JP6822668B2 - Method for Embryoid Body Formation of Pluripotent Stem Cell and Composition for Embryoid Body Formation of Pluripotent Stem Cell - Google Patents

Description

The present invention relates to a method for forming an embryoid body of pluripotent stem cells and a composition for forming an embryoid body of pluripotent stem cells.

Collagen is one of the proteins that make up the dermis, ligaments, tendons, bones, cartilage, etc., and is the main component of the extracellular matrix of multicellular organisms. As research progressed, it became clear that collagen has various physiological functions, and even now, research for discovering new physiological functions of collagen molecules and research for discovering new uses for collagen molecules. Is underway.

Studies to date have revealed that one collagen molecule is composed of three polypeptide chains, and that these three polypeptide chains form a helical structure to form one collagen molecule. It has become.

The region within each polypeptide chain for forming a helical structure is called a triple helical domain, and the triple helical domain has a characteristic amino acid sequence. Specifically, the triple helical domain has a characteristic amino acid sequence in which the amino acid sequence represented by "Gly-XY" appears repeatedly and continuously. In the amino acid sequence consisting of the above-mentioned three amino acids, amino acids other than glycine, that is, X and Y can be various amino acids.

At the amino terminus and / or the carboxyl terminus of the collagen molecule (in other words, the amino terminus and the carboxyl terminus of each polypeptide chain constituting the collagen molecule), telopeptide, which is the main antigen site of collagen, is present. The terror peptide is present in each polypeptide chain constituting the collagen molecule on the amino-terminal side and / or the carboxyl-terminal side of the triple helical domain described above.

It is known that the antigenicity of a collagen molecule can be suppressed to a low level by partially excising the terror peptide from the collagen molecule by treating it with an enzyme such as a protease. A collagen molecule in which such a terror peptide is partially excised is called atelocollagen.

Studies to date have revealed that collagen, atelocollagen, and these degradation products by proteases have various physiological functions, and collagen, atelocollagen, and these by proteases based on the physiological functions. Various uses have been developed for the degradation products of (see, for example, Patent Documents 1 and 2).

Patent Document 1 discloses a technique for using a decomposition product of collagen or atelocollagen treated with a protease (for example, pepsin and actinidine) as a medical material for hemostasis. More specifically, in Patent Document 1, first, the skin of yellowfin tuna is treated with pepsin to obtain an aqueous solution containing atelocollagen, and further, sodium chloride is added to the aqueous solution to obtain atelocollagen. Precipitated and recovered. When recovering atelocollagen as a precipitate, sodium chloride will be removed together with the supernatant. Then, in Patent Document 1, the atelocollagen recovered as a precipitate is subjected to a decomposition treatment with actinidyne to obtain a decomposition product, and the decomposition product is used as a medical material for hemostasis.

On the other hand, Patent Document 2 discloses a technique in which collagen or a degradation product obtained by treating atelocollagen with a protease is used as a composition for prevention or treatment of arteriosclerosis and diseases caused by arteriosclerosis. More specifically, in Patent Document 2, the decomposition product of collagen obtained by decomposing collagen after removing minerals by protease is used as a composition for prevention or treatment of arteriosclerosis and diseases caused by arteriosclerosis. The technology used as a thing is disclosed.

As described above, when collagen and atelocollagen are decomposed by protease, they are generally decomposed under conditions of low salt concentration. The amino acid sequence of the collagen decomposition product or the atelocollagen decomposition product under such a low salt concentration condition has already been determined, and the amino acid sequence is disclosed in Non-Patent Document 1 and the like.

By the way, as a method for inducing differentiation of embryonic stem cells (ES cells) into various cells in vitro, a method of forming an embryoid body of ES cells and inducing differentiation of the embryoid body into a target cell is widely used. (For example, Patent Document 3, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, etc.). As a method for obtaining an embryoid body of ES cells, a hanging drop method (for example, Non-Patent Document 6) and a culture method using a superhydrophilic treated culture dish (for example, Non-Patent Document 4) are generally used.

International Publication No. 2004/20470 Pamphlet (Released on March 11, 2004) Japanese Patent Publication "Japanese Patent Laid-Open No. 2001-31586 (published on February 6, 2001)" Japanese Patent Publication "Japanese Patent Laid-Open No. 2012-139246 (published on July 26, 2012)"

S. Kunii et al., Journal of Biological Chemistry, Vol.285, No.23, pp.17465-17470, June4, 2010 K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004 James A. Thomson, Joseph Itskovitz-Eldor, Sander S. Shapiro, Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall, Jeffrey M. Jones, Science 282, 1145-47 (1998) T. Nakano, S. Ando, N. Takata, M. Kawada, K. Muguruma, K. Sekiguchi, K. Saito, S. Yonemura, M. Eiraku, Y. Sasai, Cell Stem Cell, 10, 771-785, (2012) Joseph Itskovitz-Eldor, Maya Schuldiner, Dorit Karsenti, Amir Eden, OfraYanuka, Michal Amit, Hermona Soreq, Nissim Benvenisty, Molecular Medicine 6 (2), 88-95, (2000) Dang SM, Kyba M, Perlingeiro R, Daley GQ, Zandstra PW., Biotechnol Bioeng. 78, 442-53 (2002)

As mentioned above, research on the physiological functions of collagen, atelocollagen, and their decomposition products is progressing, but not all of these physiological functions have been elucidated.

It is considered that finding new physiological functions possessed by these can greatly contribute to the development of various fields such as medical field, food field, cosmetics field and basic research field.

The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a decomposition product of collagen or a decomposition product of atelocollagen having a novel physiological function.

As a result of diligent studies in view of the above problems, the present inventors can use a decomposition product of collagen having a specific structure or a decomposition product of atelocollagen as a scaffold material for forming an embryoid body of pluripotent stem cells. I found that. Then, based on this new finding, the present invention has been completed.

That is, in order to solve the above-mentioned problems, the embryoid body forming method according to the embodiment of the present invention is a method for forming an embryoid body of pluripotent stem cells, and the pluripotent stem cells are made of collagen. Including the step of culturing with a degradation product or a degradation product of atelocollagen, the degradation product is characterized by containing at least a part of the triple helical domain of the collagen or atelocollagen.

In the embryoid body forming method according to the embodiment of the present invention, the above-mentioned decomposition products contain at least one of the following collagen decomposition products (A) to (C) or atelocollagen decomposition products. Can be:
(A) of the amino acid sequence represented by the following (1) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, decomposition product or decomposition product of the atelocollagen of collagen;
(B) the amino acid sequence represented by the following (2) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between _{X 4} and G, chemical bond between _{X 6} and G, chemical bond between G and _{X 7, or,} between _{X 14} and G Degradation of collagen or atelocollagen with broken chemical bonds between;
(C) the amino acid sequence represented by the following (3) to the amino terminus of a triple helical domain of the collagen or atelocollagen, a chemical bond between Y ₁ and Y ₂ is disconnected, the collagen degradation product or atelocollagen Decomposition;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-;
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids).

In the embryoid body formation method according to the embodiment of the present invention, the amino acid sequence shown in (1) or (2) above is preferably the amino acid sequence at the amino terminus of the triple helical domain.

In the embryoid body formation method according to the embodiment of the present invention, the cleavage in the amino acid sequence represented by any of the above (1) to (3) is at least within the α1 chain and the α2 chain of the collagen or atelocollagen. On the other hand, it is preferable that it is performed.

The composition for forming an embryoid body according to an embodiment of the present invention is a composition for forming an embryoid body of pluripotent stem cells containing a decomposition product of collagen or a decomposition product of atelocollagen, and the above-mentioned decomposition product. Is characterized by containing at least a portion of the triple helical domain of the collagen or atelocollagen.

In the composition for forming an embryoid body according to an embodiment of the present invention, the above-mentioned decomposition product contains at least one of the following decomposition products of collagen (A) to (C) or decomposition products of atelocollagen. Can be:
(A) of the amino acid sequence represented by the following (1) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, decomposition product or decomposition product of the atelocollagen of collagen;
(B) the amino acid sequence represented by the following (2) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between _{X 4} and G, chemical bond between _{X 6} and G, chemical bond between G and _{X 7, or,} between _{X 14} and G Degradation of collagen or atelocollagen with broken chemical bonds between;
(C) the amino acid sequence represented by the following (3) to the amino terminus of a triple helical domain of the collagen or atelocollagen, a chemical bond between Y ₁ and Y ₂ is disconnected, the collagen degradation product or atelocollagen Decomposition;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-;
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids).

In the composition for embryoid body formation according to one embodiment of the present invention, the amino acid sequence shown in (1) or (2) above is preferably the amino acid sequence at the amino terminus of the triple helical domain.

In the composition for forming an embryoid body according to an embodiment of the present invention, the cleavage in the amino acid sequence represented by any of (1) to (3) above is within the α1 chain and the α2 chain of the collagen or atelocollagen. It is preferable that at least one of the above is performed.

According to the present invention, there is an effect that the embryoid body of pluripotent stem cells can be easily formed. Further, according to the present invention, it is possible to form an embryoid body in a shorter period of time as compared with a conventional method for forming an embryoid body.

It is a figure which shows the morphology of ES cell observed under the phase difference microscope, (a) is a gelatin-coated culture dish in the presence of MEF (MEF (+)), using DMEM medium (LIF (+)). It is a figure which shows the morphology of ES cells cultured for 3 days, (b) is a collagen decomposition product-coated culture dish, in the presence of MEF (MEF (+)), using DMEM medium (LIF (+)) 3 It is a figure which shows the morphology of ES cell culture | cultivated for 3 days, (c) is a collagen decomposition product gel culture dish in the presence of MEF (MEF (+)), and used DMEM medium (LIF (+)) for 3 days. It is a figure which shows the morphology of the cultured ES cell. MEF means mouse fetal-derived fibroblasts (Murine Embryonic Fibroblasts), and LIF means leukemia inhibitory factor (Leukemia Inhibitory Factor). In addition, (+) indicates culture in coexistence, and (-) indicates culture in non-coexistence. It is a figure which shows the morphology of ES cell observed under the phase difference microscope, (a) is a gelatin-coated culture dish in the presence of MEF (MEF (+)), using DMEM medium (LIF (-)). It is a figure which shows the morphology of ES cells cultured for 3 days, (b) is a collagen decomposition product-coated culture dish, in the presence of MEF (MEF (+)), using DMEM medium (LIF (-)) 3 It is a figure which shows the morphology of ES cell culture | cultivated for 3 days, (c) is a collagen decomposition product gel culture dish in the presence of MEF (MEF (+)), and used DMEM medium (LIF (−)) for 3 days. It is a figure which shows the morphology of the cultured ES cell. It is a figure which shows the result of having investigated the intracellular alkaline phosphatase activity of ES cell, (a) is a DMEM medium (LIF (+)) in a gelatin coat culture dish in the presence of MEF (MEF (+)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 2 days using the above, (b) is a DMEM medium (LIF) in a collagen decomposition product-coated culture dish in the presence of MEF (MEF (+)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 2 days using (+)), and (c) is a collagen decomposition product gel culture dish in the presence of MEF (MEF (+)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 2 days using DMEM medium (LIF (+)). It is a figure which shows the morphology of ES cell observed under the phase difference microscope, (a) is a gelatin-coated culture dish, in the absence of MEF (MEF (-)), using DMEM medium (LIF (+)). It is a figure which shows the morphology of the ES cell which was cultured for 3 days, and (b) is a collagen decomposition product-coated culture dish, using DMEM medium (LIF (+)) in the absence of MEF (MEF (-)). It is a figure which shows the morphology of ES cells cultured for 3 days, (c) is a collagen decomposition product gel culture dish, using DMEM medium (LIF (+)) in the absence of MEF (MEF (-)). It is a figure which shows the morphology of the ES cell which was cultured for 3 days. It is a figure which shows the morphology of ES cell observed under the phase difference microscope, (a) is a gelatin-coated culture dish, in the absence of MEF (MEF (-)), using DMEM medium (LIF (-)). It is a figure which shows the morphology of the ES cell which was cultured for 3 days, and (b) is a collagen decomposition product-coated culture dish, using DMEM medium (LIF (-)) in the absence of MEF (MEF (-)). It is a figure which shows the morphology of ES cells cultured for 3 days, (c) is a collagen decomposition product gel culture dish, using DMEM medium (LIF (-)) in the absence of MEF (MEF (-)). It is a figure which shows the morphology of the ES cell which was cultured for 3 days. It is a figure which shows the result of having observed the ES cell which contained the GFP gene which had been cultured for 4 days in a gelatin coat culture dish under the culture conditions of MEF (+) and LIF (+) under a microscope, and (a) is a phase contrast. It is a figure which shows the morphology of an ES cell observed under a microscope, (b) is a figure which shows the result of having observed the fluorescence of GFP of the ES cell shown in (a) under a fluorescence microscope, and (c) is a figure which shows. , (A) is a figure which shows the result of having Hoechst 33342 stained the nucleus of each cell of the ES cell and observed under a fluorescence microscope, and (d) is the figure of (a), (b) and (c). It is a figure which shows the result of superimposing the images. The figure which shows the result of observing the embryo-like body obtained after culturing ES cells containing the GFP gene for 4 days in a collagen decomposition product gel culture dish under the culture conditions of MEF (+) and LIF (+) under a microscope. (A) is a diagram showing the morphology of the embryo-like body observed under a phase-difference microscope, and (b) is a diagram showing the fluorescence of the embryonic body GFP shown in (a) under a fluorescence microscope. (C) is a diagram showing the results obtained by staining the nuclei of each cell of the embryonic body shown in (a) with Hoechst 33342 and observing under a fluorescence microscope, and (d) is a diagram showing the results. It is a figure which shows the result of superimposing the images of (a), (b) and (c). The figure which shows the result of observing the embryo-like body obtained after culturing ES cells containing the GFP gene for 4 days in the collagen decomposition product gel culture dish under the culture conditions of MEF (+) and LIF (-) under a microscope. (A) is a diagram showing the morphology of the embryo-like body observed under a phase-difference microscope, and (b) is a diagram showing the fluorescence of the embryonic body GFP shown in (a) under a fluorescence microscope. (C) is a diagram showing the results obtained by staining the nuclei of each cell of the embryonic body shown in (a) with Hoechst 33342 and observing under a fluorescence microscope, and (d) is a diagram showing the results. It is a figure which shows the result of superimposing the images of (a), (b) and (c). It is a figure which shows the morphology of the iPS cell observed under the phase contrast microscope, (a) is the figure which shows the morphology of the iPS cell which was cultured for 1 day in a collagen decomposition product coated culture dish, and (b) is a figure which shows the Vitronectin. It is a figure which shows the morphology of the iPS cell which was cultured for one day in a coated culture dish, and (c) is the figure which shows the morphology of the iPS cell which was cultured for one day in the iMatrix511 coated culture dish. It is a figure which shows the morphology of the iPS cell observed under the phase contrast microscope, (a) is the figure which shows the morphology of the iPS cell which was cultured for 5 days in a collagen decomposition product coated culture dish, and (b) is a figure which shows the Vitronectin. It is a figure which shows the morphology of the iPS cell which was cultured for 5 days in a coated culture dish, and (c) is the figure which shows the morphology of the iPS cell which was cultured for 5 days in the iMatrix511 coated culture dish. It is a figure which shows the morphology of the iPS cell observed under the phase contrast microscope, (a) is the figure which shows the morphology of the iPS cell which was cultured for 4 days in the collagen decomposition product coated culture dish, (b) is the figure which shows the morphology of Lipidure. It is a figure which shows the morphology of the iPS cell which was cultured for 4 days in a culture dish. It is a figure which shows the morphology of the iPS cell observed under the phase contrast microscope, (a) is the figure which shows the morphology of the iPS cell which was cultured for 12 days in the collagen decomposition product coated culture dish, and (b) is the figure which shows the Lipidure It is a figure which shows the morphology of the iPS cell which was cultured for 12 days in a culture dish. It is a figure which shows the relative expression level of each marker as a radar chart with the maximum value of the expression level obtained from the result of quantitative RT-PCR as 100, and (a) is formed in the collagen decomposition product coated culture dish. It is a figure which shows the relative expression level of each marker in the embryoid body of a mouse ES cell, and (b) is a figure which shows the relative expression level of each marker in the embryoid body of a mouse ES cell formed in a Petri dish culture dish. (C) is a diagram showing the relative expression level of each marker in the embryoid body of mouse ES cells formed in a gelatin-coated culture dish. It is a figure which shows the morphology of ES cell observed under the phase difference microscope, (a) is a gelatin-coated culture dish in the presence of MEF (MEF (+)), using DMEM medium (LIF (+)). It is a figure which shows the morphology of ES cell which was cultured for 1 day, (b) is culture | culture for 1 day using DMEM medium (LIF (+)) in the Atelocollagen coated culture dish in the presence of MEF (MEF (+)). It is a figure which shows the morphology of a petri dish, (c) is a DMEM medium (LIF (LIF (LIF)) in a collagen decomposition product coated culture dish (from the pig skin part, SEQ ID NO: 27) in the presence of MEF (MEF (+)). It is a figure which shows the morphology of ES cells cultured for 1 day using +)), and (d) is a MEF in a collagen decomposition product coated culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5). It is a figure which shows the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the coexistence (MEF (+)), (e) is a collagen decomposition product coated culture dish (pork skin part). In the origin, SEQ ID NO: 26), it is a figure which shows the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the coexistence of MEF (MEF (+)). It is a figure which shows the result of having investigated the intracellular alkaline phosphatase activity of ES cell, (a) is a DMEM medium (LIF (+)) in a gelatin coat culture dish in the presence of MEF (MEF (+)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using, (b) is a DMEM medium (LIF (+)) in the Atelocollagen coated culture dish in the presence of MEF (MEF (+)). )) Is a diagram showing the results of alkaline phosphatase activity staining of ES cells cultured for 1 day using (c) in a collagen decomposition product coated culture dish (derived from pig skin, SEQ ID NO: 27) in the presence of MEF. It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using DMEM medium (LIF (+)) in (MEF (+)), (d) is a collagen decomposition product coated culture dish (d). Alkaline phosphatase activity staining of ES cells cultured for 1 day in DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a rat tendon-derived mixture of SEQ ID NO: 24 and SEQ ID NO: 5). (E) is a DMEM medium (LIF (+)) in a collagen decomposition product-coated culture dish (derived from the petri dish, SEQ ID NO: 26) in the presence of MEF (MEF (+)). It is a figure which shows the result of the alkaline phosphatase activity staining of the ES cell cultured for 1 day using. It is a figure which shows the result of having observed under the microscope the colony obtained after culturing ES cells containing a GFP gene in a gelatin coat culture dish for 1 day under the culture conditions of MEF (+) and LIF (+). a) is a diagram showing the morphology of the colonies observed under a phase-contrast microscope, and (b) is a diagram showing the results of observing the fluorescence of GFP of the colonies shown in (a) under a fluorescence microscope. Yes, (c) is a diagram showing the result of superimposing the images of (a) and (b). Embryoid bodies obtained after culturing ES cells containing a GFP gene for 1 day in a collagen degradation product-coated culture dish (derived from pig skin, SEQ ID NO: 27) under MEF (+) and LIF (+) culture conditions. , The figure which shows the result observed under the microscope, (a) is the figure which shows the morphology of the embryoid body observed under the phase contrast microscope, (b) is the figure which shows the said embryo shown in (a). It is a figure which shows the result of observing the fluorescence of a GFP of a variant under a fluorescence microscope, and (c) is a figure which shows the result of superimposing the images of (a) and (b). Obtained after culturing ES cells containing a GFP gene for 1 day in a collagen decomposition product coated culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5) under MEF (+) and LIF (+) culture conditions. It is a figure which shows the result of observing the embryoid body under a microscope, (a) is a figure which shows the morphology of the said embryoid body observed under a phase contrast microscope, and (b) is (a It is a figure which shows the result of observing the fluorescence of the GFP of the embryoid body shown in) under a fluorescence microscope, and (c) is a figure which shows the result of superimposing the images of (a) and (b). Embryoid bodies obtained after culturing ES cells containing a GFP gene for 1 day in a collagen decomposition product-coated culture dish (derived from pig skin, SEQ ID NO: 26) under MEF (+) and LIF (+) culture conditions. , The figure which shows the result observed under the microscope, (a) is the figure which shows the morphology of the embryoid body observed under the phase contrast microscope, (b) is the figure which shows the said embryo shown in (a). It is a figure which shows the result of observing the fluorescence of a GFP of a variant under a fluorescence microscope, and (c) is a figure which shows the result of superimposing the images of (a) and (b). It is a figure which shows the morphology of ES cell observed under the phase difference microscope, (a) is a gelatin-coated culture dish, in the absence of MEF (MEF (-)), using DMEM medium (LIF (+)). It is a figure which shows the morphology of the ES cell which was cultured for 1 day, and (b) is a DMEM medium (LIF (+)) in the Atelocollagen coated culture dish in the absence of MEF (MEF (-)), and 1 It is a figure which shows the morphology of ES cell cultured for a day, (c) is a DMEM medium in a collagen decomposition product coated culture dish (from the pig skin part, SEQ ID NO: 27) in the absence of MEF (MEF (-)). It is a figure which shows the morphology of ES cells cultured for 1 day using (LIF (+)), (d) is a collagen decomposition product coated culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5). In the figure which shows the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the absence of MEF (MEF (−)), (e) is a collagen decomposition product coated culture dish. (Derived from petri dish, SEQ ID NO: 26) is a diagram showing the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the absence of MEF (MEF (−)). It is a figure which shows the result of having investigated the intracellular alkaline phosphatase activity of ES cell, (a) is a DMEM medium (LIF (+)) in a gelatin coat culture dish in the absence of MEF (MEF (−)). ) Is shown in the results of alkaline phosphatase activity staining of ES cells cultured for 1 day using DMEM medium (LIF) in an atelocollagen-coated culture dish in the absence of MEF (MEF (-)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using (+)), and (c) is the MEF in the collagen decomposition product coated culture dish (from pig skin part, SEQ ID NO: 27). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the non-coexistence (MEF (−)), (d) is a collagen decomposition product coating. ES cells cultured for 1 day in DMEM medium (LIF (+)) in a culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5) in the absence of MEF (MEF (-)). It is a figure which shows the result of alkaline phosphatase activity staining, (e) is a DMEM medium (MEF (-)) in a collagen decomposition product coated culture dish (from the pig skin part, SEQ ID NO: 26) in the absence of MEF (MEF (-)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using LIF (+)).

