Peptide for inducing cell aggregate

Abstract

An object of the present invention is to provide a cell aggregate-inducing peptide, a cell aggregate-forming agent containing the same, and a method for forming a cell aggregate using the same.SOLUTION: A cell aggregate-inducing peptide represented by General Formula (1), having hydroxypipecolic acid and lysine as constituent repeating units. (HPA-Lys) n. (1) [In the general formula (1), HPA represents hydroxypipecolic acid, Lys represents lysine, and n is an integer of 4 or more and 30 or less.]. ] SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell aggregate-inducing peptide, a cell aggregate-forming agent containing the same, and a method for forming a cell aggregate using the same. [Background technology]

[0002] In the research and development of pharmaceuticals, etc., it is common to subject cultured cells to a test substance to evaluate their response, or to induce the cultured cells to produce physiologically active substances, etc. In many cases, cells cultured in a monolayer (two-dimensional) on a substrate are used as the cultured cells. On the other hand, living organisms are composed of groups of cells arranged in three dimensions, and it is known that cells cultured in three dimensions better reflect the intrinsic functions of the living organism. For example, it is not easy to differentiate ES cells or iPS cells cultured in two dimensions, but ES cells and iPS cells cultured in a three-dimensional aggregate state can be easily differentiated. It is also known that hepatocytes cultured in a three-dimensional aggregate state have a higher ability to produce albumin, a function of hepatocytes. For this reason, it is sometimes preferable to use three-dimensionally cultured cells in the research and development of regenerative medicine, pharmaceuticals, etc. Known methods for three-dimensional cell culture include the hanging drop method, culturing on a substrate with a specific surface treatment, rotational culture, three-dimensional scaffolding, and methods for forming cell aggregates by centrifugation. However, all of these methods require complicated procedures and special equipment (instruments), and in some cases may damage the cells. As another method, a peptide for cell aggregation containing specific amino acids (lysine and proline) as constituent repeating units and a method for synthesizing the same have been reported (Patent Document 1, Patent Document 2, and Non-Patent Document 1). Non-Patent Document 1 describes a peptide (Lys-Pro) having 12 (24 amino acids) constituent repeating units of lysine and proline (KP). 12It has been reported that KP24 has cell aggregation-inducing activity. However, as reported in Patent Document 1, cell aggregation was not observed in peptides containing arginine and proline or only arginine as constituent repeating units, and only peptides containing lysine and proline as constituent repeating units have been shown to have cell aggregation activity, and the types of peptides that have been shown to have cell aggregation activity are limited. Therefore, there is a need to develop peptides other than those mentioned above that have cell aggregating ability, and to develop methods for forming cell aggregates simply and efficiently with less damage to cells. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-20961 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-218474 [Non-patent literature]

[0004] [Non-Patent Document 1] Processes 2021, 9, 538. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a cell aggregate-inducing peptide, a cell aggregate-forming agent containing the same, and a method for forming cell aggregates using them. In particular, an object of the present invention is to provide a cell aggregate-inducing peptide that causes less damage to cells and can easily and efficiently form cell aggregates, a cell aggregate-forming agent containing the same, and a method for forming cell aggregates using them. [Means for solving the problem]

[0006] The present invention has been made to solve these problems, and provides a cell aggregate-inducing peptide having an unnatural amino acid and lysine as constituent repeating units, a cell aggregate-forming agent containing the same, and a method for forming cell aggregates using the same.

[0007] That is, the present invention provides the following. Section 1. A cell aggregate-inducing peptide represented by general formula (1), which has hydroxypipecolic acid and lysine as constituent repeating units. (HPA-Lys) n ···(1) [In general formula (1), HPA represents hydroxypipecolic acid, Lys represents lysine, and n is an integer of 4 or more and 30 or less.] Section 2. Item 1. The cell aggregate-inducing peptide according to Item 1, wherein the hydroxypipecolic acid is 3-hydroxypipecolic acid, 4-hydroxypipecolic acid, and / or 5-hydroxypipecolic acid. Section 3. A method for forming a cell aggregate, comprising adding the cell aggregate-inducing peptide according to Item 1 or 2 to a culture medium for cultured cells to form a cell aggregate. Section 4. Item 1. A cell aggregate forming agent containing the cell aggregate-inducing peptide according to Item 2. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a peptide capable of forming cell aggregates (a cell aggregate-inducing peptide), a cell aggregate-forming agent containing the peptide, and a method for forming cell aggregates using them. The peptide or cell aggregate-forming agent of the present invention causes little damage to cells. Simply by adding the peptide or cell aggregate-forming agent of the present invention to a cell culture system, cell aggregates can be formed simply and efficiently while minimizing damage to cells. Furthermore, according to the present invention, it is possible to provide cell aggregates that can be effectively used in fields such as drug discovery, pharmacology, medicine, and biology, for example, in the evaluation of the efficacy and toxicity of chemical substances and pharmaceuticals, mass production of useful substances such as antibodies, and regenerative medicine. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the profiles and gradient conditions of (5HPA-Lys)12 before and after high performance liquid chromatography (HPLC) purification. [Figure 2] FIG. 1 shows the results of mass spectrometry (MALDI-TOF-MS) of (5HPA-Lys)12. [Figure 3] FIG. 1 shows the results of measuring the circular dichroism (CD) spectrum of (5HPA-Lys)12. [Figure 4] FIG. 1 shows a series of procedures for evaluating cell aggregate induction using mouse fibroblasts (L929). [Figure 5] This figure shows the results of microscopic observation of cell aggregates after the addition of (5HPA-Lys)12 in an evaluation of cell aggregate induction using L929 cells. Scale bar: 100 μm; (5HPA-Lys)12 concentrations: (1) 0.18 mM, (2) 0.27 mM, (3) 0.36 mM, (4) 0.45 mM, (5) 0.54 mM. [Figure 6] This graph shows the results of examining the cell size distribution using cell aggregates formed at (5HPA-Lys)12 concentrations of (3) 0.36 mM and (4) 0.45 mM in an evaluation of cell aggregate induction using L929 cells. The bar on the left is (3), and the bar on the right is (4). [Figure 7] This figure shows the results of three-dimensional observation of cell aggregates prepared at concentrations of (5HPA-Lys)12 (3) 0.36 mM and (4) 0.45 mM in an evaluation of cell aggregate induction using L929 cells. The left figure shows a Mil-Cell microscope stereoscopic analysis image of the cell aggregate, and the right figure shows a graph showing the results of examining the sphericity of the cell aggregate. [Figure 8]This figure shows the results of evaluating cell viability (Live / Dead staining) of L929 cell aggregates. Left: inside, right: bottom. Scale bar: 50 μm. [Figure 9] FIG. 1 shows a series of procedures for evaluating cell aggregate induction using human mesenchymal stem cells (hMSCs). [Figure 10] This figure shows the results of microscopic observation of cell aggregates after the addition of (5HPA-Lys)12 in an evaluation of cell aggregate induction using hMSCs. Scale bar: 100 μm; 3 hours (3 h), 6 hours (6 h), and 24 hours (24 hr) after addition. [Figure 11] FIG. 1 shows the results of MALDI-TOF-MS analysis of DMT-MM synthesized in Example 2 [2]. [Figure 12] FIG. 1 shows the results of MALDI-TOF-MS analysis of NH-c5HPA-COOCH3 obtained in [3-1] of Example 2. [Figure 13] FIG. 1 shows the results of measurement of NH-c5HPA(OTBS)-COOCH3 and its sodium salt (NH-c5HPA(OTBS)-COONa) obtained in [3-3] of Example 2 using a Fourier transform infrared spectrophotometer (FT-IR). [Figure 14] FIG. 1 shows the results of MALDI-TOF-MS analysis of Fmoc-c5HPA(OTBS)-OH obtained in [3-4] of Example 2. [Figure 15] HPLC analysis was performed on the crude peptide of KP24 obtained in [4] of Example 2. Figure 15 shows the HPLC elution profile of the crude peptide of KP24. [Figure 16] The purified KP24 obtained by HPLC purification in [5] of Example 2 was subjected to HPLC analysis and molecular weight measurement by MALDI-TOF-MS. The HPLC analysis results are shown in A, and the MALDI-TOF-MS measurement results are shown in B. [Figure 17]HPLC analysis was performed on the crude peptide of c5HK24 obtained in [4] of Example 2. Figure 17 shows the HPLC elution profile of the crude peptide of c5HK24. [Figure 18] The purified c5HK24 obtained by HPLC purification in [5] of Example 2 was subjected to HPLC analysis and molecular weight measurement by MALDI-TOF-MS. The results of the HPLC analysis are shown in A, and the results of the MALDI-TOF-MS measurement are shown in B. [Figure 19] HPLC analysis was performed on the crude peptide of t5HK24 obtained in [4] of Example 2. Figure 19 shows the HPLC elution profile of the crude peptide of t5HK24. [Figure 20] The purified t5HK24 obtained by HPLC purification in [5] of Example 2 was subjected to HPLC analysis and molecular weight measurement by MALDI-TOF-MS. The results of the HPLC analysis are shown in A, and the results of the MALDI-TOF-MS measurement are shown in B. [Figure 21] FIG. 1 shows the results of measuring the CD spectra of KP24, c5HK24, and t5HK24. [Figure 22] This shows the results of phase-contrast microscopic observation of L929 cells on a 96-well plate supplemented with KP24, c5HK24, t5HK24, or KA24 in "Example 2: [8-3] Evaluation of L929 cell aggregate induction." Scale bar: 200 μm. [Figure 23] 1 is a graph showing the results of examining the cell size distribution using cell aggregates produced with KP24, c5HK24, or t5HK24 in "Example 2: [8-3] Evaluation of L929 cell aggregate induction." The results for KP24, c5HK24, and t5HK24 are shown from left to right. [Figure 24] This figure shows the results of Live-Dead staining of L929 cell aggregates and observation with a confocal laser microscope in "Example 2: [8-4] Fluorescence observation of L929 cell aggregates by Live-Dead assay." Scale bar: 50 μm. [Figure 25](a) KP24, (b) c5HK24, (c) t5HK24. Three-dimensional observation results were obtained using cell aggregates prepared with (a) KP24, (b) c5HK24, and (c) t5HK24 in an evaluation of cell aggregate induction using L929 cells. A shows a stereoscopic image of the cell aggregates obtained using a Mil-Cell microscope, and B shows a graph showing the results of examining the sphericity of the cell aggregates. [Figure 26] This shows the results of observing hMSCs on a 96-well plate to which c5HK24 and t5HK24 had been added under a phase-contrast microscope in "Example 2: [9-2] Induction of hMSC cell aggregates." Scale bar: 200 μm. [Figure 27] Figure 1 shows the results of phase-contrast microscopy of hMSC aggregates at different cell densities on 96-well plates prepared by adding KP24 (A), c5HK24 (B), or t5HK24 (C) in "Example 2: [9-3] hMSC aggregate induction (evaluation of cell density)." Scale bar: 200 μm. [Figure 28A] In "Example 2: [9-3] hMSC cell aggregate induction (evaluation of cell density)," cell size distribution was examined using cell aggregates prepared using KP24, c5HK24, or t5HK24. Figure 28A is a graph showing the relationship between the diameter size of hMSC cell aggregates measured and the number of cells when the cell density was 3.0 × 104 cells / well. The results for KP24, c5HK24, and t5HK24 are shown from left to right. [Figure 28B] In "Example 2: [9-3] hMSC cell aggregate induction (evaluation of cell density)," cell size distribution was examined using cell aggregates prepared using KP24, c5HK24, or t5HK24. Figure 28B is a graph showing the relationship between the diameter size and number of hMSC cell aggregates measured at a cell density of 3.5 x 104 cells / well. The results are shown for KP24, c5HK24, and t5HK24, from left to right. [Figure 28C]In "Example 2: [9-3] hMSC cell aggregate induction (evaluation of cell density)," cell size distribution was examined using cell aggregates prepared using KP24, c5HK24, or t5HK24. Figure 28C is a graph showing the relationship between the diameter size of hMSC cell aggregates measured and the number of cells when the cell density was 4.0 × 104 cells / well. The results for KP24, c5HK24, and t5HK24 are shown from left to right. [Figure 29] This figure shows the results of Live-Dead staining of hMSC cell aggregates and observation with a confocal laser microscope in "Example 2: [9-4] Fluorescence observation of hMSC cell aggregates by Live-Dead assay." Scale bar: 50 μm. [Figure 30] (a) KP24, (b) c5HK24, (c) t5HK24. Three-dimensional observation results were obtained using hMSC cells to evaluate cell aggregate induction. (A) Mil-Cell microscope stereological analysis image of the cell aggregate, and (B) a graph showing the results of examining the sphericity of the cell aggregate. [Figure 31] This figure shows the results of observing hMSCs on a 96-well plate supplemented with KP24, c5HK24, or t5HK24 under a phase-contrast microscope in "Example 2: [9-6] Evaluation of induction of hMSC cell aggregates in serum-containing medium." Scale bar: 200 μm. [Figure 32] "Example 2: [9-7] Induction of differentiation of hMSC cell aggregates into osteoblasts" and "[9-8] Alizarin Red S staining of hMSC cell aggregates induced to differentiate into osteoblasts" were performed to induce differentiation of hMSC cell aggregates into osteoblasts and stain them with Alizarin Red S. The results of Alizarin Red S staining of hMSC cell aggregates cultured in bone differentiation medium for 10 days are shown. Scale bar: 100 μm. [Figure 33] FIG. 1 shows the profiles of KP24 obtained in [3-4] of Example 3 before (A) and after (B) HPLC purification. [Figure 34] FIG. 1 shows the profiles of t4HK24 obtained in [3-4] of Example 3 before (A) and after (B) HPLC purification. [Figure 35] This figure shows the results of phase-contrast microscopic observation of L929 cells on a 96-well plate to which KP24 had been added in "Example 3: [6-2-1] Cell aggregate induction of L929 cells in KP24." Scale bar: 200 μm. [Figure 36] This figure shows the results of phase-contrast microscopic observation of L929 cells on a 96-well plate to which t4HK24 had been added in "Example 3: [6-2-2] Induction of L929 cell aggregates in t4HK24." Scale bar: 200 μm. [Figure 37] 1 is a graph showing the relationship between the number and the results of measuring the diameter size of L929 cell aggregates formed in [6-2-1] and [6-2-2] of Example 3. The results for KP24 and t4HK24 are shown from the left. [Figure 38] This figure shows the results of Live-Dead staining of L929 cell aggregates and observation with a confocal laser microscope in "Example 3: [7] Fluorescence observation of L929 cell aggregates by Live-Dead assay." Scale bar: 50 μm. [Figure 39] 1 shows the results of phase-contrast microscopic observation of L929 cells on a 96-well plate to which t3HK24 had been added, in the induction of L929 cell aggregates in t3HK24 obtained in Example 4. Scale bar: 200 μm. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will now be described. Terms used in this specification have the meanings commonly used in the art unless otherwise specified.

