Bioactive Agents and Related Methods

Abstract

The present invention relates to methods for the detection, identification, and use of one or more bioactive agents obtained from the extracellular matrix in the presence of one or more cells, e.g., one or more cell signaling polypeptides, and to such bioactive agents. Research, diagnostic, and therapeutic methods utilizing such bioactive agents are also provided.

Description

[Technical Field]

[0001] Technical Field The present invention relates to bioactive agents, including therapeutic agents, and methods for identifying them in selected biological samples. More particularly, the methods involve the use of cells that interact with extracellular matrix, e.g., acellular or decellularized extracellular matrix, in vitro, thereby liberating one or more bioactive agents of interest, including one or more bioactive agents that may be therapeutically effective. In a further aspect, the present invention relates to the identification, characterization, and use of one such bioactive agent, namely, a polypeptide fragment of the extracellular matrix protein decorin, which has stem cell recruitment activity. [Background technology]

[0002] Background of the Invention The following contains information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or related to the presently described or claimed invention, or that any publication or document specifically or implicitly mentioned is prior art. Any discussion of prior art throughout the specification should not be construed as an admission in any way that such prior art is widely known or forms part of the common general knowledge in the art.

[0003] It will be appreciated that there is an ongoing need for bioactive agents, including therapeutically active bioactive agents, for use in a range of research and treatments. While extensive research and development efforts have focused on synthetic materials, bioactive agents derived from biological systems are also of considerable interest.

[0004] Typically, bioactive agents originating from tissues, including the extracellular matrix (ECM), have been identified from intact tissues either serendipitously or through targeted methods. However, in the case of bioactive agents derived from the extracellular matrix, these methods face many challenges. For example, tissues are complex and highly heterogeneous materials, containing not only various cell types but also the extracellular matrix itself. Therefore, identifying such agents from the complex mixture of normal tissue requires various fractionation, separation, and analytical techniques, which may result in the loss of potentially useful bioactive agents, including therapeutic agents. Furthermore, the biological properties of the extracellular matrix and its components are believed to depend on many factors, including the age of the tissue, its location, and any disease state. In addition, the extracellular matrix and its interrelated cell populations are involved in a wide variety of signaling processes mediated by various bioactive agents. These signaling events can be sustained or transient, depending on the extracellular matrix components and cell(s) involved. This raises the possibility that important bioactive agents may be only transient or short-lived within the tissue, thereby making their identification difficult. Therefore, existing methods are limited by the complexity of the tissue extracellular matrix, the relative abundance of the potential therapeutic agent within the extracellular matrix, the complex interactions that occur in the tissue extracellular matrix, and the practical difficulties of isolating and identifying the bioactive agent.

[0005] Therefore, novel methods for identifying potential therapeutic biomolecules from extracellular matrices, including acellular extracellular matrix (aECM) or decellularized extracellular matrix (dECM), are needed to overcome these limitations.

[0006] However, currently, there are no effective and convenient methods suitable for identifying bioactive agents resulting from the interaction of cells with extracellular matrix, and in particular, for identifying bioactive agents resulting from the interaction of one or more cell populations with extracellular matrix (including acellular and decellularized extracellular matrix).

[0007] Thus, there is a need to develop new and improved methods for the identification of such substances, and a related need for such substances themselves. Summary of the Invention [Problem to be solved by the invention]

[0008] It is therefore an object of the present invention to provide one or more methods for detecting and / or identifying one or more bioactive agents, e.g., bioactive polypeptides, and / or such substances, or at least to provide a useful alternative to existing methods, or at least to provide the public with a useful choice. [Means for solving the problem]

[0009] Summary of the Invention In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 188 of mammalian decorin; d) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 188 of mammalian decorin; e) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; f) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; g) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 contiguous amino acids corresponding to any amino acid sequence within residues 1 to 177 of mammalian decorin; h) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 177 of mammalian decorin; i) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; j) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; k) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 170 of mammalian decorin; l) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 170 of mammalian decorin; m) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; n) a polypeptide comprising, consisting essentially of, or consisting of at least about 10 consecutive amino acids from any one of a) to m) above; o) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) through n) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more cells (including one or more stem cells); p) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) to o) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more mesenchymal stem cells; q) a functional fragment, functional variant, peptide analog, or peptidomimetic, or derivative of any one of a) to p) above; and r) A polypeptide having at least about 70% amino acid identity to any one of a) to q) above. The present invention relates to an isolated, purified, recombinant or synthetic polypeptide selected from the group comprising:

[0010] Any of the embodiments described herein may relate to any of the aspects presented herein.

[0011] Another aspect of the present invention is a method for producing a semiconductor device comprising: a) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 188 of mammalian decorin; d) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 188 of mammalian decorin; e) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; f) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; g) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 contiguous amino acids corresponding to any amino acid sequence within residues 1 to 177 of mammalian decorin; h) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 177 of mammalian decorin; i) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; j) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; k) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 170 of mammalian decorin; l) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 170 of mammalian decorin; m) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; n) a polypeptide comprising, consisting essentially of, or consisting of at least about 10 consecutive amino acids from any one of a) to m) above; o) a polypeptide comprising or consisting of at least about 10 consecutive amino acids from any one of a) to n) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more cells (including one or more stem cells); and p) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) to o) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more mesenchymal stem cells; q) a functional fragment, functional variant, peptide analog, or peptidomimetic, or derivative of any one of a) to p) above; and r) a polypeptide having at least about 70% amino acid identity to any one of a) to q) above; s) Any combination of two or more of the above a) to r) The present invention relates to compositions, including pharmaceutical compositions, comprising one or more polypeptides selected from the group comprising:

[0012] In one embodiment, the composition comprises a pharmaceutically acceptable carrier.

[0013] Another aspect of the invention relates to a reagent comprising one or more of the polypeptides described herein and / or compositions as described herein.

[0014] Another aspect of the present invention relates to kits comprising reagents and / or compositions as described herein.

[0015] According to another aspect, the present invention provides a method for manufacturing a method of manufacturing a semiconductor device comprising: a) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 188 of mammalian decorin; d) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 188 of mammalian decorin; e) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; f) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; g) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 contiguous amino acids corresponding to any amino acid sequence within residues 1 to 177 of mammalian decorin; h) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 177 of mammalian decorin; i) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; j) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; k) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 170 of mammalian decorin; l) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 170 of mammalian decorin; m) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; n) a polypeptide comprising, consisting essentially of, or consisting of at least about 10 consecutive amino acids from any one of a) to m) above; o) a polypeptide comprising or consisting of at least about 10 consecutive amino acids from any one of a) to n) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more cells (including one or more stem cells); and p) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) to o) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more mesenchymal stem cells; q) a functional fragment, functional variant, peptide analog, or peptidomimetic, or derivative of any one of a) to p) above; and r) a polypeptide having at least about 70% amino acid identity to any one of a) to q) above; s) Any combination of two or more of the above a) to r) The present invention relates to an expression construct comprising a nucleic acid encoding a polypeptide selected from the group comprising:

[0016] Another aspect of the present invention relates to a vector comprising an expression construct as described above.

[0017] Another aspect of the present invention relates to a host cell containing an expression construct or vector as defined above.

[0018] In a further aspect, the invention relates to the use of purified, isolated, recombinant, or synthetic proteins to mediate a biological effect, wherein the proteins are a) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 188 of mammalian decorin; d) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 188 of mammalian decorin; e) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; f) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; g) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 contiguous amino acids corresponding to any amino acid sequence within residues 1 to 177 of mammalian decorin; h) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 177 of mammalian decorin; i) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; j) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; k) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 170 of mammalian decorin; l) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 170 of mammalian decorin; m) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; n) a polypeptide comprising, consisting essentially of, or consisting of at least about 10 consecutive amino acids from any one of a) to m) above; o) a polypeptide comprising or consisting of at least about 10 consecutive amino acids from any one of a) to n) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more cells (including one or more stem cells); and p) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) to o) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more mesenchymal stem cells; q) a functional fragment, functional variant, peptide analog, or peptidomimetic, or derivative of any one of a) to p) above; and r) a polypeptide having at least about 70% amino acid identity to any one of a) to q) above; s) Any combination of two or more of the above a) to r) is selected from the group comprising:

[0019] In one embodiment, the biological effect is mediated in vivo in a subject in need thereof, eg, by administration of the protein.

[0020] In another embodiment, the biological effect is mediated ex vivo, e.g., in vitro, e.g., by contacting a biological sample with the protein. For example, the biological effect is mediated in vitro by contacting one or more cells, one or more tissues, or one or more organs with the protein.

[0021] In various embodiments, the biological sample, tissue (including acellular or decellularized extracellular matrix), or cells are derived from an animal, e.g., a mammalian subject (including a human subject), or a dairy animal, e.g., a cow, sheep, or goat.

[0022] In another aspect, the present invention relates to a method of providing one or more bioactive agents, the method comprising: i. providing an extracellular matrix; ii. contacting one or more cells with an extracellular matrix in vitro; iii. optionally at least partially purifying one or more bioactive agents; and iv. recovering said one or more bioactive agents. Includes:

[0023] In another aspect, the present invention relates to a method for detecting and / or identifying one or more bioactive agents, the method comprising: i. providing an extracellular matrix; ii. contacting one or more cells in vitro with the extracellular matrix for a period of time sufficient to release one or more bioactive agents; iii. optionally at least partially purifying one or more bioactive agents; and iv. Detecting and / or identifying said one or more bioactive agents. Includes:

[0024] In various embodiments, the extracellular matrix is ​​acellular extracellular matrix, decellularized extracellular matrix, or the extracellular matrix is ​​substantially free of cells, e.g., extracellular matrix that is substantially free of viable cells. In one embodiment, the extracellular matrix is ​​substantially free of endogenous cells, e.g., completely free of endogenous cells.

[0025] In one embodiment, the acellular extracellular matrix is ​​a naturally occurring acellular extracellular matrix, for example, the acellular extracellular matrix is ​​or is derived from vitreous humor.

[0026] In one embodiment, the extracellular matrix is ​​a decellularized extracellular matrix.

[0027] In various embodiments, the extracellular matrix is ​​prepared from the dermis, pericardium, stomach, small intestine, bladder, placenta, kidney capsule, or lining of a body cavity from any type of animal, including mammals, reptiles, birds, and insects.

[0028] In one embodiment, the extracellular matrix is ​​ovine forestomach matrix (OFM).

[0029] In one embodiment, the period of time is sufficient to allow one or more cells to interact with the outside of the cell or its components.

[0030] In one embodiment, the contacting is for a period of time sufficient to release one or more bioactive agents from the extracellular matrix, hi one embodiment, the contacting is for a period of time sufficient to induce production or secretion of the bioactive agent(s) by the one or more cells.

[0031] In one embodiment, the period is from about 1 hour to about 7 days or more, hi one example, the period is at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours.

[0032] In one embodiment, the one or more cells comprise a homogeneous cell population. In one example, the cells are macrophages, such as activated macrophages.

[0033] In one embodiment, the one or more cells comprise two or more cell populations. In one example, one population comprises macrophages and / or one population comprises fibroblasts. In another example, the one or more cells comprise one or more neural cells, one or more epithelial cells, one or more endothelial cells, one or more stem cells, or one or more progenitor cells.

[0034] In various embodiments, the extracellular matrix and the one or more cells are each derived from a single tissue or are of a type that is not in contact with each other in vivo, e.g., the acellular extracellular matrix and the one or more cells are each derived from a single tissue or are of a type that is not in contact with each other in a non-pathological state in vivo.

[0035] In various embodiments, at least partial purification, if performed, includes filtration, chromatography, such as high performance liquid chromatography and / or ion exchange, gel electrophoresis, precipitation, or size exclusion (including size exclusion filtration). In certain embodiments, more than one purification method is used.

[0036] In various embodiments, purification and / or recovery is by chromatography, such as size exclusion chromatography, gel electrophoresis, or fast protein high pressure liquid chromatography.

[0037] In various embodiments, detecting and / or identifying is by assaying one or more biological functions, e.g., the ability of the bioactive agent to elicit a biological response. For example, detecting and / or identifying is by assaying chemotaxis, modulation of one or more cellular responses, e.g., modulation of gene expression, modulation of cytokine or chemokine production, modulation of cell cycle progression, modulation of cell differentiation or proliferation, modulation of cellular activation, such as activation of macrophages or neutrophils.

[0038] In various embodiments, the biological activities of bioactive agents are assessed in vitro or in vivo, and in certain embodiments, include, but are not limited to, cell migration, chemotaxis, proliferation, or inhibition of intracellular or enzymatic processes.

[0039] In various embodiments, detection and / or identification is by direct detection and / or identification of the bioactive agent, for example, by protein or nucleotide sequencing, by chromatography, by immunoassay, or by mass spectrometry.

[0040] In various embodiments, the one or more bioactive agents are polypeptides or peptides that are released by proteolytic cleavage, by intracellular processing, e.g., by endocytosis and digestion of the protein using intracellular lysosomes, by oxidative burst, or by conformational change.

[0041] Another aspect of the present invention relates to compositions comprising an extracellular matrix and one or more cells, e.g., one or more exogenous cells, including compositions for use in identifying one or more bioactive agents.

[0042] In various embodiments, the extracellular matrix is ​​acellular, decellularized, or the extracellular matrix is ​​substantially cell-free, e.g., an extracellular matrix that is substantially free of viable cells. In one embodiment, the extracellular matrix is ​​substantially free of endogenous cells, e.g., completely free of endogenous cells.

[0043] In one embodiment, the acellular extracellular matrix is ​​a naturally occurring acellular extracellular matrix, for example, the acellular extracellular matrix is ​​or is derived from vitreous humor.

[0044] In one embodiment, the acellular extracellular matrix is ​​a decellularized extracellular matrix.

[0045] In one embodiment, the one or more bioactive agents are liberated by interaction of one or more cells with an extracellular matrix, for example, an acellular or decellularized extracellular matrix.

[0046] In one embodiment of such a composition comprising one or more cells, the composition comprises a homogeneous population of cells. In one example, the cells are macrophages, e.g., activated macrophages.

[0047] In one embodiment of a composition comprising one or more cells, the composition comprises two or more cell populations, in one example, one population comprises macrophages, and / or one population comprises fibroblasts, and / or one population comprises keratinocytes.

[0048] In one example, the one or more cells are from the same species as the extracellular matrix is ​​derived from.

[0049] A further aspect of the present invention relates to compositions comprising one or more bioactive agents identified by the methods as described herein.

[0050] In one embodiment, the one or more bioactive agents are liberated by interaction of one or more cells with the extracellular matrix.

