surface of the support during the culture period. However, it can be grown for a long time.
- the polypeptide (d) can proliferate undifferentiated pluripotent stem cells in culture while suppressing peeling of the support from the cell culture surface, and improves handling in culture operations. Can do.
- the polypeptide (d) can be industrially produced without accelerating the proliferation of pluripotent stem cells in an undifferentiated state and without requiring a treatment for fixing the support to the cell culture surface by chemical bonding. It can be obtained as a polypeptide.
- polypeptide (d) eliminates the risk of contamination with antigenic substances and infectious diseases compared to natural human vitronectin, and at the same time has the same performance as natural vitronectin, ie, adhesion to pluripotent stem cells, cell proliferation, And the undifferentiation maintenance property can be maintained.
- the pluripotent stem cell can be propagated in an undifferentiated state” for the polypeptide means that the pluripotent stem cell maintains the differentiation potential during the culture period. Whether a pluripotent stem cell is in an undifferentiated state can be evaluated by a known evaluation method. For example, expression of molecular markers (expression measurement by flow cytometry such as SSEA-4 and / or Oct-4, immunostaining such as Oct-4 and / or NANOG), confirmation of pluripotent differentiation in in vitro experiments, And a method known to those skilled in the art, such as confirmation of teratoma formation by transplantation into immunodeficient mice or the like.
- molecular markers expression measurement by flow cytometry such as SSEA-4 and / or Oct-4, immunostaining such as Oct-4 and / or NANOG
- confirmation of pluripotent differentiation in in vitro experiments And a method known to those skilled in the art, such as confirmation of teratoma formation by transplantation into immunodeficient mice or the like.
- the culture period in which the pluripotent stem cells in the present invention maintain the differentiation potential varies depending on the culture conditions and the cell state of the pluripotent stem cells, but can be, for example, a one-month culture period.
- the first region in the polypeptide (d) is the same as the first region in the polypeptides (a) to (c).
- the matters described for the polypeptides (a) to (c) are applied as they are.
- the second region comprises an amino acid sequence consisting of 32 amino acid residues represented by SEQ ID NO: 3, and preferably from the viewpoint of ease of purification of the polypeptide (d), the second region in the polypeptide (d) is A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3.
- the amino acid sequence represented by SEQ ID NO: 3 is contained in a part of hemopexin-like domain II located on the C-terminal side of natural vitronectin and is composed of amino acid residues 342 to 373 of the amino acid sequence represented by SEQ ID NO: 1. Corresponds to the generated heparin-binding domain.
- the amino acid sequence represented by SEQ ID NO: 3 may be referred to as a heparin binding domain.
- Polypeptide (d) is presumed to have the ability to adsorb the support to the cell culture surface by having a heparin binding domain.
- the polypeptide (d) by using the polypeptide (d), undifferentiated pluripotent stem cells can be cultured for a long time while maintaining the undifferentiated state.
- the polypeptide (d) has a heparin-binding domain, thereby ensuring the hydrophilicity of the polypeptide (d) and suppressing the hydrophobic aggregation of the polypeptide.
- the polypeptide (d) is easy to purify and has high production efficiency.
- “having the ability to adsorb the support to the cell culture surface” means that the amino acid sequence is chemically coupled to the cell culture surface of the target incubator (hereinafter sometimes simply referred to as “culture surface”). It means physically adsorbing without a natural reaction. Whether or not the support has the ability to adsorb to the cell culture surface is determined by adding a solution containing the polypeptide to a plasma-treated polystyrene incubator at 200 pmol / cm 2 at 37 ° C., for example. It can be evaluated by whether or not the polypeptide remaining on the surface of the culture dish is present at 10 pmol / cm 2 or more when it is left for 2 hours and then washed twice with a phosphate buffer.
- the amount of the polypeptide remaining on the surface of the culture dish is the ELISA (Enzyme-Linked Immunosorbent Assay) method for quantifying the amount of binding to the antibody recognizing the polypeptide, or the amino acid produced by hydrolyzing the adsorbed polypeptide. Can be measured by quantification by HPLC or the like.
- ELISA Enzyme-Linked Immunosorbent Assay
- the heparin-binding domain has 80% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence shown in SEQ ID NO: 3, and can proliferate pluripotent stem cells in an undifferentiated state. And an amino acid sequence having the ability to adsorb the support to the cell culture surface.
- the heparin binding domain consists of an amino acid sequence in which one or several, preferably 1 to 5 amino acid residues are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 3. And an amino acid sequence having the ability to adsorb the support to the cell culture surface.
- Polypeptide (d) should just have the 1st field and the 2nd field, and there is no restriction in particular in the relative position.
- the first region is preferably located on the N-terminal side of the second region.
- the polypeptide (d) having the first region and the second region consists of 40 to 450 amino acid residues.
- 40 amino acid residues or more it is excellent in cell adhesion or cell growth or adsorbing ability of the support to the cell culture surface.
- 450 amino acid residues or less cell adhesion or cell growth is possible.
- the ability of the support to adsorb to the cell culture surface is further exhibited, and further, the formation of association, cross-linking or aggregation of proteins can be suppressed.
- Polypeptide (d) is preferably 80 or more, more preferably 90 or more, still more preferably 100 or more, from the viewpoint of making it difficult to form aggregates or the like.
- the number is preferably 400 or less, more preferably 250 or less, still more preferably 170 or less, and even more preferably 150 or less. Any of these upper limit values or lower limit values may be combined. For example, it is preferably composed of 40 to 400 amino acid residues, more preferably 80 to 250 amino acid residues, and 80 More preferably, it consists of ⁇ 150 amino acid residues, even more preferably 100-150 amino acid residues.
- Polypeptides (a) to (d) preferably have a GRAVY value of ⁇ 2.0 to ⁇ 0.95 from the viewpoint of preventing hydrophobic aggregation.
- GRAVY values (Kyte J., Doolittle R. F. (1982), J. Mol. Biol, 157: 105-132) represent the total average hydrophobicity of the polypeptides. The larger the GRAVY value, the higher the hydrophobicity. If the GRAVY value is ⁇ 0.95 or less, the occurrence of hydrophobic aggregation tends to be easily suppressed.
- the GRAVY value of the polypeptide is preferably ⁇ 1.70 to ⁇ 0.975, more preferably ⁇ 1.60 to ⁇ 1.10, in terms of achieving both suppression of aggregate formation and adsorbability or cell proliferation. . Since there is a tendency to aggregate as the number of amino acid residues is small, in the case of a polypeptide having 80 to 170 amino acid residues, the GRAVY value is obtained in that both suppression of aggregate formation and adsorption or cell proliferation are compatible. Is preferably ⁇ 1.70 to ⁇ 0.975, more preferably ⁇ 1.60 to ⁇ 1.10.
- the GRAVY value can be adjusted by, for example, increasing or decreasing the ratio of hydrophobic amino acids (eg, Trp, Tyr, Phe, Leu, Ile, Val, or Met) in the sequence, or increasing or decreasing the number of amino acid residues.
- hydrophobic amino acids eg, Trp, Tyr, Phe, Leu, Ile, Val, or Met
- polypeptides (a) to (d) preferably further have an amino acid sequence other than the amino acid sequences described above.
- Polypeptides (a) to (d) have the amino acid sequence represented by SEQ ID NO: 1, that is, the amino acid sequence of human vitronectin, from the viewpoint of appropriately exerting cell adhesion and adsorbing ability of the support to the cell culture surface. It preferably includes a partial sequence.
- the polypeptides (a) to (d) can acquire properties close to those of human vitronectin, for example, excellent adhesion and proliferation to pluripotent stem cells.
- Examples of the partial amino acid sequence of human vitronectin that can be included in the polypeptides (a) to (d) include cell adhesion and cell proliferation of the polypeptides (a) to (d), or adsorption of the support to the cell culture surface.
- the group consisting of the following third region and fourth region (3) among the amino acid sequence represented by SEQ ID NO: 1, a third region consisting of an amino acid sequence consisting of the 56th to 341st amino acid residues and an amino acid sequence selected from the partial amino acid sequence; and (4) A fourth region comprising an amino acid sequence selected from the amino acid sequence consisting of amino acid residues 374 to 459 and a partial amino acid sequence thereof among the amino acid sequence represented by SEQ ID NO: 1.
- the third region consists of the 56th to 341th amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 from the viewpoint that there is a tendency to suppress hydrophobic aggregation during the production of the polypeptide (3a).
- An amino acid sequence or a partial amino acid sequence thereof can be selected.
- An amino acid sequence consisting of the 269th to 341th amino acid residues or a partial amino acid sequence thereof can be selected.
- the polypeptides (a) to (d) further include a third region comprising any one amino acid sequence of the following (3a-i) to (3a-iii).
- (3a-iii) An amino acid sequence in which one or several, preferably 1 to 5 amino acids have been deleted, substituted or added to the amino acid sequence of (3a-i) or a partial amino acid sequence thereof.
- Polypeptides (a) to (d) preferably further comprise a third region comprising any one amino acid sequence of the following (3b-i) to (3b-iii).
- (3b-iii) An amino acid sequence wherein one or several, preferably 1 to 5 amino acids are deleted, substituted or added to the amino acid sequence of (3b-i) or a partial amino acid sequence thereof.
- the fourth region may be an amino acid sequence consisting of amino acid residues 374 to 459 or a partial amino acid sequence thereof from the viewpoint of adsorptivity to the culture dish, from the amino acid residues 374 to 409. Or a partial amino acid sequence thereof, or an amino acid sequence consisting of the 374th to 379th amino acid residues or a partial amino acid sequence thereof.
- the 374th to 379th positions are preferable in that the hydrophobic aggregation is easily suppressed at the time of producing the polypeptide, and there is a tendency that the hydrophobic aggregation is reduced by reducing the number of amino acids to be selected. is there.
- the polypeptides (a) to (d) further include a fourth region consisting of any one of the following (4a-i) to (4a-iii).
- a fourth region consisting of any one of the following (4b-i) to (4b-iii).
- Polypeptides (a) to (d) preferably further comprise a fourth region comprising any one amino acid sequence of the following (4c-i) to (4c-iii).
- (4c-i) an amino acid sequence consisting of amino acid residues 374 to 379 of the amino acid sequence represented by SEQ ID NO: 1 or a partial amino acid sequence thereof,
- (4c-ii) an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence of (4c-i) or a partial amino acid sequence thereof
- (4c-iii) An amino acid sequence wherein one or several, preferably 1 to 5 amino acids are deleted, substituted or added to the amino acid sequence of (4c-i) or a partial amino acid sequence thereof.
- the partial amino acid sequence of the amino acid sequence constituting the third region and the fourth region means an amino acid sequence composed of 3 or more consecutive amino acid residues in a predetermined range of amino acid residues. What is necessary is just to select the amino acid residue number of these partial amino acid sequences in the range which does not exceed the total amino acid residue number of polypeptide (d) mentioned above.
- Polypeptides (a) to (d) tend to have an advantage of enhancing the adsorptivity with the culture dish by including the third region. By including the fourth region, the polypeptides (a) to (d) tend to have the advantage of further increasing the adsorptivity with the culture dish. Polypeptides (a) to (d) may have only one of the third and fourth regions or both.
- the GRAVY values of the polypeptides (a) to (d) are determined from the viewpoint of ease of adjustment, increase / decrease in the number of amino acid residues in the amino acid sequences constituting the third and fourth regions, deletion of amino acid residues, It is preferable to adjust by substitution or addition, and it is particularly preferable to adjust the length of the amino acid sequence constituting the third region.
- Polypeptides (a) to (d) may not contain the amino acid residues contained in the 56th to 131st positions in the amino acid sequence shown in SEQ ID NO: 1, and the amino acid residues contained in the 56th to 268th positions.
- the amino acid residues contained in the 269th to 273rd positions may not be included, or the amino acid residues included in the 50th to 293rd positions may not be included.
- the amino acid sequence comprising these amino acid residues is presumed not to contribute to the performance of the polypeptides (a) to (d) in pluripotent cell culture, and is a sequence suitable from the viewpoint of adsorption to the culture dish. Is selected.
- the third region includes an amino acid residue corresponding to the cysteine residue of the sequence represented by SEQ ID NO: 1, it may have an amino acid residue other than the cysteine residue at the position of the cysteine residue. .
- an amino acid residue other than the cysteine residue at the position of the cysteine residue.
- a serine residue, an alanine residue, or a glycine residue can be mentioned suitably.