[0011] 1. Peptides of the present invention The peptides of the present invention have an unnatural amino acid and lysine (hereinafter sometimes referred to as "Lys") as constituent repeating units. A suitable example of the unnatural amino acid is hydroxypipecolic acid (hereinafter sometimes referred to as "HPA"). Specifically, the peptide of the present invention is a peptide represented by the following general formula (1) and has the ability to form cell aggregates. (HPA-Lys) n ···(1) [In general formula (1), HPA represents hydroxypipecolic acid, Lys represents lysine, and n is an integer of 4 or more and 30 or less.] The peptide represented by the general formula (1) is a peptide having (hydroxypipecolic acid-lysine) as a constituent repeating unit, that is, an amino acid sequence in which (HPA-Lys) is repeated. n is an integer of 4 or more and 30 or less. That is, this means that the degree of polymerization of the constituent repeating units of lysine and hydroxypipecolic acid is 4 or more and 30 or less. From the viewpoint of exhibiting a higher ability to form cell aggregates, n is preferably an integer of 6 or more, more preferably an integer of 8 or more, particularly preferably an integer of 10 or more, and is also preferably an integer of 20 or less, more preferably an integer of 16 or less, particularly preferably an integer of 14 or less. Peptides with n of less than 4 may not be able to form cell aggregates, or even if they are formed, they may not be good cell aggregates. On the other hand, when n exceeds 30, synthesis of the peptide itself may be difficult, and cell aggregates may not be able to be formed, or even if they are formed, they may not be good cell aggregates.

[0012] Hydroxypipecolic acid has a structure in which a hydroxy group (OH) is bonded to pipecolic acid, which in turn has a carboxyl group (COOH) bonded to a six-membered piperidine ring, and there are different isomers depending on the bond position. In the peptide represented by general formula (1), the n hydroxypipecolic acids may all be the same isomer or different isomers, but preferably all are the same isomer. As the hydroxypipecolic acid, 3-hydroxypipecolic acid (cis-3-hydroxypipecolic acid or trans-3-hydroxypipecolic acid), 4-hydroxypipecolic acid (cis-4-hydroxypipecolic acid or trans-4-hydroxypipecolic acid) and / or 5-hydroxypipecolic acid (cis-5-hydroxypipecolic acid or trans-5-hydroxypipecolic acid) are preferred, and from the viewpoint of exhibiting a higher cell aggregate forming ability, 5-hydroxypipecolic acid (hereinafter sometimes referred to as "5HPA") is more preferred, and cis-5-hydroxypipecolic acid (cis-5HPA) is particularly preferred. Here, an example of the constitutional repeating unit (5HPA-Lys) is represented by the following structural formula.

[0013] [ka]

[0014] (In the formula, *1 represents a bond with H or the carbonyl carbon atom of Lys, and *2 represents a bond with OH or the nitrogen atom of 5HPA.) The method for synthesizing the peptide of the present invention is not particularly limited, and any general solid-phase synthesis method or liquid-phase synthesis method can be used. For example, the general steps of solid-phase synthesis are as follows.

[0015] An amino acid whose N-terminus is protected with a protecting group (protected amino acid) is bound to a solid support via a linker at the C-terminus of the protected amino acid. Next, unreacted protected amino acids, i.e., protected amino acids not bound to the support, are removed. Next, the protecting group of the protected amino acid is removed under conditions that do not cause the support-bound protected amino acid to be released from the support. Separately, a second protected amino acid is prepared in which the N-terminus of the amino acid to be bound to the support-bound amino acid is protected with a protecting group. The second protected amino acid is added to the support-bound amino acid, and the N-terminus of the support-bound amino acid and the C-terminus of the second protected amino acid are condensed. Next, the unreacted second protected amino acid is removed, and then the protecting group of the second protected amino acid is removed. A third amino acid whose N-terminus is protected with a protecting group is added, and the N-terminus of the second amino acid and the C-terminus of the third protected amino acid are condensed. This procedure is repeated to synthesize a peptide having the desired amino acid sequence bound to the support. The desired peptide is obtained by separating this peptide from the support.

[0016] Examples of solid phase supports include, but are not limited to, styrene resin, acrylamide resin, polyethylene glycol-acrylamide composite resin, polyoxyethylene-grafted styrene resin, etc. These may be cross-linked with divinylbenzene, and may be used alone or in combination of two or more. Examples of linkers include, but are not limited to, chloromethyl, hydroxymethyl, benzhydrylamine, aminomethyl, 4-benzyloxybenzyl alcohol, 4-methylbenzhydrylamine, phenylacetamidomethyl, 4-hydroxymethylphenylacetamidomethyl, 4-(2',4'-dimethoxyphenyl-hydroxymethyl)phenoxy, 2-chlorotrityl chloride resin, 4-hydroxymethylphenoxyacetic acid (HMPA), 4-hydroxymethylbenzoic acid (HMBA), 2,4-dimethoxy-4-hydroxybenzophenone, etc. These may be used alone or in combination of two or more.

[0017] Furthermore, examples of protecting groups for amino groups in amino acids or peptides include benzyloxycarbonyl (Z), tertiary-butoxycarbonyl (Boc), tertiary-amyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl (Cl-Z), 2-bromobenzyloxycarbonyl (Br-Z), adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and the like. Examples of the protecting group for a carboxyl group include alkyl esters having an alkyl group such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, benzyl ester, 2-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, phenacyl ester, benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, and trityl hydrazide. The deprotection reaction of the protecting group can be carried out according to a conventional method, for example, a catalytic reduction method or a method using liquid ammonia / sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, trifluoromethanesulfonic acid, or the like.

[0018] On the other hand, in liquid phase synthesis, no solid phase support is used to support the resulting peptide, but reagents other than protected amino acids and / or protected peptides (e.g., condensing agents, bases, etc.) may be used in the form of resin-supported reagents, and such synthesis methods can also be used in the present invention. The same protecting groups for the amino group and the carboxyl group as those used in the solid phase synthesis can be used. The peptides of the present invention obtained as described above can be appropriately purified according to conventional methods, such as ion exchange resins, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution, and other methods commonly used in the field of peptide chemistry.

[0019] The peptides of the present invention have excellent cell aggregate-forming ability. Therefore, the peptides of the present invention are useful as active ingredients of cell aggregate-forming agents. Furthermore, the peptides of the present invention have the ability to form cell aggregates even when used in small amounts or at low concentrations, and therefore can form cell aggregates simply and efficiently while minimizing damage to cells.

[0020] 2. Cell aggregate forming agents According to the present invention, there is provided a cell aggregate forming agent containing the peptide of the present invention (hereinafter, sometimes simply referred to as "the cell aggregate forming agent of the present invention"). The cell aggregate-forming agent of the present invention may contain only the peptide of the present invention, or may contain other components as long as the cell aggregate-forming ability of the peptide of the present invention is not impaired. Examples of other components include components commonly used in research reagents, specifically water, buffers, organic solvents, chelating agents, pH adjusters, surfactants, colorants, cell culture media, etc. All of these components are commercially available or can be produced according to previously reported methods. The dosage form of the cell aggregate-forming agent of the present invention is not particularly limited as long as it allows the peptide of the present invention to come into contact with cells. Examples of dosage forms include solids and liquids. The content of the peptide of the present invention in the cell aggregate forming agent of the present invention is not particularly limited, but can usually be about 0.000001 to 10% by weight. The cell aggregate forming agent of the present invention having such a constitution can easily and efficiently form cell aggregates while minimizing damage to cells.

[0021] 3. Method for forming cell aggregates According to the present invention, there is provided a method for forming a cell aggregate (hereinafter, sometimes simply referred to as "the method of the present invention"), which comprises contacting a peptide of the present invention with a cell. In the method of the present invention, the cell aggregate-forming agent of the present invention may be used as a supply source of the peptide of the present invention. The cells to be used for forming aggregates in the method of the present invention are not particularly limited. Examples include adherent cells and non-adherent cells. Specific examples include cells involved in skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.), cells involved in muscle (muscle cells, etc.), cells involved in fat (adipocytes, etc.), cells involved in nerves (neuronal cells, etc.), cells involved in liver (hepatic parenchymal cells, etc.), cells involved in pancreas (pancreatic islet cells, etc.), cells involved in kidney (renal epithelial cells, proximal tubular epithelial cells, mesangial cells, etc.), cells involved in lungs and bronchi (epithelial cells, fibroblasts, etc.), and the like. cells, vascular endothelial cells, and smooth muscle cells, etc.), cells involved in the eye (photoreceptors, corneal epithelial cells, and corneal endothelial cells, etc.), cells involved in the prostate (epithelial cells, stromal cells, and smooth muscle cells, etc.), cells involved in bone (osteoblasts, osteocytes, and osteoclasts, etc.), cells involved in cartilage (chondroblasts and chondrocytes, etc.), cells involved in teeth (period ligament cells and osteoblasts, etc.), cells involved in blood (white blood cells and red blood cells, etc.), and stem cells {e.g., bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (oval cells, small hepatocytes, etc.), adipose tissue stem cells, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, epidermal stem cells, intestinal stem cells, spermatogonial stem cells, embryonic germ stem cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, corneal stem cells, progenitor cells (adipocyte precursor cells, vascular endothelial precursor cells, chondrocyte precursor cells, lymphocyte precursor cells, NK precursor cells, etc.), etc.

[0022] The manner in which the peptide of the present invention is brought into contact with cells is not particularly limited, and examples thereof include a method in which cells are cultured in a medium containing the peptide of the present invention. The medium is not particularly limited as long as it does not impair the cell aggregate-forming ability of the peptide of the present invention, and is appropriately selected depending on the type of cell. Specific examples include known media such as EMEM, DMEM, and RPMI. These media may also contain known additives. Examples of known additives include antibiotics. From the viewpoint of more efficient cell aggregate formation, it is preferable that the medium does not contain serum.

[0023] The content of the peptide of the present invention in the medium is not particularly limited as long as it allows the formation of cell aggregates. For example, it may be 0.0000001 to 0.01% by weight, preferably 0.000001 to 0.001% by weight, and more preferably 0.00001 to 0.001% by weight. In addition, when the peptide of the present invention is (5HPA-Lys) 12 When the peptide has (or consists of) the amino acid sequence of formula (1), where HPA is 5-hydroxypipecolic acid and n is preferably 10 to 14, particularly preferably 12, cell aggregates can be formed even at a lower concentration. In this case, the content of the peptide of the present invention in the medium can be preferably 0.000001 to 0.0001% by weight, more preferably 0.00001 to 0.0001% by weight. The culture conditions are not particularly limited as long as they allow the formation of cell aggregates, and are appropriately selected depending on the type of cell. For example, the conditions include "under saturated water vapor, 5% CO2 concentration, and 37 to 40°C."

[0024] When using adherent cells as the cells, it is desirable to allow the cells to adhere to a culture dish before contacting them with the peptide of the present invention, from the viewpoint of minimizing damage to the cells and forming cell aggregates more efficiently. The culture dish is not particularly limited as long as it can form cell aggregates. Specific examples include polystyrene dishes. The culture dish may be subjected to a cell adhesive surface treatment as long as it can form cell aggregates.