[0051] In a further aspect, the present invention provides a method for producing a composition comprising: a) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 188 of mammalian decorin; d) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 188 of mammalian decorin; e) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; f) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; g) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 contiguous amino acids corresponding to any amino acid sequence within residues 1 to 177 of mammalian decorin; h) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 177 of mammalian decorin; i) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; j) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; k) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 170 of mammalian decorin; l) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 170 of mammalian decorin; m) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; n) a polypeptide comprising, consisting essentially of, or consisting of at least about 10 consecutive amino acids from any one of a) to m) above; o) a polypeptide comprising or consisting of at least about 10 consecutive amino acids from any one of a) to n) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more cells (including one or more stem cells); and p) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) to o) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more mesenchymal stem cells; q) a functional fragment, functional variant, peptide analog, or peptidomimetic, or derivative of any one of a) to p) above; and r) a polypeptide having at least about 70% amino acid identity to any one of a) to q) above; s) Any combination of two or more of the above a) to r) The present invention relates to a method of mediating a biological effect in a biological sample or in a subject in need thereof, comprising contacting the biological sample with or administering to the subject an effective amount of a protein selected from the group comprising:

[0052] In one embodiment, the biological effect is mediated in vivo in a subject in need thereof, e.g., by administration of the protein to the subject. In one embodiment, the effective amount is a therapeutically effective amount.

[0053] In another embodiment, the biological effect is mediated ex vivo, e.g., in vitro, e.g., by contacting a biological sample with the protein. For example, the biological effect is mediated in vitro by contacting one or more cells, one or more tissues, or one or more organs with the protein.

[0054] In another aspect, the invention relates to a method of modulating tissue repair in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein as described herein.

[0055] In certain embodiments, a therapeutically effective amount is sufficient to recruit one or more stem cells, for example, to the site of administration or to the site where the administered protein is localized.

[0056] In a further aspect, the present invention relates to a method of treating a disease or disorder associated with stem cell deficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein as described herein.

[0057] In one embodiment, the disease or disorder is a disease or disorder associated with a local stem cell deficiency, for example a stem cell deficiency in a particular tissue or organ.

[0058] In yet another aspect, the present invention relates to a method of modulating stem cell mobilization or related processes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein as described herein.

[0059] In various embodiments, the associated process is angiogenesis, hematopoiesis, protein expression, induction, or deposition, tissue remodeling, repair, or regeneration, cell proliferation, cell differentiation, including stem cell differentiation, cell regulation, apoptosis, modulation of one or more immune responses, modulation of tumorigenesis, chemotaxis, or cell recruitment.

[0060] In yet another aspect, the present invention relates to a method of mediating a biological effect, modulating tissue repair in a subject in need thereof, treating a disease or disorder associated with stem cell deficiency in a subject in need thereof, or modulating stem cell mobilization or related processes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition as described herein.

[0061] In yet another aspect, the invention relates to a method of mediating a biological effect, modulating tissue repair in a subject in need thereof, treating a disease or disorder associated with stem cell deficiency in a subject in need thereof, or modulating stem cell mobilization or related processes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bioactive agent identified by a method as described herein.

[0062] In various embodiments, the polypeptides described herein are administered to a subject at a dose of about 1 ng / kg to about 1 mg / kg. For example, the polypeptides described herein are administered at a dose of about 1 ng / kg to about 100 μg / kg, or about 1 ng / kg to about 10 μg / kg, 1 ng / kg to about 1 μg / kg, or about 1 ng / kg to about 100 ng / kg.

[0063] Reference to a range of numbers disclosed herein (e.g., 1 to 10) is also meant to incorporate reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), and also to any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7). These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are considered to be equally expressly stated in this application.

[0064] Those skilled in the art will understand the meaning of various terms relating to degree as used herein. For example, the term "about," as used herein in the context of a reference to an amount (e.g., "about 9%), refers to an amount that is close to or includes the stated amount that still performs the desired function or achieves the desired result; for example, "about 9%" can include 9% and amounts close to 9% that still perform the desired function or achieve the desired result. For example, the term "about" can refer to an amount that is within less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the stated amount. Also, when the term "about" is used in reference to, for example, a figure, concentration, amount, integer, or number, the exact figure, concentration, amount, integer, or number is specifically contemplated.

[0065] Other objects, aspects, features, and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0066] [Figure 1] Figure 1 shows a schematic of a representative assay as described herein utilizing the co-culture of mammalian cells with tissue-derived decellularized extracellular matrix, where the cells modify, liberate, or process components of the decellularized extracellular matrix to produce bioactive agents, e.g., bioactive agents with therapeutic properties suitable for the treatment of human disease, which are then evaluated for bioactivity. [Figure 2] FIG. 2 shows the secondary structure and functional domains of full-length ovine decorin, along with the location of the MayDay polypeptide and the X-ray crystal structure of decorin. [Figure 3]Figure 3 presents data regarding the bioactivity of conditioned medium in a mesenchymal stem cell migration assay, as described in Example 1 herein, establishing the biological response of mesenchymal stem cells to medium produced from co-culture of ovine forestomach matrix with macrophages compared to ovine forestomach matrix alone and macrophages alone. Conditioned medium was generated from culture medium containing ovine forestomach matrix (OFM), RAW mouse macrophage cells (MΦ), and ovine forestomach matrix co-cultured with macrophages (OFM + MΦ). Transwell migration assays were performed using ovAD-MSCs with medium alone ("control") and fibroblast growth factor 2 (50 ng / mL) included as positive and negative controls, respectively. Migrated ovAD-MSCs were imaged after 6 hours. Representative photomicrographs of test groups are included in panels A to E (A = medium control; B = fibroblast growth factor 2 (50 ng / mL); C = ovine forestomach matrix; D = macrophages; E = ovine forestomach matrix + macrophages). Cell migration was quantified, and results are expressed as the mean cell migration normalized to the medium control ("normalized cell migration") (F). Error bars indicate the standard deviation of three independent experiments. Statistical significance was determined via t-test, where "*" p ≤ 0.05, "**" p ≤ 0.01, "***" p ≤ 0.001, and "****" p ≤ 0.0001. [Figure 4] Figure 4 shows mesenchymal stem cell chemotaxis in response to test samples enriched through purification for proteins subsequently identified as MayDay peptides. Peptide samples were enriched using 10 ng, 25 ng, and 100 ng of total protein. MayDay-enriched samples demonstrated a dose-dependent increase in mesenchymal stem cell chemotaxis and a significant increase in cell number compared to the medium control (0 ng) as calculated by one-way ANOVA. *p<0.05, **p≦0.01. [Figure 5]Figure 5 shows data for conditioned medium from ovine forestomach matrix FTIC10 (fluorescein isothiocyanate), macrophage + ovine forestomach matrix FTIC10, and macrophage-only cultures separated by Tris-glycine SDS-PAGE electrophoresis. Tris-glycine gels were stained with Coomassie (A) or imaged via a fluorescent scanner (B). In panel B, the approximately 12 kDa protein band of interest is highlighted (blue dotted box). [Figure 6A] Figure 6 shows two representative sequences of ovine decorin (1-360; Accession No. Q9TTE2) (gray) as discussed in Example 4 herein. Figure 6A shows peptide fragments identified by electrospray ionization and tandem mass spectrometry (ESI MS / MS), including the putative MayDay (31-189) sequence (gray underlined), as well as the locations of decorin peptide fragments identified from electrospray ionization analysis from trypsin-digested media ("blue"), size-exclusion ("yellow"), and Tris-Tricine in-gel digestion ("green") samples. [Figure 6B] Figure 6B shows the putative MayDay (31-188) sequence (gray underlined) along with the locations of protease cleavage sites predicted by MEROPS based on the human decorin sequence (1-360; accession number: P07585). Cleavage sites on the decorin sequence are shown as "bold" letters; "↑" indicates the predicted C-terminal residue of the cleavage site for each protease shown. [Figure 7A] Figure 7 shows that in vitro cleavage of decorin protein (see Figure 6A) present in the ovine forestomach matrix by macrophage-derived MMP (matrix metalloproteinase)-12 yields the MayDay peptide (Figure 7A; lane 6). [Figure 7B]The MayDay peptide exhibits higher chemotactic activity than full-length decorin, as shown in Figure 7B. Recombinant human decorin was digested with MMP-12, and then a Transwell migration assay was performed using ovAD-MSCs. Media alone ("control") and fibroblast growth factor 2 (50 ng / mL) were included as positive and negative controls, respectively. Migrated ovAD-MSCs were imaged after 6 hours. Representative photomicrographs of the test groups are included in panels A to E (A = media control; B = fibroblast growth factor 2 (50 ng / mL); C = MMP12; D = decorin; E = MMP12 + decorin). Cell migration was quantified, and results are expressed as the mean cell migration normalized to the media control ("normalized cell migration") (F). Error bars indicate the standard deviation of three independent experiments. Statistical significance was determined via t-test, where; "**" p ≤ 0.01, "***" p ≤ 0.001, "****" p ≤ 0.0001. [Figure 8] Figure 8 shows data demonstrating the bioactivity of recombinant MayDay peptides in mobilizing mesenchymal stem cells compared to the growth factor SDF1 (stromal-derived growth factor), as described in Example 6. Recombinant histidine-tagged MayDay(31-170) [rec-HISovMayDay(31-170)] was assayed at three concentrations using a Transwell assay with ocAD-MSCs. Medium alone ("control") and SDF-1 (50 ng / mL) were included as positive and negative controls, respectively. Migrated ovAD-MSCs were imaged after 6 hours. Representative photomicrographs of the test groups are included in panels A through E (A = medium control; B = SDF-1 (50 ng / mL); C = 0.05 ng / mL; D = 0.50 ng / mL; E = 5.00 ng / mL). Cell migration was quantified, and results are expressed as the mean cell migration normalized to the medium control ("normalized cell migration") (F). Error bars indicate the standard deviation of three independent experiments. Statistical significance was determined via t-test, where "***" p ≤ 0.001, "****" p ≤ 0.0001. [Figure 9A]Figure 9 shows the in vivo bioactivity of recombinant MayDay peptide in mobilizing mesenchymal stem cells in a whole animal model, as described in Example 7. Figure 9A shows representative images of animals from each treatment group (upper panels) at t = 0 and t = 24 hours after injection of labeled muBM-MSCs (murine bone marrow-mesenchymal stem cells). Arrows indicate the injection site for each treatment group. Representative images of excised "normal" and "treated" muscle tissue are also presented (lower panels). [Figure 9B] Figure 9B shows quantification of fluorescence intensity (pixels) of excised "normal" and "treated" muscle tissue for each treatment group. Error bars indicate standard error of triplicate animals. Statistical significance was determined via t-test, where "*" p < 0.05, "**" p < 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0067] Detailed Description The present invention relates to methods for identifying one or more bioactive agents, including therapeutic agents, produced during or released by the interaction of one or more cells with acellular extracellular matrix. In certain embodiments, the one or more bioactive agents are derived from the extracellular matrix of a tissue, e.g., where the agent is a component of the extracellular matrix, a fragment of a component of the extracellular matrix, or a component or fragment of the extracellular matrix that has been modified, e.g., by the interaction of one or more cells with the extracellular matrix, and therefore is in a form different from that which it has in the native extracellular matrix.

[0068] In other embodiments, the one or more bioactive agents are produced, secreted, or expressed by one or more cells as a result of their interaction with the acellular extracellular matrix.

[0069] As used herein, a "bioactive agent" is an agent capable of eliciting a biological response, e.g., stimulating a biological response in one or more cells, or indeed in one or more tissues, organs, or organisms. Particularly contemplated bioactive agents are those produced by biological systems or in response to biological interactions or stimuli, and are therefore themselves biological in character. For example, bioactive agents as contemplated herein, in certain embodiments, are proteins or polypeptides, lipids, polysaccharides, nucleic acids, chemokines, vitamins, hormones, metabolites, growth factors, cytokines, exosomes, and the like. Particularly contemplated bioactive agents are those that elicit a biological response, such as one or more of cell chemotaxis and / or recruitment, modulation of tissue remodeling and / or tissue repair, wound healing and / or regeneration, modulation of the immune response, modulation of angiogenesis, modulation of the tissue microenvironment, angiogenesis, hematopoiesis, protein expression, induction, or deposition, cell proliferation, cell differentiation (including stem cell differentiation), cell regulation (including cell cycle regulation), apoptosis, modulation of one or more immune responses, and modulation of tumorigenesis.

[0070] This description will provide those skilled in the art with a variety of uses for and of these bioactive agents in research and therapy, particularly with respect to cell chemotaxis and recruitment, e.g., tissue remodeling, modulation of tissue repair, and wound healing, modulation of the immune response, modulation of angiogenesis, and modulation of the tissue microenvironment.

[0071] Various aspects of the invention are described in further detail in the following subsections. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will control. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, examples of suitable methods and materials are described below. The materials, methods, and examples described herein are illustrative only and not limiting.

[0072] definition As used herein, the term "and / or" may mean "and" or "or."

[0073] The terms "comprise," "comprises," and "comprising" used in this specification and claims are not to be construed in an exclusive or exhaustive sense, but rather mean "consisting at least in part of." When interpreting each statement in this specification that includes the term "comprise," "comprises," or "comprising," there may be subject matter other than what the term prefaces. Related terms such as "including," "include," and "includes" are to be interpreted in the same manner.

[0074] As used herein, the term "consisting essentially of" refers to the stated object and allows for the presence of other objects that do not materially alter the essential characteristics of the stated object.

[0075] As used herein, the term "consisting of" means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0076] extracellular matrix Extracellular matrix biomaterials suitable for use herein are collagen-based biodegradable matrices containing highly conserved collagen, glycoproteins, proteoglycans, and glycosaminoglycans in their native configurations and concentrations.

[0077] One extracellular collagenous matrix for use in the present invention is the extracellular matrix of a warm-blooded vertebrate. The extracellular matrix can be obtained from a variety of sources, for example, intestinal tissue collected from animals raised for meat production, including pigs, cattle, and sheep, or other warm-blooded vertebrates. Vertebrate extracellular matrix is ​​an abundant by-product of commercial meat production operations and is therefore a low-cost tissue graft material.

[0078] Extracellular matrix biomaterials include naturally associated extracellular matrix proteins, glycoproteins, and other factors naturally found within the extracellular matrix depending on the source of the extracellular matrix.

[0079] In certain embodiments contemplated herein, the extracellular matrix used in accordance with the present disclosure is acellular extracellular matrix (aECM), i.e., the extracellular matrix of tissue that is substantially free of cells.

[0080] In various embodiments, the acellular extracellular matrix useful herein is a naturally occurring acellular extracellular matrix, i.e., an extracellular matrix that is substantially free of cells in vivo, such as the extracellular matrix of the vitreous humor, e.g., from mammalian, reptilian, or avian eyes.

