Extracellular matrix and its use to regulate the differentiation of mesenchymal stem cells (Machine-

专利摘要:
Extracellular matrix and its use to regulate the differentiation of mesenchymal stem cells. The present invention relates to an extracellular matrix comprising ECM producing cells, a lysyl oxidase (LOX), and bone morphogenetic protein 1 (BMP1), and its use to regulate the differentiation of mesenchymal stem cells and increase synthesis and/or deposits of collagen in an extracellular matrix. The present invention also relates to a method for obtaining said extracellular matrix comprising incubating cells in the presence of a composition comprising a lysyl oxidase, or a fragment thereof, and bone morphogenetic protein 1, or a fragment thereof. (Machine-translation by Google Translate, not legally binding)
公开号:ES2717374A1
申请号:ES201731441
申请日:2017-12-20
公开日:2019-06-20
发明作者:Pascual Fernando Rodriguez；Garcia Tamara Rosell
申请人:Consejo Superior de Investigaciones Cientificas CSIC；
IPC主号:

专利说明:

[0001]
[0002]
[0003]
[0004] The invention relates to an extracellular matrix (ECM) with an increase in collagen deposition comprising a lysyl oxidase (LOX) and a bone morphogenetic protein 1 (BMP1), and its use to regulate the differentiation of mesenchymal stem cells. The invention also describes an in vitro method for obtaining said extracellular matrix. Therefore, the present invention relates to cell culture methodology, more specifically, to increase the formation of extracellular matrices.
[0005]
[0006] STATE OF THE ART
[0007]
[0008] Tissue engineering is emerging as a powerful therapeutic strategy to treat injured or degenerated tissues by implanting elements that mimic natural, synthetic or semi-synthetic tissues and organs. Biomaterials based on ECM-derived cells take advantage of the inherent ability of cells to create highly sophisticated supramolecular structures.
[0009]
[0010] The extracellular matrix (ECM) is a dynamic microenvironment that significantly influences a wide variety of cellular processes, including cell proliferation, adhesion, migration and differentiation, as well as playing critical roles in homeostasis and tissue and organ regeneration. . Tissue engineering has taken advantage of these properties and ECM-based biomaterials are currently a promising therapy for tissue repair and regeneration. Compared to artificial matrices, the use of native ECM substrates allows a better conservation of the microenvironment of appropriate cell growth, thereby accelerating the repair of damaged tissue. Therefore, there is still a great interest in the development of techniques and protocols to improve the innate capacity of cells to create their own in vitro ECM . However, despite significant progress, optimal conditions for rapid and efficient deposition of ECM components, mainly collagen, the most important structural biomolecule, are still lacking. This behavior has been attributed to diluted culture media that greatly limits the post-translational extracellular modifications of collagen, in particular the cleavage of propeptide C, a reaction catalyzed by the protein C-proteinase / bone morphogenetic protein 1 (BMP1) and the formation of covalent crosslinks, initiated by members of the lysyl oxidase (LOX) family. These enzymatic activities are intimately related since BMP1 it also catalyzes the proteolytic activation of the LOX precursor to produce the active form.
[0011]
[0012] In order to facilitate the deposition of collagen in cell cultures different protocols have been developed, including ascorbic acid and serum supplement. A method of interest was designed based on the addition of inert macromolecules in culture media to mimic a high density extracellular space, a biophysical phenomenon known as macromolecular agglomeration. Under this principle, it has been described that the addition of dextran sulfate (DxS) or Ficoll ™ increases the ability to deposit an abundant extracellular matrix in different cell cultures, including fibroblasts, keratinocytes, tenocytes or chondrocytes.
[0013]
[0014] However, conventional cell culture conditions are far from ideal, due to the fact that the diluted microenvironment does not favor the production of ECM components. This is particularly evident for collagen, the most important structural biomolecule, since its synthesis and deposition in the matrix is enzymatically limited.
[0015]
[0016] MSCs are interesting candidates for biological cell-based tissue repair protocols due to their high proliferative capacity in culture, while retaining their potential for differentiation into multiple mesenchymal lineages. In addition to its undoubted scientific interest, the possibility of monitoring and controlling the MSC differentiation process is a fundamental regulatory and clinical requirement. For this reason, the molecular regulation of MSC differentiation has been studied extensively. Most studies are in vitrn, since the identity of MSCs in their tissues of in vivo origin remains undefined. Based on the information obtained from biology studies of the development of embryonic skeletongenesis, several signaling pathways and transcription factors have been investigated and have been shown to play a fundamental role in MSC differentiation. In particular, it is well known that the signaling pathways of Wnt and transforming growth factor for bone morphogenetic protein modulate the molecular differentiation of MSCs in cartilage and bone. Of great relevance to the emerging concept of stem cell niches is the demonstration that physical factors can also participate in the regulation of MSC differentiation. The knowledge of the regulation of MSC differentiation will be fundamental in the design of three-dimensional culture systems and bioreactors through mathematical models applied to systems and networks biology.