[0025] According to the method of the present invention, cell aggregates are formed after a certain period of time (e.g., 2 to 9 days) has elapsed since contacting cells with the peptide of the present invention. The presence or absence of cell aggregate formation can be evaluated by known methods, for example, by observation under an optical microscope. In this evaluation, specific criteria may be adopted, such as defining a cell aggregate with a diameter of 20 μm or more as a "cell aggregate." [Example]

[0026] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Furthermore, the reagents and materials used are commercially available unless otherwise specified. Abbreviations used in this specification are the same as those commonly used in the art unless otherwise specified.

[0027] Example 1 In this example, a peptide having cis-5-hydroxy-L-pipecolic acid and lysine as repeating units was synthesized by solid-phase synthesis and evaluated. For solid-phase synthesis, the amino acids used were N,N-α-(9-fluorenylmethoxycarbonyl)-cis-5-Ot-butoxycarbonyl-L-pipecolic acid (Fmoc-5HPA(tBu)-OH) [manufactured by API Corporation] and N-α-(9-fluorenylmethoxycarbonyl)-O-butoxycarbonyl-L-lysine (Fmoc-Lys-(Boc)-OH) [manufactured by Watanabe Chemical Industry Co., Ltd.]. The amino acid protecting group used was 9-fluorenylmethoxycarbonyl (Fmoc). The insoluble resin support used was N-(9-fluorenylmethoxycarbonyl)-t-butoxycarbonyl-lysine-alco-polyethylene glycol resin (Fmoc-Lys(Boc)-Alko-PEG Resin) [manufactured by Watanabe Chemical Industry Co., Ltd.].

[0028] [ka]

[0029] The condensation agent used was 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), and the synthetic raw materials for the condensation agent were sodium bicarbonate (Sigma-Aldrich Japan K.K.), cyanuric chloride (Tokyo Chemical Industry Co., Ltd.), and N-methylmorpholine (Sigma-Aldrich Japan K.K.). Piperidine (Sigma-Aldrich Japan) was used as a protecting group remover, and N,N-dimethylformamide (DMF) (Fujifilm Wako Pure Chemical Industries) was used for swelling and cleavage of the resin. Trifluoroacetic acid (TFA) (Watanabe Chemical Industry) was used for deprotection and resin removal. The carrier used for separation and purification was a Sephadex G-25M PD-10 column (GE Healthcare). Reagents other than the amino acids with protecting groups and the solid phase resin are as follows: N-methylmorpholine (NMM) [Fujifilm Wako Pure Chemical Industries, Ltd.] N,N-Dimethylformamide (DMF) (for peptide synthesis) [Fujifilm Wako Pure Chemical Industries, Ltd.] Dimethyl sulfoxide (DMSO) [Fujifilm Wako Pure Chemical Industries, Ltd.] Piperidine (PPD) [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0030] [(5HPA(OtBu)-Lys) 12 Fmoc solid phase synthesis method] 0.550 g (0.132 mmol) of Fmoc-Lys(Boc)-Alko-PEG Resin was placed in a column and washed three times with DMF and MeOH for 1 minute each. The column was then swollen in 25% (weight / volume) DMSO / DMF for 30 minutes. After swelling, the column was washed six times with DMF for 1 minute each to remove the 25% (weight / volume) DMSO / DMF. The column was then reacted with 20% (weight / volume) PPD / DMF for 30 minutes to deprotect the Fmoc group. After deprotection, the column was washed three times with DMF for 1 minute each to remove the 20% PPD / DMF. Completion of the Fmoc group deprotection was confirmed by the blue color change observed with a chloranil test. After washing with DMF three times for 1 minute, 3.0 equivalents of Fmoc-5HPA(tBu)-OH, 3.0 equivalents of NMM (to make the resin basic), and 6.0 equivalents of the condensation agent DMT-MM were added to the resin, and the condensation reaction was carried out for 180 minutes. Unreacted materials were removed by washing with DMF six times for 1 minute, and the condensation was completed when it was confirmed that no red color had developed in the TNBS test or blue color had developed in the chloranil test. These procedures were repeated a total of 12 times to obtain 5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-5HPA(OtBu)-Lys(Boc)-(SEQ ID NO: 1) Alko-PEG Resin (hereinafter referred to as "(5HPA-Lys 12 We synthesized a new PEG-TrtA-PEG resin (sometimes referred to as "TrtA-PEG Resin").

[0031] [Peptide purification method] (5HPA-Lys) 12 The Alko-PEG resin was stirred with the cleavage mixture (9.50 mL of trifluoroacetic acid (TFA), 0.50 mL of pure water) for 150 minutes. (5HPA-Lys) 12The peptides were dialyzed using a membrane with a molecular weight cutoff range of 100–500 Da (Spectra / Pro Dialysis Membrane Biotech CE Tubing, MWCO: 100–500D; Spectrum Laboratories Inc, CA, USA). Finally, the peptides were purified by preparative high-performance liquid chromatography (HPLC 8020 System; Tosoh Corporation, column: TSKgel-ODS-100V 5 μm; Tosoh Corporation) using water / acetonitrile containing 0.1% TFA as the eluent. The HPLC profiles and gradient conditions before and after purification are shown in Figure 1.

[0032] [Analysis method] The synthesized peptides were analyzed by mass spectrometry using a MALDI-TOF-MS system, which combines matrix-assisted laser desorption ionization (MALDI) and time-of-flight (TOF) mass spectrometry. The MALDI-TOF-MS used was a Microflex LRF System manufactured by Bruker Corporation. α-CHCA (α-cyano-4-hydroxycinnamic acid) was used as the matrix.

[0033] [Analysis results] The results of mass spectrometry are shown in Figure 2. As is clear from the figure, [M+H] + The peak appears at 3082.3, and the peak position is (5HPA-Lys) 12 This almost matches the formula weight of the peptide synthesized above, (5HPA-Lys) 12 It was confirmed that this was the case.

[0034] [CD spectrum measurement] (5HPA-Lys) 12The circular dichroism (CD) spectrum of this compound was measured using a J-1100 / 1500 circular dichroism spectrometer (manufactured by JASCO Corporation). The results are shown in Figure 3. As is clear from the figure, a negative Cotton effect was observed at 211.7 nm and a positive Cotton effect was observed at 226.1 nm. The obtained spectral pattern suggested a conformation similar to that of polyproline II helix.

[0035] [Evaluation of L929 cell aggregate induction] Cell aggregate induction was evaluated using mouse fibroblasts (L929) [manufactured by RIKEN BioResource Research Center]. 25cm 2 The medium in a tissue culture flask (for adherent cells) [manufactured by Iwaki Co., Ltd.] (code: 1123-075) was removed and washed three times with 1.0 mL of PBS. 1.0 mL of trypsin / EDTA was added and removed, followed by incubation for 1 minute at 37°C, 5% CO2, and 95% RH. 3.0 mL of serum-containing medium was added, and the cell suspension in trypsin / EDTA and serum-containing medium was transferred to a 15 mL centrifuge tube [manufactured by Iwaki Co., Ltd.] (code: 2325-015) and centrifuged at 1000 rpm for 1 minute. The supernatant was removed, and the cells were resuspended in medium. This cell suspension was used for cell testing. The number of L929 cells in the cell suspension was counted using trypan blue. 5.0 x 10 cells were placed in a 96-well plate. 4 100 μL of cells / well were seeded and (5HPA-Lys) 12 100 μL of the solution was added to the cells so that the desired concentration was reached. The cells were then incubated at 37°C, 5% CO2, and 95% RH for 7 days, with the medium replaced every two days. The cells were then observed using a phase-contrast microscope. The entire procedure is shown in Figure 4. (5HPA-Lys) 12 The results of microscopic observation of the cell aggregates on day 7 after addition are shown in Figure 5. Cell aggregates were formed at all concentrations (1) to (5), demonstrating that there was no concentration dependency. Furthermore, Non-Patent Document 1 discloses a peptide KP24 ((Lys-Pro) 12) at a concentration of 1.0 mg / mL is disclosed. On the other hand, (5HPA-Lys) 12 Cell aggregates were formed even at a low concentration of 0.18 mM (=0.5 mg / mL). 12 has a higher cell aggregate formation ability than KP24 and can form cell aggregates at low concentrations, suggesting that damage to cells can be kept low.

[0036] [Cell size evaluation] (5HPA-Lys) 12 Cell aggregates prepared at concentrations of (3) 0.36 mM and (4) 0.45 mM were observed under a microscope using an Olympus CKX41 microscope, and images were captured using a WRATYMER NOA630B camera and analyzed using WRATYMER MicroStudio software. The results are shown in Figure 6. The cell aggregate size is (5HPA-Lys) 12 When the concentration of (3) was 0.36 mM, the majority of particles had a diameter of 80 to 100 μm, and when the concentration of (4) was 0.45 mM, the majority of particles had a diameter of 60 to 80 μm.

[0037] [Three-dimensional observation and sphericity measurement of L929 cell aggregates using a cell cluster microscope (Mil-Cell)] (5HPA-Lys) 12 Three-dimensional observation of L929 cell aggregates prepared at concentrations of (3) 0.36 mM and (4) 0.45 mM was performed using a Mil-Cell (manufactured by Sumitomo Electric Industries, Ltd.). The cell aggregates were detached by pipetting and seeded in a 96-well plate (manufactured by Iwaki Corporation). A single cell aggregate detached from the 96-well plate was seeded in a Mil-Cell non-adhesive observation vessel, and then observed with Mil-Cell and analyzed using software. The results are shown in Figure 7. The sphericity of the cell aggregates produced at concentration (3) was 85.1%, and the sphericity of the cell aggregates produced at concentration (4) was 82.4%. Here, Non-Patent Document 1 describes that the sphericity of cell aggregates produced using KP24 was approximately 68%. 12 It is believed that the sphericity of the resulting cell aggregates is higher than that of KP24.

[0038] [Live / Dead Assay] Live / dead cell assessment of L929 cell aggregates was performed using a Live / Dead Viability / Cytotoxicity Assay (Invitrogen) according to the manufacturer's protocol. After 7 days of incubation, the L929 cell aggregates were rinsed once with PBS and then observed using a confocal laser scanning fluorescence microscope (LSM800 ZEN, Carl Zeiss) to observe the green fluorescence wavelength (517 nm) of live cells due to their reaction with calcein AM and the red fluorescence wavelength (617 nm) of dead cells due to their reaction with EthD-1. The results are shown in Figure 8. As can be seen from the figure, there are few red-stained cells inside the cells. Therefore, it is possible to generate cell aggregates in a viable state.

[0039] [hMSC cell aggregate induction experiment] Human mesenchymal stem cells (hMSCs) [manufactured by JCRB Cell Bank, National Institutes of Medical Infrastructure, Health and Nutrition] were cultured in a container. The mesenchymal stem cell growth medium was removed from the container and washed twice with PBS. Trypsin / EDTA was added to a 25cm culture area. 2 1.0 mL of the solution was added to the 100 mL of PBS, and the mixture was incubated for 1 minute under conditions of 5% CO2, 37°C, and 95% RH. To inhibit the action of trypsin / EDTA, three volumes of mesenchymal stem cell growth medium were added. The solution was transferred to a 15 mL PP (polypropylene) centrifuge tube and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed, and the cells were resuspended in mesenchymal stem cell basal medium. The number of hMSC cells in the cell suspension was counted using trypan blue. 1.0 × 10 hMSCs were placed in a 48-well plate. 5 cells / well, (5HPA-Lys) 12The medium was then changed every two days and incubated at 37°C, 5% CO2, and 95% RH for five days. The entire procedure is shown in Figure 9. (5HPA-Lys) 12 The progress of hMSC induction after addition (3 hours, 6 hours, and 24 hours) was observed using a phase-contrast microscope. The results are shown in Figure 10. As is clear from the figure, hMSCs gradually aggregated. In addition, hMSC cell aggregate induction experiments were carried out in the same manner as above using KP24 prepared according to the method described in Non-Patent Document 1. As a result, (5HPA-Lys) 12 These results suggest that KP24 promotes cell aggregate induction more than KP24.

[0040] Example 2 In this example, a peptide (c5HK24) in which the proline contained in KP24 was replaced with the unnatural amino acid cis-5-hydroxypipecolic acid (c5HPA) and a peptide (t5HK24) in which the proline was replaced with the unnatural amino acid trans-5-hydroxypipecolic acid (t5HPA) were synthesized by solid-phase synthesis and evaluated.

