JP2020015708A - Stem cell adhesive peptides and uses thereof - Google Patents

Abstract

To provide stem cell culture substrates with which embryonic stem cells (ES cells) and induced pluripotent cells (iPS cells) being tools for regenerative medicine can be efficiently cultured while retaining the undifferentiated state, where the substrates are stably provided in a low molecular state with little production cost.SOLUTION: The invention relates to a stem cell adhesive peptide having binding activity to dystroglycan, the peptide comprising a specific amino acid sequence derived from human laminin-511E8 or an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in the specific amino acid sequence. The invention also relates to a stem cell culture substrate comprising one, two or more of the stem cell adhesive peptides.SELECTED DRAWING: None

Description

  The present invention relates to a stem cell adhesive peptide having an activity of binding to dystroglycan (DG) and use thereof.

  Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), together with somatic stem cells, reconstitute tissues and organs inside and outside the body and remove lost tissues and organs. Instead, it is a useful tool to make regenerative medicine feasible. Pluripotent stem cells are also expected to be used in drug discovery research such as elucidation of disease mechanisms, selection of treatment methods, and application to pharmacological tests. Since the establishment of human ES cells in 1998, co-culture with human ES cells and human iPS cells (hereinafter referred to as "hESCs / hiPSCs") has been a standard culture method with feeder cells. Mitotically inactivated mouse embryonic fibroblasts (mouse embryonic fibroblasts, MEFs, feeder cells) supply humoral factors necessary for maintaining the survival, proliferation, and undifferentiation ability of hESCs / hiPSCs, It also serves as a scaffold for hESCs / hiPSCs. However, in addition to the difficulty in preparing uniform feeder cells, the co-culture system with feeder cells introduces unidentified components, which is a major problem in culturing hESCs / hiPSCs for medical applications. It has become. In order to solve such problems associated with feeder cells, it is essential to develop a culture method that does not use feeder cells. Except for suspension cells and cancerous cells, adherent cells require a culture substrate to be a scaffold, which is one of the basic characteristics called scaffold dependence of cell growth. Adhesive hESCs / hiPSCs are not only required for a scaffold for proliferation, but also essential for maintaining their characteristic undifferentiated ability.

  To date, various culture substrates have been used for culturing hESCs / hiPSCs. Culture substrates are broadly classified into naturally-derived substrates and synthetic-derived substrates. A naturally-occurring substrate is an extracellular matrix on which cells are scaffolded in vivo, and laminin is a typical example together with Matrigel and vitronectin. Laminin is a cross-linked heterotrimeric glycoprotein composed of three subunits, α-chain, β-chain, and γ-chain. Five types of α-chain, three types of β-chain, and three types of Each type has been identified, and 19 laminin isoforms from these different combinations have been localized to various cells and tissues. These isoforms have been reported to be involved in various biological processes such as cell adhesion, proliferation, migration and differentiation. So far, laminin-111 (α1β1γ1), laminin-332 (α3β3γ2), laminin-511 (α5β1γ1) recombinant protein has been shown to maintain and grow undifferentiated state of hESCs (Non-Patent Document 1 ). Among them, it has been reported that laminin-511 enables cultivation without feeder cells and maintains the undifferentiated state of hESCs / hiPSCs in the absence of serum and xenobiotic-derived components (Non-Patent Document 2). . In the production of recombinant proteins, mammalian cell expression systems are used, but full-length laminin is a 400-800 kDa giant heterotrimeric protein consisting of α-chain, β-chain and γ-chain, Mass production of these recombinant proteins has been very difficult. In contrast, laminin-E8 fragment (LM511-E8), which is a fragment of laminin-511, shows strong adhesion to hESCs / hiPSCs in recent years, and has been shown to grow while maintaining undifferentiation. (Non-Patent Document 3). Since the molecular weight of LM511-E8 is smaller than that of the full-length protein and has a higher yield than before, it has been used as a culture substrate for hESCs / hiPSCs. A technical breakthrough. However, since LM511-E8 is still a heterotrimer with a molecular weight of 150 kDa, further reduction in molecular weight is required.

  On the other hand, dystroglycan (DG), one of the non-integrin receptors of laminin-511, is responsible for cell adhesion to the basement membrane in vivo by a unique sugar chain. DG consists of two subunits, α-dystroglycan (α-DG), which has abundant sugar chains, and β-dystroglycan (β-DG), a single transmembrane protein, and binds to laminin. Has been shown to involve the O-glycan of α-DG. DG is known to be expressed in the early mouse embryo and is known to be involved in early development from analysis with KO mice, and its role in mouse ES cell expression and basement membrane formation has been reported (Non-patent Documents 4, 5). ). However, the role of DG in the maintenance and differentiation of hESCs / hiPSCs in undifferentiation has not been known.