- a serine residue or an alanine residue is preferable at the point which has a structure similar to cysteine.
- Polypeptides (a) to (d) have other optional amino acid residues other than those described above as long as the cell adhesion and the adsorption property of the support to the cell culture surface are not impaired. May be.
- a sequence consisting of any other amino acid residue include, for example, additional sequences that are added to easily produce polypeptides (a) to (d) by recombinant techniques.
- additional sequences include an N-terminal methionine residue, an N-terminal GPLG sequence, a tag sequence (eg, glutathione S-transferase (GST), FLAG tag, His tag, etc.), which can be added between the regions.
- a linker sequence for example, GGGS, GGGGS, GGGGGS, etc. can be mentioned.
- Polypeptides (a) to (d) can be produced by amino acid synthesis techniques or genetic recombination techniques known to those skilled in the art.
- the polypeptides (a) to (d) are obtained by genetic recombination technology, specifically, first, a gene encoding the target amino acid sequence is obtained and incorporated into an expression vector to produce a recombinant expression vector. This is introduced into a suitable host to produce a transformant. By culturing the obtained transformant in an appropriate medium, the target polypeptide is produced. By recovering the target polypeptide from the culture by a conventional method, the polypeptide (a) to (D) can be obtained.
- Polypeptides (a) to (c) or (d) are used from the viewpoints of cell proliferation and proliferation ability of undifferentiated pluripotent stem cells in an undifferentiated state, etc.
- a first region consisting of an amino acid sequence consisting of the first to 47th amino acid residues of the amino acid sequence represented by SEQ ID NO: 1
- a second region consisting of an amino acid sequence consisting of amino acid residues 342 to 373 of the amino acid sequence represented by SEQ ID NO: 1 (SEQ ID NO: 3, heparin binding domain), the following third region and At least one selected from the group consisting of four regions: (3) an amino acid sequence consisting of amino acid residues 269 to 341 of the amino acid sequence shown in SEQ ID NO: 1 or a third region consisting of a partial amino acid sequence thereof; and (4) an amino acid sequence shown in SEQ ID NO: 1.
- a fourth region consisting of an amino acid sequence consisting of amino acid residues 374 to 459 or a partial amino acid sequence thereof;
- polypeptides (a) to (c) or (d) can be used from the viewpoints of cell proliferation and proliferation ability of undifferentiated pluripotent stem cells in an undifferentiated state.
- a first region consisting of an amino acid sequence consisting of amino acid residues 1 to 44 of the amino acid sequence represented by SEQ ID NO: 1 (including the amino acid sequence represented by SEQ ID NO: 2 and the RGD sequence);
- From a second region heparin binding domain
- an amino acid sequence consisting of amino acid residues 342 to 373 of the amino acid sequence shown in SEQ ID NO: 3 and from the following third region and fourth region At least one selected from the group consisting of: (3) an amino acid sequence consisting of amino acid residues 269 to 341 of the amino acid sequence shown in SEQ ID NO: 1 or a third region consisting of a partial amino acid sequence thereof; and (4) an amino acid sequence shown in SEQ ID NO: 1.
- a fourth region consisting of an amino acid sequence consisting of amino acid residues 374 to 459 or
- Polypeptides (a) to (c) are cell proliferative and undifferentiated pluripotent stem cells from the viewpoint of proliferation ability in an undifferentiated state, etc.
- a polypeptide (A) consisting of 400 to 550 amino acid residues including an amino acid sequence consisting of the 374th to 459th amino acid residues
- polypeptides (a) to (c) are cell proliferating and proliferating ability of undifferentiated pluripotent stem cells in an undifferentiated state.
- a first region consisting of an amino acid sequence consisting of the first to 55th amino acid residues of the amino acid sequence represented by SEQ ID NO: 1
- a second region SEQ ID NO: 3, heparin-binding domain
- an amino acid sequence consisting of amino acid residues 342 to 373 of the amino acid sequence shown in SEQ ID NO: 1 the following third region and At least one selected from the group consisting of four regions: (3) an amino acid sequence consisting of amino acid residues 269 to 341 of the amino acid sequence shown in SEQ ID NO: 1 or a third region consisting of a partial amino acid sequence thereof; and (4) an amino acid sequence shown in SEQ ID NO: 1.
- a fourth region consisting of an amino acid sequence consisting of amino acid residues 374 to 459 or a partial amino acid sequence thereof;
- the polypeptide (A) or (B) is preferably a polypeptide having a GRAVY value of -2.0 to -0.95.
- the polypeptide (A) as the polypeptide (d) preferably has 80 to 250 amino acid residues. Further, the polypeptide (A) or (B) as the polypeptide (d) further has a GRAVY value of ⁇ 2.0 to ⁇ 0.95 and a polypeptide having 80 to 250 amino acid residues. It is preferable that Furthermore, the polypeptide (A) as the polypeptide (d) is a polypeptide having a GRAVY value of ⁇ 1.70 to ⁇ 0.975 and an amino acid residue number of 80 to 250. Is preferred.
- polypeptide (A) or (B) as the polypeptide (d) preferably has 100 to 250 amino acid residues. Further, the polypeptide (A) or (B) as the polypeptide (d) further has a GRAVY value of ⁇ 2.0 to ⁇ 0.95, and has 100 to 250 amino acid residues. It is preferable that Furthermore, the polypeptide (A) or (B) as the polypeptide (d) further has a GRAVY value of ⁇ 1.70 to ⁇ 0.975 and a number of amino acid residues of 100 to 250. A peptide is preferred.
- polypeptide (A) or (B) as the polypeptide (d) further has a GRAVY value of ⁇ 1.70 to ⁇ 0.975 and a number of amino acid residues of 100 to 170.
- a peptide is preferred. Examples of the polypeptides (a) to (c) or (d) are listed below, but the present invention is not limited thereto.
- polypeptides (a) to (c) or (d) according to the present invention, (D1) Polypeptide having the amino acid sequence shown by any of SEQ ID NO: 4 to SEQ ID NO: 23, SEQ ID NO: 38 and SEQ ID NO: 39 (d2) Any of SEQ ID NO: 4 to SEQ ID NO: 23, SEQ ID NO: 38 and SEQ ID NO: 39 A polypeptide having an amino acid sequence of 80% or more, preferably 90% or more, more preferably 95% or more, and having the ability to culture pluripotent stem cells, or (D3) In the amino acid sequence represented by any one of SEQ ID NO: 4 to SEQ ID NO: 23, SEQ ID NO: 38, and SEQ ID NO: 39, 1 or several, preferably 1 to 5 amino acids are deleted, substituted or added A polypeptide having an amino acid sequence and capable of culturing pluripotent stem cells, As a more preferable example, (D4) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NO: 4
- polypeptide (a), the polypeptide (b), the polypeptide (c), and the polypeptide (d) may be collectively referred to as “specific polypeptide”. That is, the “specific polypeptide” refers to any one or two or more of the polypeptides (a) to (d).
- the polypeptide composition includes at least one selected from the group consisting of the polypeptides (a) to (c) described above, and is at least one selected from the group consisting of the polypeptides (a) to (c).
- the dimer or higher multimeric polypeptide cross-linked via a cysteine residue contained in the first region is 20% by mass or less of the total mass of the polypeptide contained in the polypeptide composition. It is. Further, when the polypeptide composition is a composition containing the above-described polypeptide (d), the polypeptide (d) is an intermolecular cross-linkage via a cysteine residue contained in the first region.
- the dimer or higher multimeric polypeptide is 20% by mass or less of the total mass of the polypeptide contained in the polypeptide composition.
- the polypeptide composition may contain both at least one selected from the group consisting of polypeptides (a) to (c) and polypeptide (d). Includes at least one selected from the group consisting of polypeptides (a) to (d), which is a dimer or more that is intermolecularly cross-linked via a cysteine residue contained in the first region.
- the multimeric polypeptide is 20% by mass or less of the total mass of the polypeptide contained in the polypeptide composition.
- cysteine residues in the first region are present in the other polypeptides.
- an intermolecular bridge with a cysteine residue in the region By forming an intermolecular bridge with a cysteine residue in the region, a multimer of dimers or more of the polypeptide can be formed.
- the content ratio in which the multimer of polypeptides (a) to (d) cross-linked via a cysteine residue contained in the first region exceeds 20% of the total mass of the polypeptide contained in the composition In the polypeptide composition, the proliferation ability of pluripotent stem cells is impaired.
- the content of the multimer relative to the total mass of the polypeptide means that when there are a plurality of types of polypeptides in the composition, the multimer is based on the total mass of these polypeptides combined.
- the content of multimers of polypeptides (a) to (d) in the polypeptide composition is 15% by mass or less of the total mass of the polypeptide contained in the composition from the viewpoint of promoting the proliferation ability of pluripotent stem cells. It is preferably 10% by mass or less, more preferably 5% by mass or less.
- the polypeptide composition further comprises a cysteine residue at any position in the polypeptide other than a multimer by intermolecular crosslinking through the cysteine residue in the first region from the viewpoint of the culture performance of pluripotent stem cells. It is preferable that the dimer or higher multimeric polypeptide cross-linked via the molecule is 20% by mass or less, more preferably 15% by mass or less of the total mass of the polypeptide contained in the composition. It is more preferably 10% by mass or less, and further preferably 5% by mass or less.
- the content of the multimers of the polypeptides (a) to (d) in the polypeptide composition is, for example, the result of SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis) carried out by a conventional method, “ImageJ It can be calculated by analysis using image analysis software such as “provided by National Institute of Health (NIH)”.
- SDS-PAGE sodium dodecyl sulfate / polyacrylamide gel electrophoresis
- Polypeptides (a) to (d) in the polypeptide composition are contained in the first region from the viewpoint of promoting the promotion of proliferation of pluripotent stem cells, and the 25th amino acid in the amino acid sequence represented by SEQ ID NO: 1 More preferably, it comprises a polypeptide that is intramolecularly bridged between a cysteine residue corresponding to the residue and a cysteine residue corresponding to the 31st amino acid residue.
- the polypeptide composition of the present invention may contain a polypeptide other than polypeptides (a) to (d).
- the polypeptide composition is a polypeptide composition containing at least one polypeptide selected from the polypeptides (a) to (c), from the viewpoint of obtaining the effect of the present invention more efficiently, the polypeptide (a ) To (d) is preferably 85% by mass or more of the composition, more preferably 90% by mass or more, still more preferably 95% by mass or more, and 99% by mass or more.
- the polypeptide composition is a polypeptide composition containing the polypeptide (d)
- the total content of the polypeptides (a) to (d) is determined from the viewpoint of obtaining the effect of the present invention more efficiently. Is preferably 85% by mass or more, more preferably 90% by mass or more, still more preferably 95% by mass or more, and particularly preferably 99% by mass or more.
- the binding constant between the polypeptide contained in the polypeptide composition and plasminogen activator inhibitor-1 (PAI-1) is 0.06 from the viewpoint of the culture performance of pluripotent stem cells. Is also preferably large.
- a polypeptide having an intramolecular bridge formed by the 25th cysteine residue and the 31st cysteine residue exhibits a binding property to plasminogen activator inhibitor-1 (PAI-1) It has been known. For this reason, the lower the binding constant for PAI-1, the smaller the polypeptide having an intramolecular bridge formed by the 25th and 31st cysteine residues.
- the binding constant between the polypeptide in the composition and PAI-1 is more preferably 0.1 or more, and further preferably 0.22 or more.
- the upper limit of the binding constant between the polypeptide and PAI-1 in the composition is not particularly limited, but is 1.0 or less in that it corresponds to binding of one molecule of PAI-1 to all the polypeptides. Is preferred.
- Cysteine residues in polypeptides tend to crosslink with other cysteine residues in the same polypeptide or with cysteine residues in other polypeptides. It is known that the formation of intermolecular bridges formed with cysteine residues in other polypeptides can be controlled by adjusting redox conditions, for example, Sinha N. K. et al. , J Biol Chem. 250 pp.8624-8629, 1975.
- a step of preparing a reaction solution having a denaturant concentration of 4M to 8M by combining the target polypeptides (a) to (d) with a denaturant such as urea or guanidine hydrochloride is obtained.
- a reducing agent such as reduced glutathione or cysteine and an oxidizing agent such as oxidized glutathione or cystine in a predetermined ratio, for example, the ratio of reducing agent to oxidizing agent (reducing agent: oxidizing agent (molar ratio) ))
- reducing agent: oxidizing agent (molar ratio) the ratio of reducing agent to oxidizing agent
- oxidizing agent oxidized glutathione or cystine
- the polypeptide composition of the present invention comprises at least one selected from the group consisting of polypeptides (a) to (c), and is intermolecularly crosslinked through cysteine residues contained in the polypeptide.