[0041] [1] Reagents used The reagents used in this example are listed below. [1-1] Synthesis of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) Methanol (MeOH) [Fujifilm Wako Pure Chemical Industries, Ltd.] Sodium bicarbonate [manufactured by Asahi Glass Co., Ltd.] 2,4,6-trichloro-1,3,5-triazine [Fujifilm Wako Pure Chemical Industries, Ltd.] N-methylmorpholine (NMM) [Fujifilm Wako Pure Chemical Industries, Ltd.] Acetone [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0042] [1-2] Introduction of a protecting group to cis-5-hydroxypipecolic acid [1-2-1] Carboxy group protection by methylation cis-5-Hydroxypipecolic acid [UBE Corporation] Methanol (MeOH) [Fujifilm Wako Pure Chemical Industries, Ltd.] Thionyl chloride (SOCl2) [Fujifilm Wako Pure Chemical Industries, Ltd.] Toluene [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] Diethyl ether [Fujifilm Wako Pure Chemical Industries, Ltd.] Chloroform [Fujifilm Wako Pure Chemical Industries, Ltd.] [1-2-2] Hydroxy group protection with TBS group tert-Butyldimethylchlorosilane (TBSCl) [Fluorochem Ltd.] Imidazole [Fujifilm Wako Pure Chemical Industries, Ltd.] Methanol [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] Chloroform [Fujifilm Wako Pure Chemical Industries, Ltd.] [1-2-3] Saponification of methyl carboxylate with sodium hydroxide Sodium hydroxide (NaOH) [Fujifilm Wako Pure Chemical Industries, Ltd.] Methanol (MeOH) [Fujifilm Wako Pure Chemical Industries, Ltd.] [1-2-4] Protection of amino groups with Fmoc groups Tetrahydrofuran (THF) [Fujifilm Wako Pure Chemical Industries, Ltd.] Sodium hydroxide (NaOH) [Fujifilm Wako Pure Chemical Industries, Ltd.] N,N-Diisopropylethylamine (DIPEA) [Watanabe Chemical Industry Co., Ltd.] N-(9-Fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) [Watanabe Chemical Industry Co., Ltd.] Ethyl acetate [Fujifilm Wako Pure Chemical Industries, Ltd.] 0.5 mol / L hydrochloric acid [Fujifilm Wako Pure Chemical Industries, Ltd.] Anhydrous sodium sulfate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] Hexane [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0043] [1-3] Peptide synthesis [1-3-1] Fmoc solid phase synthesis method The amino acids with protecting groups and solid phase resins used in peptide synthesis are shown below. N-(9-Fluorenylmethoxycarbonyl)-proline-tritylcarboxyamidomethyl polyethylene glycol resin (Fmoc-Pro-TrtA-PEG Resin) [Watanabe Chemical Industry Co., Ltd.] N-(9-Fluorenylmethoxycarbonyl)-t-butoxycarbonyl-lysine-alco-polyethylene glycol resin (Fmoc-Lys(Boc)-Alko-PEG Resin) [Watanabe Chemical Industry Co., Ltd.] N-(9-Fluorenylmethoxycarbonyl)-N-ε-t-butoxycarbonyl-lysine (Fmoc-Lys(Boc)-OH) [Watanabe Chemical Industry Co., Ltd.] N-(9-Fluorenylmethoxycarbonyl)-proline (Fmoc-Pro-OH HO) [Watanabe Chemical Industry Co., Ltd.] N-(9-Fluorenylmethoxycarbonyl)-cis-5-t-butyl-pipecolic acid (Fmoc-c5HPA(tBu)-OH) [UBE Corporation] N-(9-Fluorenylmethoxycarbonyl)-trans-5-t-butyl-pipecolic acid (Fmoc-t5HPA(tBu)-OH) [UBE Corporation] Reagents other than the amino acids with protecting groups and the solid phase resin are shown below. N-methylmorpholine (NMM) [Fujifilm Wako Pure Chemical Industries, Ltd.] N,N-Dimethylformamide (DMF) (for peptide synthesis) [Fujifilm Wako Pure Chemical Industries, Ltd.] Dimethyl sulfoxide (DMSO) [Fujifilm Wako Pure Chemical Industries, Ltd.] Piperidine (PPD) [Fujifilm Wako Pure Chemical Industries, Ltd.] Dichloromethane (DCM) [Fujifilm Wako Pure Chemical Industries, Ltd.] Methanol (MeOH) [Fujifilm Wako Pure Chemical Industries, Ltd.] [1-3-2] TNBS test 2,4,6-Trinitrobenzene-1-sulfonic acid (TNBS) [Fujifilm Wako Pure Chemical Industries, Ltd.] Sodium bicarbonate [manufactured by Asahi Glass Co., Ltd.] Phosphate buffer solution (PBS) [DS Pharma Biomedical Co., Ltd.] [1-3-3] Chloranil test N,N-dimethylformamide (DMF) [Fujifilm Wako Pure Chemical Industries, Ltd.] Tetrachloro-p-benzoquinone (2% N,N-dimethylformamide) (chloranil) [Tokyo Chemical Industry Co., Ltd.] Acetaldehyde [Fujifilm Wako Pure Chemical Industries, Ltd.] [1-3-4] Final deprotection Trifluoroacetic acid (TFA) [Watanabe Chemical Industry Co., Ltd.] Diethyl ether [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0044] [1-4] Analysis and purification by high-performance liquid chromatography (HPLC) Trifluoroacetic acid (TFA) [Watanabe Chemical Industry Co., Ltd.] Acetonitrile [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0045] [1-5] Identification of molecular ion peaks by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) α-Cyano-4-hydroxycinnamic acid (α-CHCA) [Sigma-Aldrich Japan, LLC] Angiotensin II (human) MALDI-MS calibrant [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0046] [1-6] Measurement of circular dichroism (CD) spectra Phosphate-buffered saline (PBS) [KAC Corporation]

[0047] [1-7] Cell culture [1-7-1] Mouse fibroblast (L929) cell culture Eagle MEM Medium "Nissui" (1) (EMEM Medium) [Nissui Pharmaceutical Co., Ltd.] Fetal bovine serum (FBS) [CELL SYSTEMS CORPORATION] Glutamine "Nissui" [manufactured by Nissui Pharmaceutical Co., Ltd.] 7.5% sodium bicarbonate aqueous solution [Fujifilm Wako Pure Chemical Industries, Ltd.] Trypsin / EDTA [Fujifilm Wako Pure Chemical Industries, Ltd.] Phosphate-buffered saline (PBS) [KAC Corporation] LIVE / DEAD TM Viability / Cytotoxicity Kit, for mammalian cells [manufactured by Thermo Fisher SCIENTIFIC Inc.] [1-7-2] hMSC cell culture Mesenchymal stem cell growth medium 2 [Promo Cell] Penicillin / streptomycin (x100) [Fujifilm Wako Pure Chemical Industries, Ltd.] Trypsin / EDTA [Fujifilm Wako Pure Chemical Industries, Ltd.] Phosphate-buffered saline (PBS) [KAC Corporation] 75% ethanol [Kaneichi Pharmaceutical Co., Ltd.] LIVE / DEAD TM Viability / Cytotoxicity Kit, for mammalian cells [manufactured by Thermo Fisher SCIENTIFIC Inc.] [1-7-3] Osteoblast differentiation induction medium Dulbecco's Eagle's MEM medium "Nissui" (2) (DMEM medium) [manufactured by Nissui Pharmaceutical Co., Ltd.] 7.5% sodium bicarbonate aqueous solution [Fujifilm Wako Pure Chemical Industries, Ltd.] Penicillin / streptomycin (x100) [Fujifilm Wako Pure Chemical Industries, Ltd.] Fetal bovine serum (FBS) [CELL SYSTEMS CORPORATION] Dexamethasone [Fujifilm Wako Pure Chemical Industries, Ltd.] β-Glucerophosphate disodium pentahydrate [Fujifilm Wako Pure Chemical Industries, Ltd.] L(+)-Ascorbic acid [Fujifilm Wako Pure Chemical Industries, Ltd.] Triton X-100 [Nacalai Tesque, Inc.] Physiological saline solution [Otsuka Pharmaceutical Co., Ltd.] Alizarin Red S staining kit [Bio Mirai Kobo Co., Ltd.] Lab assays TM ALP [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0048] [2] Synthesis of DMT-MM 40.0 g of sodium bicarbonate was added to an Erlenmeyer flask and dissolved in 120 mL of methanol and 12 mL of pure water. After dissolving the sodium bicarbonate, 29.6 g of 2,4,6-trichloro-1,3,5-triazine was added and stirred in an ice bath for 15 minutes. The mixture was then stirred in a warm bath at 30-35°C for 7 hours. After stirring, pure water was added to precipitate 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), which was then filtered with an aspirator. To wash the filtered CDMT, 1000 mL of ultrapure water was added to the Erlenmeyer flask and stirred for 30 minutes. The mixture was again filtered with vacuum, and the CDMT was dried under reduced pressure. After vacuum drying, the CDMT was dissolved in acetone, and 1.5 equivalents of NMM per CDMT were added while stirring. After 30 minutes of stirring, the mixture was filtered with vacuum, yielding DMT-MM. The precipitated DMT-MM was identified using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS) manufactured by Bruker Corporation. α-CHCA was dissolved in a 1:1 mixture of acetonitrile and pure water to prepare a saturated α-CHCA solution (matrix solution). The matrix solution and the DMT-MM / methanol solution were mixed at a 1:1 ratio, and 1 μL of the mixture was dropped onto the sample stage. A mixed crystal of the sample and matrix was prepared by drying under reduced pressure. This was then used for measurement by MALDI-TOF-MS.

[0049] [3] Introduction of a protecting group to cis-5-hydroxypipecolic acid [3-1] Carboxy group protection by methylation reaction The carboxyl group of c5HPA was methyl esterified. The reaction of c5HPA with methanol is shown below.

[0050] [ka]

[0051] 300 mL of methanol was added to a three-neck flask and cooled to below -10°C. After cooling, thionyl chloride (24.6 g, 0.21 mol) was added dropwise, followed by c5HPA (20 g, 0.14 mol). The mixture was refluxed and stirred at 70°C for 6 hours, followed by stirring at room temperature for 24 hours. The solution was then transferred to a recovery flask and concentrated under reduced pressure using a rotary evaporator. After concentration, toluene was added to the precipitated white crystals, and the mixture was concentrated under reduced pressure three times. After concentration under reduced pressure, diethyl ether was added and the mixture was decanted to precipitate the target crystals. Finally, the mixture was suction filtered and dried under reduced pressure. The Rf values ​​of each reaction were measured using thin-layer chromatography (TLC) to confirm the progress of the reaction. The TLC developing solvent was a mixture of chloroform, methanol, and water in a ratio of 8:3:1. The resulting target compounds were identified by molecular weight measurement using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS) (Microflex, Bruker Corporation). [3-2] Hydroxy group protection with TBS group The TBS group was introduced to the hydroxy group of NH-c5HPA-COOCH3. The reaction of NH-c5HPA-COOCH3 with tert-butyldimethylsilyl chloride (TBSCl) is shown below.

[0052] [ka]

[0053] TBSCl (0.19 g, 6.25 mmol) was added to a recovery flask and dissolved in DMF. NH-c5HPA(OTBS)-COOCH3 (0.2 g, 1.25 mmol) and imidazole (0.085 mg, 6.25 mmol) were then added and stirred at room temperature for 14 hours. After stirring, the mixture was concentrated under reduced pressure using a rotary evaporator to obtain the product. Furthermore, the Rf values ​​of each reaction were determined using thin-layer chromatography (TLC) to confirm the progress of the reaction. The developing solvent for TLC was a mixture of chloroform, methanol, and water in a ratio of 8:3:1. [3-3] Saponification of methyl carboxylate with sodium hydroxide The methyl carboxylate of NH-c5HPA(OTBS)-COOCH3 was saponified. The reaction of NH-c5HPA(OTBS)-COOCH3 with sodium hydroxide is shown below.

[0054] [ka]

[0055] NH-c5HPA(OTBS)-COOCH3 (200 mg, 2.6 mmol) was added to a recovery flask and dissolved in methanol. Sodium hydroxide (0.11 g, 2.9 mmol) was then added and stirred for three hours. After stirring, the mixture was concentrated under reduced pressure using a rotary evaporator to obtain the product. The product was measured using a Fourier transform infrared spectrophotometer (FT-IR) FT / IR-4200 (manufactured by JASCO Corporation) and spectral analysis was performed using a waveform separation program. The detector was MCT (Hg 1-x CD x The measurement conditions were a wave number of 500 to 4000 cm. -1 So, the total number of times was 60. [3-4] Protection of amino groups with Fmoc groups The Fmoc group was introduced into the amino group of NH-c5HPA(OTBS)-COONa. The reaction of NH-c5HPA(OTBS)-COONa with Fmoc-OSu is shown below.