Miyazaki et al., Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells.Biochem Biophys Commun, 375, 27-32, 2008 Rodin et al., Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.Nat Biotechnol, 28, 611-615, 2010 Miyazaki et al., Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells.Nat Commun, 3, 1236, 2012 William et al., Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1-null mice.Hum Mol Genet, 6, 831-841, 1997 Henry and Cambell, A role for dystroglycan in basement membrane assembly.Cell, 95, 859-857, 1998

  The present invention enables efficient culture of embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) as tools for regenerative medicine while maintaining an undifferentiated state, stable supply, and no production cost An object of the present invention is to provide a stem cell culture substrate having a reduced molecular weight.

  The present inventors have focused on dystroglycan (DG), one of the receptors for laminin-511, in order to solve the above problems, and as a result, DG is expressed in human iPS cells and its undifferentiated state And that the DG was sufficiently glycosylated and had a binding activity to LM511-E8. Thus, the present inventors have prepared a peptide library covering the amino acid sequence of LM511-E8, and succeeded in identifying a peptide having DG-binding activity (DG-binding peptide) from the library. Furthermore, it was confirmed that the DG-binding peptide had an adhesive activity to human iPS cells and could be used as a culture substrate for stem cells, and that the gene could be introduced into human iPS cells by using the DG-binding peptide. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) The amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or one or several amino acids are deleted, substituted, inserted, or A stem cell adhesive peptide having an activity of binding to dystroglycan, comprising an added amino acid sequence.
(2) The stem cell adhesive peptide according to (1), wherein the stem cells are induced pluripotent stem cells, embryonic stem cells, or somatic stem cells.
(3) A stem cell culture substrate comprising one or more stem cell adhesive peptides according to (1) or (2).
(4) A kit for stem cell culture, comprising the stem cell culture substrate according to (3).
(5) A complex for introducing a gene into a stem cell, comprising the stem cell adhesive peptide according to (1) or (2) and a gene.
(6) A complex for introducing a cytotoxic substance into stem cells, comprising the stem cell adhesive peptide according to (1) or (2) and a cytotoxic substance.

  Since the stem cell adhesive peptide of the present invention has a binding activity to dystroglycan (DG) expressed on the surface of a stem cell, the stem cell adhesive peptide has high adhesiveness to a stem cell and can be proliferated while maintaining the undifferentiated state of the stem cell. It is effective as a stem cell culture substrate. Since the stem cell culture substrate of the present invention is smaller in molecular weight than recombinant laminin such as laminin-511 / -512 and laminin-511E8, it has the advantage of excellent operability, and is a synthetic substrate. It can be supplied stably and has no production cost. Further, the stem cell culture substrate of the present invention can be used as a culture substrate for elucidating the mechanism of undifferentiation of stem cells or for culturing mass-cultured stem cells to induce differentiation into target cells or tissues. Furthermore, the stem cell adhesive peptide of the present invention can be used not only to provide a scaffold for stem cell culture, but also to introduce a gene or a cytotoxic substance into stem cells using binding to dystroglycan (DG).

FIG. 1A shows immunostaining of feeder cells and colonies of human iPS cells (201B7 cells) cultured on LM511-E8 (a, d: staining with mouse anti-DG monoclonal antibody (VIA4), g, j: mouse anti-DG). Staining with DG monoclonal antibody (IIH6), b, e, h, k: staining with rabbit anti-Nanog polyclonal antibody, c, f, i, l: merge). FIG. 1B shows the results of flow cytometry analysis of 201B7 cells cultured on LM511-E8 (solid line in left panel: staining with mouse anti-DG monoclonal antibodies (VIA4, IIH6), solid line in right panel: mouse anti- Staining with SSEA-4 monoclonal antibody and TRA-1-81 monoclonal antibody (solid line)). FIG. 2A shows the results of immunoblotting with α-DG antibody (VIA4) and β-DG antibody (7D11) for human iPS cells (201B7 cells, 454E2 cells) and overlay assay with mouse laminin-111 (MsLM111). Show. FIG. 2B shows the results of the expression analysis of DG (VIA4, IIH6), SSEA-4, and SSEA-1 in differentiated cells cultured under conditions for inducing mesodermal cells, and human skin fibroblasts (HDFs 3 shows the results of expression analysis of DG in (). FIG. 3 shows the results of a binding assay between DG and human recombinant laminin (laminin-111 (LM111), laminin-511 (LM511), laminin-521 (LM521), and LM511-E8). FIG. 4 shows the amino acid sequence at positions 2534-3323 of the LM511-E8α5 chain (SEQ ID NO: 6) and peptides designed comprehensively in the amino acid sequence (arrows: range of synthesized peptides, underlined: LG module). . FIG. 5A shows the results of a binding assay between DG and a synthetic peptide. FIG. 5B shows the results of a binding assay between Fc (control) and synthetic peptides (hE8A5-20, hA5G1, hA5G4, hA5G29, hA5G47). The DG binding activity (A) and the stem cell adhesion activity (B) of the synthetic peptides (hE8A5-20, hA5G1, hA5G4, hA5G29, hA5G47) adsorbed on the ELISA plate at each concentration are shown. The schematic diagram (C) of preparation of a peptide-chitosan membrane and the result (D) of a stem cell adhesion assay for the peptide-chitosan membrane are shown. FIG. 7A shows a biotinylated DG binding peptide (hE8A5-20, A2G80). FIG. 7B shows the evaluation of the binding of the biotinylated DG-binding peptide (b-hE8A5-20, b-A2G80; solid line) to human iPS cells by flow cytometry. FIG. 8A shows a DG-binding peptide (A2G80-R9) and a Scramble peptide (Scramble-R9) to which a polyarginine sequence has been added. FIG. 8B shows a complex of a GFP expression plasmid (pEGF) and A2G80-R9 (A2G80-R9 polyplex), a complex of a GFP expression plasmid (pEGF) and Scramble-R9 (Scramble-R9 polyplex), and a complex thereof. Fig. 3 shows the results of gene transfer analysis into human iPS cells by the body.