- the polypeptide composition which is 20 mass% or less of the total mass of the polypeptide contained in a composition may be sufficient as multimer polypeptide more than a mer.
- the content of the multimer that is intermolecularly cross-linked via a cysteine residue at any position in the polypeptides (a) to (c) It is 20 mass% or less of the total mass of the peptide.
- the polypeptide composition of this form can also improve the proliferation performance of a pluripotent stem cell similarly to the polypeptide composition mentioned above.
- polypeptide (a) that can be included in the polypeptide composition of the present embodiment
- all of the matters described above with respect to the polypeptide composition of other forms are described in the polypeptide (a) in the polypeptide composition of the present embodiment.
- the matters described above are used as they are, and preferable ranges are also used as they are.
- polypeptide (b) that can be contained in the polypeptide composition of the present embodiment has an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, and the culture performance of pluripotent stem cells
- the polypeptide may have any sequence, and may not have the amino acid sequences (1-i) to (1-iii) described above.
- the amino acid sequence that the polypeptide (b) may contain, and other forms of polypeptide compositions such as GRAVY values, As long as it can be applied to the polypeptide (b) in the polypeptide composition, the above-mentioned matters are used as they are, and preferable ranges are also used as they are.
- the polypeptide (c) that can be contained in the polypeptide of this embodiment has an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 1, and the culture performance of pluripotent stem cells
- the polypeptide may have any sequence, and may not have the amino acid sequences (1-i) to (1-iii) described above.
- the above-mentioned matters are diverted as they are, and preferred ranges are diverted as they are.
- the method for culturing pluripotent stem cells of the present invention includes culturing pluripotent stem cells in the presence of the above-described polypeptide composition. According to this culture method, pluripotent stem cells can be proliferated more efficiently.
- the method for culturing pluripotent stem cells includes a polypeptide-coated culture in which the polypeptide composition according to the present invention is applied to the cell culture surface of a support from the viewpoint of enhancing the proliferation of pluripotent stem cells and handling. It includes obtaining a surface (hereinafter referred to as a culture surface preparation step), and seeding and culturing pluripotent stem cells on a polypeptide-coated culture surface (hereinafter referred to as a culture step).
- the pluripotent stem cells according to the present invention are preferably primate pluripotent stem cells, specifically embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), somatic Stem cells, fertilized egg inner cell mass cells, early embryonic cells, and the like are included, and these cells may be used alone or in admixture of two or more.
- iPS cells include those described in Nature, 2007, July 19; Vol.448, pp.313-317; Cell, 2006, August 25; Vol.126 (4), pp.663-676, or Similar cells are included. Among these, iPS cells can be mentioned as examples of pluripotent stem cells preferably applied in the present invention.
- the component or substance applied to the present invention is a component or substance derived from a primate animal, it can be preferably applied to the present invention as a component or substance derived from the same animal species.
- the culture solution used for culturing can be appropriately selected according to the type of cells to be cultured.
- the culture medium that can be used may be any known one, and examples thereof include, for example, Essential 8, DMEM, MEM, F12, DME, RPMI 1640, MCDB104, 199, MCDB153, L15, SkBM, Basal medium, and the like. Preferably, it is Essential8.
- various components that can be generally added such as glucose, FBS (fetal bovine serum) or human serum, and antibiotics (such as penicillin and streptomycin) may be added to these culture solutions.
- concentration in the case of adding serum can be suitably changed according to the culture state at that time, it can usually be 10% (v / v).
- water salts (chlorides such as sodium, potassium, magnesium and calcium, inorganic salts such as hydroxide and carbonate, and organic salts such as sodium pyruvate), amino acids (essential amino acids and Non-essential amino acids), vitamins (riboflavin, biotin, cyanocobalamin, ascorbic acid, ascorbic acid derivatives, etc.), trace elements (selenium, iron, zinc, copper, etc.), carbon sources (D-glucose, etc.), FGF (basic fibroblasts) Cell growth factor FGF-2 etc.), TGF- ⁇ , insulin and a medium containing trenspherin are preferable.
- salts chlorides such as sodium, potassium, magnesium and calcium
- inorganic salts such as hydroxide and carbonate
- organic salts such as sodium pyruvate
- amino acids essential amino acids and Non-essential amino acids
- vitamins riboflavin, biotin, cyanocobalamin, ascorbic acid, ascorbic acid derivatives, etc.
- pluripotent stem cells are preferably cultured in the absence of a heterologous animal-derived component.
- the possibility of foreign matter contamination from different animals can be eliminated with high accuracy.
- Examples of the culture in the absence of a heterologous cell-derived component include a culture using a culture solution not containing a heterologous animal-derived component, and a culture not using a feeder cell derived from a heterologous animal.
- pluripotent stem cells are preferably cultured in the absence of heterogeneous animal-derived components and serum components. Thereby, mixing of the component derived from a different animal can be eliminated further.
- the medium not containing the heterologous animal component is a low osmotic pressure medium containing at least one medium component such as non-essential amino acids, glutamic acid, ⁇ -mercaptoethanol, FGF-2, TGF- ⁇ , insulin, transferrin, etc.
- Mixed media can be used.
- media such as TeSR2 (StemCell Technologies) and Essential8 (Life Technologies) can be used, but are not limited thereto.
- normal culture conditions for example, culture in an incubator with a temperature of 37 ° C. and a concentration of 5% (v / v) CO 2 are applied.
- a normal medium used for maintaining pluripotent stem cells can be used.
- mTeSR, TeSR2 (StemCell Technologies, Inc.) etc. are mentioned, for example.
- Inoculation of pluripotent stem cells in the medium is performed by a conventional method.
- the medium used in a series of passages is not necessarily the same, and may be a different medium as long as pluripotent stem cells can be maintained in an undifferentiated state.
- the polypeptide-coated culture surface includes applying a coating solution containing a predetermined amount of the specific polypeptide to the culture surface of the support.
- a coating solution containing a predetermined amount of the specific polypeptide to the culture surface of the support.
- the total content of the specific polypeptide in the coating solution varies depending on the kind or size of the culture surface as a coating target, from the viewpoint of adsorption ability to culture surface, preferably in the 1pmol / cm 2 ⁇ 1000pmol / cm 2 100 pmol / cm 2 to 300 pmol / cm 2 is more preferable.
- aqueous medium used in order to prepare a coating solution For example, a phosphate buffer, a Tris buffer, an ultrapure water etc. can be mentioned.
- the coating may be held for a predetermined time, for example, about 30 minutes to 24 hours after the coating solution is applied, whereby the culture surface can be coated with the specific polypeptide without requiring any special treatment.
- the culturing step includes seeding and culturing pluripotent stem cells on the polypeptide-coated culture surface.
- the seeding density and culture of pluripotent stem cells are not particularly limited, and generally performed conditions may be applied as they are.
- the seeding density of about 1 ⁇ 10 3 cells / cm 2 to 1 ⁇ 10 5 cells / cm 2 may be used under the above-described culture and passage conditions.
- a cell mass of 10 ⁇ m to 100 ⁇ m may be cultured under the above-described culture and passage conditions at a seeding density of about 1 cell / cm 2 to 5 cells / cm 2 .
- pluripotent stem cells can be handled easily on the culture surface coated with the specific polypeptide, and when the polypeptide (d) is used, the undifferentiated state can be maintained and proliferated well.
- pluripotent stem cells cultured in the presence of a specific polypeptide preferably in the absence of a heterologous animal-derived component, etc.
- the pluripotent stem cells cultured by the culturing method can be sufficiently safe for use in medical applications or similar applications.
- pluripotent stem cells can be cultured at a lower cost and with a simple operation, and not only for medical use but also in the research field. Can contribute widely.
- the incubator has a support having a surface used for cell culture.
- a support those well known as cell culture supports in the art can be used as they are.
- support materials include plastics (eg, polystyrene, acrylonitrile-butadiene-styrene resin, polycarbonate resin, polyester resin), glass, microporous filter materials (eg, cellulose, nylon, glass fiber, polyester and polycarbonate). ), Bioreactor materials (may include hollow fiber tubes or microcarrier beads) used in cell culture in batch or continuous mode, or in genetic engineering (eg bioreactor etc.), and polyethylene terephthalate, Teflon (Registered trademark), ceramics and related polymer materials may be included.
- the support may be a support whose culture surface is coated with a plasma polymerization thin film.
- the form of the incubator is not particularly limited, and may be any form applicable to the culture of pluripotent stem cells.
- Examples of such containers include multi-well plates (eg 6 wells, 12 wells, 24 wells, 96 wells), culture dishes (eg petri dishes, etc.), tubes, culture flasks, roller bottles, shaking cultures. A flask etc. can be mentioned.
- the incubator according to the present invention includes a support having a cell culture surface and a polypeptide contained in a polypeptide composition disposed on the cell culture surface of the support.
- the incubator has a culture surface comprising at least one selected from the group consisting of the polypeptides in the polypeptide composition according to the present invention described above, that is, the polypeptides (a) to (d). Yes. Therefore, the specific polypeptide is favorably adsorbed to the culture surface.
- the culture surface in the incubator means a surface to which cells can adhere when cells are seeded and grown.
- the incubator according to the present invention is prepared with a support having a cell culture surface (hereinafter referred to as “preparation step”), and the cell culture surface is made of the group consisting of polypeptides (a) to (d). It can be produced by a production method including at least one selected and performing adsorption treatment (hereinafter referred to as “adsorption treatment step”). Thereby, the incubator concerning this invention can be obtained simply.
- an incubator provided with a support having a culture surface is prepared.
- a step of forming the plasma polymerization thin film on the support may be included.
- a conventional method may be applied as it is.
- the adsorption treatment step includes applying and retaining the specific polypeptide on the culture surface.
- an adsorption solution containing a specific polypeptide in a predetermined amount is prepared, applied to the culture surface, and held for a predetermined time, thereby adsorbing the specific polypeptide to the culture surface.
- the matters described in the polypeptide-coated culture surface preparation step in the culture method can be applied as they are.
- RCP-11 corresponds to the sequence of natural human vitronectin.
- the positions in the amino acid sequence (SEQ ID NO: 1) of natural human vitronectin corresponding to the amino acid sequence of each polypeptide are shown in the “NOTE” column in Tables 2 and 3.
- the amino acid sequence of each polypeptide may include an amino acid sequence added, deleted, or substituted with respect to the amino acid sequence of the natural human vitronectin in the corresponding range described in the table.
- RCP-1 to RCP-10 and RCP-17 have the same amino acid sequence except that each of the amino acid sequences represented by SEQ ID NOs: 4 to 13 and SEQ ID NO: 38 has a methionine at the N-terminus. Consists of.
- BL21 (DE3) pLysS (Novagen) was transformed by a conventional method, applied to an LB plate containing kanamycin, and incubated at 37 ° C. for 16 hours. did. After confirming the introduction of the vector by the colony direct PCR method, in kanamycin-containing LB supplemented with 1 mM IPTG (isopropyl- ⁇ -D-thiogalactopyranoside, Wako Pure Chemical Industries, Ltd.), 37 ° C., 5 hours After shaking culture, polypeptide expression was induced.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside, Wako Pure Chemical Industries, Ltd.
- the cells were collected by centrifugation, and the cells were resuspended in a washing buffer (20 mM Tris, 150 mM NaCl, pH 7.6). After disrupting the cells by sonication, the cells were centrifuged at 15000 rpm, 30 min, 4 ° C., and the insoluble fraction was collected. After washing with a washing buffer containing 0.5% by weight Triton X100, the suspension was resuspended in a low-concentration urea buffer (Low Urea Buffer: 20 mM Tris, 150 mM NaCl, 2 M urea, pH 7.6) and subjected to sonication treatment. .
- a washing buffer (20 mM Tris, 150 mM NaCl, pH 7.6
- a washing buffer 20 mM Tris, 150 mM NaCl, 2 M urea, pH 7.6
- a high concentration urea buffer (High Urea Buffer: 20 mM Tris, 150 mM NaCl, 8 M urea, pH 7.6) was added, and the insoluble fraction was solubilized by sonication. .