[0056] [ka]

[0057] NH-c5HPA(OTBS)-COONa (0.2 g, 0.73 mmol) was added to an Erlenmeyer flask and dissolved in 10 mL of THF and 10 mL of ultrapure water. NaOH (0.032 g, 0.80 mmol) was then dissolved in an ice bath. Fmoc-Osu (0.27 g, 0.80 mmol) and DIPEA (0.14 mL, 0.80 mmol) were then added and stirred at room temperature for 4 hours. After stirring, the mixture was added to ethyl acetate and stirred for 10 minutes. The target product was extracted into the organic layer, then transferred to a separatory funnel and the aqueous layer was removed. The organic layer was then washed eight times with 0.5 M HCl and once with saturated brine, transferred to an Erlenmeyer flask, and added anhydrous sodium sulfate. The mixture was then left to stand for one day. The sodium sulfate hydrate was then removed by suction filtration. The solvent was concentrated under reduced pressure using a rotary evaporator. Finally, the mixture was suction filtered and dried under reduced pressure. Thereafter, the obtained target substance was identified by measuring its molecular weight using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS) (Microflex, manufactured by Bruker Corporation).

[0058] [4] Peptide synthesis [4-1] Fmoc solid phase synthesis method The peptide was synthesized using Fmoc solid-phase synthesis. The synthesis scheme using Fmoc solid-phase synthesis is shown below.

[0059] [ka]

[0060] For KP24, 0.90 g (0.20 mmol) of Fmoc-Pro-TrtA-PEG Resin was loaded onto the column. For c5HK24 and t5HK24, 0.55 g (0.13 mmol) and 0.50 g (0.12 mmol) of Fmoc-Lys(Boc)-Alko-PEG Resin, respectively, were loaded onto the column. The column was washed three times for 1 minute with DMF and MeOH, and then swollen in 25% DMSO / DMF for 30 minutes. After swelling, the column was washed six times for 1 minute with DMF to remove the 25% DMSO / DMF. The column was then incubated for 30 minutes with 20% PPD / DMF to deprotect the Fmoc group. After deprotection, the column was washed three times for 1 minute with DMF to remove the 20% PPD / DMF. Completion of the Fmoc deprotection was confirmed by color reaction with the TNBS test and chloranil test. After washing with DMF three times for one minute, three equivalents of a protected amino acid, 95 μL of NMM, and the condensation agent DMT-MM were added to the resin in the amounts shown in Tables 1 to 3 below, and the condensation reaction was carried out for 180 minutes. Unreacted materials were removed by washing with DMF six times for one minute, and the completion of the condensation was confirmed by the TNBS test and the chloranil test. These procedures were repeated, and (Lys(Boc)-Pro) 12 -TrtA-PEG Resin (KP24-TrtA-PEG Resin), (c5HPA(tBu)-Lys(Boc)) 12 -Alko-PEG Resin (c5HK24-Alko-PEG Resin), (t5HPA(tBu)- Lys(Boc)) 12 -Alko-PEG Resin (t5HK24-Alko-PEG Resin) was synthesized. Table 1 shows the amounts of amino acid derivatives and DMT-MM used in the synthesis of KP24-TrtA-PEG Resin. In Table 1, Fmoc-Lys(Boc)-OH and Fmoc-Pro-OH were used for Lys and Pro, respectively. Table 2 shows the amounts of amino acid derivatives and DMT-MM used in the synthesis of c5HK24-Alko-PEG Resin. In Table 2, Fmoc-Lys(Boc)-OH and Fmoc-c5HPA(tBu)-OH were used for c5HPA and Lys, respectively. Table 3 shows the amounts of amino acid derivatives and DMT-MM used in the synthesis of t5HK24-Alko-PEG Resin. In Table 3, t5HPA and Lys were replaced with Fmoc-Lys(Boc)-OH and Fmoc-t5HPA(tBu)-OH, respectively.

[0061] [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] Similarly, KA24 was synthesized by substituting alanine, an amino acid that does not have a ring structure, for proline in KP24. [4-2] TNBS test The completion of deprotection and condensation was confirmed using the TNBS test. A small amount of resin was added to a solution made by mixing 200 μL of 4% aqueous sodium bicarbonate solution, 200 μL of PBS, and 100 μL of aqueous TNBS solution, and the color change of the resin was confirmed. The prepared mixed solution reacted with primary amines and turned red, so the completion of deprotection of the Fmoc group and condensation was confirmed by the presence or absence of color. [4-3] Chloranil test The chloranil test was used only to confirm the completion of the condensation and deprotection of hydroxypipecolic acid and proline with secondary amines. A small amount of resin was added to a solution containing 20 μL of 2 wt% p-chloranil / DMF and 20 μL of 2 wt% acetaldehyde / DMF, and the color change of the resin was confirmed. The resulting mixture reacted with the secondary amine, turning the resin blue, and the presence or absence of color confirmed the completion of the deprotection and condensation of the Fmoc group. [4-4] Final deprotection 9.5 mL of TFA and 0.5 mL of ultrapure water were mixed and stirred in an ice bath for 10 minutes to prepare a cleavage mixture. This cleavage mixture was added to the peptide with side chain protection and resin synthesized in [4-1] and stirred at room temperature for 150 minutes. The peptide / TFA solution and resin were then separated by filtration. Diethyl ether was added to precipitate the peptide, which was then collected. The dried peptide was dissolved in ultrapure water and dialyzed for 3 days using a dialysis membrane with a molecular weight cutoff of 100-500. The dialyzed aqueous peptide solution was lyophilized for 3 days to obtain the crude peptide as a white powder.

[0065] [5] Analysis and purification by high-performance liquid chromatography (HPLC) KP24 was analyzed by HPLC. The HPLC system used for this analysis was a Shimadzu CO-8020 column oven, SIL-20AC autosampler, SPD-10A UV-visible detector, DP-8020 data processor, CBM-20A system controller, FRC-10A fraction collector, and LC-20AD pump. A TOSOH TSK-GEL ODS-100V column was used. The measurement conditions were a flow rate of 1.00 mL / min, UV wavelength of 210 nm, and column oven temperature of 40 °C. The mobile phases were 0.1% TFA / ultrapure water (liquid A) and 0.1% TFA / acetonitrile (liquid B), with a binary solvent gradient. The sample was prepared using liquid A as the solvent. The peptide concentration was 1.0 mg / mL, UV absorption wavelength was 210 nm, flow rate was 1.0 mL / min, injection volume was 100 μL, and column oven temperature was 40 °C. Each peak was separated and collected for purification. The HPLC gradient conditions are shown below.

[0066] [Table 4]

[0067] [6] Molecular weight measurement by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) The molecular weight of each compound was confirmed using MALDI-TOF-MS (Microflex, manufactured by Bruker Corporation), and the target compounds were identified. α-CHCA was dissolved in a 1:1 mixture of acetonitrile and pure water to prepare a saturated α-CHCA solution (matrix solution). The matrix solution and DMT-MM / methanol solution were mixed at a 1:1 ratio, and 1 μL of the mixture was dropped onto the sample stage. Mixed crystals of the sample and matrix were prepared by drying under reduced pressure. These were then used for measurement using MALDI-TOF-MS.

[0068] [7] Circular dichroism (CD) spectrum measurement The secondary structures of KP24, c5HK24, and t5HK24 were analyzed using a J-1100 / 1500 circular dichroism spectrometer (JASCO Corporation). Two milliliters of peptide solution, adjusted to a peptide concentration of 100 μM in PBS, was placed in a quartz glass cell (Tosoh Quartz Corporation). The measurement conditions were: temperature 25°C, wavelength 180–250 nm, data interval 1 nm, scan speed 100 nm / min, cell length 0.5 cm, and 3 accumulations.

[0069] [8] Examination of cell aggregate-inducing activity of mouse fibroblasts (L929) by KP24, c5HK24, t5HK24 and KA24 [8-1] L929 cell culture Eagle's MEM (EMEM) medium was used for L929 culture. 2.82 g of EMEM powder was dissolved in 261 mL of ultrapure water and sterilized using an autoclave (Tomy Corporation, LBS-325). After high-pressure steam sterilization, serum-containing EMEM medium was prepared by adding 30 mL of fetal bovine serum (FBS), 0.090 g of L-glutamine, and 6 mL of 7.5% sodium bicarbonate solution. Serum-free EMEM medium was also prepared by replacing 30 mL of FBS with ultrapure water. L929 (cell number = RCB1451) was purchased from the RIKEN BioResource Research Center. Frozen L929 cells were thawed at 37°C and transferred to a 15 mL centrifuge tube (Iwaki Co., Ltd.). 10 mL of serum-containing EMEM medium was added to the tube, pipetted several times, and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed and the cells were resuspended in serum-containing EMEM. Seeding density 4.0×10 3 cells / cm 2 The cells were seeded in a culture vessel manufactured by Iwaki Co., Ltd. and cultured in an incubator set at 37°C, 5% CO2, and 95% RH. [8-2] Subculture 25cm 2 The medium in a tissue culture flask (for adherent cells) (Iwaki Co., Ltd.) (code: 1123-075) was removed and washed three times with 1.0 mL of PBS. 1.0 mL of trypsin / EDTA was added and removed, followed by incubation for 1 minute at 37°C, 5% CO2, and 95% RH. 3.0 mL of serum-containing medium was added, and the cell suspension in trypsin / EDTA and serum-containing medium was transferred to a 15 mL centrifuge tube (Iwaki Co., Ltd.) (code: 2325-015) and centrifuged at 1000 rpm for 1 minute. The supernatant was removed, and the cells were resuspended in medium. This cell suspension was used for cell testing. [8-3] L929 cell aggregate induction A cell suspension was prepared using serum-free medium in the same manner as in [8-2]. The number of L929 cells in the cell suspension was counted using trypan blue. 5.0 × 10 L929 cells were placed in a 96-well plate. 4100 μL of cells / well were seeded, and 100 μL of KP24, c5HK24, t5HK24, and KA24 were added at a concentration of 0.36 mM. The day of seeding was counted as day 0, and the cells were incubated at 37°C, 5% CO2, and 95% RH for 7 days, with the medium changed every two days. Then, the cells were observed using a phase-contrast microscope. [8-4] Fluorescence observation of L929 cell aggregates using live-dead assay Using the same procedure as in [8-3], add 5.0 × 10 L929 to a 96-well plate. 4 Cells were seeded at 1000 cells / well, and KP24, c5HK24, and t5HK24 were added at a concentration of 0.36 mM each. Cultures were then incubated for 7 days under humidified conditions at 37°C with 5% CO2. After washing once with PBS, the prepared staining solution was added and incubated for 45 minutes at 37°C with 5% CO2 and 95% RH. After incubation, the staining solution was removed, PBS was added, and the cells were observed under a confocal laser microscope. The staining solution was prepared by diluting calcein AM solution to 5.0 μM and ethidium homodimer I solution to 4.0 μM with PBS. [8-5] Three-dimensional observation of L929 cell aggregates using a cell cluster microscope (Mil-Cell) Mil-Cell (Sumitomo Electric Industries, Ltd.) was used for three-dimensional observation of cell aggregates. The culture medium was removed from cell aggregates prepared using the same procedure as in [8-3] and washed with PBS. The cell aggregates were then detached by pipetting and seeded into a 96-well plate. The concentration of the detached cell aggregates in the 96-well plate was adjusted, and they were seeded into a Mil-Cell observation container, after which they were observed and analyzed using Mil-Cell.