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be carried out in a mode in which various modifications are added within the range described. Unless otherwise specified in the present specification, "A to B" representing a numerical range means "A or more and B or less".

[1. Embryoid body formation method]
The embryoid body forming method according to one embodiment of the present invention (hereinafter, also referred to as “embryoid body forming method of the present embodiment”) is a method for forming an embryoid body of pluripotent stem cells, and is polymorphic. It is a configuration including a culture step of culturing a pluripotent stem cell together with a degradation product of collagen or a degradation product of atelocollagen.

Here, in the present specification, the above-mentioned "pluripotent stem cell" is a cell having pluripotency capable of differentiating into all cells other than placenta and self-renewal ability. Examples of such pluripotent stem cells include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation (nuclear transplanted ES cells; ntES cells), and induced pluripotent stem cells (iPS cells). ), Embryonic stem cells (EG cells), pluripotent stem cells (Multilineage-differentiating Stress Enduring cells) derived from living tissues, and the like, but are not limited thereto. In addition, examples of the above-mentioned "pluripotent stem cells" include, but are not limited to, those derived from humans, primates such as cynomolgus monkeys, and mice.

In the present specification, the above-mentioned "embryoid body" is a spherical cell mass formed from pluripotent stem cells and having an ability to differentiate into various cells under appropriate differentiation-inducing conditions. Say. The "embryoid body" is also referred to as the Embryoid Body (EB). The "embryoid body" in the present specification also includes an "embryoid body-like cell aggregate" which is an aggregate of cells in the process of forming an embryoid body from pluripotent stem cells. As used herein, the term "embryonic body" refers to pluripotency markers (eg, Nanog, OCT3 / 4 (Octamer-binding transcription factor 3/4), TRA-1-60, SSEA-3 (stage-specific embryonic antigen). -3), etc.) may be expressed, and differentiation markers (for example, SOX1 (SRY (sex determining region Y) -box 1), SOX7 (SRY (sex determining region Y) -box 7), SOX17 (SRY)) may be expressed. (sex determining region Y) -box 17), HNF-3β (Hepatocyte Nuclear Factor 3β) / FoxA2 (forkhead box protein A2), GATA4 (GATA binding protein 4), GATA6 (GATA binding protein 6), Octx2 (Orthodenticle homeobox 2) ), CXCR4 (Chemokine (CXC Motif) Receptor 4), GSC (goosecoid), etc.) may be expressed. The "embryoid body" preferably has the ability to differentiate into all three germ layers (ectoderm, endoderm, mesoderm). An embryoid body having the ability to differentiate into all three germ layers can be suitably used for inducing differentiation into a target cell.

The size of the "embryoid body" is not particularly limited, but the longest diameter is preferably 50 to 1000 μm, more preferably 100 to 500 μm. The shape of the "embryoid body" is not particularly limited, but is preferably spherical, substantially spherical, elliptical, substantially elliptical, or the like.

The formation of the embryoid body by the embryoid body forming method of the present embodiment can be confirmed by confirming the presence of spherical cell clusters with the naked eye or under a microscope. The ability of the embryoid body obtained by the embryoid body forming method of the present embodiment to differentiate into various cells means that the embryoid body is subjected to known appropriate differentiation-inducing conditions (for example, known known conditions). , Conditions for inducing differentiation into blood cells, conditions for inducing differentiation into nerve cells, etc.), and it can be confirmed by confirming that the embryoid body-derived cells have differentiated into the target cells. It can be confirmed that the embryoid body-derived cells have differentiated into the target cells by confirming the morphology, gene expression, etc. of the differentiated cells by using a known method.

In the embryoid body formation method of the present embodiment, the above-mentioned "collagen decomposition product or atelocollagen decomposition product" is a configuration containing at least a part of the triple helical domain of collagen or atelocollagen. That is, the degradation product may contain the entire triple helical domain of collagen or atelocollagen, or may contain a portion of the triple helical domain. "Collagen decomposition product or atelocollagen decomposition product" will be described in detail later.

The embryoid body formation method of the present embodiment will be described in detail below.

(1-1. Culture step)
The culturing step is a step of culturing pluripotent stem cells together with a degradation product of collagen or a degradation product of atelocollagen. "Culturing pluripotent stem cells with collagen degradation products or atelocollagen degradation products" means pluripotency in an environment in which collagen degradation products or atelocollagen degradation products are present in contact with pluripotent stem cells. It means culturing sex stem cells. For example, in one embodiment, pluripotent stem cells may be cultured using a culture vessel whose inner surface is coated with a degradation product of collagen or a degradation product of atelocollagen. In other embodiments, pluripotent stem cells may be cultured on a gel composition containing a degradation product of collagen or a degradation product of atelocollagen. The collagen degradation product or the atelocollagen degradation product preferably exists so as to prevent contact between the pluripotent stem cells and the inner surface of the culture vessel for culturing the pluripotent stem cells. As a result, the embryoid body of pluripotent stem cells can be efficiently formed. As the culture vessel for culturing pluripotent stem cells, a vessel usually used for cell culture can be used. The culture container may be a dish type, a plate type, or a bottle type.

The amount of collagen degradation product or atelocollagen degradation product used in the embryoid body formation method of the present embodiment is not particularly limited.

In one embodiment, when pluripotent stem cells are cultured using a culture vessel whose inner surface is coated with a decomposition product of collagen or a decomposition product of atelocollagen, 2 mg / mL to 20 mg / mL, preferably 3 mg / mL. An aqueous solution containing ~ 16 mg / mL, more preferably 5 mg / mL ~ 15 mg / mL, or 5 mg / mL ~ 20 mg / mL, preferably 8 mg / mL ~ 16 mg / mL collagen degradation products or atelocollagen degradation products. Can be prepared and the inner surface of the culture vessel can be coated with the aqueous solution of the decomposition product. The inner surface of the culture vessel may be coated before starting the culture of pluripotent stem cells. When the concentration of the decomposed product in the aqueous solution of the decomposed product is 2 mg / mL or more, the embryoid body can be uniformly formed. Further, when the concentration of the decomposed product in the aqueous solution of the decomposed product is 20 mg / mL or less, the reproducibility is good. On the other hand, when the concentration of the decomposition product in the aqueous solution of the decomposition product is less than 2 mg / mL, the formation of embryoid bodies may become non-uniform. Further, when the concentration of the decomposed product in the aqueous solution of the decomposed product is higher than 20 mg / mL, the viscosity may become high and the reproducibility may be poor.

In other embodiments, when pluripotent stem cells are cultured on a gel composition containing a degradation product of collagen or atelocollagen, the final concentration is 2 mg / mL to 15 mg / mL or 5 mg / mL. It is preferable that a decomposition product of collagen of ~ 15 mg / mL or a decomposition product of atelocollagen is contained in the gel composition, and a decomposition product of collagen of 5 mg / mL to 12 mg / mL or 5 mg / mL to 11 mg / mL or It is more preferable that the decomposition product of atelocollagen is contained in the gel composition. It is even more preferred that a degradation product of 7 mg / mL to 10 mg / mL or 8 mg / mL to 10 mg / mL collagen or atelocollagen degradation product is contained in the gel composition. When the concentration of the above-mentioned decomposition products contained in the gel composition is 2 mg / mL or more as the final concentration, embryoid bodies can be uniformly formed. Further, when the concentration of the decomposed product contained in the gel composition is 15 mg / mL or less as the final concentration, the reproducibility is good. On the other hand, when the concentration of the decomposition product contained in the gel composition is less than 2 mg / mL as the final concentration, the formation of embryoid bodies may become non-uniform. Further, when the concentration of the above-mentioned decomposed product contained in the gel composition is higher than 15 mg / mL as the final concentration, the viscosity may be high and the reproducibility may be poor.

A gel composition containing a decomposition product of collagen or a decomposition product of atelocollage can be prepared by, for example, combining a decomposition product of collagen or a decomposition product of atelocollagen with a concentrated culture solution (for example, 5-fold concentrated DMEM) and a buffer solution for reconstruction. It can be prepared by mixing with (50 mM sodium hydroxide, 260 mM sodium hydrogen carbonate, 200 mM HEPES), adding this mixed solution to a culture dish, and allowing it to stand in a CO ₂ incubator at 37 ° C. for about 30 minutes. ..

The medium used in the culturing step is not particularly limited as long as it is a medium for pluripotent stem cells. Such a medium can be appropriately selected depending on the type of pluripotent stem cells used. For example, a known culture medium used for maintaining the growth of pluripotent stem cells can be used. Such a culture medium can be prepared by adding non-essential amino acids, sodium pyruvate, 2-mercaptoethanol, fetal bovine serum, glutamine, nucleoside, LIF and the like to the basal medium.

As the basal medium, a conventionally known medium for animal cells can be used. For example, Ham's F12 medium, α-MEM medium, DMEM medium, RPMI-1640 medium and the like can be mentioned. These basal media may be used alone or in combination of two or more.

The basal medium may or may not contain serum. In addition, the basal medium may contain an artificial serum substitute instead of serum. Such artificial serum replacements include, for example, KnockOut® Serum Replacement (manufactured by Invitrogen).

In addition, the basal medium may or may not contain a leukemia inhibitory factor (LIF). Here, LIF is usually added to the culture medium in order to maintain the undifferentiated state of mouse ES cells. Therefore, when the embryoid body is formed from mouse ES cells, the embryoid body is usually formed in the absence of LIF. However, according to the embryoid body formation method of the present embodiment, surprisingly, even when a medium containing LIF is used as the medium for forming embryoid bodies, a medium containing no LIF is used. The embryoid body can be formed from mouse ES cells in the same manner as in the case of using.

In the culturing step, pluripotent stem cells may be co-cultured with the feeder cells, and the feeder cells may not be present. As the feeder cells, cells usually used as feeder cells in the maintenance culture of pluripotent stem cells can be used. For example, mouse embryonic fibroblast (MEF) and the like can be mentioned. When feeder cells are used, it is preferable to seed the feeder cells at least 1 day before starting the culture of pluripotent stem cells.

The seeding density of pluripotent stem cells is not particularly limited, but is preferably 1 × 10 ³ to 1 × 10 ⁶ per 1 ml of medium, and is preferably 2 × 10 ³ to 5 × 10 ^5. More preferably, the number is 5 × 10 ³ to 5 × 10 ⁴ . In general, when the number of seeds is extremely small, the viability of cells tends to decrease and it becomes difficult to form embryoid bodies. On the other hand, when the number of seeds is extremely large, uneven and large embryoid bodies are likely to be formed. In these cases, problems such as reproducibility are likely to occur when inducing differentiation. The seeding density may be appropriately determined depending on the type of pluripotent stem cells, and is not limited in any way.

The culture conditions in the culture step may be any conditions under which normal cell culture is performed. The culture temperature is, for example, 30 ° C to 40 ° C, preferably 37 ° C. The culture is carried out in an atmosphere of CO _2- containing air, and the CO ₂ concentration is, for example, 2% to 5%, preferably 5%. The culture period can be appropriately set depending on what kind of quality embryoid body is desired to be formed. For example, the longer the culture period, the larger embryoid bodies can be formed. The culture period varies depending on the type of pluripotent stem cells used, but is preferably at least 1 day, more preferably 3 days or more, and even more preferably 5 days or more. Further, it is preferably 20 days or less at the longest, more preferably 15 days or less, and further preferably 10 days or less. When the culture period is short, the embryoid body size is uneven and difficult to stabilize. On the other hand, as the number of culture days increases, the quality of the embryoid body tends to deteriorate, and heterogeneous differentiation tends to occur. The optimum number of days for culturing may be appropriately determined depending on the number of seeded cells, and is not limited in any way.

In the conventional method for obtaining an embryoid body of ES cells, for example, when mouse ES cells are used, it takes a long time (for example, about 7 days at the shortest in the hanging drop method) to obtain an embryoid body. .. On the other hand, in the embryoid body formation method of the present embodiment, for example, when mouse ES cells are used, embryoid bodies are formed on the first day after the start of culture, as shown in Examples described later. Will be done.

The conventional method for obtaining an embryoid body of ES cells has poor reproducibility and requires skill. However, in the method for forming an embryoid body of the present embodiment, a general cell culture method is used. A researcher who has various culture techniques can easily form an embryoid body.

In addition, the embryoid body formation method of the present embodiment is also excellent in that it can form not only an embryoid body but also an embryoid body having an ability to differentiate into an ectoderm, an endoderm and / or a mesoderm. ing. Such an advantage was first discovered by the present inventors. Also in the examples described later, it is shown that the embryoid body formation method of the present embodiment forms embryoid bodies that have not been observed so far. It has also been shown in the Examples that the resulting embryoid body expresses a differentiation marker.

In the culturing step, the medium may be exchanged during the culturing period. When the medium is changed, the medium may be changed every day or once every 2 to 5 days. A good culture environment can be maintained by exchanging the medium at an appropriate frequency. With the conventional hanging drop method or the superhydrophilic treatment culture dish, it is usually not possible to exchange the medium without burdening the cells. In addition, there is a high risk of aspirating and discarding cells during medium replacement. On the other hand, in the embryoid body formation method of the present embodiment, it is possible to exchange the medium without imposing a burden on the cells. Also, there is less risk of aspirating and discarding cells during medium replacement.

(1-2. Other processes)
The embryoid body formation method of the present embodiment may further include steps other than the above-mentioned culture step. For example, it may further include a confirmation step of confirming the quality of the formed embryoid body. Here, the above-mentioned "embryoid body quality" refers to the degree of undifferentiation and / or differentiation of the embryoid body, the differentiation potential of the embryoid body, the size of the embryoid body (diameter of the embryoid body), and the embryo-like body. The shape of the body is intended. That is, the confirmation step may be, for example, a step of confirming the degree of undifferentiation and / or differentiation of the embryoid body in order to confirm the quality of the embryoid body, and confirm the differentiation ability of the embryoid body. It may be a step of confirming the size (for example, diameter) of the embryoid body, or a step of confirming the shape of the embryoid body (for example, spherical shape).

The degree of undifferentiation and / or differentiation of the embryoid body can be confirmed, for example, by confirming the expression of known differentiation markers, undifferentiated markers, etc. in the embryoid body at the gene level or the protein level.

Further, for the differentiation ability of the embryoid body, for example, the embryoid body is cultured under known appropriate differentiation-inducing conditions (for example, known conditions for inducing differentiation into blood cells, conditions for inducing differentiation into nerve cells, etc.). This can be confirmed by confirming that the embryoid body-derived cells have differentiated into the cells of interest.

Further, the size and shape of the embryoid body can be confirmed, for example, by measuring the diameter and shape of the embryoid body under a phase-contrast microscope.

The above confirmation step may be performed during the above-mentioned culturing step, or may be performed after the culturing step. By performing the above confirmation step in the middle of the culturing step, the culturing period can be appropriately adjusted.

In addition, the embryoid body forming method of the present embodiment may further include a recovery step of recovering the embryoid body after the culture step.

The "collagen decomposition product or atelocollagen decomposition product" used in the embryoid body formation method of the present embodiment will be described in detail below.

<Collagen decomposition product or atelocollagen decomposition product>
In the embryoid body formation method of the present embodiment, the above-mentioned "collagen decomposition product or atelocollagen decomposition product" (hereinafter, may be simply referred to as "decomposition product") is at least a part of the triple helical domain of collagen or atelocollagen. It is a configuration that includes. That is, the degradation product may contain the entire triple helical domain of collagen or atelocollagen, or may contain a portion of the triple helical domain.

More specifically, the above-mentioned decomposition product contains at least one of the following (A) to (C) collagen decomposition products or atelocollagen decomposition products:
(A) of the amino acid sequence represented by the following (1) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, decomposition product or decomposition product of the atelocollagen of collagen;
(B) the amino acid sequence represented by the following (2) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between _{X 4} and G, chemical bond between _{X 6} and G, chemical bond between G and _{X 7, or,} between _{X 14} and G Degradation of collagen or atelocollagen with broken chemical bonds between;
(C) the amino acid sequence represented by the following (3) to the amino terminus of a triple helical domain of the collagen or atelocollagen, a chemical bond between Y ₁ and Y ₂ is disconnected, the collagen degradation product or atelocollagen Decomposition;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G- (SEQ ID NO: 1);
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G- (SEQ ID NO: 14);
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G- (SEQ ID NO: 13);
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids).

Each configuration will be described in detail below.

The collagen and atelocollagen used as materials for the decomposition products are not particularly limited, and may be any well-known collagen or atelocollagen.

Collagen used as a material for decomposition products includes mammals (eg, cows, pigs, rabbits, humans, rats or mice), birds (eg, chickens), or fish (eg, salmon, carp, eels, tuna). For example, collagen of yellowfin tuna), tilapia, tie, salmon, etc.) can be used.

More specifically, as the collagen used as the material of the decomposition product, collagen derived from the dermis, tendon, bone or fascia of the mammal or bird, or collagen derived from the skin or scale of the fish is used. be able to.

As the atelocollagen used as a material for the decomposition product, terror peptides are partially produced from the amino-terminal and / or carboxyl-terminal of the collagen molecule obtained by treating the above-mentioned mammalian, bird or fish collagen with a protease (for example, pepsin). The removed atelocollagen can be used.

Among these, chicken, porcine, bovine, human or rat collagen or atelocollagen can be preferably used as the decomposition product material, and porcine, bovine or human collagen or atelocollagen can be more preferably used as the decomposition product material. it can.

Further, by using fish collagen or atelocollagen as the material of the decomposition product, the material can be easily, safely and in large quantities, and the decomposition product of collagen or the decomposition product of atelocollagen which is safer for humans can be obtained. It can be realized.

When fish collagen or atelocollagen is used as the material for the decomposition products, it is preferable to use shark, carp, eel, tuna (for example, yellowfin tuna), tilapia, Thai or salmon collagen or atelocollagen, and tuna, tilapia, etc. It is more preferred to use tilapia or salmon collagen or atelocollagen.