- the solution containing the target peptide obtained by the above method was purified using AKTA Explorer 100 (trade name, GE Healthcare Japan Ltd.) and HiTrap Heparin HP 5 ml (trade name, GE Healthcare Japan Ltd.). . Stepwise elution was performed using the high-concentration urea buffer described above as a binding buffer and a high-salt concentration adjustment buffer (20 mM Tris, 1M NaCl, 8M urea, pH 7.6) as an elution buffer, and the target polypeptide was purified.
- BL21 (Novagen) was transformed by a conventional method, applied to an ampicillin-containing LB plate, and incubated at 37 ° C. for 16 hours. After confirming the introduction of the vector by the colony direct PCR method, the cells were cultured with shaking in ampicillin-containing LB supplemented with 100 ⁇ M IPTG at 20 ° C. for 24 hours to induce polypeptide expression.
- the cells were collected and resuspended with B-PER (registered trademark) Bacterial Protein Extraction Reagent in Phosphate Buffer (trade name, Thermo Fisher Scientific Co., Ltd.), and then the cells were disrupted by sonication.
- the insoluble fraction was removed by centrifugation at 15000 rpm, 30 min, 4 ° C., and the supernatant was purified using AKTA Explorer 100 and GSTRApHP 5 ml ⁇ 2 (trade name, GE Healthcare Japan, Inc.).
- the elution fraction was desalted using Hiprep 26/10 Desalting (trade name, GE Healthcare Japan Co., Ltd.), and a protease for cleaving GST fusion protein (PreScission Protease) was added at 1/2000 of the solution amount. And incubated at 4 ° C. for 24 hours to cleave the GST tag. It was purified again with GSRapHP 5 ml ⁇ 2, and the cleaved GST tag was adsorbed on the column and removed. The fraction passed through the column was dialyzed with Slide-A-Lizer (trade name, 3.5K MWCO .: Thermo Fisher Scientific, the same applies hereinafter) and replaced with PBS.
- Slide-A-Lizer trade name, 3.5K MWCO .: Thermo Fisher Scientific, the same applies hereinafter
- the RCP-1 polypeptide obtained as described above was run on a ready gel (12.5%, Bio-Rad) and stained with GelCode TM Blue Stain Reagent (trade name, Thermo Scientific). As a result, a single band was confirmed at a position corresponding to a molecular weight of 28.3 kDa predicted from the amino acid sequence. Similar results were obtained for other polypeptides.
- each purified polypeptide solution was dialyzed using Slide-A-Lizer (trade name, 3.5K MWCO.).
- the dialysis external solution was based on a dialysis buffer (PBS, 1.5 M NaCl, 0.5 M L-arginine, 1 mM EDTA, pH 7.4), and urea was removed by step dialysis.
- the concentration of the final dialysis product was calculated from the absorbance at 280 nm using NanoDrop (trade name, Thermo Fisher Scientific). Table 4 shows the presence or absence of aggregation after dialysis.
- the GRAVY value was calculated by adding the hydrophobicity index determined for each amino acid and dividing it by the number of amino acids (Kyte J., Doolittle RF (1982), J. Mol. Biol, 157: 105- 132).
- the GRAVY value is an index of the hydrophilicity / hydrophobicity of a polypeptide calculated from the hydrophobicity of an amino acid contained in each polypeptide. The larger the value, the more hydrophobic and the smaller the hydrophilic.
- the results are shown in Table 4.
- the presence or absence of aggregation was evaluated by the following G, A and B. The results are also shown in Table 4.
- G Aggregate formation is not seen.
- A Particle formation with a particle size of about 100 nm is observed.
- B Formation of visible aggregate having a particle diameter of 1 mm or more is observed.
- RCP-2 to RCP-5, RCP-7 to RCP-8, and RCP-17 are polypeptides having about 80 to about 170 amino acid residues and exhibit aggregation. Although it tends to occur, it can be seen that since the GRAVY value is in the range of ⁇ 1.70 to ⁇ 0.975, formation of aggregates is suppressed.
- each of boric acid buffer and 1N NaOH was applied to the surfaces coated with RCP-1 and RCP-11 to 16, and 80 ° C., humidity 100 Incubated for 24 hours. After air cooling, 75 ⁇ L of borate buffer was added to each well, and OPA (o-phthalaldehyde: Wako Pure Chemical Industries, Ltd.) / Methanol solution (160 mg / ml) and NAC (N-acetyl-L-cysteine: 50 ⁇ L of a reaction solution prepared by mixing 1: 100 (mass ratio) with a boric acid buffer solution (2 mg / ml) was added. After incubating at 40 ° C.
- OPA o-phthalaldehyde: Wako Pure Chemical Industries, Ltd.
- Methanol solution 160 mg / ml
- NAC N-acetyl-L-cysteine: 50 ⁇ L of a reaction solution prepared by mixing 1: 100 (mass ratio) with a boric acid buffer solution (2 mg / ml)
- RCP-1, 15, including PRPSLAKKQRFRHRNRKYRSQRGHSRGRNQN SEQ ID NO: 3 [amino acid sequence consisting of amino acid residues from 342 to 373 in SEQ ID NO: 1)] It can be seen that when 16 polypeptides are used, the adsorbability to the plate is as good as RCP-11 having the human vitronectin sequence. On the other hand, it can be seen that the adsorption amount of RCP-13, 14 not containing PRPSLAKKQRFRHRNRKYRSQRGHSRGRNQN is as low as about 1/4 with respect to the polypeptide containing the sequence.
- EmbryoMax registered trademark
- DMEM Invitrogen
- 10% (v / v) fetal bovine serum medium used, cultured for 24 hours using DMEM (Invitrogen), 10% (v / v) fetal bovine serum medium, and allowed to adhere on a T25 flask (trade name, Corning).
- the medium for human iPS cells the composition shown in Table 5 was added with FGF-2 (Sigma-aldrich) to a final concentration of 10 ng / ml.
- iPS cells were maintained and cultured in a 37 ° C., 5% (v / v, hereinafter the same) CO 2 incubator using the above medium.
- the medium was replaced with a new medium every day except for the day after iPS cell seeding.
- the subculture operation was performed by detaching the cells with Dispase (registered trademark) II (neutral protease Grade II, Roche) and separating the cells into an appropriate size by pipetting.
- Human iPS cells cultured as described above were treated with TrypLE Select (trade name, Invitrogen) at 37 ° C. for 5 minutes, and separated into single cells. The cells were collected by centrifugation at 300 rpm for 2 min, and a final concentration of 10 ⁇ M Y-27362 ((R)-(+)-trans-N- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide ⁇ 2HCl ⁇ H The suspension was suspended in TeSR2 (trade name, medium derived from heterogeneous animal and serum component-free medium, Stemcell Technologies) containing 2 O, Rho binding kinase inhibitor, Wako Pure Chemical Industries, Ltd.).
- TeSR2 trade name, medium derived from heterogeneous animal and serum component-free medium, Stemcell Technologies
- Prepare samples 1-17 by suspending in dialysis buffer, RCP-11, 15, 16, natural vitronectin and recombinant traminin used in the evaluation of values and aggregation properties, and add to each well of 96-well plate And 3 ° C., and adsorbed and held for 2 hours.
- iPS cells were seeded so as to have a cell density of 30000 cells / well. After culturing for 24 hours, non-adherent cells were removed by PBS washing, and only adherent cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.).
- the ALP activity was calculated with Attophos (registered trademark) AP AP Fluorescent Substrate System (Promega), and the number of undifferentiated iPS cells having ALP activity was calculated from the calibration curve.
- the results are shown in Table 6.
- RCP-1 to RCP-10, RCP-17 and RCP-11 having the 1st to 44th positions of SEQ ID NO: 1 and natural human vitronectin are cell adhesion of iPS cells.
- the rate was good.
- RCP-1 to RCP-10 and RCP-17, which do not include some or all of the 56th to 268th amino acids of the sequence shown in SEQ ID NO: 1, are natural human vitronectin and natural human vitronectin.
- the cell adhesion rate was better than RCP-11 having the same amino acid sequence. From this, it can be seen that a sequence important for cell adhesion exists in the 1st to 44th sequences of the sequence described in SEQ ID NO: 1.
- samples 1 to 12 were prepared so that RCP-1 to 10 and RCP-17 and Human Recombinant Laminin-511 as comparative controls were added at the concentrations shown in Table 8, respectively. > was seeded at a rate of 5000 cells / well on a 96-well plate on which each polypeptide was adsorbed, and cultured in a CO 2 incubator at 37 ° C. for 3 days. The number of cells after 3 days was measured by the same method as in the above ⁇ Cell adhesion evaluation 1>. The results are shown in Table 8.
- RCP-1 showed higher cell proliferation than RCP-11 having the amino acid sequence of natural vitronectin.
- the number of RCP-11 cells was about 2/3 of the number of RCP-1 cells on the 8th day of culture. The doubling time was calculated from the increase / decrease in the number of cells obtained. When RCP-1 was used, it was 46.4 ⁇ 2.1 hours, and when RCP-11 was used, 67.7 ⁇ 2.1 hours. Met.
- Table 8 also shows that RCP-1 to RCP-10 and RCP-17 all have a higher cell proliferation rate than laminin, which is the same extracellular matrix as vitronectin. It can be seen that such a high cell growth rate can be obtained similarly even if the 274th cysteine residue in SEQ ID NO: 1 is substituted with a serine residue.
- RCP-1 to RCP-10 and RCP-17 which do not contain a sequence have higher proliferation ability than RCP-11 having a sequence equivalent to that of human vitronectin and Laminin-511 which is a comparative example.
- RCP-1 to RCP-10 and RCP-17 including both CSYYQSC sequence and RGD sequence and PRPSLAKKQRFRHNRNKGYRSQRGHSRGRNQN sequence showed high cell proliferation.
- the adhesion of iPS cells to RCP-1 showed a cell adhesion rate equivalent to that of natural vitronectin when added at 125 pmol / cm 2 or more.
- FIGS. 3 and 4 (A) is an iPS cell cultured on RCP-1, (B) is an iPS cell cultured on RCP-11, and (C) is an iPS cell cultured on native human vitronectin. (D) shows iPS cells cultured on rLaminin-5, and (E) shows iPS cells cultured on rLaminin-511. Scale bar: 100 ⁇ m.
- the left column is an entire colony image
- the right column is an enlarged image
- the left column in FIG. 4 is a DAPI-stained image
- the right column is a stained image with an anti-NANOG antibody.
- Scale bar in FIGS. 3 and 4 200 ⁇ m.
- iPS cells cultured on natural human vitronectin with RCP-1,11 containing sequences effective for cell growth and adsorption to culture dishes are homogenous colonies and have high nuclear occupancy. It was characteristic of differentiated cells.
- iPS cells cultured on RCP-1 and 11 having a cell proliferation domain and an adsorption domain and natural human vitronectin strongly express NANOG throughout the colony and are in an undifferentiated state. It was found that it was maintained.
- a polypeptide consisting of 40 to 450 amino acid residues including any one of the CSYYQSC and RGD sequences and the PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN sequence is excellent in the adsorptivity of the support to the cell culture surface. It was. Further, such a polypeptide is equivalent to RCP-11 having a sequence equivalent to that of natural vitronectin and human vitronectin in maintaining cell adhesion and undifferentiated state of iPS cells under co-culture conditions with iPS cells. And iPS cell proliferation was superior to RCP-11.
- RCP-1 to RCP-10 and RCP-17 are all good in terms of maintaining cell adhesion and undifferentiated state of iPS cells. Good results in all of these capabilities were not obtained with the other polypeptides or the comparative recombinant traminin.
- a pluripotent stem cell can be proliferated in an undifferentiated state, and has excellent adsorptivity to a cell culture surface, and the polypeptide is used.
- a method and a culture device for pluripotent stem cells can be provided.
- Example 1 ⁇ Preparation of polypeptide composition> Of the RCP-1 to RCP-10 and RCP-17 obtained above, RCP-17 was used in the following examples.
- the purified polypeptide RCP-17 solution obtained in Reference Example 1 was dialyzed using Slide-A-Lizer (trade name, 3.5K MWCO.).
- the dialysis external solution was based on a dialysis buffer (PBS, 1.5 M NaCl, 0.5 M L-arginine, 1 mM EDTA, pH 7.4), and was dialyzed stepwise until the urea concentration reached 2 M to remove urea.
- PBS dialysis buffer
- the polypeptide solution is diluted 1/10 times (v / v) with a buffer for dilution (2M urea, PBS, 1.5M NaCl, 0.5M L-arginine, 1 mM EDTA, pH 7.4).