[0017]
[0018] Patent application US2012 / 178159 refers to materials and methods for culturing and expanding mammalian MSC while maintaining its undifferentiated phenotype, its capacity for self-renewal and / or differentiation potential to multiple lineages. In one embodiment, a method of the invention comprises i) seeding freshly isolated MSCs on a flat surface, such as plastic tissue culture plates, or on a 3-D scaffold and its subsequent growth under physiological or low-tension conditions. O2 (for example, less than 20 % O2) for a sufficient period of time to allow the formation of a 3-D ECM network; ii) decellularize the cultures in the plates or the 3-D framework to obtain de-cellularized ECM matrices therein; and iii) replacing the decellularized matrices in the plates or 3-D framework with the MSCs, so that the resected MSCs grow in the plate or framework comprising 3-D ECM obtained from cells and maintaining a non-phenotype. differentiated.
[0019]
[0020] The patent application US2007 / 0269886 describes a cell culture product for propagating embryonic stem cells, and keeping in cultivation its characteristics of self-renewal and pluripotency for prolonged periods of time. The cell culture product includes a substrate and a coating solution. The coating solution includes a mixture of extracellular matrix proteins and an aqueous solvent, in which the total protein concentration in the coating solution can vary from about 10 pg / ml to 1 mg / ml. Although this patent application describes a cell culture product that includes a coating solution that functions as a substitute for the extracellular matrix, capable of maintaining the undifferentiated phenotype of the stem cells, this extracellular matrix does not exhibit the usual characteristics of a matrix physiological, at both quantitative levels, that is, the extent of the components of the matrix that make up such a product, and qualitative, such as the appropriate cross-linking of collagen. Therefore, in the state of the art there is a need to develop a more physiological matrix that allows the cultivation of stem cells, and, at the same time, that is able to maintain its undifferentiated phenotype.
[0021]
[0022] DESCRIPTION OF THE INVENTION
[0023]
[0024] The inventors have found that implementing cultures of fibroblasts with supernatants rich in LOX and BMP1 from stable lines of HEK293 cells, promotes an increase in the deposition of collagen on the insoluble matrix at the expense of the fraction soluble in the extracellular medium (Example 1 ). Using decellularization protocols, the inventors also showed that matrices obtained from fibroblasts regulate the adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSCs), and this effect was modulated with LOX / BMP1 (Example 2). These results support a convenient protocol to increase the capacity of cell cultures in vitro to deposit collagen in the extracellular matrix, which represents a promising approach for its application in tissue engineering, in addition to providing evidence that matrices obtained from fibroblasts are capable of regulating the adipogenic and osteogenic differentiation of human MSCs, a cellular tool of enormous interest in regenerative medicine.
[0025]
[0026] Therefore, in one aspect, the present invention relates to an extracellular matrix (ECM), hereinafter "ECM of the invention", comprising a lysyl oxidase (LOX), or a fragment thereof. , and a bone morphogenetic protein 1 (BMP1), or a fragment thereof.