[0070] [9] Induction of cell aggregates from human mesenchymal stem cells (hMSCs) by the addition of KP24, c5HK24, and t5HK24 [9-1] hMSC cell culture Mesenchymal stem cell proliferation medium 2 (Promo Cell) was used as the hMSC culture medium. Mesenchymal stem cell proliferation medium 2 was prepared by adding 50 mL of the Supplement Mix and penicillin / streptomycin provided with the mesenchymal stem cell basal medium. Furthermore, mesenchymal stem cell basal medium was prepared by adding only penicillin / streptomycin without the Supplement Mix. hMSCs (cell number = JCRB1136) were purchased from JCRB Cell Bank. Cryopreserved hMSCs were cultured in a culture vessel at a density of 2.0 × 10 cells using the same procedure as in [8-1]. 3 cells / cm 2 The seeds were sown. [9-2] Evaluation of hMSC cell aggregate induction The mesenchymal stem cell growth medium 2 in the container in which the hMSCs were cultured was removed and washed twice with PBS. Trypsin / EDTA was added to the culture area of ​​25 cm. 2 1.0 mL of the medium was added to the cells and incubated for 1 minute under conditions of 5% CO2, 37°C, and 95% RH. To inhibit the action of trypsin / EDTA, three volumes of mesenchymal stem cell growth medium 2 were added. The solution was transferred to a 15 mL PP centrifuge tube and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed, and the cells were resuspended in mesenchymal stem cell basal medium. The number of hMSC cells in the cell suspension was counted using trypan blue. 1.0 × 10 hMSCs were placed in a 48-well plate. 5 Cells were seeded at 100 μL / well, and 100 μL of KP24, c5HK24, and t5HK24 were added at 0.36 mM each. The day of seeding was designated day 0, and the cells were incubated at 37°C, 5% CO2, and 95% RH for 5 days, with the medium changed every two days. Then, the cells were observed using a phase-contrast microscope. [9-3] hMSC cell aggregate induction (evaluation of cell density) A cell suspension was prepared using the same procedure as in [9-2] using the basal medium for mesenchymal stem cells. The number of L929 cells in the cell suspension was counted using trypan blue. 2.0 × 10 cells were placed in a 96-well plate. 4 cells / well, 3.0 × 10 4 cells / well, 3.5 × 10 4cells / well, 4.0 × 10 4 100 μL of cells / well were seeded, and 100 μL each of KP24, c5HK24, and t5HK24 was added at 0.36 mM. The medium was then changed every two days and incubated at 37°C, 5% CO2, and 95% RH for 5 days. The cells were then observed using a phase-contrast microscope. [9-4] Live-Dead staining of hMSC cell aggregates Using the same procedure as in [9-2], 3.0 × 10 hMSCs were added to a 96-well plate. 4 Cells were seeded at 1000 cells / well, and KP24, c5HK24, and t5HK24 were added at 0.36 mM. The cells were then cultured for 5 days under conditions of 5% CO2, 37°C, and 95% RH. After washing once with PBS, the prepared staining solution was added and incubated for 45 minutes under conditions of 37°C, 5% CO2, and 95% RH. After incubation, the staining solution was removed, PBS was added, and the cells were observed under a confocal laser microscope. The staining solution was prepared by diluting calcein AM solution to 5.0 μM and ethidium homodimer I solution to 4.0 μM with PBS. [9-5] Three-dimensional observation of hMSC cell aggregates using a cell cluster microscope (Mil-Cell) Mil-Cell (Sumitomo Electric Industries, Ltd.) was used for three-dimensional observation of cell aggregates. The culture medium was removed from the cell aggregates prepared using the same procedure as in [9-2] and washed with PBS. The cell aggregates were then detached by pipetting and seeded into a 96-well plate. The concentration of the detached cell aggregates in the 96-well plate was adjusted, and they were seeded into a Mil-Cell observation container, after which they were observed and analyzed using Mil-Cell. [9-6] Evaluation of hMSC cell aggregate induction in serum-containing medium The mesenchymal stem cell growth medium 2 in the container in which the hMSCs were cultured was removed and washed three times with PBS. Trypsin / EDTA was added to the culture area of ​​25 cm. 21.0 mL of the medium was added to each well and incubated for 1 minute under conditions of 5% CO2, 37°C, and 95% RH. To inhibit the action of trypsin / EDTA, serum-containing EMEM medium was added in an amount three times the amount of trypsin / EDTA. The solution was transferred to a 15 mL PP centrifuge tube and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed and Mesenchymal Stem Cell Growth Medium 2 was added to each well and the cells were resuspended. The number of hMSC cells in the cell suspension was counted using trypan blue. 3.5 x 10 hMSCs were placed in a 96-well plate. 4 100 μL of cells / well were seeded, and 100 μL each of KP24, c5HK24, and t5HK24 was added at 0.36 mM. The medium was then changed every two days and incubated at 37°C, 5% CO2, and 95% RH for 5 days. The cells were then observed using a phase-contrast microscope. [9-7] Differentiation of hMSC cell aggregates into osteoblasts Osteoblast differentiation induction medium was prepared by adding L(+)-ascorbic acid (50 μM), β-glycerophosphate disodium pentahydrate (10 mM), and dexamethasone (1 μM) to serum-containing DMEM medium. hMSCs were cultured in a 96-well plate at 3.0 × 10 4 hMSCs were seeded at 1000 cells / well, and KP24, c5HK24, and t5HK24 were added at 0.36 mM each. The plates were then cultured for 10 days under conditions of 5% CO2, 37°C, and 95% RH. The medium in the 96-well plate was then removed and 200 μM of osteoblast-inducing medium was added. The hMSCs were cultured under humidified conditions of 5% CO2, 37°C, and the osteoblast-inducing medium was replaced at 100 μL / day. [9-8] Alizarin Red S staining of osteoblast-differentiated hMSC cell aggregates Alizarin Red S staining was performed using an Alizarin Red S staining kit (Bio Mirai Kobo Co., Ltd.). The medium was removed from 96-well plates of hMSCs cultured as described in [9-2]. After washing twice with saline, 125 μL of Alizarin Red fixative was added and the plate was incubated at room temperature for 30 minutes. After removing the Alizarin Red fixative, the plate was washed twice with Alizarin Red washing solution, and 125 μL of Alizarin Red staining solution was added and incubated at room temperature for 30 minutes. After removing the Alizarin Red staining solution, the plate was washed three times with sterile water and observed under a phase-contrast microscope. After removing the sterile water, 125 μL of Alizarin Red eluate was added and incubated at room temperature for 15 minutes. Absorbance was measured using a microplate reader at an absorption wavelength of 405 nm and a reference wavelength of 620 nm.

[0071] The evaluation results of this example are shown below. The molecular weight of the DMT-MM synthesized in the above [2] Synthesis of DMT-MM was measured using MALDI-TOF-MS. The results are shown in FIG. As shown in Figure 11, a molecular ion peak at 240.9 m / z was detected, but the molecular weight of DMT-MM is 276.7 m / z. This is because the atomic weight of chlorine is 35.5 m / z, and chlorine was released during ionization of DMT-MM. Therefore, the detected peak was considered to be derived from the target product, and it was determined that the synthesis of DMT-MM was complete. In the above-mentioned [3] Introduction of a protecting group into cis-5-hydroxypipecolic acid [3-1] Protection of the carboxyl group by methylation reaction, the Rf values ​​determined by TLC in each reaction are shown in Table 5.

[0072] [Table 5]

[0073] Since the Rf value of c5HPA was 0.03, the Rf value of 3 was c5HPA. Furthermore, since the spot with Rf value 2 disappeared in the crystals after azeotropic distillation with toluene, it was determined that the Rf value of 2 was NH-c5HPA-COOCH3 hydrochloride, and the Rf value of 1 was the target substance, NH-c5HPA-COOCH3. The results of the MALDI-TOF-MS analysis of NH-c5HPA-COOCH3 are shown in Figure 12. The MALDI-TOF-MS spectrum confirmed the molecular ion peak derived from NH-c5HPA-COOCH3, which indicated that the carboxyl group protection by methylation of c5HPA was complete. In the above-mentioned [3-2] hydroxy group protection with a TBS group, the Rf values ​​determined by TLC before and after the reaction are shown in Table 6.

[0074] [Table 6]

[0075] Before the reaction, the Rf value of 3 is thought to be c5HPA, and the Rf value of 2 is thought to be NH-c5HPA-COOCH3. After the reaction, Rf value 1 appeared, so it was thought that Rf value 1 was the target compound, NH-c5HPA(OTBS)-COOCH3, and the next reaction was carried out.

[0076] In the above-mentioned [3-3] saponification reaction of methyl carboxylate with sodium hydroxide (saponification reaction of methyl carboxylate of NH-c5HPA(OTBS)-COOCH3), the FT-IR results before and after the reaction are shown in Figure 13. In the FT-IR spectrum, 1750 cm -1 The peak near the peak due to the ester bond disappeared. From this, it was determined that the methyl carboxylate of NH-c5HPA(OTBS)-COOCH3 had been eliminated, producing a carboxylate salt, and the reaction proceeded to the next step.

[0077] In the above-mentioned [3-4] protection of amino groups with Fmoc groups, the results of analysis by MALDI-TOF-MS after the reaction are shown in FIG. In the MALDI-TOF-MS spectrum, a molecular ion peak derived from Fmoc-c5HPA(OTBS)-OH was confirmed. This indicated that the Fmoc group had been successfully introduced to the amino group. Furthermore, the detection of peaks derived from the target compound confirmed the need for purification. Furthermore, the peak at m / z = 522.6 was presumed to be the impurity, potassium-added Fmoc-c5HPA(OTBS)-COOCH3.

[0078] HPLC analysis of peptides The crude peptide of KP24 obtained in the peptide synthesis described above [4] was subjected to HPLC analysis. The HPLC elution profile of the crude peptide of KP24 is shown in Figure 15. In the HPLC elution profile of KP24, a peak derived from the target product ((1) in Figure 15) was confirmed. In addition, peaks other than the target product ((2) and (3) in Figure 15) were also confirmed. These are thought to be by-products or incompletely deprotected KP24. Therefore, it was determined that isolation and purification by HPLC was necessary.

[0079] The purified KP24 obtained by the above-mentioned [5] high-performance liquid chromatography (HPLC) analysis and purification was subjected to HPLC analysis and molecular weight measurement by MALDI-TOF-MS. The HPLC analysis results (HPLC elution profile) are shown in Figure 16A, and the MALDI-TOF-MS measurement results are shown in Figure 16B. Comparing Figure 15 with Figure 16, it was confirmed that peaks other than those of the target product had disappeared. From this, it was determined that the isolation and purification of KP24 had been completed by the HPLC fractionation procedure. Furthermore, the MALDI-TOF-MS measurement results confirmed the molecular ion peak derived from the target product, which indicated that the synthesis and purification of KP24 had been completed.

[0080] The crude peptide of c5HK24 obtained in the peptide synthesis described above [4] was subjected to HPLC analysis. The HPLC elution profile of the crude peptide of c5HK24 is shown in Figure 17. In the HPLC elution profile of c5HK24, multiple peaks were confirmed. The main peak ((3) in Figure 17) was presumed to be the target product, and the product was separated, analyzed by HPLC, and its molecular weight was measured by MALDI-TOF-MS. The purified c5HK24 obtained by the above [5] high-performance liquid chromatography (HPLC) analysis and purification was analyzed by HPLC and its molecular weight was measured by MALDI-TOF-MS. The HPLC analysis results (HPLC elution profile) are shown in Figure 18A, and the MALDI-TOF-MS measurement results are shown in Figure 18B. Based on the HPLC elution profile (Figure 18A), it was determined that the isolation and purification of the main peak was complete. Furthermore, the MALDI-TOF-MS analysis results confirmed the molecular ion peak derived from the target product, which revealed that the main peak was the target product, c5HK24. Therefore, it was determined that the synthesis and purification of c5HK24 were complete.

[0081] The crude peptide of t5HK24 obtained in the peptide synthesis described above [4] was subjected to HPLC analysis. The HPLC elution profile of the crude peptide of t5HK24 is shown in Figure 19. In the HPLC elution profile of t5HK24, multiple peaks were confirmed. The main peak ((1) in Figure 19) was presumed to be the target product, and the product was separated, analyzed by HPLC, and its molecular weight was measured by MALDI-TOF-MS. The purified t5HK24 obtained by the above [5] high-performance liquid chromatography (HPLC) analysis and purification was analyzed by HPLC and its molecular weight was measured by MALDI-TOF-MS. The HPLC analysis results (HPLC elution profile) are shown in Figure 20A, and the MALDI-TOF-MS measurement results are shown in Figure 20B. Based on the HPLC elution profile (Figure 20A), it was determined that the isolation and purification of the main peak was complete. Furthermore, the MALDI-TOF-MS analysis results confirmed the molecular ion peak derived from the target product, which revealed that the main peak was the target product, t5HK24. Therefore, it was determined that the synthesis and purification of t5HK24 were complete.

[0082] Secondary structure analysis of KP24, c5HK24, and t5HK24 by circular dichroism (CD) spectroscopy The secondary structures of KP24, c5HK24, and t5HK24 in solution were analyzed using circular dichroism (CD) spectroscopy as described above [7]. The results are shown in Figure 21. In the CD spectrum, a negative maximum was observed at 210 nm for KP24, indicating that KP24 is a random coil. Furthermore, negative maxima were observed at 212 nm and 215 nm for c5HK24 and t5HK24, respectively, and a positive maximum was observed at 224 nm. These results indicate that c5HK24 and t5HK24 are polyproline II helices. The factor that affected the secondary structure is presumed to be the difference in the number of ring members between proline and hydroxypipecolic acid. Proline has a five-membered ring structure, while hydroxypipecolic acid has a six-membered ring structure. This difference in ring member number suggests that the dihedral angle of the peptide changes, affecting the secondary structure.

[0083] Evaluation of cell aggregate-inducing activity of mouse fibroblasts (L929) by KP24, c5HK24, and t5HK24 · L929 cell aggregate induction evaluation In the above-mentioned [8-3] L929 cell aggregate induction, L929 cells on a 96-well plate to which KP24, c5HK24, t5HK24, or KA24 had been added were observed under a phase contrast microscope. The results are shown in FIG. Microscopic observation of L929 cells showed that on day 1, all peptides resulted in uniformly dispersed cells, but on day 4, KP24, c5HK24, and t5HK24 cells were induced to aggregate by the peptides. Furthermore, on day 7, cell aggregates were not formed when KA24 was added, but cell aggregates were formed when KP24, c5HK24, and t5HK24 were added. These results demonstrate that c5HK24 and t5HK24, like KP24, have cell aggregate-inducing activity. Furthermore, KA24 is a peptide without a ring structure, and its lack of cell assembly-inducing activity suggests that cell assembly-inducing peptides must contain a ring structure. It is presumed that the introduction of ring structures at both ends of the lysine restricts the degree of freedom of the main chain dihedral angle due to the ring structure, stabilizing the aminobutyl group, which is the lysine side chain, in an independent state. Next, the relationship between the number of L929 cell aggregates and the results of measuring the diameter size of the L929 cell aggregates on a 96-well plate to which each of the peptides (KP24, c5HK24, and t5HK24) had been added is shown in FIG. KP24 and c5HK24 formed the most cell aggregates with diameters of 80 to 100 μm, while t5HK24 formed the most cell aggregates with diameters of 60 to 80 μm. This suggests that the cis- and trans-configured hydroxy groups of c5HPA and t5HPA, which are stereoisomers, affect cell aggregate formation in c5HK24 and t5HK24.