- a buffer for dilution 2M urea, PBS, 1.5M NaCl, 0.5M L-arginine, 1 mM EDTA, pH 7.4
- an oxidation buffer (2M urea, 2 mM oxidized glutathione [Wako Pure Chemical Industries, Ltd.], 20 mM reduced glutathione [Wako Pure Chemical Industries, Ltd.], PBS, 1.5M NaCl, 0.5M L-arginine, 1 mM EDTA, pH 7.4) was dialyzed as an external solution for dialysis for 5 days.
- dialysis was performed on a dialysis buffer (PBS, 1.5 M NaCl, 0.5 M L-arginine, 1 mM EDTA, pH 7.4) to remove urea.
- the final dialysis product was concentrated using Amicon Ultra 3K (trade name, Millipore) until the polypeptide concentration reached 0.5 mg / ml.
- the final dialyzed product was allowed to stand at 4 ° C. for 24 hours, and the oxidation was sufficiently advanced to obtain a polypeptide composition I-1 as the final product.
- the concentration of the final product was calculated from the absorbance at 280 nm using NanoDrop (trade name, Thermo Fisher Scientific).
- the purified polypeptide RCP-17 solution obtained in Reference Example 1 was prepared using Slide-A-Lizer (trade name, 3.5K MWCO.), And the dialyzed external solution was diluted with dialysis buffer (PBS). , 1.5 M NaCl, 0.5 M L-arginine, 1 mM EDTA, pH 7.4), and by step dialysis until the urea concentration reaches 0 M, the urea is removed for comparison as a final product.
- Polypeptide composition I-2 was obtained.
- VTN-N trade name, recombinant human vitronectin, Life Technologies
- Each of the peptide compositions I-3 was separated using non-reducing SDS-PAGE (15% by mass, ATTO) and stained using GelCode TM Blue Stain Reagent (Thermo Scientific). The results are shown in FIG. Further, the band intensity was quantified by analyzing the obtained stained image with ImageJ, and the abundance ratio of the monomer and the multimer was calculated. The results are shown in Table 10.
- FIG. 5 shows the formation of intermolecular cross-linked multimers in the polypeptide composition I-2 and the polypeptide composition I-3 containing VTN-N only from which urea was removed by step dialysis.
- RCP-17 multimers were not confirmed in polypeptide composition I-1 obtained through polypeptide oxidation treatment.
- the multimer was about 5% by mass or less.
- Example 2 ⁇ Cell proliferation evaluation 2> Using the polypeptide composition I-1 to polypeptide composition I-3 obtained in Example 1, the following cell proliferation evaluation was performed.
- Each polypeptide composition I-1 to I-3 is mixed with a predetermined buffer (polypeptide composition I-1 containing RCP-17) so that the concentration of the polypeptide in the composition is the final concentration shown in Table 11.
- I-2 was suspended in the dialysis buffer used in the evaluation of the GRAVY value and aggregation characteristics of Reference Example, and the polypeptide composition I-3 containing VTN-N was suspended in PBS).
- a peptide solution is prepared, added to each well of a plasma-treated polystyrene 96-well plate (BD Falcon) having a plasma-treated cell culture surface, and adsorbed by holding at 37 ° C. for 2 hours to have a polypeptide-coated surface Peptide treated 96 well plates were obtained.
- TeSR2 Stemcell Technologies Inc.
- Nutritem registered trademark, Bio Industries Inc.
- 1 volume ratio
- the ALP activity was calculated with Attophos (registered trademark) AP AP Fluorescent Substrate System (Promega), and the number of undifferentiated iPS cells having ALP activity was calculated from the calibration curve.
- Table 11 the cell proliferation rate is shown as a ratio in which the number of cells after 72 hours when cultured using recombinant traminin is 100%.
- n 3.
- polypeptide composition I-1 As shown in Table 11, it can be seen that the polypeptide composition I-1 according to the examples of the present invention is superior in cell growth to the polypeptide composition I-2. This indicates that cell proliferation activity is impaired by polypeptide multimer formation.
- the external dialysis solution is based on a dialysis buffer (PBS, 1.5 M NaCl, 0.5 M L-arginine, 1 mM EDTA, pH 7.4), urea is removed by step dialysis, and each of the polypeptide compositions II- 1 (Sample A) and II-2 (Sample B) were obtained.
- PBS dialysis buffer
- urea is removed by step dialysis, and each of the polypeptide compositions II- 1 (Sample A) and II-2 (Sample B) were obtained.
- NCP4 residues (Cys5, Cys9, Cys19, Cys21) of RCP-5 contained in polypeptide composition II-1 were intramolecularly crosslinked with Cys5 and Cys9 and Cys19 and Cys21 as a pair. Is detected as the main component. Further, only in RCP-5 contained in the polypeptide composition II-2, a component in which Cys39 was intermolecularly cross-linked was observed.
- PAI-1 Plasminogen Activator Inhibitor-1
- Blocker Casein (trade name, Thermo scientific) is added to 150 ⁇ L / well, and blocking is similarly performed by allowing to stand at 37 ° C. did.
- TBS TST
- Tween 20 was added to 150 ⁇ L / well, and the solution was continuously discarded from the well. This operation was performed 4 times in total to wash the inside of the well.
- PAI-1 (Catalog No. 1786-PI, R & D systems) solution, which was serially diluted from 170 nM to 2.6 nM in TBS, was added to each well at 50 ⁇ L / well. And allowed to stand at 37 ° C. After 1 hour, the PAI-1 solution was discarded from the well and unbound PAI-1 was removed by washing 4 times with TBST in the same manner as described above. Next, unlabeled anti-goat IgG H + L (catalog number A10537, Life technologies) stock solution was diluted 700 times with TBS, added to 150 ⁇ L / well, and further blocked by allowing to stand at 37 ° C. .
- PAI-1 Catalog No. 1786-PI, R & D systems
- HRP-labeled anti-goat IgG antibody (Catalog No. 81-1620, Life technologies) was diluted 7500 times with TBS and added to 100 ⁇ L / well. After allowing to stand at 37 ° C. for 1 hour, the antibody solution was discarded from the well, and the unbound labeled antibody was removed by washing with TBST four times in the same manner as described above. Subsequently, using a QuantaBlu Fluorogenic Peroxidase Substrate Kit (trade name, Thermo scientific), the HRP substrate was adjusted as described in the instruction, and added to 50 ⁇ L / well. After leaving still in a dark place at 37 ° C.
- FIG. 6 shows the result of plotting the obtained measured value on the X axis and the value obtained by dividing the measured value by the concentration of addition of PAI-1 (nM) on the Y axis. Since the obtained straight line corresponds to the following Scatchard Plot equation, the coupling constant (KA) can be calculated from the slope.
- PAI-1 binding amount / PAI-1 addition amount ⁇ KA ⁇ PAI-1 binding amount
- Table 12 shows the calculated results.
- RCP-5 in polypeptide composition II-1 has a larger binding constant of PAI-1 than RCP-5 in polypeptide composition II-2, that is, there are many intramolecular crosslinks of Cys25 and Cys31. I knew it existed.
- the polypeptide composition of the present invention comprises a polypeptide having an amino acid sequence having a cell adhesion ability to pluripotent stem cells, containing a predetermined partial sequence of human vitronectin N-terminal, and cysteine at the human vitronectin N-terminal side. It is a peptide composition containing a large amount in the form of a monomer that is not intermolecularly crosslinked, not in the form of a dimer or higher multimer that is intermolecularly crosslinked via a residue. It was found that pluripotent stem cells proliferate efficiently in the presence of such a polypeptide composition.