(Stem cell adhesive peptide)
The stem cell adhesive peptide of the present invention comprises the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and includes dystroglycan (hereinafter referred to as “DG” in the present specification). (In some cases). The stem cell adhesive peptide of the present invention (hereinafter sometimes referred to as “DG-binding peptide” in the present specification) is a stem cell culture medium capable of adhering stem cells and allowing them to grow and culture while maintaining their undifferentiated state. It is quality.

  The amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is an amino acid sequence derived from the amino acid sequence of human laminin-511 E8 (SEQ ID NO: 6). The following peptide (hE8A5-20, hA5G1, hA5G4, hA5G29, hA5G47) consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 Is a peptide fragment that is not stably present but is produced by artificial chemical synthesis or biosynthesis (production by a genetic engineering method) and can be stably present in a predetermined composition.

hE8A5-20: Lys Thr Leu Pro Gln Leu Leu Ala Lys Leu Ser Ile (SEQ ID NO: 1)
hA5G1: Pro Met Lys Phe Asn Gly Arg Ser Gly Val Gln Leu (SEQ ID NO: 2)
hA5G4: Thr Ala Leu Lys Phe Tyr Leu Gln Gly Pro Glu Pro (SEQ ID NO: 3)
hA5G29: Leu Arg Leu Val Ser Tyr Ser Gly Val Leu Phe Phe (SEQ ID NO: 4)
hA5G47: Ala Met Thr Phe His Gly His Gly Phe Leu Arg Leu (SEQ ID NO: 5)

  The stem cell adhesive peptide of the present invention is a mutant peptide of a peptide containing the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 as long as it has a binding activity to DG. You may. Such mutant peptides include, for example, deletion, substitution, insertion, or deletion of one to several amino acids in the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. Peptides containing the added amino acid sequence. Here, the range of "one to several" means 1 to 5, preferably 1 to 3, more preferably 1 or 2. The amino acid deletion refers to selecting and deleting an arbitrary amino acid from the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In addition, addition of an amino acid means that one to several amino acids are added to the N-terminal or C-terminal side of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. It means to let. Amino acid substitutions include, for example, conservative amino acid substitutions. In addition, the stem cell adhesive peptide of the present invention may be modified with another peptide, a sugar chain, or the like, as long as it maintains the activity of binding to DG.

  The conservative amino acid substitution described above is a hydrophobic amino acid, a polar amino acid, an acidic amino acid, a basic amino acid, an amino acid having a branched side chain, an aromatic amino acid, etc. Refers to substitution between amino acids having similar properties. Examples of hydrophobic (non-polar) amino acids include glycine, alanine, valine, leucine, isoleucine, and proline. Examples of polar amino acids include serine, threonine, cysteine, methionine, asparagine, and glutamine. Examples of acidic amino acids include asparagine. Examples of acids and glutamic acids, basic amino acids are lysine, arginine and histidine, examples of amino acids having branched side chains are valine, isoleucine and leucine, and examples of aromatic amino acids are phenylalanine, tyrosine, tryptophan and histidine. is there.