When atelocollagen is used as the material of the decomposition product, it is preferable to use atelocollagen having a heat denaturation temperature of preferably 15 ° C. or higher, more preferably 20 ° C. or higher. For example, when fish atelocollagen is used as a material for decomposition products, it is preferable to use atelocollagen such as tuna (for example, yellowfin tuna), tilapia or carp because the heat denaturation temperature is 25 ° C. or higher.

With the above configuration, the denaturation temperature of the decomposition product can be adjusted to preferably 15 ° C. or higher, more preferably 20 ° C. or higher. As a result, with the above configuration, it is possible to realize a decomposed product having excellent stability during storage and stability during use.

Collagen and atelocollagen, which are materials for decomposition products, can be obtained by well-known methods. For example, collagen can be eluted by putting collagen-rich tissues of mammals, birds or fish into an acidic solution having a pH of about 2 to 4. Furthermore, a protease such as pepsin is added to the eluate to partially remove the amino-terminal and / or carboxyl-terminal terror peptides of the collagen molecule. Further, atelocollagen can be precipitated by adding a salt such as sodium chloride to the eluate.

The above-mentioned "collagen decomposition product or atelocollagen decomposition product" contains at least one of the following (A) to (C) collagen decomposition products or atelocollagen decomposition products:
(A) of the amino acid sequence represented by the following (1) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, decomposition product or decomposition product of the atelocollagen of collagen;
(B) the amino acid sequence represented by the following (2) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between _{X 4} and G, chemical bond between _{X 6} and G, chemical bond between G and _{X 7, or,} between _{X 14} and G Degradation of collagen or atelocollagen with broken chemical bonds between;
(C) the amino acid sequence represented by the following (3) to the amino terminus of a triple helical domain of the collagen or atelocollagen, a chemical bond between Y ₁ and Y ₂ is disconnected, the collagen degradation product or atelocollagen Decomposition;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-;
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids).

As used herein, the term "triple helical domain" means that the amino acid sequence represented by "Gly-XY" (X and Y are arbitrary amino acids) is at least 3 or more, more preferably at least 80 or more. A domain containing at least 300 consecutive amino acid sequences is preferably intended, which contributes to the formation of a helical structure.

In the present specification, the above-mentioned "collagen decomposition product or atelocollagen decomposition product" is any one in the amino acid sequence shown by any one of the above (1) to (3) in the triple helical domain of collagen or atelocollagen. It may be a decomposition product in which one part of the above is cut. The above-mentioned "collagen decomposition product or atelocollagen decomposition product" may include the entire C-terminal portion of collagen or atelocollagen triple helical domain, or may contain a part thereof. There may be. If the "collagen degradation product or atelocollagen degradation product" contains a part of the C-terminal part of collagen or atelocollagen triple helical domain, the C-terminal part of collagen or atelocollagen triple helical domain is included. It may contain 3, 3 amino acids or more, 5 amino acids or more, and 10 amino acids or more. For example, sequence information of the C-terminal sequence of chicken type I collagen is described in S. Kunii et al., Journal of Biological Chemistry, Vol.285, No.23, pp.17465-17470, June4, 2010.

In one embodiment, the "collagen decomposition product or atelocollagen decomposition product" may contain at least one of the collagen decomposition products (A) to (C) or atelocollagen decomposition products. Just do it. That is, the above-mentioned "collagen decomposition product or atelocollagen decomposition product" may contain one of the collagen decomposition products (A) to (C) or the atelocollagen decomposition product alone. , One or more of the above-mentioned (A) to (C) collagen decomposition products or atelocollagen decomposition products may be contained in combination.

The polypeptide chain in which the chemical bond is cleaved in the triple helical domain may be any of collagen or a plurality of types of polypeptide chains constituting atelocollagen.

For example, the polypeptide chain in which the chemical bond is cleaved may be either an α1 chain or an α2 chain.

The polypeptide chain in which the chemical bond is cleaved is preferably at least both the α1 chain and the α2 chain among the above-mentioned polypeptide chains.

It is more preferable that the polypeptide chain in which the chemical bond is cleaved is an α1 chain among the above-mentioned polypeptide chains.

If a collagen degradation product or an atelocollagen degradation product is prepared by enzyme treatment, cleavage can easily occur only with a specific polypeptide chain.

The degradation product of collagen or the degradation product of atelocollagen may be one in which three polypeptide chains form a spiral structure. Alternatively, the collagen degradation or atelocollagen degradation is one in which the three polypeptide chains do not form a helical structure, or one in which the three polypeptide chains do not partially form a helical structure. May be good. Whether or not the three polypeptide chains form a spiral structure can be confirmed by a known method (for example, circular dichroism spectrum).

A degradation product of collagen or a degradation product of atelocollagen basically contains three polypeptide chains, but a chemical bond may be cleaved in one of the three polypeptide chains. However, the chemical bond may be cleaved in two of the three polypeptide chains, or the chemical bond may be cleaved in all three polypeptide chains.

When the three polypeptide chains form a spiral structure, the plurality of spiral structures may form a network-like aggregate or a fibrous aggregate. ..

In the present specification, the network means a structure in which molecules are connected to form a three-dimensional network by hydrogen bonds, electrostatic interactions, van der Waals bonds, etc., and gaps are formed between the networks. .. As used herein, fibrous means a substantially linear structure formed by connecting molecules by hydrogen bonds, electrostatic interactions, van der Waals bonds, or the like. Further, in the present specification, an aggregate is intended to be an aggregate in which two or more molecules of the same type interact and bind to each other without a covalent bond to form one structural unit. Whether or not a reticulated or fibrous aggregate is formed can be confirmed by observing with an electron microscope.

The decomposition product of collagen or the decomposition product of atelocollagen may have a crosslinked structure. For example, the polypeptide chain and the polypeptide chain, the spiral structure and the spiral structure, or the polypeptide chain and the spiral structure may be crosslinked by a cross-linking agent.

The crosslinked structure can be formed by a well-known crosslinking method. For example, a method of chemically cross-linking, a method of cross-linking by heat treatment, a method of cross-linking by irradiation with radiation such as ultraviolet rays, and the like can be mentioned.

Examples of the cross-linking agent used for chemical cross-linking include water-soluble carbodiimide compounds such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, diepoxy compounds such as epichlorohydrin and bisepoxydiethylene glycol, and NaBH _4. Can be mentioned.

The concentration of the cross-linking agent is preferably ^10-3 to 10% by mass with respect to the decomposition product of collagen or the decomposition product of atelocollagen. Preferably, a crosslinked structure can be formed by contacting the collagen decomposition product or the atelocollagen decomposition product with the crosslinking agent at 5 to 40 ° C. for 3 to 48 hours.

When cross-linked by ultraviolet rays, a cross-linked structure can be formed by irradiating the decomposed product of collagen or the decomposed product of atelocollagen with ultraviolet rays, for example, at room temperature for about 3 to 48 hours with an ultraviolet lamp or the like.

In the case of thermal cross-linking, a cross-linked structure can be formed by heating the decomposed product of collagen or the decomposed product of atelocollagen under reduced pressure, preferably at a temperature of about 110 to 160 ° C. for about 3 to 48 hours. it can.

A decomposition product of collagen having a crosslinked structure or a decomposition product of atelocollagen has the advantages of improved collagenase resistance and strength.

The degradation product of collagen or the degradation product of atelocollagen may be subjected to the desired chemical modification, if necessary. Examples of the type of chemical modification include acylation, myristylation, and polyethylene glycol modification.

For example, the succinylated decomposition product, which is a kind of acylation, is obtained by reacting a collagen decomposition product or an atelocollagen decomposition product with succinic anhydride in a solvent having a neutral pH such as a phosphate buffer solution. be able to. By succinylation, the solubility of the decomposition product in a solvent having a neutral pH can be improved.

Further, the decomposition product modified with polyethylene glycol can be obtained by reacting polyethylene glycol activated with cyanuric chloride with a decomposition product of collagen or a decomposition product of atelocollagen.

In the above-mentioned decomposition product of collagen or atelocollagen, the chemical bond between X ₁ and X ₂ and the chemical bond between X ₂ and G of the amino acid sequence shown in the following (1) in the above-mentioned triple helical domain. chemical bonding between the chemical bond between G and X _3, the chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, degradation product of collagen or atelocollagen Contains decomposition products of;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-:
(However, G is glycine, and X _{1 to} X ₆ are arbitrary amino acids).

Further, in the above-mentioned decomposition product of collagen or atelocollagen, the chemical bond between X ₁ and X ₂ of the amino acid sequence shown in the following (2) in the above-mentioned triple helical domain, X ₂ and G. Chemical bond between G and X ₃ , chemical bond between X ₄ and G, chemical bond between X ₆ and G, chemical bond between G and X ₇ or a chemical bond between X ₁₄ and G is disconnected, it contains decomposition product or decomposition product of atelocollagen of collagen;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-:
(However, G is glycine, and X _{1 to} X ₁₄ are arbitrary amino acids).

Further, in the above-mentioned collagen decomposition product or atelocollagen decomposition product, the chemical bond between Y ₁ and Y ₂ of the amino acid sequence shown in the following (3) at the amino terminus of the above-mentioned triple helical domain is cleaved. It also contains collagen degradation or atelocollagen degradation;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G- (However, G is glycine and Y _{1 to} Y ₉ are arbitrary amino acids).

The position of the amino acid sequence shown in (1) or (2) above in the triple helical domain is not particularly limited. For example, the amino acid sequence shown in (1) or (2) above may be present inside the triple helical domain, but is preferably present at the amino terminus of the triple helical domain (in other words). , The "G" located on the most amino-terminal side of the amino acid sequence shown in (1) or (2) above is the "G" located on the most amino-terminal side of the triple helical domain. It is preferable to match with).

When the amino acid sequence shown in (1) or (2) above exists inside the triple helical domain, the specific position of the amino acid sequence shown in (1) or (2) is not particularly limited. .. 1 or more, 5 or more, 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 on the amino-terminal side of the amino acid sequence shown in (1) or (2). There may be an amino acid sequence in which more than or more than 300 "Gly-XY" (X and Y are arbitrary amino acids) are continuous. Further, on the carboxyl-terminal side of the amino acid sequence shown in (1) or (2), 1 or more, 5 or more, 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, There may be an amino acid sequence in which 250 or more or 300 or more "Gly-XY" (X and Y are arbitrary amino acids) are continuous.

A part of the triple helical domain other than the amino acid sequence shown in (1) or (2) (that is, the amino acid sequence shown in (1) or (2) above exists inside the triple helical domain. In the case, the amino acid sequence shown in (1) or (2) is on the amino-terminal side and / or the carboxyl-terminal side. The amino acid sequence shown in (1) or (2) above is at the amino-terminal side of the triple helical domain. If it is present, the (carboxyl-terminal side of the amino acid sequence shown in (1) or (2)) may not be cleaved, or one or more of them may be cleaved.

Each of the above X _{1 to} X ₆ can be any amino acid, and the type of amino acid is not particularly limited. Further, each of X _{1 to} X ₆ may be at least a part of the same type of amino acid, or may be all different types of amino acids.

For example, each of X _{1 to} X ₆ includes glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, aspartic acid, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tyrosine, It may be any of tryptophan, hydroxyproline and hydroxylysine.

More specifically, among X _{1 to} X ₆ , X ₁ , X ₃ and X ₅ may be the same amino acid, and the others may be different amino acids.

More specifically, of X _{1 to} X ₆ , at least one selected from the group consisting of X ₁ , X ₃ and X ₅ is proline, and the others may be arbitrary amino acids.

More specifically, X ₁ may be proline and X _{2 to} X ₆ may be arbitrary amino acids.

More specifically, X ₁ and X ₃ may be proline, and X ₂ , X _{4 to} X ₆ may be arbitrary amino acids.

More specifically, X ₁ , X ₃ and X ₅ may be proline and X ₂ , X ₄ and X ₆ may be arbitrary amino acids.

More specifically, X ₁ , X ₃ and X ₅ are prolines, and X ₂ is an amino acid containing a sulfur atom in the side chain (eg, cysteine or methionine) or an amino acid containing a hydroxyl group in the side chain (eg, hydroxyproline). , hydroxy lysine or serine), X ₄ and X ₆ may be any amino acid.

More specifically, X ₁ , X ₃ and X ₅ are prolins, X ₂ is an amino acid containing a sulfur atom in the side chain (eg, cysteine or methionine), and X ₄ has an aliphatic side chain. amino acids (e.g., glycine, alanine, valine, leucine or isoleucine) containing a hydroxyl group or a side chain (e.g., hydroxyproline, hydroxylysine or serine) is, X ₆ may be any amino acid.

More specifically, X ₁ , X ₃ and X ₅ are proline, X ₂ is an amino acid containing a sulfur atom in the side chain (eg, cysteine or methionine), and X ₄ has an aliphatic side chain. amino acids (e.g., glycine, alanine, valine, leucine or isoleucine) amino acids containing a hydroxyl group or a side chain (e.g., hydroxyproline, hydroxylysine or serine), the amino acid X ₆ comprises a base on its side chain (e.g., arginine , Ricin or histidine).

More specifically, X ₁ , X ₃ and X ₅ may be proline, X ₂ may be methionine, X ₄ may be alanine or serine, and X ₆ may be arginine.

In the amino acid sequence shown in the above _(2), each of X 1 to X ₆ may be the same configuration as the _X 1 to X ₆ described above. The specific configurations of X _{7 to} X ₁₄ will be described below.

Each of the above X _{7 to} X ₁₄ can be any amino acid, and the type of amino acid is not particularly limited. Further, each of X _{7 to} X ₁₄ may be at least a part of the same type of amino acid, or may be all different types of amino acids.

For example, each of X _{7 to} X ₁₄ includes glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, aspartic acid, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tyrosine, It may be any of tryptophan, hydroxyproline and hydroxylysine.

More specifically, among X _{7 to} X ₁₄ , X ₈ , X ₉ , X ₁₀ , X ₁₂ and X ₁₃ may be the same amino acid, and the others may be different amino acids.

More specifically, of X _{7 to} X ₁₄ , at least one selected from the group consisting of X ₈ , X ₉ , X ₁₀ , X ₁₂ and X ₁₃ is proline or hydroxyproline, and the others are arbitrary amino acids. It may be.

More specifically, among the X ₇ ~X _14, X ₈ is proline or hydroxyproline, others may be any amino acid.

More _{specifically,} among the _X 7 ~X _14, X ₈ and _{X 9} is proline or hydroxyproline, others may be any amino acid.

More specifically, among X _{7 to} X ₁₄ , X ₈ , X ₉ and X ₁₀ may be proline or hydroxyproline, and the others may be arbitrary amino acids.

More _{specifically,} among the X 7 _{to X 14,} a _{X 8,} X 9, X ₁₀ and _{X 12} is proline or hydroxyproline, others may be any amino acid.

More specifically, of X _{7 to} X ₁₄ , X ₈ , X ₉ , X ₁₀ , X ₁₂ and X ₁₃ may be proline or hydroxyproline, and the others may be arbitrary amino acids.

More specifically, of X _{7 to} X ₁₄ , X ₈ , X ₉ , X ₁₀ , X ₁₂ and X ₁₃ are proline or hydroxyproline, and X ₇ is an amino acid having an aliphatic side chain (for example, Glycine, alanine, valine, leucine or isoleucine), and others may be any amino acids.

More _{specifically,} among the X 7 _{to X 14,} a _{X 8, X 9, X 10} , X 12 and _{X 13} is proline or hydroxyproline, an _amino acid _{X 7} and _{X 11} has a side chain aliphatic (For example, glycine, alanine, valine, leucine or isoleucine), and others may be any amino acids.

More _{specifically,} among the X 7 _{to X 14,} a _{X 8, X 9, X 10} , X 12 and _{X 13} is proline or hydroxyproline, an _amino acid _{X 7} and _{X 11} has a side chain aliphatic (For example, glycine, alanine, valine, leucine or isoleucine), and X ₁₄ may be an amino acid (serine, threonine, asparagine or glutamine) having a hydrophilic and non-dissociative side chain.

More specifically, of X _{7 to} X ₁₄ , X ₈ , X ₉ , X ₁₀ , X ₁₂ and X ₁₃ are proline or hydroxyproline, X ₇ is leucine, and X ₁₁ is alanine. X ₁₄ may be glutamine.

The amino acid sequence shown in (3) above is located at the amino terminus of the triple helical domain. That is, G located between Y ₃ and Y ₄ indicates glycine located on the most amino-terminal side in the triple helical domain. Then, Y ₁ , Y ₂ and Y ₃ indicate amino acids located on the amino-terminal side of the triple helical domain in a plurality of types of polypeptide chains constituting collagen or atelocollagen.

Further, on the carboxyl-terminal side of the amino acid sequence shown in (3), 1 or more, 5 or more, 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more or There may be an amino acid sequence in which 300 or more "Gly-XY" (X and Y are arbitrary amino acids) are continuous.

The part of the triple helical domain other than the amino acid sequence shown in (3) (that is, the carboxyl-terminal side of the amino acid sequence shown in (3) above) may not be cleaved, or one or more parts may be cleaved. It may be disconnected.

Each of the above Y _{1 to} Y ₉ can be any amino acid, and the type of amino acid is not particularly limited. Further, each of Y _{1 to} Y ₉ may be at least a part of the same type of amino acid, or may be all different types of amino acids.

For example, each of Y _{1 to} Y ₉ is glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, aspartic acid, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tyrosine, It may be any of tryptophan, hydroxyproline and hydroxylysine.

More specifically, Y ₃ may be proline and Y ₁ and Y ₂ may be arbitrary amino acids.

More specifically, Y ₃ is proline and Y ₁ and Y ₂ are amino acids having an aliphatic side chain (eg, glycine, alanine, valine, leucine or isoleucine) or amino acids having a hydroxyl group in the side chain (hydroxy). It may be proline, hydroxylycine or serine).

More specifically, Y ₃ may be proline, Y ₁ may be alanine or serine, and Y ₂ may be valine.

At this time, the specific composition of Y _{4 to} Y ₉ is not particularly limited, but Y ₄ and X ₁ are the same amino acids, Y ₅ and X ₂ are the same amino acids, and Y ₆ and X ₃ are _May be the same amino acid, Y ₇ and X ₄ may be the same amino acid, Y ₈ and X ₅ may be the same amino acid, and Y ₉ and X ₆ may be the same amino acid.

Conventional collagen and atelocollagen are difficult to melt at temperatures close to human body temperature. On the other hand, the decomposition product of collagen or the decomposition product of atelocollagen used in one embodiment of the present invention can be in a liquid state even at a temperature close to human body temperature.

Further, the collagen decomposition product or the atelocollagen decomposition product used in one embodiment of the present invention has a higher concentration at which gelation begins than the conventional collagen decomposition product or the atelocollagen decomposition product.

The decomposition product or decomposition product of atelocollagen collagen used in an embodiment of the present invention, the amino acid sequence shown in the above (1) in the triple helical domain of collagen or atelocollagen, between X ₁ and X ₂ A chemical bond, a chemical bond between X ₂ and G, a chemical bond between G and X ₃ , a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is broken. It contains a decomposition product of collagen or a decomposition product of atelocollagen.

The decomposition product or decomposition product of atelocollagen collagen used in an embodiment of the present invention, the amino acid sequence shown in the above (2) in the triple helical domain of collagen or atelocollagen, between X ₁ and X ₂ Chemical bond, chemical bond between X ₂ and G, chemical bond between G and X ₃ , chemical bond between X ₄ and G, chemical bond between X ₆ and G, G and X _It contains a degradation product of collagen or a degradation product of atelocollagen in which the chemical bond between ₇ or the chemical bond between X ₁₄ and G is broken.

Further, the collagen decomposition product or the atelocollagen decomposition product used in one embodiment of the present invention includes Y ₁ and Y _{2 of the} amino acid sequence shown in (3) above at the amino terminus of the triple helical domain of collagen or atelocollagen. It contains a degradation product of collagen or a degradation product of atelocollagen in which the chemical bond between and is broken.