- the present invention it is possible to provide a polypeptide composition capable of inducing cell culture performance of pluripotent stem cells, particularly excellent cell proliferation ability, and a method for culturing pluripotent stem cells using the same.
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Abstract
Description
これらの霊長類の全能性または多能性幹細胞(これらの両者を総称して、本発明では単に「多能性幹細胞」と称する。)を培養する場合には、多能性幹細胞を未分化状態で長期間にわたって維持することが要求される。未分化状態の多能性幹細胞を長期間培養するためには、一般に、マウス繊維芽細胞等のフィーダー細胞が用いられる。
しかしながら、これらの組換えペプチドでも、多能性幹細胞の細胞培養性能としては充分とはいえない。
[1] 以下のポリペプチド(a)~(c):(a)配列番号1で示されるアミノ酸配列を有するポリペプチド、(b)配列番号1で示されるアミノ酸配列との同一性が80%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、および、(c)配列番号1において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、からなる群より選択される少なくとも1種を含み、ポリペプチド(a)~(c)が、以下の(1-i)~(1-iii)のいずれか1つのアミノ酸配列:(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列で表される第1の領域を含むポリペプチドであり、かつ、ポリペプチド(a)~(c)からなる群より選択される少なくとも1種であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、ポリペプチド組成物。
[2] 40個~450個のアミノ酸残基からなるポリペプチド(d)を含み、ポリペプチド(d)が、以下の(1-i)~(1-iii)のいずれか1つのアミノ酸配列:(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列で表される第1の領域と、以下の(2-i)~(2-iii)のいずれか1つのアミノ酸配列:(2-i)PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN(配列番号3)で示されるアミノ酸配列、(2-ii)配列番号3で示されるアミノ酸配列と80%以上の同一性を有し、支持体の細胞培養表面への吸着能を有するアミノ酸配列、および(2-iii)配列番号3で示されるアミノ酸配列に対して1若しくは数個のアミノ酸が欠失、置換若しくは付加され、支持体の細胞培養表面への吸着能を有するアミノ酸配列、で表される第2の領域と、を含むポリペプチドであり、かつ、ポリペプチド(d)であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、ポリペプチド組成物。
[3] ポリペプチドにおけるいずれかの位置のシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である[1]または[2]に記載のポリペプチド組成物。
[4] 組成物中のポリペプチド(a)~(c)または(d)が、第1の領域に含まれ、配列番号1で示されるアミノ酸配列のうち25番目のアミノ酸残基に相当するシステイン残基と31番目のアミノ酸残基に相当するシステイン残基との間で分子内架橋されている少なくとも1種のポリペプチドを含む[1]~[3]のいずれか1に記載のポリペプチド組成物。
[5] 組成物中のポリペプチド(a)~(c)または(d)が、第1の領域に含まれ、配列番号1で示されるアミノ酸配列のうち、5番目のアミノ酸残基に相当するシステイン残基と9番目のアミノ酸残基に相当するシステイン残基;19番目のアミノ酸残基に相当するシステイン残基と21番目のアミノ酸残基に相当するシステイン残基;25番目のアミノ酸残基に相当するシステイン残基と31番目のアミノ酸残基に相当するシステイン残基;並びに、32番目のアミノ酸残基に相当するシステイン残基と39番目のアミノ酸残基に相当するシステイン残基;の間でそれぞれ分子内架橋されているポリペプチドを含む[1]~[3]のいずれか1に記載のポリペプチド組成物。
[6] 組成物に含まれるポリペプチドとプラスミノーゲンアクチベータインヒビター-1(Plasminogen activator inhibitor-1)との結合定数が、0.06よりも大きい[1]~[3]のいずれか1に記載のポリペプチド組成物。
[7] 組成物中のポリペプチド(a)~(c)または(d)として、下記(3a-i)~(3a-iii)のうちいずれか1のアミノ酸配列からなる第3の領域をさらに含む少なくとも1種のポリペプチドを含む[1]~[6]のいずれか1に記載のポリペプチド組成物:(3a-i)配列番号1で示されるアミノ酸配列のうち、56番目~341番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、(3a-ii)(3a-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上の同一性を有するアミノ酸配列、および、(3a-iii)(3a-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
[8] 組成物中のポリペプチド(a)~(c)または(d)として、下記(4a-i)~(4a-iii)のうちいずれか1つのアミノ酸配列からなる第4の領域をさらに含む少なくとも1種のポリペプチドを含む[1]~[7]のいずれか1に記載のポリペプチド組成物:(4a-i)配列番号1で示されるアミノ酸配列のうち、374番目~459番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、(4a-ii)(4a-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上の同一性を有するアミノ酸配列、および、(4a-iii)(4a-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
[9] 以下のポリペプチド(a)~(c):(a)配列番号1で示されるアミノ酸配列を有するポリペプチド、(b)配列番号1で示されるアミノ酸配列との同一性が80%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、および、(c)配列番号1において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、からなる群より選択される少なくとも1種を含み、ポリペプチド中に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、ポリペプチド組成物。
[10] [1]~[9]のいずれか1に記載のポリペプチド組成物の存在下で、多能性幹細胞を培養することを含む多能性幹細胞の培養方法。
[11] 細胞培養表面を有する支持体と、支持体の細胞培養表面上に配置された[1]~[9]のいずれか1に記載のポリペプチド組成物に含まれるポリペプチドと、を有する培養器。
(a)配列番号1で示されるアミノ酸配列を有するポリペプチド、
(b)配列番号1で示されるアミノ酸配列との同一性が80%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、および、
(c)配列番号1において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、
からなる群より選択される少なくとも1種を含み、ポリペプチド(a)~(c)が、以下の(1-i)~(1-iii)のいずれか1つのアミノ酸配列:
(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、
(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および
(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列
で表される第1の領域を含むポリペプチドであり、かつ、
ポリペプチド(a)~(c)からなる群より選択される少なくとも1種であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である。
(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、
(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および
(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列
で表される第1の領域と、以下の(2-i)~(2-iii)のいずれか1つのアミノ酸配列:
(2-i)PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN(配列番号3)で示されるアミノ酸配列、
(2-ii)配列番号3で示されるアミノ酸配列と80%以上の同一性を有し、支持体の細胞培養表面への吸着能を有するアミノ酸配列、および
(2-iii)配列番号3で示されるアミノ酸配列に対して1若しくは数個のアミノ酸が欠失、置換若しくは付加され、支持体の細胞培養表面への吸着能を有するアミノ酸配列、
で表される第2の領域と、を含むポリペプチドであり、かつ、
ポリペプチド(d)であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である。
以下、本発明の実施の形態について、詳細に説明する。
また、本発明の他のポリペプチド組成物は、上述したポリペプチド(d)を含み、かつ、ポリペプチド(d)であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下の組成物である。本ポリペプチド組成物は、多能性幹細胞の増殖能を、未分化状態を維持しつつ高めることができる。
また、本明細書において「~」を用いて示された数値範囲は、「~」の前後に記載される数値をそれぞれ最小値および最大値として含む範囲を示す。
また、本明細書において、組成物中の各成分の量は、組成物中に各成分に該当する物質が複数存在する場合には、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。
本明細書では、アミノ酸配列におけるアミノ酸残基を、当該技術分野で周知の一文字表記(例えば、グリシン残基を「G」)または三文字表記(例えば、グリシン残基を「Gly」)で表記する場合がある。
本発明において、ポリペプチドのアミノ酸配列に関する「%」は、特に断らない限り、アミノ酸(またはイミノ酸)残基の個数を基準とする。
本明細書におけるアミノ酸配列に関する「同一性」とは、BLASTパッケージ(Ausubelら、1999 Short Protocols in Molecular Biology、4thEd - Chapter 18を参照)を用いて計算される値を指すことができる。例えば、配列番号1に対して同一性80%以上とは、BLASTにおけるMax.Identitiesの値が80以上であることを示す。
本明細書において、アミノ酸配列における「アミノ酸の欠失、置換若しくは付加されたアミノ酸配列」との表現は、アミノ酸配列に含まれるアミノ酸の欠失、置換および付加の2つ以上の組み合わせを排除するものではない。
本発明に係るポリペプチド(a)~(c)は以下のとおりである:
(a)配列番号1で示されるアミノ酸配列を有するポリペプチド、
(b)配列番号1で示されるアミノ酸配列との同一性が80%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、および、
(c)配列番号1において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド。
配列番号1:
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL
ここで接着細胞数は、多能性幹細胞が発現するアルカリフォスファターゼの活性を定量する方法、またはMTT試験などによって定量することができる。
ポリペプチド(c)は、好ましくは、配列番号1において1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチドである。
ここで、(b1)のポリペプチドは、好ましくは、配列番号1で示されるアミノ酸配列との同一性が90%以上のアミノ酸配列からなり、かつ多能性幹細胞の培養性能を有するものであり、より好ましくは、配列番号1で示されるアミノ酸配列との同一性が95%以上のアミノ酸配列からなり、かつ多能性幹細胞の培養性能を有するものである。
また、(c1)のポリペプチドは、好ましくは、配列番号1において1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞の培養性能を有するものである。
(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、
(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および
(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列。
上記(1-i)~(1-iii)のいずれか1つのアミノ酸配列で表される第1の領域と、以下の(2-i)~(2-iii)のいずれか1つのアミノ酸配列で表される第2の領域と、を含み、40個~450個のアミノ酸残基からなるポリペプチド:
(2-i)PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN(配列番号3)で示されるアミノ酸配列、
(2-ii)配列番号3で示されるアミノ酸配列と80%以上の同一性を有し、支持体の細胞培養表面への吸着能を有するアミノ酸配列、および
(2-iii)配列番号3で示されるアミノ酸配列に対して1若しくは数個のアミノ酸残基が欠失、置換若しくは付加され、支持体の細胞培養表面への吸着能を有するアミノ酸配列。
即ち、上記(1-i)~(1-iii)のいずれか1つのアミノ酸配列で表される第1の領域は、優れた細胞接着性を有することから、細胞、特に多能性幹細胞を良好に増殖させることができる。このようなアミノ酸配列を含むポリペプチド(d)は、多能性幹細胞を、未分化状態に維持しながら長期にわたって増殖させることができる。
また、上記(2-i)~(2-iii)のいずれか1つのアミノ酸配列で表される第2の領域は、支持体の細胞培養表面への吸着性に寄与する。このようなアミノ酸配列を含むポリペプチド(d)は、支持体の細胞培養表面への良好な接着性を示す。ポリペプチド(d)は、第1の領域と第2の領域とを共に含むことによって、培養期間中に支持体の細胞培養表面から剥がれることなく、多能性幹細胞を、未分化状態を維持しながら長期にわたって増殖させることができる。また、ポリペプチド(d)は、培養中の未分化状態の多能性幹細胞を、支持体の細胞培養表面からの剥がれを抑制しつつ増殖させることができ、培養操作における取扱い性を向上させることができる。
この結果、ポリペプチド(d)は、多能性幹細胞の未分化状態での増殖を促進すると共に、化学結合による支持体の細胞培養表面への固定処理を必要とせず、工業的に生産可能なポリペプチドとして得ることができる。
また、ポリペプチド(d)は、ヘパリン結合ドメインを含むことにより、ポリペプチド(d)の親水性を担保し、ポリペプチドの疎水凝集を抑制する傾向がある。この結果、ポリペプチド(d)は精製しやすく、製造効率が高い。
ここで培養皿表面に残存するポリペプチドの量は、ポリペプチドを認識する抗体との結合量を定量するELISA(Enzyme-Linked Immunosorbent Assay)法または、吸着したポリペプチドを加水分解し、生じたアミノ酸をHPLCなどによって定量することにより測定できる。
(3)配列番号1で示されるアミノ酸配列のうち、56番目~341番目のアミノ酸残基からなるアミノ酸配列およびその部分アミノ酸配列から選択されたアミノ酸配列からなる第3の領域、および、
(4)配列番号1で示されるアミノ酸配列のうち、374番目~459番目のアミノ酸残基からなるアミノ酸配列およびその部分アミノ酸配列から選択されたアミノ酸配列からなる第4の領域。
(3a-i) 配列番号1で示されるアミノ酸配列のうち、56番目~341番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(3a-ii) (3a-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上の同一性を有するアミノ酸配列、および、
(3a-iii) (3a-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