[0081] In other embodiments, the extracellular matrix is ​​derived from an extracellular matrix that is associated with one or more cells in vivo but from which substantially all cells have been removed. As used herein, an extracellular matrix that has been decellularized, whether by active manipulation in a laboratory or by other processes, is generally referred to as decellularized extracellular matrix (dECM).

[0082] Thus, in certain embodiments contemplated herein, the extracellular matrix found in tissues is decellularized to isolate or partially purify the extracellular matrix. These types of materials are widely used in wound healing and soft tissue regeneration, typically as scaffolds to temporarily replace missing or damaged extracellular matrix in a patient. Because the decellularized extracellular matrix mimics the extracellular matrix of the tissue, the patient's cells will attach to, grow, and divide on the exogenous decellularized extracellular matrix. Over time, the decellularized extracellular matrix will be incorporated into the patient's new tissue. It has been reported that the decellularized extracellular matrix will interact with the patient's cells during regeneration through a process that mimics normal cell-extracellular matrix interactions. For example, it has been reported that the decellularized extracellular matrix stimulates angiogenesis in the new tissue and undergoes structural remodeling. Similar to the extracellular matrix found in tissues, these decellularized extracellular matrices contain a heterogeneous mixture of structural, adhesion, and signaling molecules.

[0083] The extracellular matrix can be obtained from any suitable source, for example, from the forestomach of a sheep. Typically, the extracellular matrix will be decellularized so that host cells are reduced or completely removed.

[0084] Forestomach tissue is a preferred source of extracellular matrix for use in the present invention. Suitable forestomach extracellular matrices typically comprise the lamina propria-submucosa of the ruminant forestomach. In certain embodiments of the present invention, the lamina propria-submucosa is derived from the rumen, reticulum, or omasum of the forestomach. These tissue scaffolds typically have a contoured luminal surface. Extracellular matrix tissue scaffolds may also contain decellularized tissues, such as portions of epithelium, basement membrane, or muscularis, and combinations thereof. The tissue scaffold may also comprise one or more fibrous proteins, including, but not limited to, type I collagen, type III collagen, or elastin, and combinations thereof.

[0085] In one particularly contemplated example, decellularized extracellular matrix is ​​prepared from ovine forestomach tissue using a process that removes residual ovine (sheep) cells and disinfects the resulting extracellular matrix. This decellularized extracellular matrix is ​​referred to herein as "ovine forestomach matrix" (OFM). As used herein, the term "ovine forestomach matrix" and the abbreviation OFM refer to an extracellular matrix scaffold containing the lamina propria-submucosa of the ruminant forestomach. As used herein, the term "lamina propria-submucosa" refers to a tissue structure formed by intermixing the lamina propria and submucosa of the ruminant forestomach. OFM has been shown to contain various components of tissue extracellular matrix to stimulate angiogenesis and undergo structural remodeling.

[0086] The extracellular matrices useful herein will typically be easy to handle, amenable to drying, such as lyophilization, and readily sterilized.

[0087] In various embodiments, the extracellular matrix is ​​derived from a human or animal tissue source, such as ovine, bovine, porcine, caprine, deer, or human tissue. For example, the extracellular matrix is ​​or comprises a human decellularized extracellular matrix, a ovine decellularized extracellular matrix, a bovine decellularized extracellular matrix, a porcine decellularized extracellular matrix, a deer decellularized extracellular matrix, or a caprine decellularized extracellular matrix.

[0088] In one embodiment, the extracellular matrix is ​​derived from fetal or neonatal tissue, hi another embodiment, the extracellular matrix is ​​derived from juvenile, adult, or geriatric tissue.

[0089] In one embodiment, the extracellular matrix is ​​derived from healthy tissue, while in another embodiment, the extracellular matrix is ​​derived from diseased tissue, such as extracellular matrix derived from cancerous tissue.

[0090] In one embodiment, the extracellular matrix is ​​derived from a whole organ or from a specific tissue, for example, the extracellular matrix is ​​derived from liver, kidney, lung, intestine, amniotic membrane, nerve, skin, or vascular tissue.

[0091] In particularly contemplated embodiments, the extracellular matrix is ​​derived from the forestomach of a ruminant. In certain embodiments, the extracellular matrix is ​​derived from the rumen, reticulum, or omasum of the forestomach. The extracellular matrix may, in certain embodiments, comprise portions of epithelium, basement membrane, or muscularis, and combinations thereof, derived from such tissues.

[0092] In various embodiments, the extracellular matrix also includes one or more fibrous proteins, including, but not limited to, type I collagen, type III collagen, or elastin, and combinations thereof.

[0093] It will be understood that in certain embodiments of the methods described herein, decellularized extracellular matrix derived from a particular tissue is contacted with one or more cells that are derived from or associated in vivo with that tissue. However, in other embodiments, decellularized extracellular matrix derived from a particular tissue is contacted with one or more cells that are not normally present in or associated in vivo with that tissue. For example, extracellular matrix derived from the intestine, in one such embodiment, is contacted with keratinocytes isolated from mammalian skin.

[0094] While embodiments of the methods described herein in which an extracellular matrix that is substantially free of cells and substantially free of other materials is used are specifically contemplated, it will be understood that in other embodiments, the extracellular matrix may be associated with other materials, such as one or more structural supports (including, for example, one or more polymer sheets). In the context of the identification methods described herein, it will be understood that any other materials present when contacting the extracellular matrix with one or more cells will ideally be biologically inert and / or incapable of affecting the interaction of the one or more cells with the extracellular matrix. Representative examples of polymers useful for providing structural support to the extracellular matrix include polyvinyl alcohol, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid copolymer (PLLA), and lactic-glycolic acid copolymer (PLGA).

[0095] Methods for decellularizing extracellular matrix The methods described herein benefit from the use of a substantially cell-free extracellular matrix.

[0096] Those skilled in the art will be familiar with suitable surgical methods for isolating extracellular matrix and preparing it for subsequent use, including methods suitable for isolating acellular extracellular matrix from suitable tissues within or derived from a subject animal. Methods for decellularizing extracellular matrix are well known in the art. See, e.g., U.S. Patent Nos. 4,902,508, 5,554,389, 6,099,567, and 8,415,159, each of which is incorporated herein by reference in its entirety.

[0097] In one embodiment, an extracellular matrix suitable for use as described herein is prepared by transmural osmotic flow across the wall of an organ, such as the forestomach of a ruminant. Typically, the organ is filled with one solution, sealed, and then immersed in another solution. The salinity difference between the two solutions generates transmural osmotic flow. It will be understood that an osmotic gradient can be established in either direction by alternating the placement of the solutions (i.e., hypertonic and hypotonic solutions). The gradient is preferably established in a direction that mimics the natural flow of the organ. For example, when processing tissue derived from the forestomach of a ruminant, the gradient is preferably established from the luminal surface to the abluminal surface of the tissue.

[0098] Exemplary methods for decellularization of extracellular matrix by transmural osmotic flow are presented in PCT International Patent Application No. PCT / NZ2009 / 000152, published as WO 2010 / 014021, and in U.S. Patent No. 8,415,159, each of which is incorporated herein by reference in its entirety.

[0099] Briefly, transmural osmotic flow is applied between two sides of tissue from the region of the forestomach of a vertebrate, preferably harvested from an ovine species, whereby tissue layers within all or a portion of the tissue are separated and / or decellularized. The transmural osmotic flow is directed from the luminal side of all or a portion of the tissue to the abluminal side, or from the abluminal side to the luminal side of all or a portion of the tissue. This can be achieved, for example, by separating the tissue between a hypertonic and a hypotonic solution, whereby the transmural osmotic flow is directed from the hypotonic solution toward the hypertonic solution.

[0100] The method may, in certain embodiments, further comprise removing all or part of a tissue layer, including the epithelium, basement membrane, or muscularis mucosa, and combinations thereof.

[0101] Hypertonic and hypotonic solutions typically contain, for example, water and optionally at least one buffer, detergent, or salt. Hypertonic solutions contain a higher concentration of solute than hypotonic solutions. In a particular method, the hypertonic solution contains 4 M NaCl, and the hypotonic solution contains 0.28% Triton X-200 and 0.1% EDTA. In another particular method, the hypotonic solution contains 0.1% SDS. In yet another method, the hypotonic solution contains 0.028% Triton X-200, 0.1% EDTA, and 0.1% SDS.

[0102] The extracellular matrix may be stored in a hydrated or dehydrated state. Freeze-dried or air-dried extracellular matrix may be rehydrated or partially rehydrated and used in accordance with the present invention without significant loss of its biotrophic and mechanical properties.

[0103] It will be apparent from this disclosure that the term "decellularized," as used herein, refers to the removal of cells and their associated debris from a portion of a tissue or organ, for example, from the extracellular matrix.

[0104] It will be apparent from the present disclosure that the term "decellularized extracellular matrix" (dECM), as used herein, refers to animal or human tissue that has been decellularized to provide a matrix for structural integrity and a framework for interacting with or supporting other materials.

[0105] Cells suitable for use in the methods herein Those skilled in the art will appreciate that cells of virtually all tissues, including fibroblasts, immune cells (e.g., macrophages, neutrophils, and dendritic cells), endothelial cells, and stem cells (e.g., mesenchymal stem and progenitor cells), continually interact with the extracellular matrix in vivo, e.g., via various signaling pathways, extracellular matrix adhesion molecules, and receptors. For example, fibroblasts synthesize collagen and other extracellular matrix proteins, which then assemble into fibers. Fibroblasts are intimately involved in regulating fiber contraction and matrix stiffness in a continuous feedback loop. Other cells also participate in matrix degradation and remodeling. For example, inflammatory cells, such as macrophages and mast cells, release proteases to degrade the matrix after injury. Similarly, infiltrating endothelial cells, keratinocytes, and fibroblasts also release proteases to promote tissue remodeling, while numerous cytokines and growth factors are involved in key wound healing processes, including angiogenesis, chemotaxis, proliferation, collagen synthesis, and inflammation.

[0106] In one embodiment, the one or more cells in contact with the acellular extracellular matrix in vitro are or comprise any animal cell. For example, the cell is a mammalian cell. In another example, the cell is a cell of any tissue.

[0107] In one embodiment, the one or more cells are any eukaryotic or prokaryotic cell, such as a plant cell, a bacterial cell, a fungal cell (including a yeast cell).

[0108] In one embodiment, the one or more cells are or comprise cells derived from an immortalized cell line or from a primary cell line.

[0109] In one embodiment, the one or more cells are or comprise a mixture of cells derived from a single tissue, for example, peripheral blood mononuclear cells (PMNC).

[0110] In one embodiment, the one or more cells are or comprise a mixture of different cells, whether derived from one tissue, such as epithelial cells and keratinocytes derived from skin, or from multiple tissues, such as bone marrow-derived stem cells and blood-derived macrophages.

[0111] In various embodiments, one or more cells are or have been subjected to stress or one or more special culture conditions, such as low oxygen tension, nutrient depletion, altered pH, special media, elevated CO, microbial or viral attack, etc.

[0112] In various embodiments, one or more cells are or have been induced to adopt a particular phenotype, such as an M1 or M2 macrophage phenotype, such as a phenotype induced by the addition of cytokines or lipopolysaccharide.

[0113] In various embodiments, one or more cells are or have been genetically modified, e.g., genetically modified or induced to express one or more particular proteins or to contain one or more genetic mutations.

[0114] In various embodiments, the ratio of cell to extracellular matrix surface area is 1000 cells / cm 2 to 1,000,000 cells / cm 2 can range from 100,000 cells / cm, but typically about 100,000 cells / cm 2 The co-culture is incubated under controlled temperature and humidity for a period of from 1 hour to 7 days or more. In particular examples, the period is at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours.

[0115] In one specifically contemplated embodiment, macrophage cells are cultured on ovine forestomach matrix, for example as illustrated in the Examples herein.

[0116] Identification of bioactive substances Detection and / or identification of one or more bioactive agents as contemplated herein will, in certain embodiments, utilize modern chromatographic separation techniques (e.g., size exclusion chromatography, gel electrophoresis, or fast protein high pressure liquid chromatography) to separate a starting material, such as a sample collected after interaction of one or more cells with an acellular extracellular matrix, into various fractions.

[0117] In certain embodiments, these fractions are assayed using an appropriate biological model of interest. With successful isolation and purification procedures, therapeutic agents can be identified based on the biological activity of the fractions.

[0118] In certain embodiments, detection and / or identification of one or more bioactive agents, such as one or more bioactive polypeptides described herein, involves direct detection of the bioactive agent itself. In certain embodiments, such detection is by detection methods well known in the art, such as mass spectrometry, high performance liquid chromatography, 2D SDS-PAGE, or binding with an antibody. Exemplary methods for such detection are presented herein and are readily amenable to rapid screening of samples.

[0119] In certain embodiments, detection and / or identification utilize one or more antibodies capable of selectively and specifically binding to one or more bioactive agents, such as one or more bioactive proteins described herein. In certain embodiments of the detection or diagnostic methods described herein, antigen-antibody binding is detected using an immunoassay. The design of the immunoassay may vary. For example, the immunoassay may be based on competition or on a direct reaction. Furthermore, protocols may use solid supports, or intracellular or extracellular materials. Detection of the antibody-antigen complex may involve the use of a labeled antibody (including a labeled secondary antibody). In various embodiments, the label may be, for example, an enzyme, a fluorescent label, a chemiluminescent label, a radioactive label, or a dye molecule label.

[0120] Immunoassays can include a variety of formats, such as chip-based immunoassays, enzyme-linked immunosorbent assays (ELISAs), flow-through (vertical flow) or lateral flow assays, immunofluorescence tests (IFTs), or Western blot analysis.

[0121] In certain embodiments, detecting one or more bioactive agents, e.g., one or more bioactive polypeptides described herein, includes detecting one or more biological effects mediated by the bioactive agents. Representative examples exemplified herein include the recruitment of one or more cells in an in vitro cell chemotaxis assay. Many assays for various biological effects and / or responses are known to those of skill in the art. Examples include cell proliferation assays, such as assays of fibroblast proliferation or keratinocyte proliferation; cell migration assays, such as assays of endothelial cell migration, mesenchymal stem cell migration, fibroblast migration, keratinocyte migration; cell differentiation assays, such as assays of progenitor cells derived from neural tissue, bone marrow cells, chondrocytes (including osteogenic, chondrogenic, etc.); cell polarization assays, such as macrophage polarization assays; immune cell activation assays, such as neutrophil activation assays, mast cell activation assays, T cell activation assays, B cell activation assays (including antibody production assays); phagocytosis assays, such as neutrophil phagocytosis assays, macrophage phagocytosis assays, and mast cell phagocytosis assays; cell apoptosis assays, such as tumor cell apoptosis assays and endothelial cell apoptosis assays; antibacterial, antifungal, and antiviral assays; and angiogenesis assays, such as endothelial cell migration assays and endothelial cell sprouting assays.