The above-mentioned cutting can be appropriately performed by a desired method.

For example, collagen or atelocollagen in an already cleaved state can be produced by a chemical synthesis method. As the chemical synthesis method, a general well-known chemical synthesis method can be used.

In addition, DNA encoding already cleaved collagen or atelocollagen is inserted into a well-known protein expression vector. Then, after introducing the protein expression vector into a desired host (for example, Escherichia coli, yeast, insect cells, animal cells, etc.), the expression of collagen or atelocollagen in a already cleaved state is induced in the host. This also makes it possible to produce collagen or atelocollagen that has already been cleaved.

It is also possible to perform the above cleavage by degrading collagen or atelocollagen with an enzyme (eg, protease (eg, cysteine protease)).

The details of the cutting method will be described later.

(I. Method for producing a decomposition product of collagen or a decomposition product of atelocollagen)
The method for producing collagen decomposition products or atelocollagen decomposition products is as follows.
The amino acid sequence represented by A) below in the triple helical domain of collagen or atelocollagen (1), the chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, G and X ₃ chemical bonding, between the chemical binding, X ₄ and G between or to cut the chemical bond between X ₆ and G, the cutting process, or
The amino acid sequence represented by the following (2) B) in the triple helical domain of collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, G and X ₃ chemical bond between the chemical bond between X ₄ and G, chemical bond between X ₆ and G, chemical bond between G and X _7, or, between X ₁₄ and G Breaking chemical bonds, cutting steps, or
C) A production method comprising a cleavage step of cleaving the chemical bond between Y ₁ and Y ₂ of the amino acid sequence shown in (3) below at the amino terminus of the triple helical domain of collagen or atelocollagen:
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-;
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids).

Further, the method for producing the decomposed product of collagen or the decomposed product of atelocollagen may be a production method including the following cutting steps. In other words
D) the amino acid sequence represented by the following (1) in the triple helical domain of collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, G and X ₃ chemical bond, chemical bond between X ₄ and G, and disconnects any one chemical bond selected from a chemical bond between X ₆ and G between the cutting step or,
The amino acid sequence represented by the following (2) in the triple helical domain of E) collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, G and X ₃ chemical bond between the chemical bond between X ₄ and G, chemical bond between X ₆ and G, chemical bond between G and X _7, and, between X ₁₄ and G A cutting step, or a cutting step, that cleaves any one of the chemical bonds selected from the chemical bonds
F) Cleavage step of cleaving the chemical bond between Y ₁ and Y ₂ of the amino acid sequence shown in (3) below at the amino terminus of the triple helical domain of collagen or atelocollagen:
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-:
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-:
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-:
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids).

Each configuration will be described in detail below. Since the decomposition product of collagen or the decomposition product of atelocollagen itself has already been described, the description thereof will be omitted here.

The cutting step may be any step as long as it is a step of cutting a chemical bond at a specific portion of the amino acid sequence shown in (1) to (3), and the specific configuration is not particularly limited.

The above-mentioned cleavage step may be a step of actually cleaving the chemical bond in the triple helical domain to prepare a decomposition product of collagen or a decomposition product of atelocollagen (for example, an enzymatic method).

In addition, the step of producing a degradation product of collagen or a degradation product of atelocollagen in which the chemical bond in the triple helical domain has already been cleaved (for example, a chemical synthesis method, expression of a recombinant protein) is incorporated into the concept of "cleaving step" in the present application. It can also be included.

The details of the cutting process described above will be described below.

(Ii. Cutting step based on enzymatic method)
When the cutting step based on the enzymatic method is adopted, for example, the cutting step can be configured as follows.

The cleavage step can be performed by degrading collagen or atelocollagen with an enzyme (eg, protease (eg, cysteine protease)).

The enzyme is not particularly limited, but for example, cysteine protease is preferably used.

As the cysteine protease, it is preferable to use a cysteine protease having a larger amount of acidic amino acids than a basic amino acid amount, or a cysteine protease that is active at a hydrogen ion concentration in an acidic region.

Examples of such cysteine proteases include cathepsin B [EC 3.4.22.1.], Papain [EC 3.4.22.2], ficin [EC 3.4.22.3], and actinidyne [EC 3. 4.22.14], cathepsin L [EC 3.4.22.15], cathepsin H [EC 3.4.22.16], cathepsin S [EC 3.4.22.27], bromelain [EC 3] 4.22.32], cathepsin K [EC 3.4.22.38], bromelain, calcium-dependent proteases and the like.

Among these, papain, ficin, actinidyne, cathepsin K, alloline or bromelain are preferably used, and papain, ficin, actinidyne and cathepsin K are even more preferred.

The above-mentioned enzymes can be obtained by known methods. For example, it can be obtained by producing an enzyme by chemical synthesis; extracting an enzyme from cells or tissues of bacteria, fungi, various animals and plants; producing an enzyme by genetic engineering means; and the like. Of course, it is also possible to use a commercially available enzyme.

When the cleavage step is performed by decomposing collagen or atelocollagen with an enzyme (for example, protease), the cleavage step can be performed according to the following methods (i) to (iii), for example. The following methods (i) to (iii) are merely examples of cutting steps, and the present invention is not limited to these methods (i) to (iii).

The following methods (i) and (ii) are examples of methods used for cleaving a chemical bond at a specific portion of the amino acid sequence shown in (1) or (2), and are described below. The method (iii) is an example of a method used for cleaving a chemical bond at a specific portion of the amino acid sequence shown in (3).

(I) A method of contacting collagen or atelocollagen with an enzyme in the presence of a high concentration of salt.

(Ii) A method of contacting collagen or atelocollagen with an enzyme after contact with a high concentration salt.

(Iii) A method of contacting collagen or atelocollagen with an enzyme in the presence of a low concentration of salt.

Specific examples of the method (i) described above include a method of contacting collagen or atelocollagen with an enzyme in an aqueous solution containing a high concentration of salt.

Specific examples of the above-mentioned method (ii) include, for example, a method in which an aqueous solution containing a high-concentration salt is brought into contact with an enzyme in advance, and then the enzyme is brought into contact with collagen or atelocollagen.

Specific examples of the above-mentioned method (iii) include a method in which collagen or atelocollagen is brought into contact with an enzyme in an aqueous solution containing a low-concentration salt.

The specific composition of the aqueous solution is not particularly limited, but for example, water can be used.

The specific composition of the salt is not particularly limited, but it is preferable to use chloride. The chloride is not particularly limited, but for example, NaCl, KCl, LiCl or MgCl ₂ can be used.

The concentration of the salt in the aqueous solution containing the high-concentration salt is not particularly limited, but it can be said that the higher the concentration, the more preferable. For example, the concentration is preferably 200 mM or more, more preferably 500 mM or more, more preferably 1000 mM or more, more preferably 1500 mM or more, and most preferably 2000 mM or more.

The upper limit of the salt concentration in the aqueous solution containing the high-concentration salt is not particularly limited, but may be, for example, 2500 mM. When the salt concentration is higher than 2500 mM, most of the proteins are salted out, and as a result, the efficiency of enzymatic decomposition of collagen or atelocollagen tends to decrease. On the other hand, when the salt concentration is 2500 mM or less, the enzymatic decomposition efficiency of collagen or atelocollagen can be increased.

Therefore, the salt concentration in the aqueous solution containing the high-concentration salt is preferably 200 mM or more and 2500 mM or less, more preferably 500 mM or more and 2500 mM or less, more preferably 1000 mM or more and 2500 mM or less, and more preferably 1500 mM or more. It is more preferably 2500 mM or less, and most preferably 2000 mM or more and 2500 mM or less.

The higher the salt concentration in the aqueous solution containing the high-concentration salt, the higher the specificity of the cut portion of collagen or atelocollagen by the enzyme. As a result, the collagen decomposition product or the atelocollagen decomposition product used in one embodiment of the present invention can be made more uniform and have high physiological activity.

The concentration of the salt in the aqueous solution containing the low-concentration salt is not particularly limited, but it can be said that the lower the concentration, the more preferable. For example, the concentration is preferably lower than 200 mM, more preferably 150 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, and most preferably approximately 0 mM.

The amount of collagen or atelocollagen to be dissolved in the above aqueous solution (for example, water) is not particularly limited, but for example, it is preferable to dissolve 1 part by weight of collagen or atelocollagen in 1000 parts by weight to 10,000 parts by weight of the aqueous solution.

With the above configuration, when an enzyme is added to an aqueous solution, the enzyme can be efficiently brought into contact with collagen or atelocollagen. As a result, collagen or atelocollagen can be efficiently decomposed by an enzyme (for example, protease).

The amount of the enzyme added to the aqueous solution is not particularly limited, but for example, it is preferable to add 10 parts by weight to 20 parts by weight of the enzyme with respect to 100 parts by weight of collagen or atelocollagen.

With the above configuration, since the concentration of the enzyme in the aqueous solution is high, collagen or atelocollagen can be efficiently decomposed by the enzyme (for example, protease).

Other conditions (for example, pH, temperature, contact time, etc. of the aqueous solution) when contacting collagen or atelocollagen with the enzyme in the aqueous solution are not particularly limited and can be appropriately set, but the range is as follows. Is preferable.

1) The pH of the aqueous solution is preferably pH 2.0 to 7.0, and more preferably pH 2.5 to 6.5. A well-known buffer can be added to the aqueous solution to keep the pH of the aqueous solution in the above range. At the above pH, collagen or atelocollagen can be uniformly dissolved in the aqueous solution, and as a result, the enzymatic reaction can be efficiently promoted.

2) The temperature is not particularly limited, and the temperature may be selected according to the enzyme to be used. For example, the temperature is preferably 15 ° C to 40 ° C, more preferably 20 ° C to 35 ° C.

3) The contact time is not particularly limited, and the contact time may be selected according to the amount of enzyme and / or the amount of collagen or atelocollagen. For example, the time is preferably 1 hour to 60 days, more preferably 1 day to 7 days, and even more preferably 3 to 7 days.

It is selected from the group consisting of a step of adjusting the pH, a step of inactivating the enzyme, and a step of removing impurities, if necessary, after contacting collagen or atelocollagen with the enzyme in an aqueous solution. At least one step may be performed.

In addition, the step of removing the impurities can be performed by a general method for separating substances. The step of removing the impurities can be performed by, for example, dialysis, salting out, gel filtration chromatography, or the like, electrical point precipitation, ion exchange chromatography, or hydrophobic interaction chromatography.

As described above, the cutting step can be performed by enzymatically degrading collagen or atelocollagen. At this time, the collagen or atelocollagen to be decomposed may be in a state of being contained in the living tissue. That is, the cutting step can also be performed by bringing the biological tissue into contact with the enzyme.

The living tissue is not particularly limited, and examples thereof include the dermis, tendons, bones or fascia of mammals or birds, or the skin or scales of fish.

From the viewpoint of maintaining high physiological activity and obtaining a large amount of collagen decomposition products or atelocollagen decomposition products, it is preferable to use the dermis as a biological tissue.

When the dermis is used as a living tissue, it is preferable to bring the dermis into contact with an enzyme under acidic conditions. For example, the acidic conditions are preferably pH 2.5 to 6.5, more preferably pH 2.5 to 5.0, still more preferably pH 2.5 to 4.0, and most preferably pH 2.5 to 3.5. Is.

More specifically, in the method for producing a decomposition product of collagen or a decomposition product of atelocollagen, in the cutting step, the cysteine protease is brought into contact with the dermis to bring the collagen contained in the dermis into contact with the cysteine protease. It is preferable to let it.

In addition, the method for producing a degradation product of collagen or a degradation product of atelocollagen having the amino acid sequence shown in (1) or (2) above is the dermis in the presence of a salt having a concentration of 200 mM or more in the above-mentioned cleavage step. And cysteine protease are preferably brought into contact with each other.

Further, in the method for producing a degradation product of collagen or a degradation product of atelocollagen having the amino acid sequence shown in the above (1), (2) or (3), in the above cutting step, the decomposition product is brought into contact with a salt having a concentration of 200 mM or more. It is preferable to bring the subsequent cysteine protease into contact with the dermis.

Further, in the method for producing a degradation product of collagen having the amino acid sequence shown in (3) above or a degradation product of atelocollagen, in the above-mentioned cleavage step, in the presence of a salt having a concentration lower than 200 mM, the dermis and cysteine Contact with protease is preferred.

(I-iii. Chemical synthesis method)
When the cutting step based on the chemical synthesis method is adopted, for example, the cutting step can be configured as follows.

First, information on the amino acid sequence of each polypeptide chain constituting collagen or atelocollagen is obtained from a well-known database. The polypeptide chain may be appropriately selected depending on the type of collagen or atelocollagen, and may be one type of polypeptide chain or a plurality of types of polypeptide chains.

Next, from the above-mentioned polypeptides, the polypeptide chain containing the chemical bond to be cleaved and the position of the chemical bond to be cleaved are determined, and the desired polypeptide is assumed to be cleaved. Determine the amino acid sequence of the chain.

Finally, the desired polypeptide chain is synthesized by a well-known chemical synthesis method according to the determined amino acid sequence.

As described above, the cutting step can be carried out.

The method for producing a decomposition product of collagen or a decomposition product of atelocollagen can also include a step other than the above-mentioned cutting step.

For example, a method for producing a degradation product of collagen or a degradation product of atelocollagen may include a step of synthesizing a desired polypeptide chain by a well-known chemical synthesis method and then purifying the synthesized polypeptide chain. The purification may be carried out using a well-known column as appropriate.

The method for producing a degradation product of collagen or a degradation product of atelocollagen may include a step of mixing a desired polypeptide chain with another polypeptide chain. The other polypeptide chain is not particularly limited, and may be a polypeptide chain in which a chemical bond is similarly cleaved, or a polypeptide chain in which a chemical bond is not cleaved.

(I-iii. Cleavage step based on expression of recombinant protein)
When the cleavage step based on the expression of the recombinant protein is adopted, for example, the cleavage step can be configured as follows.

First, information on the amino acid sequence of each polypeptide chain constituting collagen or atelocollagen is obtained from a well-known database. The polypeptide chain may be appropriately selected depending on the type of collagen or atelocollagen, and may be one type of polypeptide chain or a plurality of types of polypeptide chains.

Next, from the above-mentioned polypeptides, the polypeptide chain containing the chemical bond to be cleaved and the position of the chemical bond to be cleaved are determined, and the desired polypeptide is assumed to be cleaved. The amino acid sequence and DNA sequence of the strand are determined.

The DNA encoding the desired polypeptide chain is then inserted into a well-known protein expression vector. Then, after introducing the protein expression vector into a desired host (for example, Escherichia coli, yeast, insect cells, animal cells, etc.), the polypeptide chain after the chemical bond is cleaved is expressed in the host.

As described above, the cutting step can be carried out.

The method for producing a decomposition product of collagen or a decomposition product of atelocollagen can also include a step other than the above-mentioned cutting step.

For example, a method for producing a degradation product of collagen or a degradation product of atelocollagen may include a step of expressing a desired polypeptide chain in a host and then purifying the expressed polypeptide chain. The purification may be carried out using a well-known column as appropriate.

The method for producing a degradation product of collagen or a degradation product of atelocollagen may include a step of mixing a desired polypeptide chain with another polypeptide chain. The other polypeptide chain is not particularly limited, and may be a polypeptide chain in which a chemical bond is similarly cleaved, or a polypeptide chain in which a chemical bond is not cleaved.

[2. Composition for embryoid body formation]
The embryoid body-forming composition according to an embodiment of the present invention (hereinafter, also referred to as “embryoid body-forming composition of the present embodiment”) is a composition for embryoid body formation of pluripotent stem cells. The decomposition product of collagen or the decomposition product of atelocollagen is contained as a main component, and the decomposition product contains at least a part of the triple helical domain of the collagen or the atelocollagen. That is, the degradation product may contain the entire triple helical domain of collagen or atelocollagen, or may contain a portion of the triple helical domain. The above-mentioned "pluripotent stem cell", the above-mentioned "embryoid body", and the above-mentioned "collagen decomposition product or atelocollagen decomposition product" are as described in the above-mentioned "1. Embryoid body formation method". , The description is omitted here.

The composition for embryoid body formation of the present embodiment contains a decomposition product of collagen or a decomposition product of atelocollagen as a main component having embryoid body formation activity. The amount of collagen decomposition products or atelocollagen decomposition products contained in the embryoid body-forming composition of the present embodiment is not particularly limited. For example, the composition for embryoid body formation of the present embodiment may contain a decomposition product of collagen or a decomposition product of atelocollagen in an amount of 0.1% by weight or more, and among them, 50% by weight or more is contained. It is more preferable that the content is 90% by weight or more, and 100% by weight is most preferable.

When the inner surface of the culture vessel is coated with the composition for forming an embryo-like body of the present embodiment, the composition for forming an embryo-like body of the present embodiment is diluted with water or the like, and the final concentration is 2 mg / mL or more. 20 mg / mL, preferably 3 mg / mL to 16 mg / mL, more preferably 5 mg / mL to 15 mg / mL, or 5 mg / mL to 20 mg / mL, preferably 8 mg / mL to 16 mg / mL collagen degradation or atelocollagen A solution containing the decomposition product of the above can be prepared, and the inner surface of the culture vessel can be coated with the solution. When the concentration of the decomposition product in the solution is 2 mg / mL or more, embryoid bodies can be uniformly formed. Further, when the concentration of the decomposition product in the solution is 20 mg / mL or less, the reproducibility is good. On the other hand, when the concentration of the decomposition product in the solution is less than 2 mg / mL, the formation of embryoid bodies may become non-uniform. Further, when the concentration of the decomposition product in the solution is higher than 20 mg / mL, the viscosity may be high and the reproducibility may be poor.

In addition, a component other than a decomposition product of collagen or a decomposition product of atelocollagen may be added to the composition for embryoid body formation of the present embodiment. These components are not particularly limited, and desired components can be added as appropriate. For example, a collagen degradation product or atelocollagen degradation product is mixed with a concentrated culture solution (for example, 5-fold concentrated DMEM, etc.) and a reconstitution buffer solution (50 mM sodium hydroxide, 260 mM sodium hydrogen carbonate, 200 mM HEPES). It may be a gelling composition. In this case, the decomposition product of collagen or the decomposition product of atelocollagen may be contained in the gelation composition as a final concentration of 2 to 15 mg / mL, 5 to 15 mg / mL, or 5 to 15 mg / mL. It is preferably contained at 12 mg / mL, more preferably at 7 to 10 mg / mL. When the concentration of the above-mentioned decomposition products contained in the gel composition is 2 mg / mL or more as the final concentration, embryoid bodies can be uniformly formed. Further, when the concentration of the decomposed product contained in the gel composition is 15 mg / mL or less as the final concentration, the reproducibility is good. On the other hand, when the concentration of the decomposition product contained in the gel composition is less than 2 mg / mL as the final concentration, the formation of embryoid bodies may become non-uniform. Further, when the concentration of the above-mentioned decomposed product contained in the gel composition is higher than 15 mg / mL as the final concentration, the viscosity may be high and the reproducibility may be poor.

As described above, the collagen decomposition product or the atelocollagen decomposition product used in one embodiment of the present invention has a higher concentration at which gelation begins than the conventional collagen decomposition product or the atelocollagen decomposition product. Therefore, the collagen decomposition product or the atelocollagen decomposition product used in one embodiment of the present invention having the same concentration as the conventional collagen decomposition product or the atelocollagen decomposition product gelling can be stably stored at room temperature. .. Therefore, the composition for embryoid body formation of the present embodiment can be stably stored at room temperature.

By using the embryoid body forming composition of the present embodiment, the embryoid body of pluripotent stem cells can be easily formed. Further, by using the composition for forming an embryoid body of the present embodiment, an embryoid body can be formed in a shorter period of time as compared with a conventional method for forming an embryoid body.

The composition for embryoid body formation of the present embodiment can be used in the embryoid body forming method of the present embodiment described above regardless of the origin animal, the origin site and the N-terminal (amino terminal) cleavage site.

The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. Is also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to Examples.