[0027]
[0028] The term "extracellular matrix" or "ECM" refers to a collection of extracellular molecules secreted by cells from mammalian tissues that provides structural and biochemical support to the surrounding cells, in particular connective tissue cells, e.g. cells such as fibroblasts , osteoblasts, chondrocytes, epithelial cells, smooth muscle cells, adipocytes, and mesenchymal cells, and whose material in vivo surrounds and supports these cells. In general, the NDE is made up of fibers embedded in what is commonly referred to as a 'ground substance'. The fibers are formed by structural proteins, generally collagen and / or elastin. In aspects of the present invention, the fibers of the matrix are preferably collagen. Particularly suitable collagens are fibril-forming collagens. Particularly preferred are type I collagen, type II collagen, type III collagen, type IV collagen or type X collagen. Type I collagen is most preferred. The 'substance' is composed of proteoglycans (or mucopolysaccharides). ) and may comprise proteins that provide functionality such as fibrillin, fibronectin, and / or laminin. In aspects of the invention, the ECM suitably comprises at least one proteoglycan as a component of the fundamental substance. Proteoglycan is formed by a core protein with branches of glycosaminoglycan (GAG) molecules. Suitable GAGs are for example hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate, heparin sulfate, and keratan sulfate. GAGs are preferably linked to the core protein through a trisaccharide bond (eg, a GalGalXyl bond). Examples of proteoglycans are decorin, biglycan, versican and aggrecan. The proteoglycans may be optionally interconnected by hyaluronic acid molecules. Alternatively, multiple proteoglycans can be attached to a single main structure of hyaluronic acid. In both cases, the fundamental substance forms a polymer network or a gel capable of retaining water. This network may also comprise proteins such as: glycoproteins, as for example. laminin, entactin, tenascin, fibrillin or fibronectin, to improve the structural integrity of the network and for cell attachment to the ECM; osteocalcin (GIa protein), as a protein that binds to calcium during the mineralization; osteonectin, which fulfills a function of bridging between collagen and mineral component; and sialoproteins, such as bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoroprotein (DSPP) and extracellular matrix phosphoglycoprotein (MEPE). The matrix may further comprise cytokines and growth factors. Suitable cytokines and growth factors include osteoprotegerin (OPG), epidermal growth factor (EGF), fibroblast growth factors (bFGF, FGF-I and FGF-2), interferon-a (IFN-a), interleukins (IL) -1, IL-4, IL-6, IL-10, and IL-11), growth factor obtained from platelets (PDGF), transforming growth factors (TGF-a and TGF-P), necrosis factors tumor (TNF), insulin-like growth factors (IGF-I and IGF-II), osteoclast differentiation factor (ODF, also known as OPGL [osteoprotegerin ligand], RANKL [NFB ligand receptor activator], TRANCE [activation-induced cytokine related to TNF]) and macrophage colony stimulating factor (M-CSF). Most of these (IL-1, IL-4, IL-6, IL-11, TNF, EGF, bFGF, FGF-2, PDGF, and M-CSF) stimulate bone resorption. Some (IGF-1 and IGF-11, FGF-2 and TGF-3) improve bone formation, while others (OPG) inhibit bone resorption. In addition, others (PDGF and TGF-P) also stimulate the proliferation and differentiation of collagen-producing cells.
[0029]
[0030] The surface on which the ECM of the present invention is deposited may be able to adhere the cells to be cultured and be able to release the cells when the culture process is complete. It is also known that animal cells in particular adhere well to surfaces that carry high densities of sodium ions. Therefore, they adhere to materials that tend to acquire a negative charge and thus bind to sodium ions. The surface can be formed by a material to which the cells adhere or can be manufactured from an inert support and coated with material of this type. Suitable materials include plastics, materials such as nylon, polycarbonate, polystyrene, epoxy resins, silicone rubber, cellulose acetate, cellulose nitrate, cellophane, polyethylene terephthalate, polyformaldehyde, fluorinated ethylene and propylene copolymer, polyphenylene oxide, mica polypropylene, carbon, collagen, inert insoluble metal oxides, phosphates, silicates or carbides, silicon carbide, inert metals such as stainless steel, aluminum, titanium or palladium, or ceramic or glass. Sometimes it is necessary to modify the characteristics of the surface by applying a coating of less adhesive material to the cells, which facilitates their removal. The coating materials which make it easier to remove the cells from the surfaces of the matrix are polyfluorinated hydrocarbons such as polytetrafluoroethylene, or silicones such as polymethylhydrogensiloxane. On the other hand, a surface of low adhesive capacity can be altered to give better adhesion by applying a suitable coating.
[0031] Therefore, the coating of the particular surface that is used will depend on the type of cells to be cultivated and on whether the collection of the matrix is needed. A coating or combination of coatings suitable for any particular application can be determined empirically. Materials that may be suitable for matrix formation for use in the present invention include polycarbonate, nylon 6, nylon 11, nylon 12, glass, polyformaldehyde, polypropylene, and 2,6-dimethylphenylene oxide. Coated matrices which may be suitable for the formation of matrices for use in the present invention include polycarbonate coated with polytetrafluoroethylene, silicone, polymethylhydrogensiloxane; glass coated with silicone, polytetrafluoroethylene or stearic acid; or polyethylene terephthalate, nylon 6, nylon 11 and nylon 12, each coated with polytetrafluoroethylene. In any specific application of the present method, the material of choice in particular can be determined by additional factors such as the method by which the matrix is to be sterilized.