[0122] Bioactive Agents Proteins present in or derived from the extracellular matrix, including modified or fragmented proteins and peptides, such as those produced through proteolytic activity generated by one or more cells, are examples of bioactive agents amenable to identification, isolation, and use in accordance with the present disclosure.

[0123] In one example, the protein is a) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 188 of mammalian decorin; d) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 188 of mammalian decorin; e) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; f) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; g) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 contiguous amino acids corresponding to any amino acid sequence within residues 1 to 177 of mammalian decorin; h) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 177 of mammalian decorin; i) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; j) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; k) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 1 to 170 of mammalian decorin; l) an N-terminal fragment of mammalian decorin comprising, consisting essentially of, or consisting of at least 10 consecutive amino acids corresponding to any amino acid sequence within residues 31 to 170 of mammalian decorin; m) a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; n) a polypeptide comprising, consisting essentially of, or consisting of at least about 10 consecutive amino acids from any one of a) to m) above; o) a polypeptide comprising or consisting of at least about 10 consecutive amino acids from any one of a) to n) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more cells (including one or more stem cells); and p) a polypeptide comprising or consisting of at least about 10 contiguous amino acids from any one of a) to o) above, wherein the polypeptide comprises a motif or region capable of interacting with or recruiting one or more mesenchymal stem cells; q) a functional fragment, functional variant, peptide analog, or peptidomimetic, or derivative of any one of a) to p) above; and r) A polypeptide having at least about 70% amino acid identity to any one of a) to q) above. The protein is selected from the group comprising:

[0124] In other examples, the bioactive agent is a matrikine, matricryptin, a subdomain of an extracellular matrix protein, or a ligand present in or derived from an extracellular matrix protein.

[0125] Proteins present in or derived from the extracellular matrix, including modified or fragmented proteins and peptides such as those produced through proteolytic activity, have been reported to be involved in many processes, including tissue remodeling, angiogenesis, and cell proliferation. Examples of such proteins include the so-called matrikines and matricryptins, which are polypeptides derived from or containing subdomains of extracellular matrix proteins and can regulate cell signaling.

[0126] Matrikines are specific regions of extracellular matrix proteins that act as ligands and alter cellular behavior following ligand-receptor binding. For example, the discoidin domain receptor (DDR) binding sequence of collagen is a well-characterized matrikine (Bellon, Martiny et al. 2004, Maquart, Pasco et al. 2004, Tran, Lamb et al. 2005). Binding of this ligand to the receptor DDR-1 in epithelial cells reportedly induces smooth muscle cell migration. Binding of the same consensus sequence to the receptor DDR-2 in mesenchymal stem cells (MSCs) reportedly induces the production of MMP-1, which degrades nearby fibrillar collagen.

[0127] In contrast, matricryptins are ligands that must be released from their parent proteins, e.g., by proteolytic cleavage, intracellular processing, or after a conformational change, in order to elicit their biological activity and / or bind to their target receptors.

[0128] Ligands for matrikines and matricryptins have been reported to often act with lower binding affinities in the mM range compared to cytokines, which typically act in the nM range. It has been suggested that this lower binding affinity is compensated for by the fact that the ligands may exist as tandem repeats within the parent protein and because these ligands are not typically internalized or depleted by target cells. Furthermore, because these ligands are derived from the extracellular matrix, cells within the matrix are likely to be close to their production site, and the local concentrations of these ligands in cells present within the matrix may be relatively high, unlike secreted growth factors that form gradients from a distance.

[0129] Matricryptin or cryptic peptide fragments may, in certain cases, have bioactive functions that are different or altered from those of the parent proteins from which they are derived. For example, collagen and elastin fragments have been reported to promote the migration, differentiation, and proliferation of wound-healing cells. Disruption of basement membranes has also been reported to disrupt adhesion molecules between keratinocytes and proteins (e.g., fibronectin, type IV collagen, and laminin), activating keratinocytes. Destruction of transient matrix proteins, particularly glycosaminoglycans and proteroglycans, has been reported to release signaling molecules that alter fibroplasia, angiogenesis, and even inflammatory responses. Heparin sulfate can be degraded by heparinase to produce low-molecular-weight fragments that promote the activity of fibroblast growth factor 2, and degradation products of hyaluronic acid have been reported to induce angiogenesis in a chick chorioallantoic membrane (CAM) model. The epidermal growth factor-like repeats of matricryptin are present on laminin-5, but this ligand must be made available by the action of MT1-MMP (membrane-type matrix metalloproteinase) and MMP-2 before it can exert its biological function. Other examples of matricryptin include endostatin, released from type XVIII collagen by elastase, cathepsins, and MMPs; tumstatin, released from type IV collagen by MMP-9; and the XGXXPG consensus sequence of elastin, released by MMP-2, MMP-9, MMP-7, and MMP-12.

[0130] Those skilled in the art will recognize, upon reading this description, that these proteins are representative of bioactive agents that can be identified by the methods contemplated herein, thus providing a variety of uses of and for these proteins in research and therapy, particularly with respect to, for example, cell chemotaxis and recruitment, modulation of tissue remodeling, tissue repair and wound healing, modulation of the immune response, modulation of angiogenesis, and modulation of the tissue microenvironment.

[0131] Proteins suitable for use herein include naturally occurring proteins and peptides, as well as derivatives thereof, including proteins and peptides that have one or more amino acid variations from the naturally occurring protein or peptide.

[0132] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs. Unless otherwise specified, the term "amino acid" includes both D- and L-stereoisomers, if the respective structures allow for such stereoisomeric forms.

[0133] Naturally occurring amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Tip or W), tyrosine (Tyr or Y), and valine (Val or V).

[0134] Unnatural amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyproline ("4Hyp"). N-methylglycines include, but are not limited to, N-methyl-, N-isoleucine ...

[0135] The term "amino acid analog" refers to a natural or unnatural amino acid in which one or more of the C-terminal carboxyl group, the N-terminal amino group, and side chain functional groups have been reversibly or irreversibly chemically blocked or otherwise modified to another functional group. For example, aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)cysteine ​​sulfoxide, and S-(carboxymethyl)-cysteine ​​sulfone.

[0136] The term "expression construct" refers to a genetic construct that contains sequences that allow for the transcription of a polynucleotide molecule of interest, and optionally the translation of the transcript into a polypeptide. Expression constructs typically contain, in the 5' to 3' direction: (1) a promoter that is functional in the host cell into which the construct will be introduced; (2) a polynucleotide to be expressed, and (3) A termination factor that functions in the host cell into which the construct will be introduced.

[0137] As used herein, the term "vector" refers to a polynucleotide molecule, usually but not limited to double-stranded DNA, that is amenable to use in molecular biological techniques, for example, to modify, manipulate, replicate, amplify, or transport polynucleotide molecules. In certain embodiments, vectors are used to transport polynucleotide molecules (e.g., but not limited to, genetic constructs, such as expression constructs) into host cells or organisms. In certain instances, vectors are capable of replicating and / or maintaining in more than one host system.

[0138] A "fragment" of a polypeptide is a subsequence of the polypeptide, typically a sequence that performs a function required for an activity, such as an enzymatic or binding activity, and / or that provides the three-dimensional structure of the polypeptide or a portion thereof, e.g., an epitope. It will be understood that a fragment of a polypeptide may have or elicit a function or functions that are different from those possessed or exhibited by the full-length polypeptide from which it is derived.

[0139] As used herein, the term "peptide" refers to a short polymer of amino acids linked together by peptide bonds. While the names associated with various classes of amino acid polymers (e.g., peptides, proteins, polypeptides, etc.) are somewhat arbitrary, it will be recognized that peptides are generally about 50 amino acids or more in length. Peptides can include naturally occurring amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides can be subsequences of naturally occurring proteins or unnatural (including synthetic) sequences.

[0140] As used herein, the term "synthetic peptide" encompasses peptides having amino acid sequences that differ distinctly from those found in natural peptides and / or natural proteins. As used herein, a "synthetic peptide" can be produced or synthesized by any suitable method (e.g., recombinant expression, chemical synthesis, enzymatic synthesis, etc.) and can include any chemical modification to the parent peptide, including, but not limited to, methods such as truncation, deletion, cyclization, or synthetic or semi-synthetic non-peptide derivatives that retain the same biological function(s) of the starting peptide. Protein synthesis methods, such as solid-phase synthesis, are well known in the art.

[0141] The term "peptide mimetic" or "peptidomimetic" refers to a peptide-like molecule that mimics a sequence derived from a protein or peptide. A peptide mimetic or peptidomimetic can contain amino acids and / or non-amino acid components. Examples of peptidomimetics include chemically modified peptides, peptoids (wherein side chains are attached to nitrogen atoms of the peptide backbone rather than the α-carbon), β-peptides (wherein amino groups are attached to the β-carbon rather than the α-carbon), and the like. A chemical modification includes one or more modifications at the amino acid side chain, the α-carbon atom, the terminal amine group, or the terminal carboxy group. A chemical modification can be the addition of chemical moieties, the creation of new bonds, or the removal of chemical moieties. Modifications of amino acid side chains include, but are not limited to, acylation of the ε-amino group of lysine, N-alkylation of arginine, histidine, or lysine, alkylation of the carboxylic acid group of glutamic acid or aspartic acid, lactam formation with the side chain carboxyl group of glutamic acid or aspartic acid via cyclization of the ε-amino group of lysine, hydrocarbon "stapling" (e.g., to stabilize an α-helical conformation), and deamidation of glutamine or asparagine. Modifications of terminal amino groups include, but are not limited to, desamino, N-lower alkyl, N-di-lower alkyl, constrained alkyl (e.g., branched, cyclic, fused, adamantyl), and N-acyl modifications. Modifications of terminal carboxyl groups include, but are not limited to, amide, lower alkyl amide, constrained alkyl (e.g., branched, cyclic, fused, adamantyl) alkyl, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Additionally, one or more side chains or terminal groups may be protected by protecting groups known to the ordinarily skilled peptide chemist. The α-carbon of the amino acid may be mono- or dimethylated.

[0142] It will be understood that any one of the proteins or peptides described herein, in certain embodiments, will comprise one or more naturally occurring amino acids, one or more amino acid analogs, or will be or comprise a synthetic peptide or synthetic polypeptide or peptide mimetic. Likewise, it will be understood that any one of the proteins or peptides described herein, in certain embodiments, will be the starting point for one or more modifications, synthetic methods, or protein engineering methods to develop a peptide analog having a desired biological activity (i.e., a biological activity that is qualitatively similar to that of the parent protein or peptide, but that is of a quantitatively different order of magnitude than that elicited by the parent protein or peptide, or that is actually a different biological activity).

[0143] As used herein, the term "fusion polypeptide" refers to a polypeptide comprising two or more amino acid sequences, e.g., two or more polypeptide domains, fused by peptide bonds through their respective amino and carboxyl residues to form a single contiguous polypeptide. It should be understood that the two or more amino acid sequences may be directly fused or indirectly fused through their respective amino and carboxyl termini through a linker or spacer or additional polypeptide.

[0144] As used herein, the term "polypeptide" encompasses an amino acid chain of any length, but preferably at least 10 amino acids, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds. The polypeptides described herein are purified natural products or are produced partially or completely using recombinant or synthetic techniques. The term can refer to polypeptides, polypeptide aggregates, such as dimers or other multimers, fusion polypeptides, polypeptide variants, or derivatives thereof.

[0145] It will be understood that natural variants may exist between individual bacterial strains for the specific polypeptides and proteins contemplated herein. These variants may be demonstrated by amino acid differences(s) in the overall sequence or by deletions, substitutions, insertions, inversions, or additions of amino acid(s) within the sequence. Amino acid substitutions that do not substantially alter biological and immunological activity are well known. Amino acid substitutions between related amino acids, or substitutions that occur frequently during evolution, include Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val, among others. Other amino acid addition substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / Ile, Leu / Val, and Ala / Glu. Based on this information, methods have been developed for rapid and sensitive protein comparison and for determining functional similarity between homologous proteins. Mutations of the exemplary embodiments described herein, including substitutions, deletions, and / or insertions of such amino acids, are within the scope of the present invention, so long as the resulting protein maintains its immunoreactivity. This explains why one or more proteins described herein may have a level of identity of less than 100% when isolated from different wild isolates, while still exhibiting the same protein with the same immunological characteristics. Such mutations in the amino acid sequence of a particular protein described herein, which still provide a protein capable of reacting with antibodies specific for the protein specifically identified herein, are considered to be immunologically functional equivalents of the protein identified herein and, therefore, do not substantially affect the immunogenicity of the protein.

[0146] When a protein is to be used, for example, for diagnostic or therapeutic purposes, e.g., to react with an antibody, or to mediate a biological effect, e.g., one or more biological functions associated with the native protein in vivo, it may be convenient, but is not necessary, to use the entire protein. It is also possible to use a polypeptide fragment of the protein (e.g., intact, bound to a carrier, or as a component in a fusion polypeptide), or a polypeptide fragment derived from the protein or related amino acid sequence that has a desired biological effect, e.g., that is capable of eliciting an immune response against the protein or that is capable of being recognized by antibodies specific for the protein, mediating a cell signaling effect, etc. Such a polypeptide fragment may be referred to with reference to a function it possesses, e.g., a function it shares with the full-length protein from which it was derived. For example, a polypeptide fragment that has an immunological effect may be referred to as an immunogenic fragment, where an "immunogenic fragment" is understood to be a fragment of a full-length protein that retains its ability to elicit an immune response in a vertebrate host or be recognized by antibodies specific for the parent protein. Similarly, a polypeptide fragment that retains or has one or more biological effects elicited by the full-length protein from which it is derived, or that has a related or different biological effect, is referred to herein as a "bioactive fragment" or "bioactive polypeptide fragment." Similarly, a polypeptide that has a biological effect, for example, a polypeptide that is capable of stimulating a biological response in a cell or eliciting a therapeutic effect, may be referred to herein as a "bioactive fragment" or "bioactive polypeptide fragment" or grammatical equivalents thereof.

[0147] Various techniques are available for identifying such polypeptide fragments, as well as DNA fragments encoding such fragments. For example, in the case of immunogenic fragments, such fragments may contain one or more determinants or epitopes. Well-established empirical and computational methods for epitope detection exist and are well known to those skilled in the art. For example, computer algorithms can designate particular protein fragments as immunologically significant epitopes based on their sequence and / or structural identity with known epitopes. The determination of these regions is typically based on a combination of hydrophilicity criteria and secondary structure features. Immunogenic fragments (or epitopes) are usually at least 6 amino acids in length, more commonly 8 amino acids in length, and preferably greater than 8, e.g., 9, 10, 12, 15, or even 20 or more amino acids in length. Thus, nucleic acid sequences encoding such fragments are at least 18, more commonly 24, and preferably 27, 30, 36, 45, or even 60 nucleic acids in length.