<1. Effect of salt concentration on porcine-derived α1 chain cleavage>
A 50 mM citrate buffer (pH 3.0) having a sodium chloride concentration of 0 mM, 200 mM, 1000 mM, 1500 mM or 2000 mM was prepared. Water was used as the solvent for the aqueous solution.

To activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes. As the actinidine, a product purified by a well-known method was used (see, for example, Non-Patent Document 2).

Then, type I collagen derived from pig was dissolved in 50 mM citrate buffer (pH 3.0) containing a salt. An aqueous solution containing actinidyne and the solution containing porcine-derived type I collagen were contacted at 20 ° C. for 10 days or longer to prepare a decomposition product of type I collagen. Type I collagen derived from pigs was purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of the α1 chain transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Table 1 shows the amino acid sequences of the amino terminus of the α1 chain degradation product and its vicinity when the salt concentration is 0 mM, 200 mM, 1000 mM, 1500 mM or 2000 mM.

As shown in Table 1, it was clarified that the cleavage sites in the α1 chain differed when the salt concentration was different. More specifically, low salt concentrations (eg, 0 mM) can result in cleavage outside the triple helical domain, and high salt concentrations (eg, 200 mM and above) can result in cleavage inside the triple helical domain. It was revealed.

The cutting site when the salt concentration was high was a novel cutting site found by the present inventor.

When the salt concentration is changed, the amount of the decomposition product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 2 and the decomposition product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 3 contained in the decomposition product The amount and the ratio of were different depending on the conditions such as reaction time, reaction pH, and reaction temperature. When a large amount of decomposition products was to be prepared, NaCl was insolubilized when the salt concentration exceeded 2000 mM. When preparing a large amount of decomposition products, it is considered preferable to set the upper limit of the salt concentration to 500 mM or 800 mM.

<2. Effect of salt concentration on cleavage of α1 chains from rats and chickens>
A 50 mM citrate buffer (pH 3.0) having a sodium chloride concentration of 0 mM and 2000 mM was prepared. Water was used as the solvent for the aqueous solution.

To activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.

Then, type I collagen derived from the rat tail or type I collagen derived from the chicken skin was dissolved in 50 mM citrate buffer (pH 3.0) containing a salt. An aqueous solution containing actinidyne and type I collagen derived from the rat tail or type I collagen derived from the chicken skin were brought into contact with each other for 10 days or more at 20 ° C. to prepare a decomposition product of type I collagen. As the actinidine, the same one used in the above-mentioned Example <1> was used. In addition, type I collagen derived from the rat tail and type I collagen derived from the chicken skin were purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of the α1 chain transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Table 2 shows the amino acid sequences of the amino terminus of the α1 chain degradation product derived from rats when the salt concentration is 0 mM and 2000 mM and its vicinity, and the partial structure of the α1 chain derived from undegraded rats (salt concentration column). Refer to the data in which is "-").

Table 3 shows the amino acid sequences of the amino terminus of the α1 chain degradation product derived from chickens when the salt concentration is 0 mM and 2000 mM and its vicinity, and the partial structure of the α1 chain derived from undegraded chickens (salt concentration column). Refer to the data in which is "-").

As shown in Tables 2 and 3, even in α1 chains derived from different species, low salt concentration (eg, 0 mM) causes cleavage outside the triple helical domain, and high salt concentration results in triple. It was revealed that cutting is likely to occur inside the helical domain.

The cutting site when the salt concentration was high was a novel cutting site found by the present inventor.

When the salt concentration is 2000 mM, the amount of the decomposition product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 24 and the decomposition product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 5 contained in the decomposition product The ratio of to the amount of was different depending on the conditions such as reaction time, reaction pH, and reaction temperature.

<3. Examination of salt types>
A salt-free solution, an aqueous solution having a MgCl ₂ concentration of 500 mM, and an aqueous solution having a KCl concentration of 200 mM were prepared. Water was used as the solvent for the aqueous solution.

Actinidyne and porcine-derived type I collagen were mixed with each of the above aqueous solutions and then reacted at 20 ° C. for 10 days or more to prepare a decomposition product of type I collagen. As the actinidine, the same one used in the above-mentioned Example <1> was used. In addition, type I collagen derived from pigs was purified based on a well-known method (see, for example, Non-Patent Document 2).

Each of the above-mentioned decomposition products was subjected to polyacrylamide gel electrophoresis to separate the decomposition products of α1 chain.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of the α1 chain transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Table 4 shows the α1 chains derived from pigs when an aqueous solution having a salt concentration of 0 mM was used, when an aqueous solution having a MgCl ₂ concentration of 500 mM was used, and when an aqueous solution having a KCl concentration of 200 mM was used. The amino acid sequences at and near the amino end of the degradation product and the partial structure of the undegraded porcine-derived α1 chain (see data in which the salinity column is "-") are shown.

As shown in Table 4, even with different types of salts, low salt concentrations (eg 0 mM) cause cleavage outside the triple helical domain, and high salt concentrations (eg 200 mM KCl, 500 mM MgCl). ₂ ), it became clear that cleavage occurs inside the triple helical domain. In the case of 200 mM KCl and 500 mM MgCl ₂ , the amount of the degradation product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 2 and the amino terminus of the amino acid sequence shown in SEQ ID NO: 9 are included in the degradation product. The ratio with the amount of the decomposition product was different depending on the conditions such as reaction time, reaction pH, and reaction temperature.

The cutting site when different types of salts were used was a novel cutting site found by the present inventor.

<4. Examination of types of cysteine protease>
In this example, cathepsin K, which is a kind of cysteine protease, was used to examine the cleavage site of α1 chain under high salt concentration conditions. The test method and test results will be described below.

A 50 mM citrate buffer (pH 3.0) having a sodium chloride concentration of 2000 mM was prepared. Water was used as the solvent for the aqueous solution.

To activate cathepsin K, cathepsin K was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 45 minutes. As the cathepsin K, a commercially available product was used.

Then, type I collagen derived from chicken or type I collagen derived from pig was dissolved in 50 mM citrate buffer (pH 3.0) containing a salt. A decomposition product of type I collagen was prepared by contacting an aqueous solution containing cathepsin K with a chicken-derived type I collagen or a pig-derived type I collagen-containing solution at 20 ° C. for 10 days or longer. .. In addition, chicken-derived type I collagen and pig-derived type I collagen were purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of the α1 chain transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Refer to Table 5 for the amino acid sequences of the amino terminus of the α1 chain degradation product derived from pigs and their vicinity, and the partial structure of the α1 chain derived from pigs that has not been degraded (the column of salinity is “-”. ) Is shown.

As shown in Table 5, it was revealed that even cathepsin K, which is a kind of cysteine protease, cleaves inside the triple helical domain when the salt concentration is high.

Further, as shown in Table 5, in the case of cathepsin K, which is a kind of cysteine protease, a plurality of types of cleavage sites were confirmed.

In the case of the decomposition product of type I collagen derived from chicken, the decomposition product of α1 chain derived from chicken is the decomposition product of α1 chain derived from chicken corresponding to the following SEQ ID NOs: 11 and 12 and the amino terminal in the following SEQ ID NO: 10. A chicken-derived α1 chain degradation product corresponding to the broken chemical bond between the 10th “S” and the 11th “G” was confirmed.

<5. Effect of dialysis salt concentration on porcine-derived α1 chain cleavage>
Actinidyne was placed in a dialysis tube, and the actinidyne was dialyzed against a dialysis external solution having a sodium chloride concentration of 2000 mM. Then, the external dialysis solution was changed to distilled water, and dialysis was continued to obtain actinidine. As the actinidine, a product purified by a well-known method was used (see, for example, Non-Patent Document 2). To activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.

Then, type I collagen derived from pig was dissolved in 50 mM citrate buffer (pH 3.0) containing a salt. An aqueous solution containing actinidyne and type I collagen derived from pigs were brought into contact with each other for 3 days or more at 20 ° C. to prepare a decomposition product of type I collagen. In addition, type I collagen derived from pigs was purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of the α1 chain transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Table 6 shows the amino acid sequences of the amino terminus of the α1 chain degradation product derived from pigs when the dialysis salt concentration is 2000 mM and its vicinity, and the partial structure of the undegraded α1 chain derived from pigs (the column of salt concentration is shown. Refer to the data that is "-").

As shown in Table 6, it was revealed that high dialysis salt concentration causes cleavage outside or inside the triple helical domain. In the case of 2000 mM, the amount of the decomposition product having the amino end of the amino acid sequence shown in SEQ ID NO: 26 and the amount of the decomposition product having the amino end of the amino acid sequence shown in SEQ ID NO: 27 contained in the decomposition product. , The ratio of the amount of the decomposition product having the amino end of the amino acid sequence shown in SEQ ID NO: 15 was different depending on the conditions such as reaction time, reaction pH, and reaction temperature.

The cut portion when the salt concentration was high included a novel cut portion found by the present inventor.

<6. Effect of dialysis salt concentration on cleavage of human-derived α1 chain>
Actinidyne was placed in a dialysis tube, and the actinidyne was dialyzed against a dialysis external solution having a sodium chloride concentration of 2000 mM. Then, the external dialysis solution was changed to distilled water, and dialysis was continued to obtain actinidine. As the actinidine, a product purified by a well-known method was used (see, for example, Non-Patent Document 2). To activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.

Then, human-derived type I collagen was dissolved in 50 mM citrate buffer (pH 3.5) containing a salt. An aqueous solution containing actinidyne and human-derived type I collagen were brought into contact with each other for 10 days or more at 20 ° C. to prepare a decomposition product of type I collagen. In addition, human-derived type I collagen was purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of the α1 chain transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

As shown in Table 7, it was revealed that high dialysis salt concentration causes cleavage outside or inside the triple helical domain. In the case of 2000 mM, the amount of the decomposition product having the amino end of the amino acid sequence shown in SEQ ID NO: 29 and the amount of the decomposition product having the amino end of the amino acid sequence shown in SEQ ID NO: 30 contained in the decomposition product. , The ratio of the amount of the decomposition product having the amino end of the amino acid sequence shown in SEQ ID NO: 16 was different depending on the conditions such as reaction time, reaction pH, and reaction temperature.

The cut portion when the salt concentration was high included a novel cut portion found by the present inventor.

<7. Cleavage of α1 chain derived from fish>
Actinidyne was placed in a dialysis tube, and the actinidyne was dialyzed against a dialysis external solution having a sodium chloride concentration of 2000 mM. Then, the external dialysis solution was changed to distilled water, and dialysis was continued to obtain actinidine. To activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.

Next, type I collagen derived from fish (specifically, yellowfin tuna) was dissolved in 50 mM citrate buffer (pH 3.0) containing a salt. An aqueous solution containing actinidyne and type I collagen derived from fish were brought into contact with each other at 20 ° C. for 3 days or longer to prepare a decomposition product of type I collagen. As the actinidine, the same one used in the above-mentioned Example <1> was used. In addition, fish-derived type I collagen was purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of α1 chain (type I collagen derived from fish) transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory according to a well-known method.

Table 8 shows the amino acid sequences of the amino terminus of the α1 chain degradation product derived from fish and its vicinity when the salt concentration of the dialysis external solution is 2000 mM. As shown in Table 8, three types of α1 chain (type I collagen derived from fish) degradation products were detected, and the amino acid sequences at the amino terminus of each of these degradation products were SEQ ID NO: 18 and SEQ ID NO: 19. And succeeded in identifying the amino acid sequence shown in SEQ ID NO: 31.

<8. Cleavage of human-derived α2 chain>
In order to activate cathepsin K in the same manner as in <4>, cathepsin K was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 45 minutes.

Then, human-derived type I collagen was dissolved in 50 mM phosphate buffer (pH 6.0) containing a salt. An aqueous solution containing cathepsin K and human-derived type I collagen were brought into contact with each other for 10 days or more at 20 ° C. to prepare a decomposition product of type I collagen. As the cathepsin K, the same one used in the above-mentioned example <1> was used. In addition, human-derived type I collagen was purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of α2 chain (type I collagen derived from human) transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Table 9 shows the amino acid sequences of the amino terminus of the α2 chain degradation product derived from humans and its vicinity when the salt concentration of the reaction solution is 200 mM.

<9. Cleavage of α2 chain derived from chicken>
Actinidyne was placed in a dialysis tube, and the actinidyne was dialyzed against a dialysis external solution having a sodium chloride concentration of 2000 mM. Then, the external dialysis solution was changed to distilled water, and dialysis was continued to obtain actinidine. To activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.

Next, type I collagen derived from chicken was dissolved in 50 mM citrate buffer (pH 3.0) containing a salt. An aqueous solution containing actinidyne and type I collagen derived from chicken were brought into contact with each other for 7 days or more at 20 ° C. to prepare a decomposition product of type I collagen. As the actinidine, the same one used in the above-mentioned Example <1> was used. In addition, chicken-derived type I collagen was purified based on a well-known method (see, for example, Non-Patent Document 2).

The above-mentioned decomposition products were subjected to polyacrylamide gel electrophoresis to separate the decomposition products of type I collagen.

Then, the decomposition product of type I collagen was transferred to the PVDF (Polyvinylidene Difluoride) membrane by a conventional method. Then, the amino acid sequence of the amino terminal of the degradation product of α2 chain (type I collagen derived from chicken) transferred to the PVDF membrane was determined by the Edman degradation method.

The actual Edman analysis was performed according to a well-known method by requesting Approscience Co., Ltd. or Kinki University School of Medicine Analytical Instruments Joint Laboratory.

Table 10 shows the amino acid sequences of the amino terminus of the α2 chain degradation product derived from chicken and its vicinity when the salt concentration of the dialysis external solution is 2000 mM.

As shown in the table, it was revealed that even in the α1 chain or α2 chain derived from different species, cleavage occurs inside the triple helical domain at high salt concentration. The cutting site when the salt concentration was high was a novel cutting site found by the present inventor.

<10. Test on embryoid body formation ability of collagen degradation product 1>
(Culture dish)
The above-mentioned collagen degradation products (the above-mentioned collagen degradation products derived from pigs at a dialysis salt concentration of 2000 mM, that is, the amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27 ((1), (2) and (3)). A commercially available culture dish contacted with a mixture of degradation products (corresponding to the amino acid sequence shown in) was used for the test. Specifically, 60 μL of an aqueous solution of a 10 mg / mL collagen decomposition product was added to a 35 mm culture dish, and the mixture was sufficiently blended into the culture dish and allowed to stand at room temperature for 5 hours to prepare a collagen decomposition product-coated culture dish.

In addition, a 10 mg / mL aqueous solution of a decomposition product of collagen is mixed with a 5-fold concentrated DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and a buffer solution for reconstruction (50 mM sodium hydroxide, 260 mM sodium hydrogen carbonate, 200 mM HEPES). Then, 200 μL of this mixed solution was added to a 35 mm culture dish, spread and coated uniformly on the culture dish, and allowed to stand in an incubator at 37 ° C. for 30 minutes. This was used as a collagen decomposition product gel culture dish.

In addition, 1 mL of 0.1% gelatin solution was added to a 35 mm culture dish, and the mixture was allowed to fully blend into the culture dish and allowed to stand at room temperature for 4 hours. After 4 hours, the gelatin solution was removed to prepare a gelatin-coated culture dish.

(Mouse ES cells)
Mouse ES cells transferred from Professor Teruhiko Wakayama, Faculty of Life and Environmental Sciences, University of Yamanashi, were used. These mouse ES cells are labeled with GFP (green fluorescent protein), and the gene encoding the GFP protein is inserted into the chromosome.

(Preparation of feeder cells)
Mitomycin-treated mouse embryo-derived fibroblasts (MEF) (CMPMEFCF, manufactured by DS Pharma Biomedical) were prepared at 1 × 10 ⁵ cells / mL. 7 × 10 ⁴ MEFs were seeded in each of a gelatin-coated culture dish, a collagen decomposition product-coated culture dish, and a collagen decomposition product gel culture dish, and a DMEM medium (KnockOutDMEM, manufactured by Thermo Fisher Science) containing 10% fetal bovine serum was applied. The cells were cultured for 1 day under the conditions of 37 ° C. and 5% CO ₂ .

(Formation of embryoid body)
DMEM (KnockOutDMEM, manufactured by Thermo Fisher Science) containing 500 U / mL leukemia inhibitory factor (LIF, product number 199-16501, Wako Pure Chemical Industries, Ltd.) and 20% fetal bovine serum (hereinafter, "DMEM medium"). (LIF (+)) ”) was used to prepare 2.5 × 10 ⁴ mouse ES cells / mL. 2 mL of mouse ES cell suspension was seeded in a gelatin-coated culture dish, a collagen degradation product-coated culture dish, and a collagen degradation product gel culture dish in which MEF had been seeded in advance, and DMEM medium (LIF (+)) was used. Was cultured under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (+) and LIF (+)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment. In the examples, "MEF (+)" indicates culture in the presence of MEF, and "MEF (-)" indicates culture in the absence of MEF. Further, "LIF (+)" indicates culture in the coexistence of LIF, and "LIF (-)" indicates culture in the absence of LIF.

During the culture period, the medium was replaced with fresh DMEM medium (LIF (+)) every day.

Three days after the start of the culture, the morphology of ES cells was observed under a phase-contrast microscope. The morphology of each cell is shown in FIG. In FIG. 1, (a) is a diagram showing the morphology of ES cells cultured in a gelatin-coated culture dish in the presence of MEF (MEF (+)) using DMEM medium (LIF (+)) for 3 days. , (B) are diagrams showing the morphology of ES cells cultured for 3 days in DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a collagen decomposition product-coated culture dish. (C) is a diagram showing the morphology of ES cells cultured in a collagen decomposition product gel culture dish in the presence of MEF (MEF (+)) for 3 days using DMEM medium (LIF (+)).

When cultured in a gelatin-coated culture dish, the MEF adhered to and extended to the bottom surface of the gelatin-coated culture dish. During that time, ES cells were forming colonies. It had the same morphology as general ES cell culture findings ((a) in FIG. 1).

When cultured in a collagen degradation product coated culture dish, the MEF did not adhere to the bottom surface of the collagen degradation product coated culture dish. The observed cells formed a floating three-dimensional cell mass (embryoid body), and had a completely different morphology from the general ES cell culture findings of a gelatin-coated culture dish (FIG. 1 (b)). ).

When cultured in the collagen degradation product gel culture dish, the MEF was adhered to the bottom surface of the collagen degradation product gel culture dish. The observed cells adhered to the collagen degradation product gel to form a three-dimensional cell mass (embryoid body). The morphology was completely different from the general ES cell culture findings of the gelatin-coated culture dish. It seemed that cells extended from the embryoid body adhering to the collagen degradation product gel and connected to another embryoid body ((c) in FIG. 1).

The morphology of ES cells cultured in a collagen degradation product-coated culture dish or a collagen degradation product gel culture dish was completely different from the morphology of ES cells cultured in a gelatin-coated culture dish. The ES cells cultured in the gelatin-coated culture dish only formed colonies of general ES cells, whereas the ES cells cultured in the collagen degradation product-coated culture dish were floating embryos despite the addition of LIF. Formed a shape. That is, it was shown that culturing ES cells in a collagen decomposition product-coated culture dish can be a new method for forming embryoid bodies. On the other hand, in the ES cells cultured in the collagen degradation product gel culture dish, embryoid bodies that had not been observed so far were formed in the presence of LIF. Specifically, the embryoid body was shown to adhere to the collagen degradation product gel.

A similar experiment was performed using DMEM medium (KnockOutDMEM, manufactured by Thermo Fisher Scientific) (hereinafter referred to as "DMEM medium (LIF (-))") containing 20% fetal bovine serum without LIF. Specifically, DMEM medium (LIF (−)) was used to prepare 2.5 × 10 ⁴ mouse ES cells / mL. 2 mL of mouse ES cell suspension was seeded in a gelatin-coated culture dish, a collagen degradation product-coated culture dish, and a collagen degradation product gel culture dish in which MEF had been seeded in advance, and DMEM medium (LIF (-)) was applied. The cells were cultured under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (+) and LIF (−)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment.

During the culture period, the medium was replaced with fresh DMEM medium (LIF (−)) every day.