The stem cell adhesive peptide of the present invention can be synthesized using a known peptide synthesis technique based on its amino acid sequence. For example, it is prepared by a solid phase synthesis method or a liquid phase synthesis method, and the solid phase synthesis method is simple in operation. For the peptide synthesis method, specifically, for example, the Boc (t-butoxycarbonyl) method, the Fmoc (9-fluorenylmethoxycarbonyl) method, or the like can be used depending on the type of the amino acid protecting group. After binding the Boc-amino acid or Fmoc-amino acid to the resin, deprotection and washing of the Boc group or Fmoc group, and condensation and washing of the Boc-amino acid or Fmoc-amino acid are repeated. Condensation, when the Boc method, can be carried out using dicyclohexylcarbodiimide (DCC) / methylene chloride (CH ₂ Cl _2), also in the case of Fmoc method, N, N'-diisopropylcarbodiimide (DIPCDI) / N- It can be performed using hydroxybenzotriazole (HOBt) / dimethylformamide (DMF). Such a condensation method is called a mixed acid anhydride method, a DCC-additive method, and the like. Other condensation methods include an active esterification method, an azide method, and the like. The deprotection can be performed by treatment with 50% trifluoroacetic acid (TFA) / CH ₂ Cl _{2 in} the case of the Boc method, and by treatment with 20% piperidine / DMF in the case of the Fmoc method. The amino acid to be bound may be a known protecting group, for example, benzyloxy, benzyl, tosyl, 2,6-dichlorobenzyl, 2-bromobenzyloxycarbonyl, Boc, mesitylene-2-sulfonyl, depending on the type of the side chain functional group. And the like. In addition, when a peptide synthesizer is used, deletion, substitution, insertion, or addition can be easily performed by changing the type of protected amino acid.

(Stem cell culture substrate)
The stem cell adhesive peptide of the present invention can be used as a culture substrate when culturing stem cells expressing DG. The stem cell adhesive peptide used in the stem cell culture substrate of the present invention may be one of the above stem cell adhesive peptides, or may be two or more.

  In the present invention, the term “stem cell culture substrate” refers to a culture substrate (substrate for supporting cells) that serves as a scaffold when culturing stem cells instead of feeder cells. When stem cells are seeded and cultured on the stem cell culture substrate of the present invention, the stem cells can be proliferated while maintaining an undifferentiated state. Here, the stem cells are not particularly limited as long as they are mammalian stem cells expressing DG. The stem cells may be pluripotent stem cells or somatic stem cells, but pluripotent stem cells are preferred. In the present invention, “pluripotent stem cells” have the ability to differentiate into all cells present in a living body derived from the ectoderm, mesoderm and endoderm (pluripotency), and also have the proliferative ability. Stem cells Pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by transplantation (ntES cells), and pluripotent germ stem cells (mGS cells) And sperm stem cells (GS cells), adult pluripotent stem cells (MAPC cells), muse cells (MUSE cells), and the like, with iPS cells and ES cells being preferred. Somatic stem cells include neural stem cells, mesenchymal stem cells, hematopoietic stem cells, cardiac stem cells, liver stem cells, small intestine stem cells, and the like. Examples of mammals from which stem cells are derived include humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, cows, and the like, with humans being preferred.

  The stem cell culture substrate of the present invention is used by binding to the surface of a culture vessel (support, carrier) when culturing stem cells. The method of bonding to the surface of the culture vessel is not particularly limited, and examples thereof include known methods such as a method using physical bonding and a method using chemical bonding, or a combination thereof. The physical binding is performed by applying, coating, or adsorbing the stem cell culture substrate on the surface of the culture vessel, and drying it. For example, a solution obtained by diluting a stem cell culture substrate with an appropriate solvent (for example, water, PBS, physiological saline, ethanol, or the like) is added to a culture container, and left to dry. Can be coated. On the other hand, chemical bonding is performed by forming a covalent bond or a non-covalent bond (for example, an ionic bond) on the stem cell culture substrate via a bonding group on the surface of the culture vessel. In the case of covalent bonding, a spacer can be bound to the amino terminus or carboxyl terminus of the stem cell adhesive peptide, which is a stem cell culture substrate, and can be bound to the culture vessel surface via this spacer. The spacer is preferably a hydrophilic spacer in order to improve the adsorption efficiency by reducing the steric hindrance of the peptide and to suppress non-specific adsorption, for example, an amino group, a carboxyl group, an aldehyde group, an epoxy group and the like at both ends. And polyalkylene oxide derivatives substituted with

  As one embodiment of the present invention, the surface of the culture vessel to which the stem cell culture substrate is bound is coated with another cell culture substrate capable of controlling the orientation of the peptide after being coated on the surface of the culture vessel in advance, so that the stem cell adhesion is improved. The orientation of the peptide can be enhanced. Examples of other cell culture substrates that can be used in combination with the stem cell culture substrate of the present invention include, but are not limited to, polysaccharides such as chitosan membrane, agarose, hyaluronic acid, and alginic acid.

  The material and shape of the culture vessel are not particularly limited as long as the culture of stem cells can be performed, and those well known in the art can be used. Examples of the material include natural polymers, synthetic polymers, metals, ceramics, and glass, and shapes include, for example, films, films, plates, gels, beads, and the like, depending on the material. Specific examples of the culture container include a flask, a Petri dish, a dish, a Petri dish, a microplate, a microwell plate, a multiplate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, a culture bag, a roller bottle, and the like. .