(3b-i) 配列番号1で示されるアミノ酸配列のうち、269番目~341番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(3b-ii) (3b-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上の同一性を有するアミノ酸配列、および、
(3b-iii) (3b-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
中でも、培養皿への吸着性に加えて、ポリペプチド作製時に疎水凝集を抑制しやすいという点で、374番目~379番目が好ましく、選択するアミノ酸数を減らすことによって疎水凝集が軽減される傾向がある。
(4a-i)配列番号1で示されるアミノ酸配列のうち、374番目~459番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(4a-ii) (4a-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上の同一性を有するアミノ酸配列、および、
(4a-iii) (4a-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
また、下記(4b-i)~(4b-iii)のうちいずれかひとつのアミノ酸配列からなる第4の領域をさらに含むものも好ましい。
(4b-i) 配列番号1で示されるアミノ酸配列のうち、374番目~409番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(4b-ii) (4b-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上の同一性を有するアミノ酸配列、および、
(4b-iii) (4b-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
(4c-i)配列番号1で示されるアミノ酸配列のうち、374番目~379番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(4c-ii)前記(4c-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上の同一性を有するアミノ酸配列、および、
(4c-iii)(4c-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。
また、ポリペプチド(a)~(d)のGRAVY値は、調整容易性の観点から、第3および第4の領域を構成するアミノ酸配列におけるアミノ酸残基数の増減、アミノ酸残基の欠失、置換若しくは付加等により調整されることが好ましく、特に、第3の領域を構成するアミノ酸配列の長さを調整することがより好ましい。
遺伝子組み換え技術によりポリペプチド(a)~(d)を得る場合、具体的にはまず、対象となるアミノ酸配列をコードする遺伝子を取得し、これを発現ベクターに組み込んで、組換え発現ベクターを作製し、これを適当な宿主に導入して形質転換体を作製する。得られた形質転換体を適当な培地で培養することにより、目的とするポリペプチドが産生されるので、培養物から目的とするポリペプチドを常法により回収することにより、ポリペプチド(a)~(d)を得ることができる。
(1)配列番号1で示されるアミノ酸配列の1番目~47番目のアミノ酸残基からなるアミノ酸配列からなる第1の領域と、
(2)配列番号1で示されるアミノ酸配列の342番目~373番目のアミノ酸残基からなるアミノ酸配列(配列番号3、ヘパリン結合ドメイン)からなる第2の領域と、以下の第3の領域および第4の領域からなる群より選択される少なくとも1つと:
(3)配列番号1で示されるアミノ酸配列の269番目~341番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第3の領域;および
(4)配列番号1で示されるアミノ酸配列の374番目~459番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第4の領域、
を含む80個~450個のアミノ酸残基からなるポリペプチド(A)であることが好ましい。
(1)配列番号1で示されるアミノ酸配列の1番目~44番目のアミノ酸残基からなるアミノ酸配列(配列番号2で示されるアミノ酸配列とRGD配列を含む)からなる第1の領域と、
(2)配列番号3で示されるアミノ酸配列の342番目~373番目のアミノ酸残基からなるアミノ酸配列からなる第2の領域(ヘパリン結合ドメイン)と、以下の第3の領域および第4の領域からなる群より選択される少なくとも1つ:
(3)配列番号1で示されるアミノ酸配列の269番目~341番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第3の領域;および
(4)配列番号1で示されるアミノ酸配列の374番目~459番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第4の領域と、
を含む100個~450個のアミノ酸残基からなるポリペプチド(B)であることが好ましい。
(1)配列番号1で示されるアミノ酸配列の1番目~47番目のアミノ酸残基からなるアミノ酸配列からなる第1の領域と、
(2)配列番号1で示されるアミノ酸配列の342番目~373番目のアミノ酸残基からなるアミノ酸配列からなる第2の領域(配列番号3、ヘパリン結合ドメイン)と、以下の第3の領域および第4の領域からなる群より選択される少なくとも1つと:
(3)配列番号1で示されるアミノ酸配列の269番目~341番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第3の領域;および
(4)配列番号1で示されるアミノ酸配列の374番目~459番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第4の領域、を含む400個~550個のアミノ酸残基からなるポリペプチド(A)であることが好ましい。
(1)配列番号1で示されるアミノ酸配列の1番目~55番目のアミノ酸残基からなるアミノ酸配列からなる第1の領域と、
(2)配列番号1で示されるアミノ酸配列の342番目~373番目のアミノ酸残基からなるアミノ酸配列からなる第2の領域(配列番号3、ヘパリン結合ドメイン)と、以下の第3の領域および第4の領域からなる群より選択される少なくとも1つ:
(3)配列番号1で示されるアミノ酸配列の269番目~341番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第3の領域;および
(4)配列番号1で示されるアミノ酸配列の374番目~459番目のアミノ酸残基からなるアミノ酸配列若しくはその部分アミノ酸配列からなる第4の領域と、
を含む400個~550個のアミノ酸残基からなるポリペプチド(B)であることが好ましい。
また、ポリペプチド(d)としての上記ポリペプチド(A)は、アミノ酸残基数が80個~250個であることが好ましい。
さらに、ポリペプチド(d)としての上記ポリペプチド(A)または(B)は、さらにGRAVY値が-2.0~-0.95であり、アミノ酸残基数が80個~250個のポリペプチドであることが好ましい。
またさらに、ポリペプチド(d)としての上記ポリペプチド(A)は、さらにGRAVY値が-1.70~-0.975であり、アミノ酸残基数が80個~250個のポリペプチドであることが好ましい。
また、ポリペプチド(d)としての上記ポリペプチド(A)または(B)は、アミノ酸残基数が100個~250個であることが好ましい。
さらに、ポリペプチド(d)としての上記ポリペプチド(A)または(B)は、さらにGRAVY値が-2.0~-0.95であり、アミノ酸残基数が100個~250個のポリペプチドであることが好ましい。
またさらに、ポリペプチド(d)としての上記ポリペプチド(A)または(B)は、さらにGRAVY値が-1.70~-0.975であり、アミノ酸残基数が100個~250個のポリペプチドであることが好ましい。
またさらに、ポリペプチド(d)としての上記ポリペプチド(A)または(B)は、さらにGRAVY値が-1.70~-0.975であり、アミノ酸残基数が100個~170個のポリペプチドであることが好ましい。
ポリペプチド(a)~(c)または(d)の一例を以下に挙げるが、本発明はこれらに限定されない。
(d1)配列番号4~配列番号23、配列番号38および配列番号39のいずれかで示されるアミノ酸配列を有するポリペプチド
(d2)配列番号4~配列番号23、配列番号38および配列番号39のいずれかで示されるアミノ酸配列との同一性が80%以上、好ましくは90%以上、より好ましくは95%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、または、
(d3)配列番号4~配列番号23、配列番号38および配列番号39のいずれかで示されるアミノ酸配列において1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、
を挙げることができ、より好ましい例として、
(d4)配列番号4~配列番号23、配列番号38および配列番号39のいずれかで示されるアミノ酸配列からなるポリペプチド
(d5)配列番号4~配列番号23、配列番号38および配列番号39のいずれかで示されるアミノ酸配列との同一性が80%以上、好ましくは90%以上、より好ましくは95%以上のアミノ酸配列からなり、かつ多能性幹細胞の培養性能を有するポリペプチド、または、
(d6)配列番号4~配列番号23、配列番号38および配列番号39のいずれかで示されるアミノ酸配列において1若しくは数個、好ましくは1個~5個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞の培養性能を有するポリペプチドを挙げることができる。
なお、本明細書においては、ポリペプチド(a)、ポリペプチド(b)、ポリペプチド(c)、及びポリペプチド(d)を総称して「特定ポリペプチド」ということがある。つまり、「特定ポリペプチド」とはポリペプチド(a)~(d)のうちの任意の一つ又は二つ以上を指す。
ポリペプチド組成物は、上述したポリペプチド(a)~(c)からなる群より選択される少なくとも1種を含み、ポリペプチド(a)~(c)からなる群より選択される少なくとも1種であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、ポリペプチド組成物に含まれるポリペプチドの全質量の20質量%以下である。また、ポリペプチド組成物が、上述したポリペプチド(d)を含む組成物である場合には、ポリペプチド(d)であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、ポリペプチド組成物に含まれるポリペプチドの全質量の20質量%以下である。ポリペプチド組成物は、それぞれのポリペプチド組成物において、ポリペプチド(a)~(c)からなる群より選択される少なくとも1種と、ポリペプチド(d)との双方を含んでもよく、この場合には、ポリペプチド(a)~(d)からなる群より選択される少なくとも1種であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、ポリペプチド組成物に含まれるポリペプチドの全質量の20質量%以下である。
本発明の多能性幹細胞の培養方法は、上述したポリペプチド組成物の存在下で、多能性幹細胞を培養することを含む。本培養方法によれば、多能性幹細胞をより効率よく増殖させることができる。
なかでも、本発明において好ましく適用される多能性幹細胞の例には、iPS細胞を挙げることができる。
なお、霊長類動物としては、ヒト、サル、ゴリラなどが挙げられ、使用されるポリペプチド(a)~(d)と同属となるヒトであることが特に好ましい。本発明に適用される成分または物質が、霊長類動物に由来する成分または物質であれば、同種動物由来の成分または物質として本発明に好ましく適用可能である。
さらに、本発明の培養方法では、多能性幹細胞を異種動物由来成分および血清成分の非存在下で培養することが好ましい。これにより、よりいっそう、異種動物由来成分の混入を排除することができる。
コーティング溶液における特定ポリペプチドの総含有量は、コーティング対象となる培養表面の種類または大きさによって異なるが、培養表面に対する吸着能の観点から、1pmol/cm2~1000pmol/cm2とすることが好ましく、100pmol/cm2~300pmol/cm2とすることがより好ましい。コーティング溶液を調製するために用いられる水性媒体としては特に制限はなく、例えば、リン酸緩衝液、トリス緩衝液、超純水等を挙げることができる。
コーティングは、コーティング溶液を付与した後、所定時間、例えば30分~24時間程度保持すればよく、これにより特別な処理を要せずに、培養表面に特定ポリペプチドを被覆することができる。
多能性幹細胞の播種密度および培養については、特に制限はなく、一般に行われる条件をそのまま適用すればよい。例えば、1×103個/cm2~1×105個/cm2程度の播種密度として、上述した培養および継代条件にて培養すればよい。また10μm~100μmの細胞塊を1個/cm2~5個/cm2程度の播種密度として上述した培養および継代条件にて培養してもよい。
さらに、特定ポリペプチドの存在下(好ましくは、さらに異種動物由来成分等の非存在下)で培養した多能性幹細胞は、試料等に由来する抗原性物質等の異物の混入可能性を、ほとんど完全に、または大幅に排除することが可能であり、当該培養方法にて培養した多能性幹細胞は医療用途、もしくはそれに準じた用途への使用に対し、充分に安全性を担保することができる。
さらにまた、特定ポリペプチドを用いた培養方法によれば、多能性幹細胞をより低コスト、かつ簡便な操作にて培養することができ、医療用途のみならず研究分野での需要に対しても広く貢献することができる。
本発明において培養器とは、細胞培養に用いられる表面を有する支持体を有するものである。このような支持体としては、当業界で細胞培養用支持体として周知のものをそのまま用いることができる。支持体の素材の例には、プラスチック(例えば、ポリスチレン、アクリロニトリル-ブタジエン-スチレン樹脂、ポリカーボネート樹脂、ポリエステル樹脂)、ガラス、微孔質のフィルター材料(例えば、セルロース、ナイロン、ガラス繊維、ポリエステルそしてポリカーボネート)、バッチ式または連続式で細胞培養において、あるいは遺伝子工学(例えばバイオリアクター等)において用いられるバイオリアクター用材料(中空繊維チューブまたはマイクロキャリアビーズを含めてもよい。)、および、ポリエチレンテレフタレート、Teflon(登録商標)、セラミックおよびその関連ポリマー材料等を含めてもよい。
また、支持体は、プラズマ重合薄膜により培養表面が被覆された支持体であってもよい。
本培養器は、前述した本発明にかかるポリペプチド組成物中のポリペプチド、即ち、ポリペプチド(a)~(d)からなる群より選択される少なくとも1種を備えた培養表面を有している。そのため、培養表面に対して特定ポリペプチドが良好に吸着しており、特定ポリペプチド上に多能性幹細胞を播種した場合には、取扱い性よく、また、ポリペプチド(d)を用いた場合には未分化状態を維持させた状態で、多能性幹細胞を増殖させることができる。
ここで、培養器における培養表面とは、細胞を播種し成育する際に、細胞が付着し得る表面を意味する。
吸着処理工程については、培養方法においてポリペプチド被覆培養表面調製工程で説明した事項をそのまま適用することができる。
<ポリペプチドの調製>
PCRを利用した常法にて、表2および表3に示すアミノ酸配列を有するRCP-1~RCP-17の各ポリペプチドをコードする遺伝子配列を増幅した。なお、RCP-11は天然ヒトビトロネクチンの配列に相当する。なお、各ポリペプチドのアミノ酸配列に対応する天然ヒトビトロネクチンのアミノ酸配列(配列番号1)における位置を、表2および表3中、「NOTE」欄に示す。ただし、各ポリペプチドのアミノ酸配列には、表中に記載された対応する範囲の天然ヒトビトロネクチンのアミノ酸配列に対して付加、削除、または置換されたアミノ酸配列が含まれる場合がある。なお、RCP-1~RCP-10およびRCP-17は、上述した配列番号4~13および配列番号38で示されるアミノ酸配列のそれぞれに対して、N末端にメチオニンを有する以外は、同一のアミノ酸配列からなる。
また凝集有無については、以下のG、AおよびBで評価した。結果を表4に併せて示す。
G:凝集物形成が見られない。
A:粒径100nm程度の粒子形成が認められる。
B:粒径1mm以上の可視凝集の形成が認められる。
<培養皿への吸着性評価>
上記の方法により得られた各ポリペプチドを、所定のバッファにて、0~200pmol/cm2の所定の最終濃度でウェルへ添加できるように希釈し、プラズマ処理済ポリスチレン製96ウェルプレート(Tissue Culture―Treated、Falcon社)に64μLずつ添加した。37℃、2時間インキュベートして各ポリペプチドをプレートに吸着させた後、PBSで2回洗浄し、RCP-1~RCP-16の各ポリペプチド被覆表面を得た。
<細胞接着性評価1>
上記ポリペプチドへのヒトiPS細胞(「Tic」:細胞番号No.JCRB1331:独立行政法人 医薬基盤研究所[567-0085 大阪府茨木市彩都あさぎ7丁目6番8号]より分譲)の細胞接着性評価は、以下のとおりに行った。