Three days after the start of the culture, the morphology of ES cells was observed under a phase-contrast microscope. The morphology of each cell is shown in FIG. In FIG. 2, (a) is a diagram showing the morphology of ES cells cultured in a gelatin-coated culture dish in the presence of MEF (MEF (+)) using DMEM medium (LIF (−)) for 3 days. , (B) are views showing the morphology of ES cells cultured in a collagen decomposition product-coated culture dish in the presence of MEF (MEF (+)) for 3 days using DMEM medium (LIF (−)). (C) is a diagram showing the morphology of ES cells cultured in a collagen decomposition product gel culture dish in the presence of MEF (MEF (+)) for 3 days using DMEM medium (LIF (−)).

When cultured in a gelatin-coated culture dish, the MEF adhered to and extended to the bottom surface of the gelatin-coated culture dish. During that time, ES cells were forming colonies. It had the same morphology as general ES cell culture findings ((a) in FIG. 2).

When cultured in a collagen degradation product coated culture dish, the MEF did not adhere to the bottom surface of the collagen degradation product coated culture dish. The observed cells formed a floating three-dimensional cell mass (embryoid body), and had a completely different morphology from the general ES cell culture findings of a gelatin-coated culture dish (FIG. 2 (b)). ).

When cultured in the collagen degradation product gel culture dish, the MEF was adhered to the bottom surface of the collagen degradation product gel culture dish. The observed cells adhered to the collagen degradation product gel to form a three-dimensional cell mass (embryoid body). The morphology was completely different from the general ES cell culture findings of the gelatin-coated culture dish. It seemed that cells extended from the embryoid body adhering to the collagen degradation product gel and connected to another embryoid body ((c) in FIG. 2).

The morphology of ES cells cultured in a collagen degradation product-coated culture dish or collagen degradation product gel culture dish using DMEM medium (LIF (-)) was completely different from the morphology of ES cells cultured in a gelatin-coated culture dish. .. However, there was no clear difference in morphological findings compared to ES cells cultured in collagen decomposition product coated culture dishes or collagen decomposition product gel culture dishes using DMEM medium (LIF (+)). The ES cells cultured in the gelatin-coated culture dish only formed colonies of general ES cells, whereas the ES cells cultured in the collagen degradation product-coated culture dish did not contain LIF. , Formed a floating embryonic body. That is, it was shown that culturing ES cells in a collagen decomposition product-coated culture dish can be a new method for forming embryoid bodies. On the other hand, in the ES cells cultured in the collagen decomposition product gel culture dish, embryoid bodies that had not been observed so far were formed in the absence of LIF. Specifically, the embryoid body was shown to adhere to the collagen degradation product gel.

<11. Test on embryoid body formation ability of collagen degradation product 2>
Next, in order to examine the undifferentiated ability of ES cells cultured in a gelatin-coated culture dish, a collagen decomposition product-coated culture dish, or a collagen decomposition product gel culture dish, the intracellular alkaline phosphatase activity was examined.

Mouse ES cells 2.5 × using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Scientific) containing 500 U / mL LIF and 20% fetal bovine serum (hereinafter referred to as “DMEM medium (LIF (+))”). 10 ⁴ pieces / mL was prepared. 2 mL of mouse ES cell suspension was seeded in a gelatin-coated culture dish, a collagen degradation product-coated culture dish, and a collagen degradation product gel culture dish in which MEF had been seeded in advance, and DMEM medium (LIF (+)) was used. Was cultured under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (+) and LIF (+)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment. During the culture period, the medium was replaced with fresh DMEM medium (LIF (+)) every day.

After culturing for 2 days, the culture supernatant of the cells was removed and washed with sterile PBS. Subsequently, a 4% paraformaldehyde solution was added to fix the cells until the cells were immersed, and the cells were allowed to stand at room temperature for 30 minutes. Then, the operation of adding sterile distilled water until the cells were immersed and washing was repeated twice. After removing the washing liquid, 250 μL of an alkaline phosphatase stain attached to a TRACP & ALP double-stain kit (product code MK300, manufactured by Takara Bio Inc.) was added to a culture dish and allowed to stand at 37 ° C. for 45 minutes. To stop the reaction, the stain was removed and washed 3 times with sterile distilled water. The results are shown in FIG.

FIG. 3 is a diagram showing the results of examining the intracellular alkaline phosphatase activity of ES cells cultured for 2 days under MEF (+) and LIF (+) culture conditions, and FIG. 3 (a) is a diagram in FIG. It is a figure which shows the presence or absence of alkaline phosphatase activity of ES cells cultured for 2 days using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a gelatin-coated culture dish, FIG. , A graph showing the presence or absence of alkaline phosphatase activity in ES cells cultured for 2 days using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a collagen decomposition product-coated culture dish. c) is a diagram showing the presence or absence of alkaline phosphatase activity in ES cells cultured for 2 days in DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a collagen decomposition product gel culture dish. is there.

It was shown that embryonic bodies formed from ES cells cultured in collagen-degraded product-coated culture dishes and collagen degradation product gel culture dishes were positive for alkaline phosphatase activity, similar to colonies of ES cells cultured in gelatin-coated culture dishes. ((A) to (c) in FIG. 3). On the other hand, the co-cultured MEF was negative for alkaline phosphatase activity. These results indicate that the embryoid bodies formed from ES cells cultured in the collagen degradation product coated culture dish and the collagen degradation product gel culture dish maintain the undifferentiated ability.

<12. Test on embryoid body formation ability of collagen degradation product 3>
Using a culture dish not seeded with MEF instead of the culture dish seeded with MEF, a similar experiment was performed in DMEM medium containing LIF and 20% fetal bovine serum (KnockOutDMEM, Thermofisher Science). (Hereinafter referred to as "DMEM medium (LIF (+))"). Specifically, DMEM medium (LIF (+)) was used to prepare 2.5 × 10 ⁴ mouse ES cells / mL. 2 mL of mouse ES cell suspension was seeded in a gelatin-coated culture dish, a collagen degradation product-coated culture dish, and a collagen degradation product gel culture dish in which MEF was not seeded, and DMEM medium (LIF (+)) was used. , 37 ° C., 5% CO ₂ (culture conditions: MEF (−) and LIF (+)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment.

During the culture period, the medium was replaced with fresh DMEM medium (LIF (+)) every day.

Three days after the start of the culture, the morphology of ES cells was observed under a phase-contrast microscope. The morphology of each cell is shown in FIG. In FIG. 4, (a) is a diagram showing the morphology of ES cells cultured in a gelatin-coated culture dish in a DMEM medium (LIF (+)) for 3 days, and (b) is a collagen decomposition product-coated culture. It is a figure which shows the morphology of ES cells cultured for 3 days using DMEM medium (LIF (+)) in a dish, (c) is the DMEM medium (LIF (+)) in a collagen decomposition product gel culture dish. It is a figure which shows the morphology of the ES cell which was cultured for 3 days using.

When cultured in a gelatin-coated culture dish, ES cells spread in a single layer, and colonized cells and single-adhered cells were observed separately. There were also cells showing a morphology showing cell processes. Compared to the culture findings (FIG. 1 (a)) of general ES cells in the presence of MEF and LIF, the number of colonies is smaller and the size is smaller, and the cell-cell adhesion is such that each cell can be distinguished. Was shown ((a) in FIG. 4).

When cultured in a collagen decomposition product-coated culture dish, the observed ES cells form an adherent or floating three-dimensional cell mass (embryoid body) ((b) in FIG. 4), which is a general gelatin-coated culture dish. The morphology was completely different from the typical ES cell culture findings ((a) in FIG. 4). If anything, the proportion of floating embryoid bodies was high.

When cultured in a collagen degradation product gel culture dish, the observed ES cells formed a three-dimensional cell mass (embryoid body) adhering to or floating on the collagen degradation product gel. The morphology was completely different from the general ES cell culture findings of the gelatin-coated culture dish. The embryoid bodies adhering to the collagen degradation gel were relatively larger than the floating embryoid bodies. ((C) in FIG. 4).

The morphology of ES cells cultured in a collagen degradation product-coated culture dish or a collagen degradation product gel culture dish using DMEM medium (LIF (+)) in the absence of MEF (MEF (-)) is expressed in a gelatin-coated culture dish. The morphology of the cultured ES cells was completely different. However, in the presence of MEF (MEF (+)), MEF does not coexist as compared with ES cells cultured in a collagen degradation product coated culture dish or a collagen degradation product gel culture dish using DMEM medium (LIF (+)). The morphology of ES cells cultured below (MEF (-)) in a collagen degradation product-coated culture dish or collagen degradation product gel culture dish using DMEM medium (LIF (+)) is slightly different in size and shape. It was observed, but there was no clear difference. ES cells cultured in gelatin-coated culture dishes under MEF (−) and LIF (+) culture conditions formed colonies of common ES cells or were single adherent cells. It should be noted that the ES cells cultured in the collagen decomposition product-coated culture dish under the culture conditions of MEF (−) and LIF (+) formed embryoid bodies even though the MEF was not co-cultured. That is, it was shown that culturing ES cells in a collagen decomposition product-coated culture dish can be a new method for forming embryoid bodies. Similarly, in ES cells cultured in a collagen degradation product gel culture dish under MEF (-) and LIF (+) culture conditions, embryoid bodies that have not been observed so far are formed in the absence of MEF. It was. Specifically, the embryoid body formed from ES cells cultured in a collagen degradation product gel culture dish under MEF (-) and LIF (+) culture conditions adheres to or floats on the collagen degradation product gel. It has been shown.

<13. Test on embryoid body formation ability of collagen degradation product 4>
Using a culture dish not seeded with MEF instead of the culture dish seeded with MEF, the same experiment was performed in DMEM medium containing 20% fetal bovine serum without LIF (KnockOutDMEM, manufactured by Thermo Fisher Science). (Hereinafter referred to as "DMEM medium (LIF (-))"). Specifically, DMEM medium (LIF (−)) was used to prepare 2.5 × 10 ⁴ mouse ES cells / mL. 2 mL of mouse ES cell suspension was seeded in a gelatin-coated culture dish, a collagen degradation product-coated culture dish, and a collagen degradation product gel culture dish in which MEF was not seeded, and DMEM medium (LIF (-)) was used. , 37 ° C., 5% CO ₂ (culture conditions: MEF (−) and LIF (−)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment.

During the culture period, the medium was replaced with fresh DMEM medium (LIF (−)) every day.

Three days after the start of the culture, the morphology of ES cells was observed under a phase-contrast microscope. The morphology of each cell is shown in FIG. In FIG. 5, (a) is a diagram showing the morphology of ES cells cultured in a gelatin-coated culture dish in a DMEM medium (LIF (−)) for 3 days, and (b) is a collagen decomposition product-coated culture. It is a figure which shows the morphology of ES cells cultured for 3 days using DMEM medium (LIF (−)) in a dish, (c) is the DMEM medium (LIF (−)) in a collagen decomposition product gel culture dish. It is a figure which shows the morphology of the ES cell which was cultured for 3 days using.

When cultured in a gelatin-coated culture dish, ES cells spread in a single layer, and colonized cells and single-adhered cells were observed separately. There were also cells showing a morphology showing cell processes. Compared to the culture findings (FIG. 1 (a)) of general ES cells in the presence of MEF and LIF, the number of colonies is smaller and the size is smaller, and the cell-cell adhesion is such that each cell can be distinguished. Was shown ((a) in FIG. 5).

When cultured in a collagen-degraded product-coated culture dish, the observed ES cells form an adherent or floating three-dimensional cell mass (embryoid body) (FIG. 5 (b)), which is a general gelatin-coated culture dish. The morphology was completely different from the typical ES cell culture findings ((a) in FIG. 5). If anything, the proportion of floating embryoid bodies was high.

When cultured in a collagen degradation product gel culture dish, the observed ES cells formed a three-dimensional cell mass (embryoid body) adhering to or floating on the collagen degradation product gel. The morphology was completely different from the general ES cell culture findings of the gelatin-coated culture dish. The embryoid bodies adhering to the collagen degradation gel were relatively larger than the floating embryoid bodies. ((C) in FIG. 5).

The morphology of ES cells cultured in a collagen degradation product-coated culture dish or a collagen degradation product gel culture dish using DMEM medium (LIF (-)) in the absence of MEF (MEF (-)) is expressed in a gelatin-coated culture dish. The morphology of the cultured ES cells was completely different. However, in the presence of MEF (MEF (+)), a collagen degradation product coated culture dish (FIG. 1 (b)) or a collagen degradation product gel culture dish (FIG. 1 (c)) using DMEM medium (LIF (+)). ) Was cultured in a collagen degradation product-coated culture dish or a collagen degradation product gel culture dish using DMEM medium (LIF (-)) in the absence of MEF (MEF (-)) as compared with the ES cells cultured in. The morphology of ES cells was slightly different in size and shape, but there was no clear difference. Under MEF (−) and LIF (−) culture conditions, ES cells cultured in gelatin-coated culture dishes either formed colonies of common ES cells or were single adherent cells. It should be noted that the ES cells cultured in the collagen decomposition product-coated culture dish under the culture conditions of MEF (-) and LIF (-) formed embryoid bodies even though the MEF was not co-cultured. That is, it was shown that culturing ES cells in a collagen decomposition product-coated culture dish can be a new method for forming embryoid bodies. Similarly, in ES cells cultured in a collagen degradation product gel culture dish under MEF (-) and LIF (-) culture conditions, embryoid bodies that have not been observed so far are formed in the absence of MEF. It was. Specifically, the embryoid body formed from ES cells cultured in a collagen degradation product gel culture dish under MEF (-) and LIF (-) culture conditions adheres to or floats on the collagen degradation product gel. It has been shown.

<14. Test on embryoid body formation ability of collagen degradation product 5>
Next, it was confirmed by culturing GFP-expressing ES cells whether or not the obtained embryoid body consisted only of ES cells. Specifically, mouse ES using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Scientific) containing 500 U / mL LIF and 20% fetal bovine serum (hereinafter referred to as "DMEM medium (LIF (+))"). Cells 2.5 × 10 ⁴ cells / mL were prepared. 2 mL of mouse ES cell suspension was seeded in a gelatin-coated culture dish or a collagen decomposition product gel culture dish in which MEF had been seeded in advance, and the DMEM medium (LIF (+)) was used at 37 ° C. Cultured under 5% CO ₂ conditions for 4 days (culture conditions: MEF (+) and LIF (+)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment.

Further, using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Scientific) containing 20% fetal bovine serum without LIF (hereinafter referred to as "DMEM medium (LIF (-))"), mouse ES cells 2.5 × 10 ⁴ pieces / mL was prepared. 2 mL of mouse ES cell suspension was seeded in a collagen decomposition product gel culture dish in which MEF had been seeded in advance, and DMEM medium (LIF (-)) was used under the conditions of 37 ° C. and 5% CO ₂ . (Culture conditions: MEF (+) and LIF (-)).

After culturing for 4 days, the nuclei of cells obtained by culturing under the respective culture conditions were stained with Cellstain®-Hoechst 33342 solution (product number H342, Dojin Chemical Laboratory Co., Ltd.).

The results are shown in FIGS. 6 to 8. FIG. 6 is a diagram showing the results of observing ES cells cultured in a gelatin-coated culture dish for 4 days under a microscope under MEF (+) and LIF (+) culture conditions, and FIG. 6A is a phase contrast diagram. It is a figure which shows the morphology of ES cell observed under a microscope, (b) is a figure which shows the result of observing the fluorescence of GFP of ES cell shown in (a) under a fluorescence microscope, and (c) is a figure which shows. , (A) is a diagram showing the results of staining the ES cells shown in (a) with Hoechst 33342 and observing them under a fluorescence microscope, and (d) is a superposition of the images of (a), (b) and (c). It is a figure which shows the result. In addition, FIG. 7 shows the results of observing the embryonic bodies obtained after culturing ES cells in a collagen degradation product gel culture dish for 4 days under the culture conditions of MEF (+) and LIF (+) under a microscope. It is a figure, (a) is a figure which shows the morphology of the embryo-like body observed under the phase-contrast microscope, (b) is the fluorescence of the GFP of the embryo-like body shown in (a) under the fluorescence microscope. It is a figure which shows the observation result, (c) is the figure which showed the result of having Hoechst 33342 stained the embryo-like body shown in (a), and observed under the fluorescence microscope, (d) is the figure which shows (a). It is a figure which shows the result of superimposing the images of (b) and (c). In addition, FIG. 8 shows the results of observing the embryonic bodies obtained after culturing ES cells in a collagen degradation product gel culture dish for 4 days under the culture conditions of MEF (+) and LIF (-) under a microscope. It is a figure, (a) is a figure which shows the morphology of the embryo-like body observed under the phase-contrast microscope, (b) is the fluorescence of the GFP of the embryo-like body shown in (a) under the fluorescence microscope. It is a figure which shows the observation result, (c) is the figure which showed the result of having Hoechst 33342 stained the embryo-like body shown in (a), and observed under the fluorescence microscope, (d) is the figure which shows (a). It is a figure which shows the result of superimposing the images of (b) and (c).

As a result, it was confirmed that when ES cells were cultured in a gelatin-coated culture dish under MEF (+) and LIF (+) culture conditions, GFP-positive ES cells spread in a single layer and formed colonies (FIG. 6). (A) to (d)).

In addition, when ES cells are cultured in a collagen degradation product gel culture dish using DMEM medium (LIF (+)) or DMEM medium (LIF (-)) in the presence of MEF (MEF (+)), it is observed. The GFP-positive ES cells formed a three-dimensional cell mass (embryonic stem) adhering to the collagen degradation product gel. The morphology was completely different from the general ES cell culture findings of the gelatin-coated culture dish. Since the cells other than the embryoid body were GFP negative, it was inferred that the cells other than the embryoid body were MEF ((a) to (d) in FIG. 7 and (a) to (d) in FIG. 8). ). In addition, it was presumed that the part surrounded by the broken line in FIG. 8A was collagen and secretions from cells.

From the above results, it was shown that the embryoid bodies formed in the collagen degradation product gel culture dish in the presence of MEF were derived from ES cells regardless of the presence or absence of LIF in the culture medium.

<15. Test on embryoid body formation ability of collagen degradation product 6>
(Human iPS cells)
Human iPS cells (201B7 strain, RIKEN BRC) established by Professor Shinya Yamanaka's laboratory at Kyoto University were used. In the iPS cell line, all cells were 46XX by chromosomal analysis and no chromosomal abnormality was observed.

(Preparation of collagen decomposition product coated culture dish)
A decomposition product of porcine-derived collagen was prepared at 10 mg / mL, coated with 50 μL on a 6-well plate, and allowed to stand for 3 hours. As the collagen decomposition product, the above-mentioned collagen decomposition product (among the collagen decomposition products derived from pigs at a salt concentration of 0 mM, the amino acid sequence shown in SEQ ID NO: 2 (the amino acid sequence shown in (3)) Decomposition product with equivalent) was used. As a control group 1, 1 mL of Vitronectin (Thermo Fisher Scientific Co., Ltd.) diluted 100-fold with D-PBS was added to 1 well of a 6-well plate, and the mixture was allowed to stand for 1 hour or more. As a control group 2, iMatrix 511 (Nippi Co., Ltd.) was coated at 0.5 μg / cm ² .

(Embryoid formation of human iPS cells)
Human iPS cells were adjusted to 1.0 × 10 ⁵ cells using a NutriStem medium (Cosmo Bio Co., Ltd.) dedicated to iPS cells, and seeded in the culture dish prepared above. Culturing was carried out under the conditions of 37 ° C. and 5% CO ₂ for 5 days. During the culture period, the medium was replaced with fresh NutriStem medium daily.

The morphology of iPS cells was observed under a phase-contrast microscope 1 day and 5 days after the start of culturing. The morphology of each cell is shown in FIGS. 9 and 10. In FIG. 9, (a) is a diagram showing the morphology of iPS cells cultured for 1 day in a collagen decomposition product-coated culture dish, and (b) is a diagram showing the morphology of iPS cells cultured for 1 day in a Vitronectin-coated culture dish. (C) is a diagram showing the morphology of iPS cells cultured for 1 day in an iMatrix511 coated culture dish. In FIG. 10, (a) is a diagram showing the morphology of iPS cells cultured for 5 days in a collagen decomposition product-coated culture dish, and (b) is a diagram showing the morphology of iPS cells cultured for 5 days in a Vitronectin-coated culture dish. (C) is a diagram showing the morphology of iPS cells cultured for 5 days in an iMatrix511 coated culture dish.