[0148] Similarly, one of skill in the art will know how to identify bioactive fragments using a variety of assays targeted at identifying or detecting specific biological responses. Exemplary methods suitable for use in identifying or detecting bioactive fragments contemplated herein are presented below, including in the Examples.

[0149] The term "variant" with respect to a polypeptide encompasses naturally occurring polypeptides, recombinant polypeptides, and synthetically produced polypeptides, such as polypeptides containing one or more non-naturally occurring amino acids, one or more amino acid analogs, and peptidomimetics. Variant polypeptide sequences preferably have a similar or similar nucleotide sequence to a sequence of the invention, at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least The polypeptides of the present invention may exhibit 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, at least 100 amino acid positions, or the entire length of the polypeptides of the present invention.

[0150] Polypeptide sequence identity can be determined in the following manner: A subject polypeptide sequence is compared to a candidate polypeptide sequence using bl2seq in BLASTP (from the BLAST suite of programs, version 2.2.10 [October 2004]), which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are used, except that filtering of low-complexity regions should be disabled.

[0151] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate polynucleotide sequence and a subject polynucleotide sequence using a global sequence alignment program. EMBOSS-needle (available at http: / / www.ebi.ac.uk / emboss / align / ), as discussed above, and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0152] Polypeptide variants contemplated herein also include those that exhibit similarity to one or more specifically identified sequences that are likely to preserve the functional equivalence of such sequences and that are not reasonably believed to have occurred by chance. Such sequence similarity for polypeptides can be determined using the publicly available bl2seq program from the BLAST suite of programs (NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ) version 2.2.10 [October 2004]). Polypeptide sequence similarity can be determined using the following unix command line parameters:

[0153] bl2seq-i peptideseq1-j peptideseq2-FF-pblastp

[0154] The variant polypeptide sequence preferably has a nucleotide sequence that is greater than 1×10 compared to any one of the specifically identified sequences. -10 less than 1×10 -20 Less than 1×10 -30 Less than 1×10 -40 Less than 1×10 -50 Less than 1×10 -60 Less than 1×10 -70 Less than 1×10 -80 Less than 1×10 -90 Less than 1×10 -100Less than 1×10 -110 Less than 1×10 -120 Less than 1×10 -123 indicates an E value less than

[0155] The parameter -FF disables filtering of low-complexity segments. The parameter -p selects the appropriate algorithm for sequence pairs. The program finds regions of similarity between sequences and reports an "E-value" for each such region, which is the expected number of times one can expect to find such a match by chance in a database of a given standard size containing random sequences. For small E-values, much lower than 1, this is the approximate probability of such a random match.

[0156] Conservative substitutions of one or several amino acids of the described polypeptide sequences without significant alteration of its biological activity are also included in the present invention. Those skilled in the art will know how to make phenotypically silent amino acid substitutions (see, for example, Bowie et al., 1990, Science 247, 1306).

[0157] Polypeptide variants contemplated herein also include those produced from nucleic acids that encode a polypeptide that differs from the wild-type polypeptide in that it has been processed differently to have a different amino acid sequence. For example, in one embodiment, the variant is produced by alternative splicing patterns of a primary RNA transcript relative to that which produces the wild-type polypeptide.

[0158] In other embodiments, the bioactive agent is a lipid, polysaccharide, nucleic acid, chemokine, vitamin, hormone, or the like.

[0159] Therapeutic Methods and Compositions The methods and bioactive agents described herein are, in certain embodiments, used in therapy, eg, for the treatment of one or more diseases, disorders, conditions, or states, in a subject in need thereof.

[0160] As used herein, a "subject" is an animal, usually a mammal (including a mammalian pet animal or human). Exemplary pet animals include cats, horses, and dogs. Exemplary agricultural animals include cattle, sheep, goats, deer, and pigs.

[0161] It will be understood that the various treatments contemplated herein will typically embody the administration of an effective amount of a bioactive agent.

[0162] An "effective amount" is an amount sufficient to effect beneficial or desired results (including clinical results). An effective amount can be administered in one or more administrations by various routes of administration. The effective amount will vary depending on, among other factors, the disease or condition being treated, the severity of the disease or condition, the age and relative health of the subject, the potency of the agent being administered, the form of administration, and the treatment desired. One of ordinary skill in the art will be able to determine the appropriate dosage, taking into account any of these other relevant factors.

[0163] Particularly contemplated diseases, disorders, conditions, or states to be treated include those that would benefit from modulation of stem cell responses in a subject.

[0164] As used herein, the term "treatment" and related terms, such as "treating" and "treat," generally refer to the treatment of a human or non-human subject in which some desired therapeutic effect is achieved. The therapeutic effect can be, for example, the prevention, reduction, amelioration, arrest, or prevention of a disease or condition.

[0165] As used herein, the term "stem cell" refers to a cell that is capable of self-renewal without differentiation and is capable of differentiating into other cell types. The term "stem cell" encompasses totipotent, pluripotent, and multipotent cells, as provided by the context.

[0166] Representative stem cells particularly contemplated for use herein include stem cells cultured in vitro after isolation from a stem cell-containing tissue source. In one embodiment, the stem cells are mesenchymal stem cells.

[0167] Stem cells can differentiate into other cell types and also have the ability to self-renew without differentiation. By providing various differentiated functional cells as needed, stem cells are important for the formation of new tissues and the repair of damaged or diseased tissues [Li, L., & Xie, T. (2005). Stem cell niche: structure and function. Annu. Rev. Cell Dev. Biol., 21, 605-631.] In addition to this progenitor function, stem cells also exhibit their own functions to promote tissue regeneration and repair, for example, through the secretion of bioactive factors and by regulating the behavior of other cell types [Duscher, D., Barrera, J., Wong, VW, Maan, ZN, Whittam, AJ, Januszyk, M., & Gurtner, GC (2016). Stem cells in wound healing: the future of regenerative medicine? A mini-review. Gerontology, 62(2), 216-225.] As part of normal tissue growth and repair, local endogenous stem cells perform these functions; however, in the setting of extensive tissue injury or when coexisting disease factors exist, the normal endogenous stem cell population may not be sufficient [Kanji, S., & Das, H. (2017). Advances of stem cell therapeutics in cutaneous wound healing and regeneration. Mediators of inflammation, 2017.]

[0168] Stem cell therapy has been recognized as having great potential in regenerative medicine, and the administration of additional stem cells has been shown to be effective in treating skin wounds [Falanga, V., Iwamoto, S., Chartier, M., Yufit, T., Butmarc, J., Kouttab, N., & Carson, P. (2007). Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue engineering, 13(6), 1299-1312.], nervous system injuries [di Summa, PG, Kingham, PJ, Raffoul, W., Wiberg, M., Terenghi, G., & Kalbermatten, DF (2010). Adipose-derived stem cells enhance peripheral nerve regeneration. Journal of Plastic, Reconstructive & Aesthetic Surgery, 63(9), 1544-1552.], and myocardial infarction [Berry, MF, Engler, AJ, Woo, YJ, Pirolli, TJ, Bish, LT, Jayasankar, V., & Sweeney, HL (2006). Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. American Journal of Physiology-Heart and Circulatory Physiology, 290(6), H2196-H2203.

[0169] However, current stem cell administration techniques are inefficient and invasive, requiring the collection, extraction, enrichment, and re-administration of isolated stem cell populations from donor tissue. Therefore, therapeutic agents capable of inducing local mobilization of endogenous stem cells would be of considerable utility in treating conditions that benefit from stem cell activity.

[0170] For example, in adult bone marrow, mesenchymal stem cells (MSCs) are a pool of regenerative cells capable of self-renewal and playing an important role in tissue repair. First, mesenchymal stem cells can differentiate into cells needed at the injury site. They support hematopoiesis by maintaining the hematopoietic stem cell niche. They release cytokines that regulate inflammatory responses and trophic factors that promote healing processes, such as cell recruitment, angiogenesis, and collagen synthesis. Along with fibroblasts, fibrocytes, and pericytes, mesenchymal stem cells can differentiate into myofibroblast progenitor cells, which, stimulated by cell-matrix interactions, matrix stiffness, and mechanical stress, become myofibroblasts (the primary matrix producing cells in wound healing).

[0171] Bone marrow-derived stem cells have also been reported to form pericytes, endothelial cells, and keratinocytes. Mesenchymal stem cells not only differentiate into the cell types required at the injury site, but also play an important role in regulating other wound-healing cells. Mesenchymal stem cells increase the rates of fibroblast migration, proliferation, and collagen synthesis, as well as endothelial cell tube formation. During the inflammatory phase of wound healing, mesenchymal stem cells regulate the immune response by producing IL-10 and IL-4 and blocking T cell proliferation. During the proliferation phase, mesenchymal stem cells release paracrine signals to recruit keratinocytes, dermal fibroblasts, and other nearby stem cells. They secrete important wound-healing growth factors, such as keratinocyte growth factor (KGF), vascular endothelial growth factor, and platelet-derived growth factor. Finally, during remodeling, mesenchymal stem cells regulate matrix metalloproteinase expression and collagen deposition.

[0172] Thus, the administration of bioactive agents capable of eliciting the mobilization of mesenchymal stem cells to a particular site is useful for treating diseases, disorders, pathologies, or conditions that would benefit from the myriad biological responses that mesenchymal stem cells can elicit. The mobilization of other stem cell types to the site in question may have therapeutic utility as well.

[0173] One such bioactive agent is the MayDay peptide described herein. Accordingly, specifically contemplated herein are detection methods, diagnostic methods, research and treatment methods, compositions, reagents, and kits that utilize one or more of the proteins described herein.

[0174] Accordingly, the present invention relates to kits for detecting and / or identifying one or more bioactive agents, e.g., via the methods as described herein, wherein the kits comprise an acellular extracellular matrix and optionally one or more exogenous cells, optionally together with one or more compositions as described herein.

[0175] In the present invention, the physiological effect of MayDay or a fragment, variant, peptide analog, or derivative thereof is to mobilize stem cells in the broader context of soft tissue regeneration, which may also be involved in downstream biological processes including, but not limited to, angiogenesis, vasculogenesis, tissue remodeling, enrichment of stem cells in the circulation, and muscle tissue regeneration.

[0176] Mobilization of stem cells is broadly beneficial for soft tissue repair and the management or intervention of diseases, disorders, or conditions associated with soft tissue, including muscle, which may include, but are not limited to, myocardial infarction, tissue loss due to surgical intervention or trauma, and enrichment of stem cells, including progenitor cells, in the peripheral blood system.

[0177] In one aspect, the invention relates to a pharmaceutical composition comprising an effective amount of a bioactive agent identified by a method as described herein, such as an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt or solvent thereof, and a pharmaceutically acceptable carrier.

[0178] A pharmaceutical composition may contain effective amounts of two or more substances in combination, such as two or more peptides described herein.

[0179] Compositions suitable for the administration of bioactive agents such as bioactive proteins, including therapeutic administration of bioactive agents comprising proteins or peptides, in a subject in need thereof, are known in the art.

[0180] For example, a bioactive agent, such as a polypeptide as described herein, e.g., a so-called MayDay peptide, or a fragment, variant, peptide analog, or derivative thereof, is in certain embodiments formulated with a pharmaceutically acceptable carrier, excipient, or combined with other substances to improve the bioavailability, half-life, or efficacy of the bioactive agent, and the composition is administered to a subject.

[0181] The term "pharmaceutically acceptable carrier" refers to a carrier (adjuvant or vehicle) that can be administered to a subject together with a biologically active agent, such as a MayDay peptide described herein or a pharmaceutically acceptable salt or solvate thereof.

[0182] Traditionally, such a carrier has been saline (0.9% sodium chloride), but other carriers are also applicable.

[0183] Pharmaceutically acceptable carriers that may be used in the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), such as da-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as Tween, or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0184] Cyclodextrins, such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyalkyl cyclodextrins (including 2- and 3-hydroxypropyl-3-cyclodextrin), or other solubilizing derivatives, may also be advantageously used to enhance delivery. The oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethylcellulose or similar dispersing agent, commonly used in formulating pharmaceutically acceptable dosage forms such as emulsions or suspensions.

[0185] The compositions are formulated to allow administration to a subject by any selected route, including, but not limited to, oral or parenteral (including topical, subcutaneous, intramuscular and intravenous) administration.

[0186] For example, the compositions can be formulated using appropriate pharmaceutically acceptable carriers (including excipients, diluents, adjuvants, and combinations thereof) selected according to the intended route of administration and standard pharmaceutical practice. For example, the compositions can be administered orally as a powder, liquid, tablet, or capsule, or topically as an ointment, cream, or lotion. Suitable formulations can contain additional substances, including emulsifiers, antioxidants, flavorings, or coloring agents, as needed, and can be adapted for immediate, delayed, modified, sustained, pulsed, or sustained release.

[0187] In certain embodiments relating to administration of polypeptides as described herein, administration will typically involve injection or deposition directly into the site of interest, for example, into soft tissue at the site of injury.

[0188] The compositions can be formulated to optimize bioavailability or activity, or to maintain plasma, blood, and tissue concentrations within a therapeutic range, for example, for an extended period of time. For example, sustained release delivery formulations may be used to optimize the concentration of the bioactive agent at the site of action.

[0189] The compositions may be formulated for periodic administration, for example to provide sustained exposure.

[0190] The composition may be administered parenterally. Examples of parenteral dosage forms include aqueous solutions of the active substance, isotonic saline, or 5% glucose, or other well-known pharmaceutically acceptable excipients. For example, cyclodextrins or other solubilizing agents known to those skilled in the art may be used as pharmaceutical excipients for delivery of therapeutic agents.

[0191] Examples of dosage forms suitable for oral administration include, but are not limited to, tablets, capsules, lozenges, or similar dosage forms, or any liquid dosage form, such as syrup, aqueous solution, emulsion, etc., that can provide a therapeutically effective amount of the composition. Capsules may contain any standard pharmaceutically acceptable material, such as gelatin or cellulose. Tablets can be prepared by compressing a mixture of active ingredients, solid carriers, and lubricants according to conventional procedures. Examples of solid carriers include starch and sugar bentonite.

[0192] The active ingredient may also be administered in the form of a hard-shell tablet or capsule containing a binder, such as lactose or mannitol, a conventional filler, and a tableting agent. Dosage forms for oral administration can be formulated with an enteric coating to prevent dissolution or disintegration of the dosage form in the stomach, allowing for delayed release of the drug and / or release of the drug after the stomach (e.g., in the upper intestine).

[0193] Examples of dosage forms suitable for transdermal administration include, but are not limited to, transdermal patches, transdermal bandages, and the like.

[0194] Examples of dosage forms suitable for topical administration of the composition include any lotion, stick, spray, ointment, paste, cream, gel, etc., whether applied directly to the skin or via a vehicle such as a pad or patch.