[0084] · Live-Dead staining evaluation of L929 cell aggregates In the above-mentioned [8-4] Live-Dead assay fluorescence observation of L929 cell aggregates, L929 cell aggregates were Live-Dead stained with calcein AM and ethidium homodimer I and observed with a confocal laser microscope. The results are shown in Figure 24. In Figure 24, (a) is KP24, (b) is c5HK24, and (c) is t5HK24. In this Live-Dead assay observation, live cells emit green light and dead cells emit red light when observed with a confocal laser microscope, allowing for the distinction between live and dead cells. As shown in Figure 24, almost no dead cells were observed at the top, center, or bottom of the cell aggregates formed with c5HK24 and t5HK24. This confirmed that, like KP24, cells within the cell aggregates were viable, and that c5HK24 and t5HK24 were able to form cell aggregates large enough to allow oxygen and nutrients to reach the inner cells. Furthermore, it was found that c5HK24 and t5HK24 were not cytotoxic.

[0085] · Measurement and comparison of sphericity of L929 cell aggregates using a cell cluster microscope (Mil-Cell) In the three-dimensional observation of L929 cell aggregates using the cell cluster microscope (Mil-Cell) described above [8-5], stereoscopic images of L929 cell aggregates observed using Mil-Cell are shown in Figure 25. In Figure 25, (a) is KP24, (b) is c5HK24, and (c) is t5HK24. From the stereoscopic images taken with the Mil-Cell microscope (FIG. 25A), it was observed that the L929 cell aggregates were present in a shape very similar to a sphere, regardless of the peptide used. Furthermore, the results of measuring the sphericity of the cell aggregates from the stereoscopic images are shown in Figure 25B. The sphericity of the cell aggregates was 80.3% for KP24, 80.8% for c5HK24, and 80.7% for t5HK24. These results indicate that the L929 cell aggregates induced by KP24, c5HK24, and t5HK24 maintained a relatively similar morphology with no significant differences between the peptides, and that they were nearly perfectly spherical, as their sphericity was all above 80%. Furthermore, it was suggested that the sphericity converged regardless of the inducing activity of KP24, c5HK24, and t5HK24. These results suggest that c5HK24 and t5HK24 have cell aggregate-inducing activity in L929.

[0086] Evaluation of cell aggregate induction and osteoblast induction of human mesenchymal stem cells (hMSCs) by KP24, c5HK24, and t5HK24 · hMSC cell aggregate induction evaluation In the above-mentioned [9-2] hMSC cell aggregate induction, hMSCs on a 96-well plate to which c5HK24 and t5HK24 had been added were observed under a phase contrast microscope. The results are shown in FIG. As shown in Figure 26, c5HK24 formed several cell aggregates, demonstrating that c5HK24 induces cell aggregate formation in hMSCs. However, in t5HK24, cells detached from the edge of the well and did not form cell aggregates. This is likely due to the fact that hMSCs are larger than L929 cells, resulting in a reduced intercellular distance, leading to excessive induction. Therefore, we next investigated the optimal cell density for inducing hMSCs.

[0087] · hMSC cell aggregate induction (evaluation of cell density) Figure 27 shows the results of phase-contrast microscopy of hMSC cell aggregates with different cell densities on 96-well plates prepared by adding KP24, c5HK24, or t5HK24 in the above-mentioned [9-3] hMSC cell aggregate induction (evaluation of cell density). As shown in Figure 27, KP24 (Figure 27A), c5HK24 (Figure 27B), and t5HK24 (Figure 27C) were 3.0 × 10 4 Cell aggregates were formed at a cell density of 2.0 × 10 cells / well or more. 4 hMSC cell aggregates did not form under the condition of 100 cells / well, suggesting that t5HK24 had weak activity in inducing hMSC cell aggregates and that the cells were not spaced far enough apart to induce aggregation. The relationship between the diameter size of hMSC cell aggregates measured at each cell density and the number of cells is shown in Figure 28A (3.0 × 10 4 cells / well), Figure 28B (3.5 × 10 4 cells / well) and Figure 28C (4.0 × 10 4 cells / well). From Figures 28A, 28B, and 28C, it can be seen that the variation in diameter size of the cell aggregates was small at a cell density of 3.0 x 10 4 cells / well, the cell density was 3.5 × 10 4 The cell density was 4.0×10 4 It can be seen that the cell density is even greater at 4.0 × 10 cells / well. 4At cells / well, c5HK24 produced cell aggregates with diameters of 60–120 μm, and t5HK24 produced cell aggregates with diameters of 80–140 μm. However, KP24 produced cell aggregates with diameters of 180 μm or greater. It has been reported that the diffusion limit for oxygen, nutrients, and waste products within cell aggregates is 150–200 μm. Therefore, it was suggested that cell aggregates produced with KP24 may not receive sufficient oxygen and nutrients. However, c5HK24 and t5HK24 could form cell aggregates of appropriate size that receive sufficient oxygen and nutrients to the interior, compared to KP24. Based on the above, the optimal cell density for inducing hMSC cell aggregates is 3.0 × 10 4 Since the cell density was estimated to be 100 cells / well, subsequent evaluations were performed at this cell density.

[0088] · Live-Dead staining evaluation of hMSC cell aggregates In the above [9-4] Live-Dead staining of hMSC cell aggregates, hMSC cell aggregates were stained with calcein AM and ethidium homodimer I and observed with a confocal laser microscope. The results are shown in Figure 29. In Figure 29, (a) is KP24, (b) is c5HK24, and (c) is t5HK24. As shown in Figure 29, c5HK24 and t5HK24 showed almost no dead cells at the top, center, or bottom of the hMSC cell aggregates, as was the case with L929 cell aggregates. This confirmed that cells within the cell aggregates were viable, and that c5HK24 and t5HK24 were able to form hMSC cell aggregates large enough to deliver oxygen and nutrients to the inner cells. Furthermore, c5HK24 and t5HK24 were found to be non-cytotoxic.

[0089] · Measurement and comparison of sphericity of hMSC cell aggregates using Mil-Cell In the three-dimensional observation of hMSC cell aggregates using the cell cluster microscope (Mil-Cell) described above [9-5], stereoscopic images of hMSC cell aggregates observed using Mil-Cell are shown in Figure 30A. In Figure 30A, (a) is KP24, (b) is c5HK24, and (c) is t5HK24. From the stereoscopic analysis images (Figure 30A) of the Mil-Cell microscope, it was observed that hMSC cell aggregates existed in a shape very close to spheres for all peptides. Furthermore, the results of measuring the sphericity of the cell aggregates from the stereoscopic images are shown in Figure 30B. The sphericity of the cell aggregates was 76.1% for KP24, 78.1% for c5HK24, and 77.7% for t5HK24. This indicates that the hMSC cell aggregates induced by KP24, c5HK24, and t5HK24 had a shape close to perfect spheres, with no significant difference observed between the peptides.

[0090] · Evaluation of hMSC cell aggregate induction in serum-containing medium In the evaluation of induction of hMSC cell aggregates in serum-containing medium described above in [9-6], hMSCs on 96-well plates supplemented with KP24, c5HK24, and t5HK24 were observed under a phase-contrast microscope. The results are shown in Figure 31. As shown in Figure 31, none of the peptides formed cell aggregates in serum-containing medium. This is thought to be because the inclusion of serum in the medium increased the adhesion of the cells to the substrate, making cell migration difficult and preventing the peptides from inducing cell migration. This suggests that serum inhibits the peptide's inducing ability. Therefore, when inducing differentiation of hMSC cell aggregates into osteoblasts, we decided to first create hMSC cell aggregates and then induce differentiation.

[0091] · Alizarin Red S staining evaluation of osteogenic differentiation-induced hMSC cell aggregates To characterize the hMSC aggregates, we performed the above-mentioned [9-7] hMSC aggregate differentiation into osteoblasts and [9-8] Alizarin Red S staining of osteoblast-induced hMSC aggregates. The results of staining hMSC cell aggregates cultured in bone differentiation medium for 10 days with Alizarin Red S are shown in FIG. The calcium deposits in the hMSC aggregates were stained red in KP24, c5HK24, and t5HK24, confirming their differentiation into osteoblasts. hMSC aggregates induced by c5HK24 and t5HK24 were shown to differentiate into osteoblasts, just like KP24.

[0092] Example 3 In this example, a peptide (t4HK24) in which the proline contained in KP24 was replaced with the unnatural amino acid trans-4-hydroxypipecolic acid (t4HPA) was synthesized by solid-phase synthesis and evaluated.

[0093] [1] Reagents used In this example, the same reagents as in Example 2 were used, except that in the Fmoc solid-phase synthesis, N-(9-fluorenylmethoxycarbonyl)-trans-4-t-butyl-pipecolic acid (Fmoc-t4HPA(tBu)-OH) [manufactured by UBE Corporation] was used as the unnatural amino acid.

[0094] [2] Synthesis of DMT-MM DMT-MM was synthesized in the same manner as in Example 2.

[0095] [3] Peptide synthesis [3-1] Fmoc solid phase synthesis method Peptide synthesis was carried out using the Fmoc solid phase synthesis method according to the same synthesis scheme as in Example 2. The amounts of amino acids used in the synthesis of KP24 are shown in Table 7, and the amounts of amino acids used in the synthesis of t4HK24 are shown in Table 8. [3-1-1] Solid-phase synthesis of KP24 Fmoc-Pro-TrtA-PEG Resin 1.0g (0.20mmolg -1The column was loaded with 1000-mM HCl and washed three times with DMF and MeOH for 1 minute each. The column was then swollen in 25% DMSO / DMF for 30 minutes. After swelling, the 25% DMSO / DMF was removed by six 1-minute washes with DMF. The column was then incubated with 20% PPD / DMF for 30 minutes to deprotect the Fmoc group. After deprotection, the column was washed three times with DMF for 1 minute to remove the 20% PPD / DMF. The deprotection of the Fmoc group was confirmed by the development of blue color in the chloranil test. After three 1-minute washes with DMF, the resin was added with three equivalents of the protected amino acid, 95 μL of NMM (to make the resin basic), and 0.17 g of DMT-MM (a condensation agent), and the condensation reaction was carried out for 180 minutes. Unreacted material was removed by six 1-minute washes with DMF, and the completion of the condensation was confirmed by the development of red color in the TNBS test and blue color in the chloranil test. Repeat these steps to obtain (Lys(Boc)-Pro) 12 -TrtA-PEG Resin (KP24-TrtA-PEG Resin) was synthesized.

[0096] [Table 7]

[0097] [3-1-2] Solid-phase synthesis of t4HK24 Fmoc-Lys(Boc)-Alko-PEG Resin 0.50g (0.23mmolg -1The column was loaded with 1000-mcg HCl and washed three times with DMF and MeOH for 1 minute each. The column was then swollen in 25% DMSO / DMF for 30 minutes. After swelling, the 25% DMSO / DMF was removed by six 1-minute washes with DMF. The column was then incubated with 20% PPD / DMF for 30 minutes to deprotect the Fmoc group. After deprotection, the column was washed three times with DMF for 1 minute to remove the 20% PPD / DMF. The deprotection of the Fmoc group was confirmed by the development of blue color in the chloranil test. After three 1-minute washes with DMF, the resin was added with 3 equivalents of a protected amino acid, 95 μL of NMM (to make the resin basic), and 0.095 g of DMT-MM (a condensation agent), and the condensation reaction was carried out for 180 minutes. Unreacted materials were removed by six 1-minute washes with DMF, and the completion of the condensation was confirmed by the development of red color in the TNBS test and blue color in the chloranil test. These steps were repeated to obtain (t4HPA(tBu)-Lys(Boc)) 12 -Alko-PEG Resin (t4HK24-Alko-PEG Resin) was synthesized.

[0098] [Table 8]

[0099] [3-2] TNBS test A TNBS test was carried out in the same manner as in Example 2 to confirm the completion of deprotection of the Fmoc group and condensation.

[0100] [3-3] Chloranil test A TNBS test was carried out in the same manner as in Example 2 to confirm the completion of deprotection of the Fmoc group and condensation.