When cultured in a collagen decomposition product-coated culture dish, it was observed that iPS cells were gathered after 1-day culture. After culturing for 5 days, embryoid bodies were formed (FIG. 9 (a) and FIG. 10 (a)).

When cultured in a Vitronectin-coated culture dish, it had the same morphology as general iPS cell culture findings. That is, after culturing for one day, the iPS cells individually adhered to the bottom surface of the culture dish and were in the cell morphology of monolayer culture. After culturing for 5 days, the iPS cells became hyperproliferative and were shown to adhere to all of the bottom surface of the culture dish (FIG. 9 (b) and FIG. 10 (b)).

When cultured in the iMatrix511 coated culture dish, the iPS cells adhered to the bottom surface of the culture dish. The observation images 1 day after culturing and 5 days after culturing were similar to the general iPS cell culture findings of the Vitronectin-coated culture dish.

The morphology of iPS cells cultured in collagen degradation coated dishes was quite different from the morphology of iPS cells cultured in Vitronectin-coated dishes or iMatrix511-coated dishes. The iPS cells cultured in the Vitronectin-coated culture dish only showed general iPS cell proliferation, but the iPS cells cultured in the collagen degradation product-coated culture dish did not add LIF or the like. Formed an embryoid body. That is, it was shown that culturing iPS cells in a collagen decomposition product-coated culture dish can be a new method for forming embryoid bodies.

<16. Test on embryoid body formation ability of collagen degradation product 7>
(Culturing iPS cells in a medium containing no basic fibroblast growth factor)
The same experiment as in <15> was extended to the 12th day of culture using a medium containing no basic fibroblast growth factor (bFGF), and the morphology of the embryoid body was observed. That is, as the human iPS cells and collagen degradation products, the same ones as in <15> were used.

(Preparation of collagen decomposition product coated culture dish)
The collagen degradation product was prepared at 10 mg / mL, coated with 50 μL on a 6-well plate, and allowed to stand for 3 hours. As a control group, Lipdure (NOF CORPORATION) whose plastic surface was processed with 2-methacryloyloxyethyl phosphorylcholine monomer was used. It is widely known that iPS cells form floating embryoid bodies when cultured in Lipdure culture dishes.

(Embryoid formation of human iPS cells)
Human iPS cells were adjusted to 3.0 × 10 ⁵ cells using a NutriStem medium (Cosmo Bio Co., Ltd.) dedicated to iPS cells, and seeded in the culture dish prepared above. Culturing was carried out under the conditions of 37 ° C. and 5% CO ₂ for 12 days. During the culturing period, half the amount was replaced with fresh NutriStem medium every day until the 4th day of culturing. After the 5th day of culturing, half of the amount was exchanged every other day using hiPS medium, which is a medium for iPS cells containing no basic fibroblast growth factor (bFGF).

The morphology of iPS cells was observed under a phase-contrast microscope 4 days and 12 days after the start of culturing. The morphology of each cell after 4 and 12 days is shown in FIGS. 11 and 12, respectively. In FIG. 11, (a) is a diagram showing the morphology of iPS cells cultured for 4 days in a collagen decomposition product-coated culture dish, and (b) shows the morphology of iPS cells cultured for 4 days in a Lipidure culture dish. It is a figure which shows. FIG. 12 (a) is a diagram showing the morphology of iPS cells cultured for 12 days in a collagen decomposition product-coated culture dish, and FIG. 12 (b) is a diagram of iPS cells cultured for 12 days in a Lipidure culture dish. It is a figure which shows the form.

When cultured in a collagen decomposition product-coated culture dish, the appearance of iPS cells forming embryoid bodies was observed after 4 days of culture (FIG. 11 (a)). Furthermore, as a result of culturing in a special medium containing no bFGF until the 12th day, it was shown that the embryoid body was maintained ((a) in FIG. 12).

When cultured in a Lipidure culture dish for 4 days, a floating embryoid body was shown as in the case of general iPS cell culture findings (FIG. 11 (b)). Furthermore, as a result of culturing in a special medium containing no bFGF until the 12th day, it was shown that the embryoid body became enormous ((b) in FIG. 12).

The embryoid body formation of human iPS cells using a collagen decomposition product-coated culture dish is easier to exchange the medium because it adheres to the bottom surface of the culture dish as compared with the embryoid body formation of human iPS cells in a Lipidure culture dish. The advantage of having it became clear. The Lipidure culture dish is prone to accidental loss of cells due to suction when changing the medium. In addition, there was a concern that the embryoid bodies recovered from the Lipidure culture dish would come into contact with each other and become huge during the centrifugation operation in the medium exchange. On the other hand, it was found that such a problem is unlikely to occur in the collagen decomposition product coated culture dish. Furthermore, it was clarified that the embryoid bodies of iPS cells formed in the collagen degradation product-coated culture dish did not collapse even when cultured in a special medium containing no bFGF.

Embryoid bodies of human iPS cells cultured in collagen decomposition dish-coated culture dishes did not show a clear difference in morphological findings as compared with human iPS cells in Lipidure culture dishes. That is, it was shown that culturing the embryoid body of human iPS cells in a collagen decomposition product-coated culture dish can be a new method for maintaining the embryoid body.

<17. Test on induction of embryoid body differentiation of collagen degradation products>
Next, it was confirmed that the embryoid body of mouse ES cells obtained by using a degradation product of collagen can be induced to differentiate. Specifically, EB-dedicated medium containing no LIF (20% fetal bovine serum, 1 mM MEM Sodium Pyruvate Solution, 1/100 diluted MEM Non Essential Amino Acid Solution, 10 μM β-mercaptoethanol added to DMEM medium) ( Hereinafter, referred to as “EB medium”) was used to prepare mouse ES cells 5.0 × 10 ⁴ cells / well. Next, a 12-well plate collagen decomposition product-coated culture dish or a Lipidure culture dish prepared in advance by the same method as above was prepared, and mouse ES cell suspensions were seeded respectively. The cells were cultured in EB medium under the conditions of 37 ° C. and 5% CO ₂ for 15 days. The medium was replaced with new EB medium every day after 2 days of culturing. ES cells before seeding cultured in a gelatin-coated culture dish were used as controls in this experiment.

(Extraction of RNA from mouse ES cell embryoid body)
After culturing for 15 days, embryoid bodies of mouse ES cells obtained by culturing under the respective culture conditions were collected by centrifugation, and the respective RNAs were collected in total RNA purification buffer Lysis Buffer RA1 (Takara Bio Co., Ltd.) and β. -Mixed with mercaptoethanol, further 70% ethanol was added and mixed well. The obtained solution was centrifuged on a spin column for RNA purification, adsorbed on a gel, washed, and further treated with DNase to elute from the column to purify the total RNA of embryoid bodies in each culture dish.

(Preparation of RNA cDNA from mouse ES cell embryoid body)
By a conventional method, cDNA was synthesized by the RT-PCR method using total RNA as a template using PrimeScript RT Master Mix (Perfect Real Time) manufactured by Takara Bio Inc. The cDNA solution after the reaction was stored at −20 ° C. or −80 ° C. until the next experiment.

(Analysis of embryoid body RNA of mouse ES cells)
In order to investigate the effect of culturing with collagen degradation products on differentiation, the RNA expression levels of the following three differentiation markers were examined. GATA4 (GATA Binding Protein 4) involved in mesoderm, endoderm and mesoderm differentiation, AFP (α-fetoprotein) specific to endoderm hepatocytes, NKX2.5 specific to myocardial progenitor cells The RNA expression level was quantified using the primers of NK-2 differentiation factor protected, locus 5). Using the cDNA obtained above as a template, each primer, a reagent such as SYBR Fast qPCR Mix, and an appropriate amount of ultrapure water are mixed, and using Thermal Cycler Dice Real Time System (Takara Bio Inc.), a conventional method is used. Quantitative RT-PCR was performed.

FIG. 13 shows a radar chart of the relative expression levels of each marker, with the maximum expression level obtained from the RT-PCR results as 100. It was revealed that the embryoid bodies of mouse ES cells formed in the collagen decomposition product coated culture dish had a low relative expression level of NKX2.5 but a high relative expression level of GATA4 and AFP (FIG. 13 (a)). ). The RNA relative expression level of the embryoid body formed in the Lipidure culture dish was similar to that of the embryoid body in the collagen decomposition product coated culture dish (FIG. 13 (b)). On the other hand, in the mouse ES cells of the control gelatin-coated culture dish, on the contrary, the relative expression level of NKX2.5 was shown to be higher than that of GATA4 and AFP (FIG. 13 (c)). That is, it was clarified that the embryoid body of the collagen decomposition product-coated culture dish has a great influence on the differentiation of ES cells as well as the embryoid body of the Lipidure culture dish. The opposite of the results of the cells in the gelatin-coated culture dish indicates that it is suitable for the induction of differentiation peculiar to the embryoid body.

Induction of differentiation of ES cells and iPS cells is essential through the embryoid body, and it is important to satisfy appropriate conditions for inducing differentiation after embryoid body formation. Therefore, it was proved that the embryoid body of the collagen decomposition product-coated culture dish also has a sufficient ability to induce differentiation. Since the embryoid body of the conventional Lipidure culture dish is floating, it is easy to form a large agglomerate. Therefore, the differentiation of ES cells and iPS cells in the Lipidure culture dish greatly affects the quality of differentiation. On the other hand, the embryoid body of the collagen decomposition product coated culture dish can improve the reproducibility and quality of differentiation.

<18. Test on embryoid body formation ability of collagen degradation product 8>
(Culture dish)
The above-mentioned collagen degradation product (type I collagen degradation product derived from pig skin (SEQ ID NO: 27), or type I collagen degradation product derived from rat tendon (mixture of SEQ ID NO: 24 and SEQ ID NO: 5), or pig A commercially available culture dish in contact with a degradation product of type I collagen derived from the skin (SEQ ID NO: 26) was used for the test. Specifically, the concentration of collagen degradation product in a 35 mm culture dish (type I collagen degradation product derived from pig skin (SEQ ID NO: 27) is 3.2 mg / mL to 9.6 mg / mL, type I derived from rat tendon). Collagen degradation product (mixture of SEQ ID NO: 24 and SEQ ID NO: 5) is 2.8 mg / mL to 8.4 mg / mL, and type I collagen degradation product derived from pig skin (SEQ ID NO: 26) is 3.3 mg / mL. mL) was changed and added, and the mixture was sufficiently blended in a culture dish and allowed to stand at room temperature for 5 hours to prepare a collagen decomposition product-coated culture dish.

(Mouse ES cells)
Mouse ES cells transferred from Professor Teruhiko Wakayama, Faculty of Life and Environmental Sciences, University of Yamanashi, were used. These mouse ES cells are labeled with GFP (green fluorescent protein), and the gene encoding the GFP protein is inserted into the chromosome.

(Preparation of feeder cells)
Mitomycin-treated mouse embryo-derived fibroblasts (MEF) (CMPMECFF, manufactured by DS Pharma Biomedical) were prepared at 6.1 × 10 ⁴ cells / mL. 1.2 x 10 ⁵ MEFs were sown in a gelatin-coated culture dish, an atelocollagen-coated culture dish (porcine tendon-derived pepsin-solubilized type I collagen, COL 1, manufactured by Asahi Technograss), and a collagen decomposition product-coated culture dish. Then, the cells were cultured in DMEM medium (DMEM, manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% bovine fetal serum under the conditions of 37 ° C. and 5% CO ₂ for 1 day.

(Formation of embryoid body)
Using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Science) containing 500 U / mL of Leukemia Inhibitory Factor (LIF) and 20% fetal bovine serum (hereinafter referred to as "DMEM medium (LIF (+))"). , 2.5 × 10 ⁵ mouse ES cells / mL were prepared. 5.0 × 10 ⁴ cells / cm ^{2 of} mouse ES cells were seeded in each of a gelatin-coated culture dish, an atelocollagen-coated culture dish, and a collagen decomposition product-coated culture dish in which MEF had been seeded in advance, and the DMEM medium (LIF (LIF) +)) Was used to culture under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (+) and LIF (+)). ES cells cultured in a gelatin-coated culture dish and an atelocollagen-coated culture dish were used as controls in this experiment. In the examples, "MEF (+)" indicates culture in the coexistence of MEF. Further, "LIF (+)" indicates culture in the coexistence of LIF.

One day after the start of culturing, the morphology of ES cells was observed under a phase-contrast microscope. The morphology of each cell is shown in FIG. In FIG. 14, (a) is a diagram showing the morphology of ES cells cultured in a gelatin-coated culture dish in the presence of MEF (MEF (+)) for 1 day using DMEM medium (LIF (+)). , (B) are diagrams showing the morphology of ES cells cultured for 1 day in DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in an Atelocollagen-coated culture dish. ) Are ES cells cultured for 1 day in DMEM medium (LIF (+)) in a collagen decomposition product-coated culture dish (derived from pig skin, SEQ ID NO: 27) in the presence of MEF (MEF (+)). It is a figure which shows the morphology, (d) is a DMEM medium (from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5) in the presence of MEF (MEF (+)) in a collagen decomposition product coated culture dish. It is a figure which shows the morphology of ES cells cultured for 1 day using LIF (+)), (e) is a collagen decomposition product coated culture dish (derived from the pig skin part, SEQ ID NO: 26) in the presence of MEF (MEF). (+)) Is a diagram showing the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)). When cultured in a gelatin-coated culture dish and an atelocollagen-coated culture dish, the MEF adhered to the bottom surface of the gelatin-coated culture dish and extended. During that time, ES cells were forming colonies. It had the same morphology as general ES cell culture findings (FIGS. 14 (a) and 14 (b)).

When cultured in a collagen degradation product-coated culture dish, the ES cells form a three-dimensional cell mass (embryonic stem cell), which is completely different from the general ES cell culture findings of the gelatin-coated culture dish and the atelocollagen-coated culture dish. It had a different form ((c) to (e) in FIG. 14). In addition, MEF adhered to the bottom surface of the collagen decomposition product-coated culture dish, but did not extend to the entire bottom surface unlike the gelatin-coated culture dish and the atelocollagen-coated culture dish, and mainly with collagen on the bottom surface of the embryoid body of ES cells. It was glued.

The morphology of ES cells cultured in collagen degradation-coated culture dishes was completely different from the morphology of ES cells cultured in gelatin-coated culture dishes and atelocollagen-coated culture dishes. The ES cells cultured in the gelatin-coated culture dish and the atelocollagen-coated culture dish only formed colonies of general ES cells, whereas the ES cells cultured in the collagen degradation product-coated culture dish formed adherent embryonic stem cells. Was formed. That is, animal species of collagen degradation products (eg, pigs, rats), origin sites (eg, skin and tendons) and N-terminal cleavage sites (eg, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: 5). Regardless, culturing ES cells in a collagen degradation-coated dish has been shown to be a new way to form embryoid bodies.

<19. Test on embryoid body formation ability of collagen degradation product 9>
In order to examine the undifferentiated ability of ES cells cultured in a gelatin-coated culture dish, an atelocollagen-coated culture dish, and a collagen degradation product-coated culture dish, the intracellular alkaline phosphatase activity was examined.

(Culture dish)
The above-mentioned collagen degradation product (type I collagen degradation product derived from pig skin (SEQ ID NO: 27), or type I collagen degradation product derived from rat tendon (mixture of SEQ ID NO: 24 and SEQ ID NO: 5), or pig A commercially available culture dish in contact with a degradation product of type I collagen derived from the skin (SEQ ID NO: 26) was used for the test. Specifically, the concentration of collagen degradation product in a 35 mm culture dish (type I collagen degradation product derived from pig skin (SEQ ID NO: 27) is 3.2 mg / mL to 9.6 mg / mL, type I derived from rat tendon). Collagen degradation product (mixture of SEQ ID NO: 24 and SEQ ID NO: 5) is 2.8 mg / mL to 8.4 mg / mL, and type I collagen degradation product derived from pig skin (SEQ ID NO: 26) is 3.3 mg / mL. mL) was changed and added, and the mixture was sufficiently blended in a culture dish and allowed to stand at room temperature for 5 hours to prepare a collagen decomposition product-coated culture dish.

(Formation of embryoid body)
2.5 x 10 ⁵ pieces using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Scientific) containing 500 U / mL LIF and 20% fetal bovine serum (hereinafter referred to as "DMEM medium (LIF (+))"). / ML was prepared. 5.0 × 10 ⁴ cells / cm ^{2 of} mouse ES cells were seeded in each of a gelatin-coated culture dish, an atelocollagen-coated culture dish, and a collagen decomposition product-coated culture dish in which MEF had been seeded in advance, and the DMEM medium (LIF (LIF) +)) Was used to culture under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (+) and LIF (+)). ES cells cultured in a gelatin-coated culture dish and an atelocollagen-coated culture dish were used as controls in this experiment.

After culturing for 1 day, the culture supernatant of the cells was removed and washed with sterile PBS. Subsequently, a 4% paraformaldehyde solution was added to fix the cells until the cells were immersed, and the cells were allowed to stand at room temperature for 30 minutes. Then, the operation of adding sterile distilled water until the cells were immersed and washing was repeated twice. After removing the washing liquid, the alkaline phosphatase stain attached to the TRACP & ALP double-stain kit (product code MK300, manufactured by Takara Bio Inc.) was added to the culture dish and allowed to stand at 37 ° C. for 45 minutes. To stop the reaction, the stain was removed and washed 3 times with sterile distilled water. The results are shown in FIG.

FIG. 15 is a diagram showing the results of examining the intracellular alkaline phosphatase activity of ES cells cultured for 1 day under MEF (+) and LIF (+) culture conditions, and FIG. 15A is a diagram showing the results of examining the intracellular alkaline phosphatase activity. It is a figure which shows the result of alkaline phosphatase staining of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a gelatin-coated culture dish, (b) is a figure which shows. , A figure showing the results of alkaline phosphatase staining of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in an Atelocollagen coated culture dish, (c). Is alkaline of ES cells cultured for 1 day in DMEM medium (LIF (+)) in the presence of MEF (MEF (+)) in a collagen decomposition product coated culture dish (derived from pig skin, SEQ ID NO: 27). It is a figure which shows the result of phosphatase staining, (d) is a collagen decomposition product coated culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5) in the presence of MEF (MEF (+)). It is a figure which shows the result of alkaline phosphatase staining of ES cells cultured for 1 day using DMEM medium (LIF (+)), (e) is a collagen decomposition product coated culture dish (from pig skin part, SEQ ID NO: 26). It is a figure which shows the result of alkaline phosphatase staining of ES cells cultured for 1 day using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)).

Embryonic stems formed from ES cells cultured in collagen degradation-coated culture dishes were shown to be alkaline phosphatase activity-positive, similar to colonies of ES cells cultured in gelatin-coated culture dishes and atelocollagen-coated culture dishes. ((C) to (e) in FIG. 15). On the other hand, the co-cultured MEF was negative for alkaline phosphatase activity. These results show that the collagen degradation product species (eg, pigs, rats), origin sites (eg, skin and tendons) and N-terminal cleavage sites (eg, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: Regardless of No. 5), it is shown that the embryoid bodies formed from ES cells cultured in the collagen decomposition product coated culture dish maintain the undifferentiated ability.

<20. Test on embryoid body formation ability of collagen degradation product 10>
Next, it was confirmed whether or not the embryoid body obtained by culturing the GFP-expressing ES cells consisted only of ES cells.