[0195] Examples of dosage forms suitable for suppository administration of the composition include any solid dosage form for insertion into a body orifice, particularly those inserted rectally, intravaginally, and intraurethrally.

[0196] Examples of dosage forms suitable for injection of the composition include delivery via a bolus, such as single or multiple doses by intravenous injection, subcutaneous, subdermal, and intramuscular administration, or oral administration.

[0197] Examples of dosage forms suitable for sustained-release administration of the composition include pellets or solid formulations of the peptide, in which the peptide is entrapped in a matrix of biodegradable polymers, microemulsions, liposomes, or is microencapsulated.

[0198] Examples of infusion devices for the compositions include infusion pumps, including implantable drug pumps, to provide a desired number of doses or steady state administration.

[0199] Examples of implantable injection devices for the compositions include any solid dosage form in which the bioactive agent is encapsulated or dispersed in a biodegradable or synthetic polymer, such as silicone, silicone rubber, silastic, or similar polymers.

[0200] Examples of dosage forms suitable for transmucosal delivery of the composition include enemas, suppositories, tampons, creams, gels, pastes, foams, atomized solutions, powders, and similar reservoir solutions containing carriers known in the art in addition to the active ingredient. These dosage forms include dosage forms suitable for inhalation or insufflation of the composition, including compositions comprising solutions and / or suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and / or powders. Transmucosal administration of the composition can use any mucous membrane, but typically utilizes nasal, buccal, vaginal, and rectal tissues. Formulations suitable for intranasal administration of the composition can be administered in liquid form, for example, as nasal sprays, nasal drops, or by aerosol administration via a nebulizer containing an aqueous or oily solution of polymer particles. Formulations may be prepared, for example, as aqueous solutions in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0201] Examples of dosage forms suitable for buccal or sublingual administration of the composition include lozenges, tablets, etc. Examples of dosage forms suitable for intraocular administration of the composition include inserts and / or compositions comprising solutions and / or suspensions in pharmaceutically acceptable aqueous or organic solvents.

[0202] Examples of formulations of the composition can be found, for example, in Sweetman, SC (ed.), Martindale. The Complete Drug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, p. 2483; Aulton, ME (ed.), Pharmaceutics. The Science of Dosage Form Design, Church Hill Livingston, Edinburgh, 2000, p. 734; and Ansel, H. C, Allen, LV and Popovich, NG, Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott, 1999, p. 676. Excipients used in drug delivery systems are described in various publications known to those skilled in the art, including, for example, Kibbe, EH, Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, 2000, p. 665. The United States Pharmacopoeia also provides examples of controlled-release oral dosage forms, including those formulated as tablets or capsules. See, for example, United States Pharmacopeia 23 / National Formulary 18, United States Pharmacopeial Convention, Rockville, MD, 1995 (hereinafter "USP"), which also describes specific tests for determining the drug release potential of extended-release and delayed-release tablets and capsules. The USP test for drug release from extended-release and delayed-release articles is based on the dissolution of drug from a dosage unit over an elapsed test time. Descriptions of various test equipment and procedures can be found in the USP. Further guidance on the analysis of extended-release dosage forms is provided by the U.S. Food and Drug Administration (see Industry Guide: Extended-Release Oral Dosage Forms: Development, Evaluation, and Application of In Vitro / In Vivo Correlations, Rockville, MD: Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 1997).

[0203] When two or more substances are administered or used, the two or more substances may be administered or used simultaneously, sequentially, or separately.

[0204] The invention will be further described with reference to the following examples, it being understood that the invention as claimed is not in any way limited by these examples. [Example]

[0205] Example Example 1: Representative Assays for Bioactive Agents This example describes the use of ovine forestomach matrix (OFM) in combination with macrophages (MΦ) in a Transwell migration bioassay to identify potential bioactive agents (in this case, bioactive agents with chemotactic activity).

[0206] method Isolation of ovAD-MSCs Ovine adipose-derived stromal cells (ovAD-MSCs) were isolated from the subcutaneous adipose tissue of donor animals according to Li et al. (Li, Curley et al. 2018). Adipose tissue was aseptically excised from the shoulders of adult female sheep. Tissue specimens were cut into approximately 1 x 1 cm pieces and then rinsed with Dulbecco's phosphate-buffered saline (DPBS) (Gibco) (three times, 20 mL) at room temperature for 10 minutes. The tissue was minced and then digested with 0.1% collagenase from Clostridium histolyticum in DPBS (10 mL) (Sigma-Aldrich, St. Louis, MI, USA) at 37°C for 1 hour with gentle shaking at 50 rpm. An equal volume of DMEM10 was added and the mixture was incubated overnight on a 100 mm cell culture plate (Corning, NY, USA). Adherent cells were rinsed with (DMEM2, 10 mL) and passaged in DMEM2 for 3 passages. Cells were maintained in DMEM2 (10 mL), with medium changes every 3 days, and trypsinized weekly using TrypLE™ Express (1.5 mL) (Gibco).

[0207] Differentiation of ovAD-MSCs Cells (ovAD-MSCs, passage 3) were split and seeded at a concentration of 100,000 cells / mL (0.5 mL) in DMEM2 in 24-well plates (Corning) until the monolayer reached 80% confluency. The medium was then replaced with osteogenic, chondrogenic, or adipogenic differentiation medium (1 mL) (StemPro™ Osteogenic Differentiation Kit, Adipogenic Differentiation Kit, Chondrogenic Differentiation Kit, Life Technologies, Carlsbad, CA, USA). Cells were maintained in the respective medium for 2 weeks, with the medium being replaced every 3 days. The differentiated cell monolayer was rinsed with DPBS (1 mL) and then fixed with 10% neutral buffered formalin (Sigma-Aldrich) (1 mL) at room temperature for 10 minutes. Monolayer adipocytes were stained with Oil Red O (0.5% w / v isopropanol, 1 mL, room temperature, 10 min) (Sigma-Aldrich) and counterstained with 0.1% hematoxylin solution (1 mL, room temperature, 10 min) (Sigma-Aldrich). Osteocytes were stained with 2% Alizarin Red S (1 mL, room temperature, 10 min) (Sigma-Aldrich). Chondrocytes were stained with Alcian Blue 8XG solution (1 mL, 1% w / v 3% acetic acid, pH 2.5, room temperature, 10 min) (Sigma-Aldrich). The culture medium was rinsed with ROHO (3 times, 1 mL) and then imaged using an Olympus inverted phase-contrast microscope (IX51, Olympus, Tokyo, Japan; data not shown).

[0208] Co-culture of macrophages and ovine forestomach matrix Ovine forestomach matrix was cut into approximately 4 x 4 cm samples and preconditioned in 2 mL of DMEM in 100 mm culture plates (Corning) at 37°C for 16 hours. RAW264.7 (ATCC, TIB-71) (Raschke, Baird et al. 1978) macrophages (MΦ) in 1 mL of DMEM (100,000 cells / mL) were seeded onto the ovine forestomach matrix (approximately 100,000 cells) and incubated (30 minutes at 37°C) to allow cell attachment. Additional DMEM was added to a final volume of 5 mL per well. Samples were incubated at 37°C for 24 hours. As a control, macrophages were seeded at the same concentration into blank plates. The medium was aspirated and collected, and phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich) was added to a final concentration of 10 μM. Samples of conditioned medium from each sample (macrophages only, ovine forestomach matrix only, macrophages + ovine forestomach matrix) were sterile filtered (0.22 μm) and stored at −20° C. before use.

[0209] Transwell migration assay Transwell migration assays were performed using a 24-well Transwell system (6.5 mm Transwell®, Corning) according to the method of Boyden et al. (Boyden 1962). Conditioned medium samples (ovine forestomach matrix, macrophages, and ovine forestomach matrix + macrophages) were diluted 1:1 with DMEM supplemented with fetal bovine serum to a final concentration of 0.5% (DMEM 0.5). DMEM 0.5 and recombinant human fibroblast growth factor-2 (Sigma-Aldrich) (50 ng / mL in DMEM 0.5) were used as negative and positive controls, respectively. For each well, 400 μl of each test sample was added to the lower chamber in triplicate. ovAD-MSCs (passage 5) were trypsinized using TrypLE™ Express (1.5 mL) (Gibco), counted, and resuspended in DMEM 0.5 at 100,000 cells / mL. A volume of 100 μL of the cell suspension was plated onto the insert (upper chamber). The culture was incubated for 6 hours, after which the Transwell membrane was removed from the plate and rinsed with DPBS (500 μL). Non-migrating cells were removed from the insert using a cotton swab, and the insert was then fixed for 10 minutes with ice-cold methanol (0.5 mL) (Sigma) diluted to 80% v / v with RHO. The fixed insert was transferred to a new plate containing 0.5 mL of 0.5% (w / v) crystal violet (Sigma-Aldrich) staining solution in 20% methanol / ROH2O (v / v) for 30 minutes. The inserts were rinsed with RHO (3 times, 100 mL) and then dried. Cells were imaged under an inverted microscope (IX51, Olympus, Tokyo, Japan) at 400x magnification, with five representative images taken per insert across the entire insert. The number of migrated cells was manually counted using ImageJ (NIH, Bethesda, USA) and calculated based on the membrane area (0.33 cm). 2) to determine the total number of migrated cells per insert. The number of migrated cells was expressed relative to the number of cells migrated in the medium-only control. Results are expressed as normalized cell migration compared to the medium-only control and represent the mean value of three independent experiments. Statistical analysis (t-test) was performed using GraphPad Prism (version 8.4.1); * ", p<0.05; " ** ", p<0.01; " *** ", p<0.001; " **** ”, p<0.0001.

[0210] result Quantification of mesenchymal stem cell (MSC) chemotaxis in response to ovine forestomach matrix alone, macrophages alone, or ovine forestomach matrix-macrophage co-cultures is shown in FIG.

[0211] As can be seen, in this ovAD-MSC migration assay, ovine forestomach matrix plus macrophage-conditioned medium resulted in a statistically significant increase in relative cell migration compared to the medium-only control (2.14 ± 1.19 and 0.98 ± 0.47, respectively; Figure 3). Conditioned medium derived from macrophages alone (MΦ, 1.21 ± 0.57; Figure 3) and ovine forestomach matrix alone (OFM, 1.27 ± 0.55; Figure 3) showed significant increases in relative cell migration, but the combination of both ovine forestomach matrix and macrophages resulted in the greatest relative cell migration.

[0212] Thus, culturing macrophages in the presence of ovine forestomach matrix was significantly better at recruiting mesenchymal stem cells than ovine forestomach matrix alone or macrophages alone.

[0213] While not wishing to be bound by any theory, the inventors believe that the observed increase in efficacy obtained from the ovine forestomach matrix + macrophage co-culture is related to the interaction of macrophages with the ovine forestomach matrix producing one or more liberated bioactive substances from the decellularized extracellular matrix, wherein the liberated bioactive substance(s) exhibit improved mesenchymal stem cell recruitment compared to the two individual components when separated.

[0214] Example 2: Isolation of biologically active polypeptides This example illustrates the isolation of a bioactive polypeptide using representative methods as described herein.

[0215] The ovine forestomach matrix was labeled with a fluorescent tag, fluorescein isothiocyanate (FITC), allowing tracking of peptides derived from the ovine forestomach matrix during purification and isolation of the various fractions.

[0216] A stock solution of FITC was made at 10 mg / mL in DMSO and kept in aliquots in light-proof containers at −20° C. A fresh solution of 1 mg / mL FITC was made fresh in 0.1 M NaHCO, pH 9.3; this was diluted to give a final working concentration of 10 μg / mL in 10 mL.

[0217] Ovine forestomach matrix (16 mm dish or 4 × 4 cm 2) were stained by incubation with a 10 μg / mL FITC solution in NaHCO3 for 16 hours at 4°C in a light-tight box on a gentle shaker. Unbound FITC was removed by washing three times with 10 mL of PBS for 20 minutes with shaking (50 rpm). Ovine forestomach matrix was conditioned for 16 hours in cell culture medium containing 1% fetal bovine serum and washed three more times in PBS to remove as much fetal bovine serum as possible in preparation for cell inoculation. Macrophage cells were washed with PBS to remove all fetal bovine serum and spun down to a density of 1 million cells per mL. Cells in 1 mL were plated in a 4 × 4 cm 2 The cells were seeded onto sheep forestomach matrix and incubated in a Petri dish for 30 minutes to allow cell attachment, after which 10 mL of serum-free medium was added to each dish. The cultures were incubated for 48 hours, after which the medium was collected, filtered using a 0.22 μm syringe-driven filter, and stored at 4°C.

[0218] Gel filtration was used to isolate all released peptides from the filtered medium. The buffer for anion exchange was 10 mM Tris pH 8.4 or "DEAE buffer." Diethylaminoethyl (DEAE) beads were swollen in 10 mM Tris, and 1 mL of beads was added to a 15 mL tube using a pipette with the end cut off. The tube was filled with 10 mM Tris, after which the beads were washed three times by centrifuging at 200 rpm for 10 minutes and replacing with 10 mL of Tris buffer each time.

[0219] A 0.5 mL sample of filtered media solution containing the candidate protein of interest was added to 1 mL of DEAE beads. The tube was vortexed for 2 minutes and then left at room temperature for 5 minutes to allow protein binding. The tube was spun down for 2 minutes, and the supernatant containing unbound protein was collected. An equal volume of DEAE buffer (0.5 mL of 10 mM Tris pH 8.4) was added to the DEAE-sample mixture, and the tube was vortexed for 1 minute. The tube was centrifuged for 2 minutes, and the supernatant containing the wash buffer and unbound protein was collected. 0.4 mL of 10 mM Tris solution containing 150 mM NaCl was added to the beads, and the tube was vortexed for 2 minutes and then left at room temperature for 5 minutes to allow protein elution. The beads were centrifuged for 2 minutes, and the supernatant containing the 150 mM salt eluate was collected. This process was repeated using a 10 mM Tris solution containing 400 mM NaCl to collect a 400 mM salt eluate. Fluorescence measurements for each collected fraction provided a fluorescence index relative to the initial sample and the volume of the collected fraction. Samples were separated by SDS-PAGE in Tricine as undiluted samples and concentrated to one-tenth of their original volume using a speed vacuum.

[0220] Fluorescently labeled fractions containing the ovine forestomach matrix-derived peptides of interest were assayed using the agar plug chemotaxis assay as described above in Example 1.

[0221] result The bioactivity of various amounts of partially purified polypeptide is shown in Figure 4. As can be seen, 10 ng of partially purified protein is sufficient to elicit a significant increase in chemotaxis of mesenchymal stem cells compared to the negative control. Further increased chemotaxis was observed with higher amounts (25 ng and 100 ng) of partially purified polypeptide.