  The stem cell culture substrate of the present invention, and the culture vessel to which the stem cell culture substrate is bound (coated) may be made into a kit together with a medium and reagents necessary for stem cell culture.

The use amount of the stem cell culture substrate of the present invention is not particularly limited, for example, about 0.01 μg / ml or more, preferably about 0.01 to 10 μg / ml, more preferably about 0.01 to 2 μg / ml of a stem cell adhesive peptide solution. Treating the culture vessel. About 0.01~10μg / ml, stem cell adhesive peptide from about 0.01~2μg / ml as the amount of stem cell adhesive peptide per area of the culture vessel, approximately 0.0015~1.5μg / cm ^2, from about 0.0015 to about 0.3 [mu] g / corresponding to the cm ^2.

  The method of culturing stem cells using the stem cell culture substrate of the present invention can be performed according to the usual methods and conditions for culturing stem cells. The medium used for the culture is not particularly limited, and a medium generally used for maintaining and growing the undifferentiated state of the stem cells may be used. Is also good. For example, a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) necessary for survival and proliferation of stem cells, specifically, Dulbecco's Modified Eagle's Medium (D-MEM) medium , Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (D-MEM / F-12) medium, Glasgow MEM (G-MEM) medium, Basal Medium Eagle (BME) medium, Minimum Essential Medium (MEM) medium, Eagle's minimal essential medium (EMEM) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, RPMI 1640 medium, Medium 199 medium, αMEM medium, ham medium, Fischer medium, and a mixed medium thereof. In addition, the medium includes growth factors (FGF, EGF, etc.), tumor necrosis factor (TNF), interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement , Antibiotics and the like. Serum may or may not be added to the medium.

The conditions for culturing the stem cells may be in accordance with the usual conditions used for culturing stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. The medium is preferably changed once every two to three days, and more preferably every day. The culture conditions can also be set by appropriately varying the range in which the stem cells can survive and proliferate.

(Complex for introducing genes or cytotoxic substances into stem cells)
In another aspect of the present invention, the stem cell adhesive peptide can be used as a carrier peptide for efficiently introducing a desired gene or cytotoxic substance into stem cells. Thus, according to the present invention, there is provided a complex for introducing a gene or a cytotoxic substance into a stem cell, comprising the stem cell adhesive peptide and a gene or a cytotoxic substance.

  The gene to be introduced is not particularly limited, for example, a DNA repair gene, a cancer-related gene, a differentiation-related gene, a nerve-related gene, an immune-related gene, a marker gene for evaluating gene transfer efficiency or expression stability, etc. (Eg, a gene encoding GFP, luciferase, β-galactosidase) and the like. Complexation (conjugation) of the stem cell adhesive peptide and the gene can be performed by any method known in the art. The method of adding amino acids is preferred. Examples of the cationic polyamino acid include polyarginine, polylysine, polyornithine, polycitrulline, and the like, with polyarginine being preferred. The degree of polymerization of the cationic polyamino acid is not particularly limited, but is preferably 3 or more, more preferably 5 or more.

The cytotoxic substance to be introduced is not particularly limited as long as it is a substance that is toxic to undifferentiated stem cells expressing DG, and examples thereof include anticancer agents (emtansine, doxorubicin, bleomycin, and olimycin). statins, maytansine, etc.), toxin (diphtheria toxin, saporin toxin, Pseudomonas toxin, abrin toxin, ricin A toxin, ribosome inactivating protein, etc.), radioactive materials ^{(90 Y, 186 Re, 212} Bi, 131 I, 111 In, ⁶⁴ Cu, etc.), and cytotoxic nucleic acids (RNAi, antisense nucleic acids, ribozymes, etc.). Complex formation (conjugation) between the stem cell adhesive peptide and the above-mentioned cytotoxic substance can also be performed by any method known in the art. For example, when the cytotoxic substance is a low molecular weight compound, the stem cell adhesive peptide and the low molecular weight compound may be directly bonded by a functional group of the stem cell adhesive peptide, but may be indirectly bonded via a crosslinking agent. May be. The cross-linking agent is not particularly limited as long as it has one or more functional groups that react with the stem cell adhesive peptide and the cytotoxic substance, and can form a stable complex by binding them. Generally, a bifunctional crosslinking agent having functional groups at both ends is used. The bifunctional crosslinking agent may be either a homobifunctional crosslinking agent having the same functional group at both terminals or a heterobifunctional crosslinking agent having different functional groups at both terminals. The functional groups target the amino group, carboxyl group, and sulfhydryl group of the stem cell adhesive peptide, and can bind to these groups, such as N-succinimidyl ester (NHS ester) group, maleimide group, aldehyde group, thiol group, and sulfhydryl group. Group, carbodiimide group, mercapto group, pyridinyl group and the like. Specific examples of the crosslinking agent include N-hydroxysuccinimide (NHS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), glutaraldehyde (GA), 1-ethyl-3- (3-dimethylamino Propyl) carbodiimide hydrochloride (EDAC), dithiobis (succinimidylpropionate) (DSP), N, N'-dichlorohexylcarbodiimide (DCC) and the like, and commercially available products thereof may be used. In addition, when the siRNA is bound, it can be carried out by a method of adding the above-mentioned cationic polyamino acid to the stem cell adhesive peptide or a method of biotin-streptavidin binding. When the toxin is a protein toxin, it is preferable to bind at the gene level, in which case, a method of directly binding as a fusion gene or a method of indirect binding using a general DNA linker may be used. it can.