[0101] [3-4] Final deprotection 9.5 mL of TFA and 0.5 mL of ultrapure water were mixed and stirred in an ice bath for 10 minutes to prepare a cleavage mixture. The cleavage mixture was added to the resin synthesized in [3-1] and stirred at room temperature for 150 minutes. The peptide / TFA solution and resin were then separated by filtration. Diethyl ether was added to precipitate the peptide, which was then collected. The dried peptide was dissolved in ultrapure water and dialyzed for 3 days using a dialysis membrane with a molecular weight cutoff of 100-500. The dialyzed peptide solution was lyophilized for 3 days to obtain crude peptide as a white powder.

[0102] [4] Analysis and purification by high-performance liquid chromatography (HPLC) [4-1] Analysis by high-performance liquid chromatography (HPLC) KP24 and t4HK24 were analyzed by HPLC. The HPLC equipment used for the analysis was a CO-8020 column oven, a SIL-20AC autosampler, an SPD-10A UV-visible detector, a DP-8020 data processor, a CBM-20A system controller, an FRC-10A fraction collector, and an LC-20AD pump, all manufactured by Shimadzu Corporation. A TOSOH TSK-GEL ODS-100V column was used. The measurement conditions were a flow rate of 1.00 mL / min, a UV wavelength of 210 nm, and a column oven temperature of 40°C. A binary solvent gradient was used as the mobile phase, consisting of 0.1% TFA / ultrapure water (liquid A) and 0.1% TFA / acetonitrile (liquid B). Samples were prepared using liquid A as the solvent.

[0103] [4-2] Purification of KP24 and t4HK24 by high-performance liquid chromatography (HPLC) KP24 and t4HK24 were purified by HPLC. The HPLC equipment used for the analysis was a Shimadzu CO-8020 column oven, SIL-20AC autosampler, SPD-10A UV-visible detector, DP-8020 data processor, CBM-20A system controller, FRC-10A fraction collector, and LC-20AD pump. The column used was a TOSOH TSK-GEL ODS-120T. The measurement conditions were a flow rate of 2.87 mL / min, UV wavelength of 210 nm, and column oven temperature of 40°C. A binary solvent gradient was used as the mobile phase: 0.1% TFA / ultrapure water (liquid A) and 0.1% TFA / acetonitrile (liquid B). The sample was prepared using liquid A as the solvent.

[0104] [5] Identification by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) The molecular weights of the crude peptides of KP24 and t4HK24 and the crystals obtained by freeze-drying after isolation and purification by HPLC were confirmed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, and the target compounds were identified.

[0105] [6] Induction of cell aggregates in mouse fibroblasts (L929) by KP24 and t4HK24 [6-1] L929 cell culture Eagle's MEM (EMEM) medium was used for L929 culture. 2.82 g of EMEM powder was dissolved in 261 mL of ultrapure water and sterilized using an autoclave (Tomy LBS-325). After high-pressure steam sterilization, serum-containing EMEM medium was prepared by adding 30 mL of fetal bovine serum (FBS), 0.090 g of L-glutamine, and 6.0 mL of 7.5% sodium bicarbonate solution. Serum-free EMEM medium was also prepared by replacing 30 mL of FBS with ultrapure water. L929 (cell number = RCB1451) was purchased from the RIKEN BioResource Research Center. Frozen L929 cells were thawed at 37°C and transferred to a 15 mL centrifuge tube (Iwaki Co., Ltd.). 10 mL of serum-containing EMEM medium was added to the tube, pipetted several times, and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed and the cells were resuspended in serum-containing EMEM. Seeding density 4000cells / cm 2 The cells were seeded in a culture vessel manufactured by Iwaki Co., Ltd. and cultured in an incubator set at 37°C, 5% CO2, and 95% RH. [6-2] L929 cell aggregate induction [6-2-1] Cell aggregation induction of L929 cells in KP24 The serum-containing EMEM medium in the container in which L929 cells were cultured was removed and washed twice with PBS. Trypsin / EDTA was added to a 25cm culture area. 2 1.0 mL of the cells was added to the 1000-well plate and incubated for 1 minute under conditions of 5% CO2, 37°C, and 95% RH. To inhibit the action of trypsin / EDTA, serum-containing EMEM medium was added in an amount three times the amount of trypsin / EDTA. The solution was transferred to a 15 mL PP centrifuge tube and centrifuged at 1000 rpm for 4 minutes. The supernatant was removed and resuspended in serum-free EMEM. 5.0 × 10 L929 cells were placed in a 96-well plate. 4 100 μL of cells / well were seeded, and 100 μL of 1.0 mg / mL KP24 was added. The day of seeding was counted as day 0, and the cells were incubated at 37°C, 5% CO2, and 95% RH for 7 days, with the medium changed every two days. After that, the cells were observed using a phase-contrast microscope. [6-2-2] Induction of L929 cell aggregates in t4HK24 The L929 cell aggregate induction in t4HK24 was carried out in the same manner as described above in [6-2-1] KP24, except that t4HK24 was used instead of KP24. Then, observation was performed using a phase-contrast microscope.

[0106] [7] Live / Dead staining of L929 cell aggregates [7-1] Live / Dead staining of L929 cell aggregates in KP24 5.0 × 10 L929 cells were cultured in a 96-well plate. 4 100 μL of cells / well were seeded, and 100 μL of 1.0 mg / mL KP24 was added. The cells were then incubated for 7 days under conditions of 5% CO2, 37°C, and 95% RH to induce cell aggregation. The staining solution was prepared by diluting calcein AM solution to 5 μM and ethidium homodimer I solution to 4 μM in PBS. After washing twice with PBS, the prepared staining solution was added. The cells were incubated for 40 minutes under conditions of 37°C, 5% CO2, and 95% RH. The cells were then observed under a confocal laser scanning microscope. [7-2] Live / Dead staining of L929 cell aggregates in t4HK24 Live-dead staining of L929 cell aggregates in t4HK24 was performed in the same manner as described above for [7-1] Live-dead staining of L929 cell aggregates in KP24, except that t4HK24 was used instead of KP24. Then, the cells were observed using a confocal laser microscope.

[0107] [8] Three-dimensional analysis of cell aggregates using cell cluster microscope (Mil-Cell) Mil-Cell (Sumitomo Electric Industries, Ltd.) was used for three-dimensional analysis of cell aggregates. The observed cell aggregates were measured for sphericity using Mil-Cell. The culture medium from the prepared cell aggregates was removed and washed with PBS. The cell aggregates were then detached by pipetting and seeded in a 96-well plate. The concentration of the detached cell aggregates in the 96-well plate was adjusted, and they were seeded in a Mil-Cell observation container, after which they were observed and analyzed using Mil-Cell.

[0108] The evaluation results of this example are shown below. HPLC analysis of peptides The results of HPLC analysis of KP24 that was subjected to final deprotection in the above-mentioned [3-4] final deprotection are shown in Figure 33A. The HPLC gradient conditions were as follows:

[0109] [Table 9]

[0110] The sample solvent used was 0.1% TFA / ultrapure water, and the mobile phases used were 0.1% TFA / ultrapure water (liquid A) and 0.1% TFA / acetonitrile (liquid B). As shown in Figure 33A, peaks corresponding to the target product were confirmed, but peaks other than the target product were also observed. These were thought to be by-products or incompletely deprotected KP24. Therefore, it was determined that separation and purification by HPLC was necessary. Therefore, the peak believed to be the target product was isolated and purified under the following gradient conditions and reanalyzed by HPLC. The results are shown in Figure 33B.

[0111] [Table 10]

[0112] As shown in Figure 33B, only peaks derived from the target product were observed. Furthermore, the peak area ratio confirmed that the purity was 97%, and therefore it was determined that the synthesis and purification of KP24 was complete. The results of HPLC analysis of t4HK24 that was subjected to final deprotection in the above-mentioned [3-4] final deprotection are shown in Figure 34A. The HPLC gradient conditions were as follows:

[0113] [Table 11]

[0114] The sample solvent used was 0.1% TFA / ultrapure water, and the mobile phases used were 0.1% TFA / ultrapure water (liquid A) and 0.1% TFA / acetonitrile (liquid B). As shown in Figure 34A, a peak corresponding to the target product was confirmed, but several other peaks were also observed. These were thought to be by-products or incompletely deprotected t4HK24. Therefore, it was determined that separation and purification by HPLC was necessary. Therefore, the peak believed to be the target product was isolated and purified under the following gradient conditions and reanalyzed by HPLC. The results are shown in Figure 34B.

[0115] [Table 12]

[0116] As shown in Figure 34B, only peaks derived from the target product were observed. Furthermore, the peak area ratio confirmed that the purity was 98%, and therefore it was determined that the synthesis and purification of t4HK24 was complete.

[0117] Evaluation of cell aggregate induction activity of mouse fibroblasts (L929) by KP24 and t4HK24 · Evaluation of L929 cell aggregate induction by KP24 In the above-mentioned [6-2-1] cell aggregate induction of L929 cells with KP24, L929 cells on a 96-well plate to which KP24 had been added were observed with a phase contrast microscope. The results are shown in FIG. Microscopic observation of L929 cells showed that on day 1, the cells were dispersed and uniformly present, regardless of whether KP24 was added or not. However, on day 3, the cells in the wells to which KP24 was added gradually gathered together, forming cell aggregates. On day 7, it was confirmed that the cells had aggregated more than on day 3. This demonstrated that the synthesized KP24 has cell aggregate-inducing activity.

[0118] · Evaluation of L929 cell aggregate induction by t4HK24 In the above-mentioned [6-2-2] L929 cell aggregate induction by t4HK24, L929 cells on a 96-well plate to which t4HK24 had been added were observed using a phase contrast microscope. The results are shown in FIG. Microscopic observation of L929 cells showed that on day 1, the cells were dispersed and uniformly present regardless of whether t4HK24 was added, but on day 3, the cells in the wells to which t4HK24 was added gradually began to gather. On day 7, cell aggregates were confirmed to have formed. This demonstrated that t4HK24 has cell aggregate-inducing activity.

[0119] · Size comparison of cell aggregates The relationship between the number of L929 cell aggregates and the results of measuring the diameter size of the L929 cell aggregates formed in the above-mentioned [6-2-1] and [6-2-2] is shown in FIG. For both KP24 and t4HK24, the largest number of cell aggregates was 60-80 μm in diameter, followed by cell aggregates with diameters of 60 μm or less, 80-100 μm, and 100-120 μm. This suggests that the size of the aggregates can be controlled to ensure the survival of cells. Furthermore, it was found that a large amount of aggregates were formed per well. This suggests that three-dimensional culture using KP24 and t4HK24 as liquid factors is possible for mass production.

[0120] · Live / Dead staining assessment of L929 cell aggregates [7] Figure 38 shows the results of live-dead staining of L929 cell aggregates using calcein AM and ethidium homodimer I. The results were observed using a confocal laser microscope. As shown in Figure 38, KP24 and t4HK24 showed almost no dead cells at the top, center, or bottom of the cell aggregates. This confirmed that the cells inside the cell aggregates were viable, and that they were able to form cell aggregates large enough to deliver oxygen and nutrients to the inner cells. These peptides were also found to be non-cytotoxic.

[0121] Example 4 According to the method of Example 3, a peptide (t3HK24) in which the proline contained in KP24 was replaced with the unnatural amino acid trans-3-hydroxypipecolic acid (t3HPA) was synthesized and evaluated. Cell aggregate induction of L929 cells in t3HK24 was carried out in the same manner as in Example 3, and observation was carried out using a phase-contrast microscope. The results are shown in Figure 39. As shown in Figure 39, the cells were dispersed and uniformly present on day 1, but cell aggregates were confirmed to have formed on day 7. This demonstrated that t3HK24 has cell aggregate-inducing activity. [Industrial Applicability]

[0122] The cell aggregate-inducing peptide or cell aggregate-forming agent of the present invention causes little damage to cells. Simply by adding the peptide or cell aggregate-forming agent of the present invention to a cell culture system, cell aggregates can be formed simply and efficiently while minimizing damage to cells. According to the present invention, it is possible to provide cell aggregates that can be effectively used in fields such as drug discovery, pharmacology, medicine, and biology, for example, in the evaluation of the efficacy and toxicity of chemical substances and pharmaceuticals, the mass production of useful substances such as antibodies, and regenerative medicine.

Claims

1. A cell aggregate-inducing peptide represented by general formula (1), which has hydroxypipecolic acid and lysine as constituent repeating units. (HPA-L]3) ｎ ・・・(1) [In general formula (1), HPA represents hydroxypipecolic acid, Lys represents lysine, and n is an integer of 4 or more and 30 or less.]

2. The cell aggregate-inducing peptide according to claim 1, wherein the hydroxypipecolic acid is 3-hydroxypipecolic acid, 4-hydroxypipecolic acid and / or 5-hydroxypipecolic acid.

3. A method for forming cell aggregates, comprising adding the cell aggregate-inducing peptide according to claim 1 or 2 to a culture medium for cultured cells to form cell aggregates.

4. A cell aggregate forming agent comprising the cell aggregate-inducing peptide according to claim 1 or 2.

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