(Culture dish)
The above-mentioned collagen degradation product (type I collagen degradation product derived from pig skin (SEQ ID NO: 27), or type I collagen degradation product derived from rat tendon (mixture of SEQ ID NO: 24 and SEQ ID NO: 5), or pig A commercially available culture dish in contact with a degradation product of type I collagen derived from the skin (SEQ ID NO: 26) was used for the test. Specifically, the concentration of collagen degradation product in a 35 mm culture dish (type I collagen degradation product derived from pig skin (SEQ ID NO: 27) is 3.2 mg / mL to 9.6 mg / mL, type I derived from rat tendon). Collagen degradation product (mixture of SEQ ID NO: 24 and SEQ ID NO: 5) is 2.8 mg / mL to 8.4 mg / mL, and type I collagen degradation product derived from pig skin (SEQ ID NO: 26) is 3.3 mg / mL. mL) was changed and added, and the mixture was sufficiently blended in a culture dish and allowed to stand at room temperature for 5 hours to prepare a collagen decomposition product-coated culture dish.

(Formation of embryoid body)
Specifically, using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Scientific) containing 500 U / mL LIF and 20% fetal bovine serum (hereinafter referred to as "DMEM medium (LIF (+))"), 2. 5 × 10 ⁵ pieces / mL was prepared. 5.0 × 10 ⁴ cells / cm ^{2 of} mouse ES cells were seeded in a gelatin-coated culture dish and a collagen degradation product-coated culture dish in which MEF had been seeded in advance, and DMEM medium (LIF (+)) was used. The cells were cultured under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (+) and LIF (+)). ES cells cultured in a gelatin-coated culture dish were used as controls for this experiment.

The results are shown in FIGS. 16 to 19. FIG. 16 shows the results of microscopic observation of colonies obtained after culturing ES cells containing the GFP gene in a gelatin-coated culture dish for 1 day under MEF (+) and LIF (+) culture conditions. It is a figure, (a) is a figure which shows the morphology of the said colony observed under a phase contrast microscope, (b) is a figure which observed the fluorescence of GFP of the said colony shown in (a) under a fluorescence microscope. It is a figure which shows the result, (c) is a figure which shows the result of superimposing the images of (a) and (b). In addition, FIG. 17 is obtained after culturing ES cells containing a GFP gene in a collagen decomposition product-coated culture dish (derived from pig skin, SEQ ID NO: 27) under MEF (+) and LIF (+) culture conditions for 1 day. It is a figure which shows the result of observing the embryoid body under a microscope, (a) is a figure which shows the morphology of the said embryoid body observed under a phase contrast microscope, and (b) is (a It is a figure which shows the result of observing the fluorescence of the GFP of the embryoid body shown in) under a fluorescence microscope, and (c) is a figure which shows the result of superimposing the images of (a) and (b). .. In addition, FIG. 18 shows ES cells containing a GFP gene in a collagen decomposition product-coated culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5) under MEF (+) and LIF (+) culture conditions. It is a figure which shows the result of observing the embryoid body obtained after culturing for one day under a microscope, and (a) is the figure which shows the morphology of the said embryoid body observed under a phase-contrast microscope. b) is a diagram showing the result of observing the fluorescence of the GFP of the embryoid body shown in (a) under a fluorescence microscope, and (c) is a diagram in which the images of (a) and (b) are superimposed. It is a figure which shows the result. Further, FIG. 19 is obtained after culturing ES cells containing a GFP gene for 1 day in a collagen decomposition product-coated culture dish (derived from pig skin, SEQ ID NO: 26) under MEF (+) and LIF (+) culture conditions. It is a figure which shows the result of observing the embryoid body under a microscope, (a) is a figure which shows the morphology of the said embryoid body observed under a phase contrast microscope, and (b) is (a It is a figure which shows the result of observing the fluorescence of the GFP of the embryoid body shown in) under a fluorescence microscope, and (c) is a figure which shows the result of superimposing the images of (a) and (b). ..

As a result, it was confirmed that when ES cells were cultured in a gelatin-coated culture dish under MEF (+) and LIF (+) culture conditions, GFP-positive ES cells spread in a single layer and formed colonies (FIG. 16). (A) to (c)).

In addition, when ES cells were cultured in a collagen degradation product-coated culture dish using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)), the observed GFP-positive ES cells were collagen. A three-dimensional cell mass (embryoid body) adhered to the decomposition product coat was formed. This was a completely different form from the general ES cell culture findings of the gelatin-coated culture dish. Since the cells other than the embryoid body were GFP negative, it was inferred that the cells other than the embryoid body were MEF ((a) to (c) in FIGS. 17 and (a) to (c) in FIGS. 18). And (a) to (c) of FIG.

From the above results, the animal species of collagen degradation products (eg, pigs, rats), origin sites (eg, skin and tendons) and N-terminal cleavage sites (eg, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: Regardless of No. 5), the embryoid bodies formed in the collagen degradation product-coated culture dish in the presence of MEF were shown to be derived from ES cells.

<21. Test on embryoid body formation ability of collagen degradation product 11>
(Culture dish)
The above-mentioned collagen degradation product (type I collagen degradation product derived from pig skin (SEQ ID NO: 27), or type I collagen degradation product derived from rat tendon (mixture of SEQ ID NO: 24 and SEQ ID NO: 5), or pig A commercially available culture dish in contact with a degradation product of type I collagen derived from the skin (SEQ ID NO: 26) was used for the test. Specifically, the concentration of collagen degradation product in a 35 mm culture dish (type I collagen degradation product derived from pig skin (SEQ ID NO: 27) is 3.2 mg / mL to 9.6 mg / mL, type I derived from rat tendon). Collagen degradation product (mixture of SEQ ID NO: 24 and SEQ ID NO: 5) is 2.8 mg / mL to 8.4 mg / mL, and type I collagen degradation product derived from pig skin (SEQ ID NO: 26) is 3.3 mg / mL. mL) was changed and added, and the mixture was sufficiently blended in a culture dish and allowed to stand at room temperature for 5 hours to prepare a collagen decomposition product-coated culture dish.

(Formation of embryoid body)
Using a culture dish not seeded with MEF instead of the culture dish seeded with MEF, a similar experiment was performed in DMEM medium containing LIF and 20% fetal bovine serum (KnockOutDMEM, Thermofisher Science). (Hereinafter referred to as "DMEM medium (LIF (+))"). Specifically, using DMEM medium (LIF (+)), mouse ES cells 2.5 × 10 ⁵ cells / mL were prepared. 5.0 × 10 ⁴ cells / cm ^{2 of} mouse ES cells were seeded in each of a gelatin-coated culture dish, an atelocollagen-coated culture dish, and a collagen decomposition product-coated culture dish in which MEF was not seeded, and the DMEM medium (LIF (+)) was seeded. ) Was cultured under the conditions of 37 ° C. and 5% CO ₂ (culture conditions: MEF (−) and LIF (+)). ES cells cultured in a gelatin-coated culture dish and an atelocollagen-coated culture dish were used as controls in this experiment. In the examples, "MEF (-)" indicates culture in the absence of MEF. Further, "LIF (+)" indicates culture in the coexistence of LIF.

One day after the start of culturing, the morphology of ES cells was observed under a phase-contrast microscope. The morphology of each cell is shown in FIG. In FIG. 20, (a) is a diagram showing the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)) in a gelatin-coated culture dish, and (b) is a diagram showing the morphology of ES cells cultured in an atelocollagen-coated culture dish. , DMEM medium (LIF (+)) is a diagram showing the morphology of ES cells cultured for 1 day, in which (c) is a DMEM in a collagen decomposition product coated culture dish (derived from pig skin, SEQ ID NO: 27). It is a figure which shows the morphology of ES cell culture | culture | culture | culture | culture medium (LIF (+)) for 1 day, (d) is the collagen decomposition product coated culture dish (derived from rat tendon, the mixture of SEQ ID NO: 24 and SEQ ID NO: 5 ) Shows the morphology of ES cells cultured for 1 day using DMEM medium (LIF (+)), and (e) is a collagen decomposition product coated culture dish (derived from pig skin, SEQ ID NO: 26). It is a figure which shows the morphology of the ES cell which was cultured for 1 day using DMEM medium (LIF (+)).

When cultured in a gelatin-coated culture dish and an atelocollagen-coated culture dish, ES cells spread in a single layer, and single adherent cells were observed. There were also cells showing a morphology showing cell processes. Compared with the culture findings of general ES cells in the presence of MEF and in the presence of LIF ((a) and (b) in FIG. 14), it is shown that the cell-cell adhesion is typical enough to distinguish each cell. ((A) and (b) in FIG. 20).

When cultured in a collagen decomposition product-coated culture dish, the observed ES cells formed adherent or floating three-dimensional cell clusters (embryonic stem cells) (FIGS. 20 (c) to (e)), and were gelatin-coated. The morphology was completely different from the general ES cell culture findings ((a) and (b) in FIGS. 14) of the culture dish and the atelocollagen-coated culture dish.

In the absence of MEF (MEF (-)), the morphology of ES cells cultured in a collagen degradation product-coated culture dish using DMEM medium (LIF (+)) is the morphology of collagen degradation product animal species (eg, pigs, rats). ), Origin sites (eg, skin and tendons) and N-terminal cleavage sites (eg, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: 5) in gelatin-coated and atelocollagen-coated dishes. The morphology of the cultured ES cells was completely different. However, in comparison with ES cells ((c) to (e) in FIG. 14) cultured in a collagen decomposition product-coated culture dish using DMEM medium (LIF (+)) in the presence of MEF (MEF (+)). The forms of ES cells ((c) to (e) in FIG. 20) cultured in a collagen decomposition product-coated culture dish using DMEM medium (LIF (+)) in the absence of MEF (MEF (-)). Although some differences were observed in size, shape, adhesion, floating, etc., animal species of collagen degradation products (eg, pigs, rats), origin sites (eg, skin and tendons) and N-terminal cut sites (eg, skin and tendons) For example, there was no clear difference regardless of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, and SEQ ID NO: 5).

The ES cells cultured in the gelatin-coated culture dish and the atelocollagen-coated culture dish under the culture conditions of MEF (−) and LIF (+) were single adherent cells. Notably, ES cells cultured in collagen degradation-coated culture dishes include collagen degradation animal species (eg, pigs, rats), origin sites (eg, skin and tendons) and N-terminal cleavage sites (eg, eg). Regardless of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: 5), embryoid bodies were formed even under the condition that MEF was not co-cultured. That is, culturing ES cells in a collagen degradation-coated culture dish means that the collagen degradation product animal species (eg, pigs, rats), origin sites (eg, skin and tendons) and N-terminal cleavage sites (eg, sequences) It has been shown that regardless of No. 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: 5), it can be a new method for forming an embryoid body.

<22. Test on embryoid body formation ability of collagen degradation product 12>
Next, in order to examine the undifferentiated ability of ES cells cultured in a gelatin-coated culture dish, an atelocollagen-coated culture dish, and a collagen degradation product-coated culture dish, the intracellular alkaline phosphatase activity was examined.

(Culture dish)
The above-mentioned collagen degradation product (type I collagen degradation product derived from pig skin (SEQ ID NO: 27), or type I collagen degradation product derived from rat tendon (mixture of SEQ ID NO: 24 and SEQ ID NO: 5), or pig A commercially available culture dish in contact with a degradation product of type I collagen derived from the skin (SEQ ID NO: 26) was used for the test. Specifically, the concentration of collagen degradation product in a 35 mm culture dish (type I collagen degradation product derived from pig skin (SEQ ID NO: 27) is 3.2 mg / mL to 9.6 mg / mL, type I derived from rat tendon). Collagen degradation product (mixture of SEQ ID NO: 24 and SEQ ID NO: 5) is 2.8 mg / mL to 8.4 mg / mL, and type I collagen degradation product derived from pig skin (SEQ ID NO: 26) is 3.3 mg / mL. mL) was changed and added, and the mixture was sufficiently blended in a culture dish and allowed to stand at room temperature for 5 hours to prepare a collagen decomposition product-coated culture dish.

(Formation of embryoid body)
2.5 x 10 ⁵ pieces using DMEM (KnockOutDMEM, manufactured by Thermo Fisher Scientific) containing 500 U / mL LIF and 20% fetal bovine serum (hereinafter referred to as "DMEM medium (LIF (+))"). / ML was prepared. 5.0 × 10 ⁴ cells / cm ^{2 of} mouse ES cells were seeded in each of a gelatin-coated culture dish, an atelocollagen-coated culture dish, and a collagen decomposition product-coated culture dish in which MEF was not seeded, and the DMEM medium (LIF (+)) was seeded. ) Was used for culturing under the conditions of 37 ° C. and 5% CO ₂ . ES cells cultured in a gelatin-coated culture dish and an atelocollagen-coated culture dish were used as controls in this experiment.

After culturing for 1 day, the culture supernatant of the cells was removed and washed with sterile PBS. Subsequently, a 4% paraformaldehyde solution was added to fix the cells until the cells were immersed, and the cells were allowed to stand at room temperature for 30 minutes. Then, the operation of adding sterile distilled water until the cells were immersed and washing was repeated twice. After removing the washing liquid, the alkaline phosphatase stain attached to the TRACP & ALP double-stain kit (product code MK300, manufactured by Takara Bio Inc.) was added to the culture dish and allowed to stand at 37 ° C. for 45 minutes. To stop the reaction, the stain was removed and washed 3 times with sterile distilled water. The results are shown in FIG.

FIG. 21 is a diagram showing the results of examining the intracellular alkaline phosphatase activity of ES cells cultured for 1 day under MEF non-coexistence (MEF (−)) and LIF (+) culture conditions. , (A) show the results of alkaline phosphatase activity staining of ES cells cultured for 1 day in DMEM medium (LIF (+)) in a gelatin-coated culture dish in the absence of MEF (MEF (-)). FIG. 3 (b) shows alkaline phosphatase activity staining of ES cells cultured for 1 day in DMEM medium (LIF (+)) in a petri dish in an atelocollagen-coated culture dish in the absence of MEF (MEF (−)). It is a figure which shows the result, (c) is a DMEM medium (LIF (+)) in a collagen decomposition product coated culture dish (from the pig skin part, SEQ ID NO: 27) in the absence of MEF (MEF (−)). It is a figure which shows the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using, (d) is a collagen decomposition product coated culture dish (derived from rat tendon, a mixture of SEQ ID NO: 24 and SEQ ID NO: 5). , MEF non-coexistence (MEF (−)), the result of alkaline phosphatase activity staining of ES cells cultured for 1 day using DMEM medium (LIF (+)), (e) is collagen degradation. Alkaline phosphatase activity staining of ES cells cultured for 1 day in DMEM medium (LIF (+)) in a petri dish (from pig skin, SEQ ID NO: 26) in the absence of MEF (MEF (-)). It is a figure which shows the result of.

Single ES cells cultured in gelatin-coated and atelocollagen-coated dishes under MEF (-) and LIF (+) culture conditions are colonies of ES cells under MEF (+) and LIF (+) culture conditions. (Similar to (a) and (b) in FIG. 15, it was shown to be positive for alkaline phosphatase activity ((a) and (b) in FIG. 21). Adherent and floating embryoid bodies formed from ES cells cultured under MEF (-) and LIF (+) culture conditions in a collagen degradation-coated culture dish are MEF (+) and LIF (+) culture conditions. It was shown that the embryoid bodies to which the cells were adhered ((c) to (e) in FIG. 15) were positive for alkaline phosphatase activity ((c) to (e) in FIG. 21). These results show that embryoid bodies formed from ES cells cultured in a collagen degradation product-coated culture dish in the absence of MEF are also collagen degradation product animal species (eg, pigs, rats), origin sites (eg, skin). And tendon) and N-terminal cleavage sites (eg, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 24, SEQ ID NO: 5), indicating that the undifferentiated ability is maintained.

From the above, it is clear that the collagen degradation product of this example has the ability to form embryoid bodies in mouse ES cells and human iPS cells, and can be utilized for the subsequent differentiation induction required. became. By long-term culturing the embryo-like body obtained by the method of one embodiment of the present invention in an appropriate differentiation-inducing medium, it is differentiated into endoderm, mesoderm, and ectoderm, and further neural tissue, bone / cartilage tissue. , Fat tissue, muscle tissue, etc. are expected to be reconstructed.

The present invention can be used in industries related to regenerative medicine.

Claims (8)

A method of forming embryoid bodies of pluripotent stem cells
Including the step of culturing pluripotent stem cells with a degradation product of collagen or a degradation product of atelocollagen,
The degradation product contains the amino acid sequence set forth in SEQ ID NO: 2, 24, 25, 26 or 27 at the amino terminus as at least part of the triple helical domain of the collagen or atelocollagen .
The decomposition product further contains 100 or more "Gly-XY" (X and Y are arbitrary amino acids) on the carboxyl-terminal side of the amino acid sequence shown in SEQ ID NO: 2, 24, 25, 26 or 27. A method for forming an embryoid body, which comprises a continuous amino acid sequence . The decomposition product according to claim 1, wherein the decomposition product contains at least one of the following decomposition products of collagen (A) to (C) or decomposition products of atelocollagen. Embryoid body formation method:
(A) of the amino acid sequence represented by the following (1) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, decomposition product or decomposition product of the atelocollagen of collagen;
(B) the amino acid sequence represented by the following (2) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between _{X 4} and G, chemical bond between _{X 6} and G, chemical bond between G and _{X 7, or,} between _{X 14} and G Degradation of collagen or atelocollagen with broken chemical bonds between;
(C) the amino acid sequence represented by the following (3) to the amino terminus of a triple helical domain of the collagen or atelocollagen, a chemical bond between Y ₁ and Y ₂ is disconnected, the collagen degradation product or atelocollagen Decomposition;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-;
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids). The embryoid body formation method according to claim 2, wherein the amino acid sequence shown in (1) or (2) is the amino acid sequence at the amino terminus of the triple helical domain. 2. The invention is characterized in that the cleavage in the amino acid sequence represented by any of (1) to (3) above is performed in at least one of the α1 chain and the α2 chain of the collagen or atelocollagen. The method for forming an embryoid body according to. A composition for embryoid body formation of pluripotent stem cells containing a degradation product of collagen or a degradation product of atelocollagen.
The degradation product contains the amino acid sequence set forth in SEQ ID NO: 2, 24, 25, 26 or 27 at the amino terminus as at least part of the triple helical domain of the collagen or atelocollagen .
The decomposition product further contains 100 or more "Gly-XY" (X and Y are arbitrary amino acids) on the carboxyl-terminal side of the amino acid sequence shown in SEQ ID NO: 2, 24, 25, 26 or 27. A composition for embryoid body formation, which comprises a continuous amino acid sequence . The decomposition product according to claim 5, wherein the decomposition product contains at least one of the following decomposition products of collagen (A) to (C) or decomposition products of atelocollagen. Composition for embryoid body formation:
(A) of the amino acid sequence represented by the following (1) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between X ₄ and G, or a chemical bond between X ₆ and G is disconnected, decomposition product or decomposition product of the atelocollagen of collagen;
(B) the amino acid sequence represented by the following (2) in the triple helical domain of the collagen or atelocollagen, a chemical bond between X ₁ and X _2, the chemical bond between X ₂ and G, and G chemical bond between X _3, a chemical bond between _{X 4} and G, chemical bond between _{X 6} and G, chemical bond between G and _{X 7, or,} between _{X 14} and G Degradation of collagen or atelocollagen with broken chemical bonds between;
(C) the amino acid sequence represented by the following (3) to the amino terminus of a triple helical domain of the collagen or atelocollagen, a chemical bond between Y ₁ and Y ₂ is disconnected, the collagen degradation product or atelocollagen Decomposition;
(1) -G-X _1- X _2- G-X _3- X _4- G-X _5- X ₆ -G-;
(2) -G-X _1- X _2- G-X _3- X _4- G-X _5- X _6- G-X _7- X _8- G-X _9- X _10- G-X _11- X _12- G-X _13- X ₁₄ -G-;
(3) -Y _1- Y _2- Y _3- G-Y _4- Y _5- G-Y _6- Y _7- G-Y _8- Y ₉ -G-;
(However, G is glycine, and X _{1 to} X ₁₄ and Y _{1 to} Y ₉ are arbitrary amino acids). The composition for embryoid body formation according to claim 6, wherein the amino acid sequence shown in (1) or (2) is an amino acid sequence at the amino terminal of the triple helical domain. 6. The invention is characterized in that the cleavage in the amino acid sequence represented by any of (1) to (3) above is performed in at least one of the α1 chain and the α2 chain of the collagen or atelocollagen. The composition for embryoid body formation according to.