[0222] Thus, the methods and assays described in this example are useful for detecting biologically active polypeptides and are suitable for preparing samples that allow identification of the biologically active polypeptide(s) in subsequent methods.

[0223] Example 3: Identification of bioactive peptides This example describes further identification of the bioactive polypeptide partially purified in Example 2 above.

[0224] method Separation of conditioned medium samples by Tris-glycine SDS-PAGE A sample of conditioned medium (30 μL) was diluted 3:1 with 4x Laemmli buffer (100 mM Tris, pH 6.8, 8% w / v SDS, 40% v / v glycerol, 20% w / v β-mercaptoethanol, 0.2% w / v bromophenol blue). The sample was boiled in a 100°C water bath for 10 min. Tris-glycine gels (4% acrylamide stacking gel and 20% acrylamide separating gel) were prepared using a Bio-Rad gel system and run in glycine running buffer (25 mM Tris, 192 mM glycine, 0.1% w / v SDS, pH 8.3) (Sigma-Aldrich). A total volume of 15 μL of each sample and 8 μL of protein standard ladder (Precision Plus Protein™ 2-Color Standard, Bio-Rad, Hercules, CA, USA) were loaded per well. Tris-glycine gels were run at 100 V for 1 hour. Fluorescent protein bands were visualized with a Fluoroscan Ascent FL (Thermo Fisher Scientific) and then stained with Coomassie Brilliant Blue (0.1% w / v Coomassie Brilliant Blue R-250, 50% v / v methanol, 10% glacial acetic acid) for 3 hours at room temperature with gentle shaking. Coomassie-stained gels were imaged using a Typhoon FLA9500 (GE Healthcare, Chicago, IL, USA).

[0225] result Ovine forestomach matrix FITC10, macrophages, and ovine forestomach matrix FITC10 A sample of conditioned medium made from macrophages was separated on a Tris-glycine gel and imaged via fluorescence of the resulting protein bands (Figure 5B). Cultures of macrophages alone did not yield fluorescently labeled proteins (lane 4 in Figure 5B), whereas ovine forestomach matrix FITC10 Conditioned medium prepared from only the co-culture medium (ovine forestomach matrix) mainly yielded high molecular weight protein bands (approximately 75–250 kDa, lane 2 in Figure 5B). FITC10 + macrophages) gave rise to a distinct fluorescently labeled protein band at approximately 12 kDa (blue dotted box, lane 3 in Figure 5B).

[0226] Example 4: Identification of the physiologically active peptide "MayDay" This example describes the identification via mass spectrometry (MS) of the bioactive polypeptide (referred to herein as MayDay), partially purified in Examples 2 and 3 above.

[0227] method Sample preparation Ovine forestomach matrix (4 × 4 cm) was labeled with 0 and 10 μg / mL FITC in 0.1 M sodium bicarbonate as described above. The resulting labeled and unlabeled materials (ovine forestomach matrix and ovine forestomach matrix) were analyzed. FITC10 ) were allowed to acclimate in DMEM (5 mL) for 16 hours. Co-culture of ovine forestomach matrix and macrophages was performed as described above, but the method was modified to use approximately 50,000 cells of RAW265.7 macrophages per ovine forestomach matrix sample (4 × 4 cm) in a final volume of 0.5 mL of DMEM. Cultures were incubated for 24 hours, and these samples (ovine forestomach matrix + macrophages, ovine forestomach matrix) were cultured for 16 hours. FITC10Conditioned media from the cells (+macrophages, and macrophages only) was collected. Samples were sterile filtered using a 0.22 μm filter and treated with PMSF as described above. Samples were desalted with PBS (5 mL), concentrated by ultrafiltration using Amicon Ultra-15 centrifugal filters (Ultracel-PL membrane, 3 kDa, Merck / Millipore, Burlington, MA, USA), and stored at −20°C before use.

[0228] Protein quantification was performed using a bicinchoninic acid (BCA) kit for protein determination (Sigma-Aldrich) according to the manufacturer's instructions.

[0229] In-solution trypsin digestion Macrophage + sheep forestomach matrix samples were resuspended in PBS to a final concentration of 0.1 mg / mL. Samples (20 μg) were reduced with 10 mM 1,4-dithiothreitol (DTT) (Sigma-Aldrich) (20 μL, 60 min, 60°C) and then alkylated with 20 mM iodoacetamide (Sigma-Aldrich) (20 μL, 30 min, room temperature, in the dark). Samples (60 μL) were digested overnight (37°C) with 0.1 μg trypsin (Sigma-Aldrich). Samples were dried and then reconstituted in loading buffer (0.1 M sodium bicarbonate) before undergoing ESI MS / MS analysis.

[0230] Size exclusion chromatography Lyophilized samples of macrophage + sheep forestomach matrix-conditioned medium were resuspended in PBS to a final protein concentration of 0.1 mg / mL. Samples (100 μl, approximately 100 μg) were fractionated into a 96-well plate by size exclusion chromatography (SEC) (GE Superdex 75 10 / 300GL, GE Healthcare, MA, USA) using a mobile phase of 50 mM sodium phosphate (pH 7), 150 mM NaCl, and a flow rate of 0.35 mL / min. The eluate was monitored at 214 nm, 220 nm, and 280 nm. Fractions were pooled based on known retention times and molecular weights of the following standards: aldolase, conalbumin, carbonic anhydrase, ribonuclease A, and aprotinin. Pooled samples (approximately 1 mL) were reduced with 10 mM DTT at 60°C for 1 hour and then alkylated with 25 mM iodoacetamide (30 minutes at room temperature). Trypsin (500 ng) was added to each sample, followed by overnight digestion at 37°C. Samples were analyzed using OMIX C. 18 The samples were desalted using a 100 μL tip (Agilent / Varian, A57003100K, Santa Clara, CA, USA) and then eluted with 100 μL of acetonitrile (ACN) / formic acid. The samples were dried and then reconstituted in loading buffer prior to ESI MS / MS analysis.

[0231] Tris-tricine SDS-PAGE and in-gel protein digestion Samples (macrophages + sheep forestomach matrix, macrophages + sheep forestomach matrix) FITC10, and macrophages only) were prepared as described above, then diluted 3:1 with 4x Laemmli buffer and denatured as described above. A total volume of 15 μL of each sample and 8 μL of protein standard ladder (Precision Plus Protein™ 2-Color Standard, Bio-Rad) were loaded per well. Tris-Tricine gels (4% acrylamide stacking gel and 16% acrylamide separating gel) were run using anode buffer (100 mM Tris, 100 mM Tricine, 0.1% w / v SDS, pH 8.25) and cathode buffer (100 mM Tris, pH 8.9). Gels were run on ice (approximately 4°C) at 60 V for 2 hours. Tris-Tricine gels were visualized by fluorescence using a Fluoroscan Ascent FL (Thermo Fisher Scientific) and then stained with Coomassie Brilliant Blue (0.1% Coomassie Brilliant Blue R-250, 50% v / v methanol, and 10% glacial acetic acid) for 3 hours at room temperature with gentle shaking. Coomassie-stained gels were imaged using a Typhoon FLA9500 scanner (GE Healthcare, Chicago, IL, USA).

[0232] The area of ​​interest corresponding to the 12 kDa molecular weight band was divided into macrophages + sheep forestomach matrix, macrophages + sheep forestomach matrix, and FITC10 A lane containing both ATP and ATP was excised from the gel. The sample was reduced with dithiothreitol (DTT) (10 mM, 20 μL) at 60°C for 1 hour and then alkylated with iodoacetamide (20 mM, 20 μL) at room temperature in the dark for 30 minutes. The protein was digested with 100 ng of trypsin overnight at room temperature. The sample was concentrated to 30 μL and then subjected to ESI MS / MS.

[0233] ESI MS / MS analysis The digested samples were injected into an Eksigent Ultranano LC system (Eksigent, Livermore, CA, USA) interfaced with a Triple TOF 5600 (AB Sciex, Redwood City, CA, USA). The digested samples (10 μL, 20 μL, or 40 μL) were injected into a peptide trap (Peptide Cap Trap, Michrom Bioresources, Auburn, CA, USA) and desalted for 5 min with 0.1% formic acid / 2% acetonitrile (ACN) at 10 μl / min. The peptide trap was then switched to an analytical column (Halo C18, 160 Å, 2.7 μm, 75 μm × 10 cm, Advances Materials, Wilmington, DE, USA). Peptides from trypsin-digested and in-gel-digested samples were eluted from the column using a solvent gradient of 95% (0.1% formic acid in water) / 5% (99.9% acetonitrile / 0.1% formic acid) to 60% (0.1% formic acid in water) / 40% (99.9% acetonitrile / 0.1% formic acid) at a flow rate of 550 nL / min over a 42-minute period. Peptides from size-exclusion chromatography were eluted from the column using a solvent gradient of HO:acetonitrile (95:5; +0.1% formic acid) to HO:acetonitrile (5:95; +0.1% formic acid) at a constant flow rate (500 nL / min) over an 80-minute period.

[0234] The eluate was subjected to positive-ion nanoflow electrospray analysis in information-dependent acquisition (IDA) mode. A time-of-flight mass spectrometry survey scan was acquired (m / z 350–1500, 0.25 s). The 10 most intense multiply charged ions (counts >150) in the survey scan were sequentially subjected to MS / MS analysis. MS / MS spectra were accumulated for 200 ms over the mass range of m / z 100–1500, with a total cycle time of 2.3 s. Raw data files (.wiff) were converted to mascot generic files (.mgf) using AB SCIEX Command Driver software (AB SCIEX, Redwood City, CA, USA). Data files were run in Mascot (Matrix Science, UK) and searched against the Swissprot database (sheep (Ovis aries) [sp_sheep_140625]).

[0235] result Ovine forestomach matrix FITC10 Large volumes of conditioned medium prepared from macrophages and sheep forestomach matrix macrophages were prepared, and mass spectrometry was performed on samples prepared via three different methods: 1. in-solution trypsin digestion of conditioned medium; 2. purification by size exclusion chromatography; and 3. separation on a one-dimensional Tris-Tricine gel followed by in-gel trypsin digestion. The approximately 12 kDa protein of interest was clearly separated via the Tris-Tricine gel (data not shown). In each approach, sheep forestomach matrix FITC10 Both the +macrophage and ovine forestomach matrix +macrophage samples were analyzed using FITC as a means of tracking the protein(s) of interest via fluorescence. ESI MS / MS analysis was performed on the ovine forestomach matrix +macrophage sample only, avoiding any complications resulting from FITC labeling. The MASCOT database was used to identify all protein fragments identified from the three sample preparation methods.

[0236] The extracellular matrix protein decorin (DCN) was consistently identified in MASCOT search results. Database searches identified several unique peptides (Uniprot; Q9TTE2, also known as bone proteoglycan II, PG-S2, PG40, mass 39947 Da) shown below as matches to the ovine protein decorin (emPAI; 0.08).

[0237] Peptides identified via the three different methods are shown in Figure 6A. The in-solution trypsin-digested sample yielded the most protein hits ("blue," Figure 6A), spanning much of the decorin sequence. This most likely arises from the relative impurity of the trypsin-digested sample. The sample prepared via size-exclusion chromatography yielded one sequence that aligned to the N-terminal sequence of decorin ("yellow," Figure 6A), and the sample prepared from the Tris-Tricine gel yielded an additional sequence that also aligned to the N-terminal region of decorin ("green," Figure 6A). The sequences of these latter two unique peptides are presented below: 1.KISPGAFAPLVKL 2.RVVQCSDLGLEKV

[0238] Table 1 below presents the amino acid sequence of the full-length decorin protein [SEQ ID NO: 1], with the amino acid sequences corresponding to these two unique peptides underlined. Figure 6A shows the location of another unique peptide within the N-terminal portion of decorin within the decorin sequence, while the location of the MMP12 proteolytic cleavage site is shown in Figure 6B. The amino acid sequences corresponding to the recombinant MayDay31-170 polypeptide [SEQ ID NO: 2] and to two N-terminal fragments of decorin (each terminating at the MMP12 cleavage site, MayDay31-177 [SEQ ID NO: 3] and MayDay31-188 [SEQ ID NO: 4], respectively) are shown in Table 1. Again, the locations of the amino acid sequences corresponding to these two unique peptides shown above are underlined.

[0239] [Table 1]

[0240] Computational analysis was performed using the MERPOS database to predict proteolytic cleavage sites in ovine decorin based on sequence homology to known human decorin cleavage sites. As shown in Figure 6B, decorin contains many predicted protease sites, including those for MMP-2, -3, -7, -12, and -13, as well as ADAMTs 5. In particular, two MMP-12 sites were predicted to reside between residues 177-178 and 188-189.

[0241] Example 5: Production and evaluation of bioactive MayDay peptides via digestion of decorin by MMP12 This example describes the production of the MayDay polypeptide identified in Example 4 and evaluation of its bioactivity in cleaving the parent protein using MMP12.

[0242] Stock solutions of MMP-12 catalytic domain (Sino Biologicals, Beijing, China) and decorin (Sino Biologicals) were prepared at 0.25 mg / mL in ROH2O and stored at -20°C before use. Decorin samples (10 μL) were digested with 0, 0.1, 5, or 10 μL of MMP-12 solution to obtain protein:enzyme ratios of 1:1, 2:1, or 100:1. The final sample volume was brought to 40 μL with MMP-12 buffer (50 mM Tris, 100 mM NaCl, 0.05% w / v Bridge 35, pH 8.0). Samples were incubated at 37°C for 16 hours with gentle shaking.

[0243] The digested sample (30 μL) was diluted 3:1 with 4x Laemmli buffer and denatured as described above. A total volume of 30 μL was loaded onto a precast Bis-Tris gel (4-12% Bolt NuPAGE, Invitrogen, Carlsbad, CA, USA). The Bis-Tris gel was run with 5 μL of Tan protein standard solution (SeeBlue protein standard, Invitrogen). The gel was run at 100 V for 90 minutes using an Invitrogen Minigel System (Invitrogen) in Bolt running buffer (Bolt™ MES SDS running buffer, Invitrogen™). The gel was rinsed (three times, 10 mL RHO) and then stained with Coomassie as described above.

[0244] Decorin and decorin digested with MMP12 were analyzed on an SDS page gel, demonstrating protein fragmentation, as shown in Figure 7A.

[0245] The samples were then added to a Transwell assay using decorin plus MMP12 to determine the bioactivity of the three samples, along with controls of decorin only (250 ng) and MMP12 only (25 ng).