  The complex for gene transfer into stem cells of the present invention can be used for gene therapy of a specific genetic disease or the like. For example, a gene that repairs the causative gene in iPS cells (disease-derived iPS cells) produced from somatic cells or a biological sample collected from a patient with a disease known to be caused by an abnormality or defect in a specific gene Or a normal gene can be introduced using the complex, and the cells and tissues that have been induced to differentiate therefrom can be used for the treatment of returning to a patient.

  In addition, the complex for introducing a cytotoxic substance into stem cells of the present invention is a target for removing undifferentiated stem cells that may become cancerous after transplantation from a cell population that has been induced to differentiate into target cells. It can be used to provide an implant material. Specifically, in the medium after differentiation induction of stem cells, such as by adding a complex for introducing a cytotoxic substance of the present invention, by contacting the complex with the cells induced to differentiate from stem cells, Since the expression of DG is reduced in cells after differentiation induction, the complex recognizes only undifferentiated cells expressing DG and damages undifferentiated stem cells by cytotoxic substances. Can be killed. Furthermore, if there is a possibility that undifferentiated cells may remain in the culture solution, if necessary, the labeled DG-binding peptide of the present invention can be added to the treated medium, and completely removed using a cell sorter or the like. . When the complex for introducing a cytotoxic substance into stem cells of the present invention is allowed to act on floating cells, dead stem cells can be separated and removed using a density gradient medium. As a result, according to the present invention, it is possible to obtain desired differentiation-inducing cells without contamination of undifferentiated stem cells without damage. Differentiation-inducing cells after treatment can be subcultured several times to completely remove dead stem cells, or washed several times with a buffer such as PBS, and used as transplant cells for regenerative therapy. Can be used.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, these examples do not limit the present invention.

(Example 1) Expression of dystroglycan (DG) in human iPS cells 201B7 cells were used as human iPS cells, cultured on feeder cells or LM511-E8, and the expression of dystroglycan (DG) was determined by immunofluorescence staining. Observed. When feeder cells are used, Primate ES Cell Medium (ReproCELL, Yokohama, Japan) is used, and when LM511-E8 is used, StemFitAK02N (Ajinomoto, Tokyo, Japan) is used as a culture medium, and 201B7 cells are cultured. Colonies were formed in 8 days. For immunofluorescence staining, a lumox 24 well plate (Sarstedt, Numbrecht, Germany) was used. Colonies were collected by primary antibody: mouse anti-DG monoclonal antibody (VIA4, IIH6), antibody against undifferentiated marker Nanog (rabbit anti-Nanog polyclonal antibody), secondary antibody: Alexa488-labeled anti-mouse IgG antibody and Alexa594-labeled anti-rabbit IgG antibody. Double staining was performed. Nuclei were stained with Hoechst as counterstain. As a result, it was confirmed that DG was strongly expressed in any of the 201B7 cells cultured on feeder cells or LM511-E8 (FIG. 1A).

  Furthermore, the expression of DG was analyzed by flow cytometry. The 201B7 cells cultured on LM511-E8 were transformed with primary antibodies: mouse anti-DG monoclonal antibodies (VIA4, IIH6) and antibodies against undifferentiation markers SSEA-4 and TRA-1-81 (mouse anti-SSEA-4, TRA-81). 1-81 monoclonal antibody), secondary antibody: stained with Alexa488-labeled anti-mouse IgG antibody. As a result, it was confirmed that DG was strongly expressed in 201B7 cells (FIG. 1B, left panel). The expression of the undifferentiated markers (SSEA-4, TRA-1-81) also confirmed that the stem cells were in an undifferentiated state (FIG. 1B, right panel). In addition, the reaction with anti-DG (IIH6) antibody indicated that a sugar chain having laminin binding ability was added to DG.

(Example 2) Analysis of dystroglycan (DG) expressed in human iPS cells Using 201B7 cells and 454E2 cells as human iPS cells, α-dystroglycan (α-DG) expressed in human iPS cells was anti-α-DG Antibody (VIA4) and β-dystroglycan (β-DG) were detected by immunoblotting using anti-β-DG antibody (7D11). As a result, α-DG and β-DG were detected at 120 to 200 kDa and 38 kDa, respectively (FIG. 2A, left panel and middle panel). α-DG is known to retain laminin binding properties even after being transferred to a PVDF membrane after separation by SDS-PAGE (Ibraghimov-Beskrovnaya et al., Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature, 355, 696-702, 1992). An overlay assay using mouse laminin-111 (MsLM-111) showed that MsLM-111 bound at the same 120-200 kDa as α-DG, and that α-DG of human iPS cells has laminin binding activity. (FIG. 2A, right panel).