ヒトiPS細胞を維持するためのフィーダー細胞として、EmbryoMax(登録商標)(初代マウス胚性線維芽細胞:ハイグロマイシン耐性、マイトマイシンC処理済み, C57/BL6由来、継代3代目)(Millipore社)を使用し、DMEM(Invitrogen社)、10% (v/v) ウシ胎児血清培地を使用して24時間培養し、T25フラスコ(商品名、Corning社)上に接着させた。ヒトiPS細胞用培地は、表5の組成のものに、FGF-2(Sigma-aldrich社)を最終濃度10ng/mlとなるように添加したものを使用した。
<細胞接着性評価2>
表7に示すポリペプチドをFmoc固相合成法にて合成した。天然ビトロネクチンを130pmol/cm2の濃度で吸着させた表面を作製した後、100μMの上記合成ペプチドを添加した細胞懸濁液を30,000cells/ウェルの割合で播種した。播種24h後の接着細胞数を、<細胞接着性評価1>と同様の手法にて算出した。結果を表7に示す。表7中、細胞接着率については、合成ペプチドを含まない培養液での細胞接着率を100とした相対値とした。n=3。
<増殖評価>
上記<細胞接着性評価1>と同様に回収したiPS細胞を、RCP-1、11および天然ヒトビトロネクチンを吸着させた96ウェルプレートに対し、250cells/ウェルの割合で播種し、37℃、5%CO2インキュベーター内で8日間培養した。各経時後の接着細胞数を、上記<細胞接着性評価1>と同様の方法で計測し、増殖曲線を得た。増殖曲線を図2に示す。なお図2において黒菱形はRCP-1を用いた例、黒四角はRCP-11を用いた例を示す。
同様にRCP-1~10およびRCP-17と、比較対照として、Human Recombinant Laminin-511をそれぞれ表8に示す添加濃度となるように、試料1~12を調製し、上記<細胞接着性評価1>と同様にして各ポリペプチドを吸着させた96ウェルプレートに対して5000cells/ウェルの割合で播種し、37℃、CO2インキュベーター内で3日間培養した。3日後の細胞数を上記<細胞接着性評価1>と同様の方法で計測した。結果を表8に示す。
また表8より、RCP-1~RCP-10およびRCP-17はいずれも、ビトロネクチンと同じ細胞外マトリクスであるラミニンよりも細胞増殖率が高いことがわかる。このような高い細胞増殖率は、配列番号1における274番目のシステイン残基をセリン残基に置換しても同様に得られることがわかる。
さらに、表8より、CSYYQSCの配列およびRGD配列と、PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNの配列の双方を含むRCP-1~RCP-10およびRCP-17はいずれも高い細胞増殖性を示すことがわかった。
<細胞接着性評価3>
RCP-1を、125pmol/cm2~1000pmol/cm2の濃度にPBSで調整して使用した以外は、上記<細胞接着性評価1>と同様にして細胞接着性を評価した。結果を表9に示す。表9中、細胞接着率については、天然ビトロネクチンを130pmol/cm2の濃度で吸着させた培養器に対する細胞接着率を100とした場合の相対値とした。n=3。
<未分化維持評価>
上記<細胞接着性評価1>と同様に回収したiPS細胞を、TeSR2中に懸濁した。上記<細胞接着性評価1>で用いた試料1、試料2、試料5、試料6および試料7をそれぞれ、上記<細胞接着性評価1>と同様にして吸着させた6ウェルプレート(Tissue culture-treated, Falcon社)にiPS細胞を播種し、37℃、CO2インキュベーター内で培養した。培地は播種翌日を除き、毎日新たな培地に交換した。6日毎に前述と同様の方法にて継代を行った。それぞれの試料上で培養したiPS細胞の形態を図3に示す。
<ポリペプチド組成物の調製>
上記で得られたRCP-1~RCP-10およびRCP-17のうち、RCP-17を以下の実施例に用いた。
参考例1において得られた精製後のポリペプチドRCP-17の溶液を、Slide-A-Lizer(商品名、3.5K MWCO.)を用いて透析した。透析外液は、透析用バッファ(PBS、1.5M NaCl、0.5M L-アルギニン、1mM EDTA、pH7.4)を基本とし、尿素濃度が2Mになるまで段階透析し、尿素を除去した。
透析後、ポリペプチド溶液を希釈用バッファ(2M 尿素、PBS、1.5M NaCl、0.5M L-アルギニン、1mM EDTA、pH7.4)で1/10倍(v/v)に希釈し、次にポリペプチドの酸化を進行させるために、酸化バッファ(2M 尿素、2mM 酸化型グルタチオン[和光純薬工業株式会社]、20mM 還元型 グルタチオン[和光純薬工業株式会社]、PBS、1.5M NaCl、0.5M L-アルギニン、1mM EDTA、pH7.4)を透析外液として5日間透析した。続いて透析用バッファ(PBS、1.5M NaCl、0.5M L-アルギニン、1mM EDTA、pH7.4)に透析をして尿素を除去した。最終透析産物はアミコンウルトラ 3K(商品名、ミリポア社)を使って、ポリペプチド濃度が0.5mg/mlになるまで濃縮した。
最終透析産物を4℃で24h静置し、酸化を十分に進行させ、最終産物としてポリペプチド組成物I-1を得た。最終産物の濃度はNanoDrop(商品名、Thremo Fisher Scientific社)を用いて280nmの吸光度より算出した。
上記のようにして得られたRCP-17のポリペプチドを含有するポリペプチド組成物I-1およびI-2と、比較例としてVTN-N(商品名、リコンビナントヒトビトロネクチン、Life Technologies)を含むポリペプチド組成物I-3をそれぞれ、非還元SDS-PAGE(15質量%,ATTO社)を用いて分離し、GelCodeTM Blue Stain Reagent(Thermo Scientific社)を用いて染色した。結果を図5に示す。 また、得られた染色像をImageJにて解析することによりBand強度を定量し、単量体および多量体の存在比を算出した。結果を表10に示す。
また、表10に示されるように、実施例に係るポリペプチド組成物I-1では、多量体が約5質量%以下であった。
<細胞増殖性評価2>
実施例1で得られたポリペプチド組成物I-1~ポリペプチド組成物I-3を用いて、以下の細胞増殖性評価を行った。
<ポリペプチド組成物の調製>
上記で得られたRCP-1~RCP-10およびRCP-17のうち、RCP-5に対して、以下の方法で還元処理を施した。
RCP-5を含むポリペプチド溶液に、Tris(2-carboxyethyl)phosphine Hydrochloride(和光純薬工業株式会社)を終濃度100mMとなるように添加し、4℃で24h静置し、試料Aを調製した。
比較として、RCP-5を含むポリペプチド溶液を、未処理のまま-80℃で凍結させて、試料Bを調製した。
それぞれの試料を、Slide-A-Lizer(商品名、3.5K MWCO.)を用いて透析した。透析外液は、透析用バッファ(PBS、1.5M NaCl、0.5M L-アルギニン、1mM EDTA、pH7.4)を基本とし、段階透析にて尿素を除去し、それぞれポリペプチド組成物II-1(試料A)およびII-2(試料B)を得た。
Peptide Mass Fingerprint法を用いてジスルフィド結合の解析を行った。詳細を以下に示す。
50mM重炭酸アンモニウム緩衝液(pH7.8)900μLをエッペンドルフチューブに入れ、ポリペプチド組成物II-1およびII-2をそれぞれ100μL加えた。さらに、10μLの100μg/mlトリプシン(和光純薬工業株式会社)または500ng/mlのGlu-C(Promega)を添加し、37℃で一晩静置した。消化された断片化ペプチドを遠心エバポレーター(Buchi)にて100μLまで濃縮した。
上記消化物10μLをziptip C15チップ(商品名、ミリポア)を用いて脱塩し、測定試料とした。
UltraflexTOF/TOF(商品名、Bruker Daltonics)を用い、レーザー波長337nmにより試料を測定した。リフレクターモードにて測定を行い、同じくPeptide calibration standard II(商品名、Bruker Daltonics、製品番号222570)を用いて、m/z 700~4000の範囲を質量校正した。非還元測定では、ペプチド消化物1μLとCHCAマトリクス(α-シアノ-4-ヒドロキシケイヒ酸)の0.1% TFA/H2O-MeCN(2:1)の飽和溶液4μLを混合し、ターゲットプレートへ載せた。還元測定では、ペプチド消化物0.5μLと40mM DTT溶液(pH9.0)0.5μLをターゲットプレート上で混合し、20分静置した後にCHCAマトリクス飽和溶液0.5μLを載せ、乾燥させた。試料をターゲットプレートに載せてからのオンターゲット洗浄(脱塩)は、Bruker Daltonics社より入手した手順書に従い実施した。
得られた質量分析値は、Mascot server(商品名、マトリックスサイエンス株式会社)にて検索を行い、それぞれの酵素による断片化ペプチドの配列を同定した。同様に、還元処理による断片化ペプチドの質量変化を調べることで断片化ペプチド内に含まれるシステインが関わるジスルフィド結合の架橋位置を同定した。
Cys25とCyc31が架橋した構造を持つポリペプチドと結合することが知られているPlasminogen Acitivator Inhibitor-1(PAI-1)を用い、Cys25とCyc31の架橋構造の検出を試みた。
PAI-1結合量/PAI-1添加量 = -KA×PAI-1結合量
実施例3の<細胞増殖性評価2>に記載の方法と同様にして、ポリペプチド組成物II-1およびII-2の細胞増殖性を評価した。結果を表13に示す。ポリペプチド組成物II-1はポリペプチド組成物II-2と比較して細胞増殖活性に優れる、つまりCys25とCys31と分子内架橋体が結合しているポリペプチドは細胞増殖活性に優れることがわかった。
本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に援用されて取り込まれる。
Claims (11)
- 以下のポリペプチド(a)~(c):
(a)配列番号1で示されるアミノ酸配列を有するポリペプチド、
(b)配列番号1で示されるアミノ酸配列との同一性が80%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、および、
(c)配列番号1において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、
からなる群より選択される少なくとも1種を含み、ポリペプチド(a)~(c)が、以下の(1-i)~(1-iii)のいずれか1つのアミノ酸配列:
(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、
(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および
(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列
で表される第1の領域を含むポリペプチドであり、かつ、
ポリペプチド(a)~(c)からなる群より選択される少なくとも1種であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、ポリペプチド組成物。 - 40個~450個のアミノ酸残基からなるポリペプチド(d)を含み、ポリペプチド(d)が、以下の(1-i)~(1-iii)のいずれか1つのアミノ酸配列:
(1-i)配列番号1で示されるアミノ酸配列のうち1番目~44番目のアミノ酸残基からなるアミノ酸配列、
(1-ii)アミノ酸配列(1-i)からなるアミノ酸配列との同一性が80%以上のアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列、および
(1-iii)アミノ酸配列(1-i)において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ多能性幹細胞に対する細胞接着能を有するアミノ酸配列
で表される第1の領域と、以下の(2-i)~(2-iii)のいずれか1つのアミノ酸配列:
(2-i)PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN(配列番号3)で示されるアミノ酸配列、
(2-ii)配列番号3で示されるアミノ酸配列と80%以上の同一性を有し、支持体の細胞培養表面への吸着能を有するアミノ酸配列、および
(2-iii)配列番号3で示されるアミノ酸配列に対して1若しくは数個のアミノ酸が欠失、置換若しくは付加され、支持体の細胞培養表面への吸着能を有するアミノ酸配列、
で表される第2の領域と、を含むポリペプチドであり、かつ、
ポリペプチド(d)であって、第1の領域に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、ポリペプチド組成物。 - ポリペプチドにおけるいずれかの位置のシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、請求項1又は請求項2記載のポリペプチド組成物。
- 組成物中のポリペプチド(a)~(c)または(d)が、第1の領域に含まれ、配列番号1で示されるアミノ酸配列のうち25番目のアミノ酸残基に相当するシステイン残基と31番目のアミノ酸残基に相当するシステイン残基との間で分子内架橋されている少なくとも1種のポリペプチドを含む請求項1~請求項3のいずれか一項記載のポリペプチド組成物。
- 組成物中のポリペプチド(a)~(c)または(d)が、第1の領域に含まれ、配列番号1で示されるアミノ酸配列のうち、
5番目のアミノ酸残基に相当するシステイン残基と9番目のアミノ酸残基に相当するシステイン残基;
19番目のアミノ酸残基に相当するシステイン残基と21番目のアミノ酸残基に相当するシステイン残基;
25番目のアミノ酸残基に相当するシステイン残基と31番目のアミノ酸残基に相当するシステイン残基;並びに、
32番目のアミノ酸残基に相当するシステイン残基と39番目のアミノ酸残基に相当するシステイン残基;
の間でそれぞれ分子内架橋されているポリペプチドを含む請求項1~請求項3のいずれか一項記載のポリペプチド組成物。 - 組成物に含まれるポリペプチドとプラスミノーゲンアクチベータインヒビター-1(Plasminogen activator inhibitor-1)との結合定数が、0.06よりも大きい請求項1~請求項3のいずれか一項記載のポリペプチド組成物。
- 組成物中のポリペプチド(a)~(c)または(d)として、下記(3a-i)~(3a-iii)のうちいずれか1のアミノ酸配列からなる第3の領域をさらに含む少なくとも1種のポリペプチドを含む請求項1~請求項6のうちいずれか一項記載のポリペプチド組成物:
(3a-i)配列番号1で示されるアミノ酸配列のうち、56番目~341番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(3a-ii)(3a-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上の同一性を有するアミノ酸配列、および、
(3a-iii)(3a-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。 - 組成物中のポリペプチド(a)~(c)または(d)として、下記(4a-i)~(4a-iii)のうちいずれか1つのアミノ酸配列からなる第4の領域をさらに含む少なくとも1種のポリペプチドを含む請求項1~請求項7のうちいずれか一項記載のポリペプチド組成物:
(4a-i)配列番号1で示されるアミノ酸配列のうち、374番目~459番目のアミノ酸残基からなるアミノ酸配列またはその部分アミノ酸配列、
(4a-ii)(4a-i)のアミノ酸配列またはその部分アミノ酸配列と80%以上の同一性を有するアミノ酸配列、および、
(4a-iii)(4a-i)のアミノ酸配列またはその部分アミノ酸配列に対して、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列。 - 以下のポリペプチド(a)~(c):
(a)配列番号1で示されるアミノ酸配列を有するポリペプチド、
(b)配列番号1で示されるアミノ酸配列との同一性が80%以上のアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、および、
(c)配列番号1において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ多能性幹細胞の培養性能を有するポリペプチド、
からなる群より選択される少なくとも1種を含み、ポリペプチド中に含まれるシステイン残基を介して分子間架橋されている二量体以上の多量体ポリペプチドが、組成物に含まれるポリペプチドの全質量の20質量%以下である、ポリペプチド組成物。 - 請求項1~請求項9のいずれか一項記載のポリペプチド組成物の存在下で、多能性幹細胞を培養することを含む多能性幹細胞の培養方法。
- 細胞培養表面を有する支持体と、支持体の細胞培養表面上に配置された請求項1~請求項9のいずれか一項記載のポリペプチド組成物に含まれるポリペプチドと、を有する培養器。
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