[0246] Samples for the Transwell migration assay were prepared as follows: decorin (20 μL at 0.25 mg / mL) and MMP-12 (10 μL at 0.25 mg / mL) were brought to 40 μL with digestion buffer, as described above, to give a 2:1 protein:enzyme ratio. As controls, decorin alone (20 μL at 0.25 mg / mL) and MMP-12 alone (10 μL at 0.25 mg / mL) were brought to 40 μL with digestion buffer, and a digestion buffer-only sample ("control") was incubated for the same time. The samples were incubated overnight at 37°C for 16 hours. After incubation, each solution was combined with 0.5 ml of DMEM 0.5 to quench the enzyme digestion. Transwell migration assays using ovAD-MSC cells were performed as described above. Membranes were imaged, and migrated cells were counted using ImageJ. Results are expressed as normalized cell migration compared to medium-only samples and represent the mean values ​​of three independent experiments. Statistical analysis (t-test) was performed using GraphPad Prism (version 8.4.1); * " p < 0.05, " ** ” p ≤ 0.01, p ≤ 0.001; **** ” p≦0.0001.

[0247] result The number of migrated cells present on the lower chamber of the Transwell membrane is shown in Figure 7B. As can be clearly seen, significantly more cells migrate to the lower chamber when digested with MMP12 in the presence of decorin. In comparison, fewer cells were observed with the negative control sample of medium only and with undigested decorin protein.

[0248] This example demonstrates the ability of enzymatically produced MayDay protein to elicit cell recruitment comparable to that observed with MayDay polypeptides obtained from ovine forestomach matrix+macrophage cultures. Thus, this example establishes that the biological activity of bioactive polypeptides identified in assays such as those described herein can be reproduced by synthetically produced polypeptides.

[0249] Example 6: Production and evaluation of recombinant bioactive MayDay peptides This example describes the recombinant production of the MayDay polypeptides identified herein, and evaluation of their biological activity.

[0250] method Recombinant histidine-tagged MayDay(31-170) (rec-HISovMayDay(31-170)) was expressed and purified by Biomatik (Ontario, Canada) according to established procedures. Briefly, the ovine decorin sequence 31-170 fused to a 6x histidine tag at its N-terminus was cloned into the pET30a cloning vector. The expression plasmid was transformed into Escherichia coli (E. coli) BL21 cells, and the cells were grown at 37°C in Luria Broth (LB) medium supplemented with 50 μg / mL kanamycin (Sigma-Aldrich) until the absorbance at 600 nm reached 0.6. Isopropyl-β-d-1-thiogalactopyranoside (IPTG) (0.2 mM) (Sigma-Aldrich) was then added to the medium, and the cells were further incubated at 15°C for 16 hours. The cell suspension was sonicated with lysis buffer (50 mM Tris, pH 8.5, 300 mM NaCl, 20 mM histidine) (Sigma-Aldrich). The supernatant was loaded onto a nickel-IDA affinity column pre-equilibrated with lysis buffer, centrifuged, and the supernatant was collected. Fractions were analyzed by SDS-PAGE. Fractions were pooled and dialyzed against the final buffer (50 mM Tris, pH 8.5, 150 mM NaCl).

[0251] Recombinant histidine-tagged MayDay(31-171) (rec-HISovMayDay(31-170)) was tested in a Transwell migration assay using ovAD-MSCs as described above. rec-HISovMayDay(31-170) was prepared in PBS (0.1 mg / mL) and then diluted in DMEM 0.5 to final concentrations of 0.05, 0.50, and 5.00 ng / mL. Human recombinant SDF-1 (Sigma) was prepared in PBS (0.1 mg / mL) and diluted in DMEM 0.5 to a final concentration of 50 ng / mL. Membrane images were acquired, and migrated cells were counted using ImageJ. Results are expressed as normalized cell migration compared to media-only samples and represent the mean of three independent experiments. Statistical analysis (t-test) was performed using GraphPad Prism (version 8.4.1); * " p < 0.05, " ** " p ≤ 0.01, " *** ” p ≤ 0.001; **** ” p≦0.0001.

[0252] result The number of migrated cells present on the chamber below the Transwell membrane is shown in Figure 8. As can be clearly seen, the presence of only 5 ng / mL of recombinant MayDay polypeptide elicited significant cell migration, comparable to that observed with 50 ng / mL of SDF-1. In contrast, very few cells were observed using the negative control sample of medium alone.

[0253] This example demonstrated that recombinantly produced MayDay protein had the ability to induce cell recruitment comparable to that observed with MayDay polypeptides obtained from ovine forestomach matrix+macrophage cultures, thus establishing that the biological activity of bioactive polypeptides identified in assays such as those described herein can be reproduced by recombinantly expressed polypeptides.

[0254] Example 7: In vivo evaluation of the bioactivity of recombinant MayDay peptides This example describes the evaluation of recombinant MayDay polypeptide in an in vivo mesenchymal stem cell mobilization model.

[0255] method Expression of recombinant MayDay(31-170) The untagged protein (rec-ovMayDay(31-170)) was expressed in BL21 E. coli cells using the pSUMO vector as described above. After amplification and expression, the supernatant was loaded onto Q Sepharose™ fast flow pre-equilibrated with lysis buffer, centrifuged, and the supernatant fraction was analyzed by SDS-PAGE. Purity (>85%) was confirmed by SDS-PAGE. Lyophilized fractions were stored at -20°C.

[0256] Fluorescent labeling of mouse bone marrow mesenchymal stem cells (muBM-MSCs) muBM-MSCs were labeled using the Cellvue NIR815 fluorescent cell labeling kit (Licor, Lincoln, USA) immediately prior to injection into Balb / c mice. An 80% confluent plate of muBM-MSCs was resuspended in DMEM (5 mL), centrifuged, and resuspended in Diluent C (Licor, Lincoln, USA) at a concentration of 2 × 10 7 A final concentration of 4 × 10 cells / mL was obtained. Cells were labeled with a near-infrared dye (NIR815) according to the manufacturer's instructions. Briefly, Cellvue dye stock solution (2 μL, 4 × 10 cells / mL) was added. -6 MuBM-MSCs (M) was added to Diluent C (1 mL). The dye was then added to muBM-MSCs in Diluent C (1 mL) and incubated at 37°C for 5 minutes. The reaction was quenched with FBS (2 mL). Cells were pelleted by centrifugation at 400 rpm for 10 minutes and then rinsed with PBS (3 times, 10 mL). After the final wash, cells were resuspended in complete medium (5 mL) and kept at 37°C before use.

[0257] Mesenchymal stem cell chemotaxis in vivo Test articles, recombinant MayDay (rec-ovMayDay31-170), and human recombinant SDF-1 (Sigma) were prepared in 0.9% sterile saline (Braun, Melsungen, Germany). Five treatment groups were used: rec-ovMayDay(31-170) [1 μg / animal (approximately 0.05 μg / kg)], 10 μg / animal (approximately 0.5 mg / kg), and 25 μg / animal (approximately 1.25 mg / kg)]; SDF-1 10 μg / animal (approximately 0.5 mg / kg), and 0.9% sterile saline. Balb / c mice were anesthetized using isoflurane and placed in a ventral recumbent position. The anesthetic gas was administered via a nasal cone. The injection sites (hind paws and tail) were prepared by wiping with chlorhexidine. Test articles were administered to Balb / c mice (n=3 per test article) via 30 μL intramuscular injection into the right hind leg muscle ("treated").

[0258] After 5–10 min, NIR815-labeled muBM-MSCs (approximately 5 × 10 6 Each animal was injected with 5 mL / kg of labeled muBM-MSCs into its tail vein. Animals were imaged at predetermined time points using an optical imaging system (Pearl Trilogy, Licor, Lincoln, USA): 0, 3, 6, 12, and 24 hours after administration of labeled muBM-MSCs. After 24 hours, the animals were euthanized by cervical dislocation. The hindlimb musculature from the "treated" site and the corresponding "normal" tissue from the left hindlimb of each animal were dissected. Furthermore, major organs (brain, spleen, liver, intestine, kidney, and lung) were collected from all animals.

[0259] The explanted "treated" and "normal" muscle tissues were imaged using a Pearl Trilogy imaging system using the 800 nm channel (excitation: 786 nm, emission: 814 nm), and the fluorescent signal (pixels) for each was determined using Image studio software (version 5.2, Licor). For each sample, the background fluorescent signal (pixels) was also calculated over an equal area (cm). 2) tissue samples. Sample fluorescence was determined based on the signal of the test samples ("treated" and "normal") minus the corresponding background fluorescence.

[0260] The multipotency of isolated muBM-MSCs was confirmed by three-lineage differentiation assays (osteogenesis, adipogenesis, and chondrogenesis) (data not shown).

[0261] result Animals injected with recombinant MayDay peptide demonstrated donor cell recruitment to the injection site, as can be readily seen in Figure 9. No significant cell recruitment to the injection site was observed in the vehicle-only controls.

[0262] At all concentrations of rec-ovMayDay(31-170) tested, muBM-MSC localization to the injection site significantly increased ("normal" vs. "treated," Figure 9A), suggesting mobilization of muBM-MSC to the site of rec-ovMayDay(31-170) administration. Sites receiving higher concentrations of 10 and 25 μg of rec-ovMayDay(31-170) ("treated," Figure 9A) showed significantly higher localized muBM-MSC mobilization compared to sites receiving the vehicle-only control. Significantly increased fluorescence resulting from cell mobilization was observed in excised muscle tissue from animals injected with rec-ovMayDay(31-170) compared to normal controls, and the fluorescence observed in the excised tissue increased with increasing rec-ovMayDay(31-170) concentration (Figure 9A, lower panel).

[0263] Signal intensity at the injection site was quantified, and the results, presented in Figure 9, clearly demonstrated increased cell recruitment upon administration of recombinant MayDay peptide, and to a lesser extent with administration of SDF1. The highest dose of 25 μg of rec-ovMayDay(31-170) resulted in the localization of significantly more muBM-MSCs than the control, 10 μg of SDF-1.

[0264] These examples clearly demonstrate that bioactive agents can be prepared, isolated, identified, and produced (e.g., by recombinant methods) using the methods described herein, and that the biological activity of such materials can be evaluated and utilized to provide bioactive agents with research, diagnostic, and therapeutic value.

[0265] Publications [Table 2]

[0266] The words "comprise," "comprises," "including," and similar words used herein are not to be construed in an exclusive or exhaustive sense. In other words, they mean "including, but not limited to." When interpreting each statement herein that includes the term "comprise," "comprises," or "including," there may be other objects in addition to the object preceded by the term.

[0267] The entire disclosures of all applications, patents and publications, cited above and below (if any), are hereby incorporated by reference.

[0268] Where reference is made in the preceding description to integers or components that have known equivalents thereof, such integers are incorporated herein as if individually set forth.

[0269] It should be noted that various changes and modifications to the preferred embodiments of the present invention described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. Therefore, such changes and modifications are intended to be included in the present invention.

[0270] The invention may also be broadly said to consist of the parts, elements, and features referred to or shown in the application specification individually, or collectively, any or all combinations of two or more of said parts, elements, or features.

[0271] Aspects of the present invention have been described by way of example only, and it should be understood that changes, modifications, and additions may be made thereto without departing from the scope of the present invention, provided that the invention exists as defined in the appended claims. Furthermore, where known equivalents exist to particular features, such equivalents are incorporated as if specifically referred to herein.

Claims

1. 1. A pharmaceutical composition for treating a disease or disorder associated with stem cell deficiency in a subject in need thereof, or for modulating stem cell mobilization in a subject in need thereof, comprising: a) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; d) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; e) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; f) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; g) a polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4; ;and h) A polypeptide having at least 95% amino acid identity to any one of a) to g) above. comprising at least one isolated, purified, recombinant, or synthetic polypeptide selected from the group consisting of: Pharmaceutical compositions.

2. 1. A pharmaceutical composition for treating a disease or disorder associated with stem cell deficiency in a subject in need thereof, or for modulating stem cell mobilization in a subject in need thereof, comprising: An expression construct or vector comprising a nucleic acid encoding a polypeptide as defined in claim 1. Pharmaceutical compositions.

3. 1. A pharmaceutical composition for treating a disease or disorder associated with stem cell deficiency in a subject in need thereof, or for modulating stem cell mobilization in a subject in need thereof, comprising: A host cell comprising an expression construct or vector as defined in claim 2. Pharmaceutical compositions.

4. 1. A pharmaceutical composition for modulating tissue repair or treating a disease, disorder or condition associated with soft tissue in a subject in need thereof, comprising: comprising a therapeutically effective amount of one or more polypeptides as defined in claim 1. Pharmaceutical compositions.

5. The pharmaceutical composition of claim 4, wherein the therapeutically effective amount is sufficient to recruit one or more stem cells to the administration site where the pharmaceutical composition is administered or to the site where the administered polypeptide is localized.

6. The pharmaceutical composition of claim 1 , wherein the disease or disorder is a disease or disorder associated with a local stem cell deficiency.

7. The pharmaceutical composition of claim 6, wherein the local stem cell deficiency is a stem cell deficiency in a specific tissue or organ.

8. 2. The pharmaceutical composition of claim 1, wherein the stem cell mobilization comprises one or more of angiogenesis, hematopoiesis, protein expression, induction, or deposition, tissue remodeling, repair, or regeneration, cell proliferation, cell differentiation including stem cell differentiation, cell regulation, apoptosis, modulation of one or more immune responses, modulation of tumorigenesis, chemotaxis, or cell mobilization.

9. 9. The pharmaceutical composition of claim 1 or any one of claims 4 to 8, comprising an effective amount of the polypeptide to induce one or more processes selected from the group consisting of angiogenesis, vasculogenesis, tissue remodeling, enrichment of circulating stem cells, and muscle tissue regeneration.

10. The pharmaceutical composition of claim 4, wherein the disease, disorder or condition associated with soft tissue is a tissue defect.

11. The pharmaceutical composition of claim 10, wherein the tissue loss results from a surgical procedure or trauma.

12. 12. A pharmaceutical composition according to claim 1 or any one of claims 4 to 11, comprising an amount of said polypeptide effective to promote wound healing and / or soft tissue repair.

13. a medicament for treating a disease or disorder associated with stem cell deficiency in a subject in need thereof; a medicament for modulating stem cell mobilization in a subject in need thereof; a medicament for regulating tissue repair in a subject in need thereof; Medicaments for treating diseases, disorders or conditions associated with soft tissue in a subject in need thereof In the production of a) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 1 to 188 of mammalian decorin; b) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 31 to 188 of mammalian decorin; c) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 1 to 177 of mammalian decorin; d) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 31 to 177 of mammalian decorin; e) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 1 to 170 of mammalian decorin; f) a polypeptide comprising or consisting of an amino acid sequence corresponding to residues 31 to 170 of mammalian decorin; g) a polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4; ;and h) A polypeptide having at least 95% amino acid identity to any one of a) to g) above.

2. Use of at least one isolated, purified, recombinant or synthetic polypeptide selected from the group consisting of:

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