  According to a previous report (Miyazaki et al., Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells., Nat. Commun. 3, 1236, 2012), human iPS cells (201B7 cells) were induced to differentiate into mesoderm. After 14 days of culture, analysis by flow cytometry was performed. As a result, in cells after induction of differentiation, the expression of SSEA-4, a marker of human iPS cells, decreases, while the expression of SSEA-1, a marker of undifferentiation, increases, and the direction of differentiation from undifferentiated state to (FIG. 2B, left panel). At the same time, it was confirmed that when undifferentiation was lost, the expression of α-DG having laminin binding ability decreased (FIG. 2B, middle lower panel). This result suggested that DG may be a molecule associated with the undifferentiated state of human iPS cells. In addition, DG expression was not observed in the human skin fibroblasts (HDFs), which are the original 201B7 cells, suggesting a relationship with undifferentiated human iPS cells (FIG. 2B, right panel).

(Example 3) Analysis of binding of dystroglycan (DG) to LM511-E8
To evaluate the binding of DG to LM511-E8, a recombinant protein (MsDG-Fc) was prepared by fusing mouse α-dystroglycan (MsDG) with the IgG ₁ Fc domain of human immunoglobulin. MsDG-Fc was expressed in Expi293 cells, and the culture supernatant containing MsDG-Fc was used for the binding assay. The ELISA plate was coated with 20 nM of human recombinant laminin-111 (LM111), laminin-511 (LM511), laminin-521 (LM521) and LM511-E8. Bound MSDG-Fc was detected by antibodies to IgG ₁ Fc domain. As a result, MsDG-Fc bound to LM111, LM511, and LM521 coated at 20 nM at the same level. On the other hand, no significant binding was observed for LM511-E8, but significant binding was observed at 60 nM (FIG. 3). This indicated that the LM511-E8 region had DG binding activity, although weaker than LM511.

(Example 4) Identification of dystroglycan (DG) -binding amino acid sequence of LM511-E8
(1) Construction of a peptide library covering the α5 chain of LM511-E8
A peptide library was constructed to identify the DG-binding amino acid sequence of LM511-E8. LM511-E8 is composed of Ala2534-Ala3323 of human α5 chain, Leu1561-Leu1786 of β1 chain, and Asn1364-Pro1609 of γ1 chain (Ido et al., The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin γ chains in integrin binding by laminins, J Biol Chem, 282, 11144-11154, 2007). Since the binding of the receptor mainly suggests the involvement of the α chain, a peptide covering the Ala2534-Ala3323 region of the α5 chain was designed (FIG. 4). The peptides were designed to be based on 12 amino acids in length and overlap by 4 residues with adjacent peptides. However, Cys in the sequence was excluded from the peptide design in consideration of formation of disulfide bonds by oxidation and nonspecific cell adhesion. When the N-terminal of the peptide was Glu or Gln, Gly was added to the N-terminal in consideration of the influence of pyroglutamine formation by the cyclization reaction. All peptides were synthesized by the Fmoc solid phase synthesis method. The synthesized peptide-resin was deprotected with TFA, purified by HPLC, and freeze-dried. After purification, the purity was analyzed by HPLC, and the molecular weight was confirmed using mass spectrometry. By the above method, peptides covering a total of the α5 chain of LM511-E8 (96 kinds in total) were synthesized.

(2) Screening for peptides that bind to dystroglycan (DG)
Using the peptide library covering the α5 chain of LM511-E8 constructed in (1), a peptide binding to DG was screened. Peptides covering the α5 chain of LM511-E8 were coated on an ELISA plate and evaluated by a binding assay using MsDG-Fc. As a result, the following five peptides (hE8A5-20: KTLPQLLAKLSI (SEQ ID NO: 1), hA5G1: PMKFNGRSGVQL (SEQ ID NO: 2), hA5G4: TALKFYLQGPEP (SEQ ID NO: 3), hA5G29: LRLVSYSGVLFF (SEQ ID NO: 4), hA5G47: AMTFHGHGFLRL (SEQ ID NO: 5)) showed binding activity to MsDG-Fc (FIG. 5A). The remaining 91 peptides showed no binding activity to MsDG-Fc.

  The five types of peptides having DG binding activity showed no binding to the control Fc, indicating that they were DG-specific binding (FIG. 5B).

(Example 5) DG binding activity and cell adhesion activity of dystroglycan (DG) binding peptide
The concentration dependence of five peptide peptides (hE8A5-20, hA5G1, hA5G4, hA5G29, hA5G47) for which DG binding activity was observed was evaluated. As a result, it was shown that five types of peptides bound to MsDG-Fc in a concentration-dependent manner. Above all, hE8A5-20 and hA5G29 showed strong binding activity to DG (FIG. 6A, A).

  Further, the DG-binding peptide was coated on an ELISA plate in the same manner as in the binding assay of Example 4 (2), and the cell adhesion activity was evaluated using human iPS cells (201B7 cells). As a result, hE8S5-20 showed a remarkable adhesive activity to human iPS cells in a concentration-dependent manner, and the remaining peptides showed the adhesive activity at a high concentration (FIG. 6-1, B).

  A peptide-chitosan membrane in which a peptide is immobilized on chitosan, a high-molecular-weight polysaccharide matrix, has been reported as a biomaterial with cell adhesion ability (Mochizuki et al, Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering. FASEB J. 17, 875-877, 2003). Peptide-chitosan membranes, unlike the random coating of ELISA plates, allow peptides to be oriented and adsorbed. Therefore, Cys-Gly-Gly (CGG) was added to the N-terminal of the DG-binding peptide (hE8A5-20, hA5G1, hA5G4, hA5G29, hA5G47) to prepare a peptide-chitosan matrix (FIG. 6-2, C). Cell adhesion activity to human iPS cells was evaluated. As a result, in the CGG-hE8A5-20 peptide, strong adhesive activity to human iPS cells was observed in a concentration-dependent manner (FIG. 6-2, D). These results indicated that the DG-binding peptide can be used as a stem cell culture substrate.

(Example 6) Analysis of binding of biotinylated dystroglycan (DG) -binding peptide to human iPS cells
In order to examine the use of the DG-binding peptide for substance introduction, the binding to human iPS cells in a liquid phase was evaluated. The hE8A5-20 peptide identified in the present invention and the A2G80 peptide (VQLRNGFPYFSY) showing DG binding activity in previous studies were biotinylated using Biotin-OSu (Dojindo, Kumamoto, Japan) and purified by HPLC. (FIG. 7A). Human iPS cells (201B7 cells) cultured on LM511-E8 were detached with a Cell dissociation buffer (ThermoFisher Scientific), and a biotinylated peptide was added at 100 μg / ml and reacted. Next, streptavidin labeled with Alexa488 was reacted and analyzed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). As a result, the hE8A5-20 peptide was shown to bind to human iPS cells, and its binding activity was higher than that of the A2G80 peptide (FIG. 7B).

(Example 7) Analysis of gene transfer into human iPS cells by DG-binding peptide
Gene transfer into human iPS cells was attempted using DG-binding peptide. To bind plasmid DNA to A2G80, which is the same DG-binding peptide as the above five peptides identified as DG-binding peptides, A2G80-R9 was added, in which 9 arginines were added to the C-terminal side with 2 glycines as linkers ( (FIG. 8A). As a control, Scramble-R9 in which 9 arginines were similarly added to the scrambled peptide (Scramble) peptide instead of A2G80 and two glycines were added to the C-terminal side as a linker (FIG. 8A).

  PEGFP-N3 (Takara Bio, Shiga, Japan) was used as the gene to be introduced. Human iPS cells (201B7 cells) were seeded on a lumox 24 well plate (Sarstedt, Numbrecht, Germany) coated with LM511-E8, and colonies were formed by culturing for 7-8 days. A complex (A2G80-R9 polyplex) of the above plasmid pEGFP-N3 (4 μg) and peptide A2G80-R9 (15.5 μg) was added to each well, and after 48 hours of culture, Keyence BZ-X700 (Keyence, Osaka, Japan) was observed. As a result, with the A2G80-R9 peptide, EGFP fluorescence was observed at the periphery of the colony, which revealed that the gene was introduced into human iPS cells via DG (FIG. 8B, arrow). In contrast, no fluorescence of EGFP was observed in Scramble-R9.

  INDUSTRIAL APPLICABILITY The present invention can be used in the field of producing regenerative medical materials.

Claims (6)

  The amino acid sequence shown in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence A stem cell adhesive peptide having an activity of binding to dystroglycan, comprising an amino acid sequence.   The stem cell adhesive peptide according to claim 1, wherein the stem cell is an induced pluripotent stem cell, an embryonic stem cell, or a somatic stem cell.   A stem cell culture substrate comprising one or more of the stem cell adhesive peptides according to claim 1 or 2.   A kit for stem cell culture, comprising the stem cell culture substrate according to claim 3.   A complex for introducing a gene into a stem cell, comprising the stem cell adhesive peptide according to claim 1 or 2 and a gene.   A complex for introducing a cytotoxic substance into stem cells, comprising the stem cell adhesive peptide according to claim 1 and a cytotoxic substance.

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