[0032]
[0033] The ECM of the invention comprises a protein lysyl oxidase (LOX). The LOX protein, also known as protein lysine 6-oxidase, is a protein that, in humans, is encoded by the LOX gene. In humans, the LOX gene is located on chromosome 5q23.3-31.2. The DNA sequence encodes a polypeptide of 417 amino acids, whose first 21 residues constitute a signal peptide, weighing approximately 32 kDa. The carboxy terminus contains the active copper (II) ion, lysine, tyrosine and cysteine residues comprising the catalytically active site. Although alternative splicing forms have been described to give rise to multiple transcript variants, the present mention only refers to isoform 1 of the LOX protein (preprotein, NCBI Reference Sequence NP_002308.2 (SEQ ID NO: 1)), the only one that maintains the processing site by protease BMP1, and therefore is processed and activated proteolytically with BMP1.
[0034]
[0035] HE
[0036]
[0037] In a particular embodiment of the ECM of the invention, the LOX protein comprises a sequence of amino acids with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 1.
[0038] In the present invention, it is understood that "identity" or "sequence identity" refers to the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a different degree of identity will be obtained, expressed as a percentage. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by conventional sequence alignment algorithms known in the state of the art, such as, for example, BLAST [Altschul SF et al. Basic local alignment search tool. J Mol Biol.
[0039] 19905 of Oct; 215 (3): 403-10]. BLAST programs, for example, BLASTN, BLASTX, and T BLASTX, BLASTP, and TBLASTN, are in the public domain on the National Center for Biotechnology Information (NCBI) website. The person skilled in the art understands that mutations in the nucleotide sequence of genes that give rise to conservative amino acid substitutions at non-critical positions for the functionality of the protein are evolutionarily neutral mutations that do not influence its overall structure or functionality. Therefore, the LOX protein variants of SEQ ID NO: 1 are included within the context of the present invention. Apart from humans, other sources from which variants of the LOX protein can be obtained include, but are not limited to, non-human animals such as non-human primates, pigs, mice and rats among others. In a more particular embodiment, the LOX protein comprises the sequence SEQ ID NO: 1. The present invention also includes fragments of the LOX protein, said fragments being considered variants of the LOX protein. As used herein, the term "LOX protein fragment" or "LOX protein fragment" refers to a polypeptide having one or more (several) amino acids deleted from the amino and / or carboxyl terminal end of the protein LOX; or a homologous sequence thereof; wherein the fragment has lysyl oxidase activity. Therefore, the LOX protein variants show lysyl oxidase activity. Assays for evaluating the lysyl oxidase activity of a given protein (or variant) are widely known in the state of the art. Examples of these assays include, but are not limited to, a method based on the measurement of tritiated water released by enzymatic action on lysine and hydroxylysine bound to labeled proteins (Melet, J. et al., 1977. Analytical Biochemistry, vol 77 ( 1): 141-146) and a fluorescence assay using 1,5-diaminopentane as a substrate and releasing hydrogen peroxide which is detected using Amplex red in reactions coupled to horseradish peroxidase (Palamakumbura AH1 and Trackman PC, 2002. Anal Biochem, 300 (2): 245 51). In addition, commercial kits, such as the Lisil Oxidase Activity Assay Kit from BioVision, Inc., San Francisco, or Abcam PLC, Cambridge, UK, can also be used to measure lysyl oxidase activity.
[0040] The NDE of the invention also comprises a bone morphogenetic protein 1 or BMP1. BMP1 is a protein that in humans is encoded by the BMP1 gene. The BMP1 locus encodes a protein that is capable of inducing cartilage formation in vivo. The BMP1 protein cleaves the C-terminal propeptides of procollagen I, II, and III and its activity increases with the endopeptidase C enhancer protein of procollagen. The BMP1 gene variants is expressed as splicing (splicing) alternative that share a N-terminal protease domain but differ in their C-terminal region. There are five isoforms of the protein created by alternative splicing: precursor of isoform 3 (NCBI Reference Sequence: NP_006120 (SEQ ID NO: 2)), precursor of isoform 1 (NCBI Reference Sequence: NP_001190 SEQ ID NO: 3)), isoform X2 (NCBI Reference Sequence: XP_016869227, SEQ ID NO: 4)), isoform X3 (NCBI Reference Sequence: XP_011542919 (SEQ ID NO: 5)), isoform X1 (Reference Sequence) from NCBI: XP_006716449 (SEQ ID NO: 6)
[0041]
[0042] SEQ ID NO: 2
[0043]
[0044]
[0045] SEQ ID NO: 3
[0046]
[0047] SEQ ID NO: 4
[0048]
[0049] SEQ ID NO: 5
[0050]
[0051]
[0052]
[0053]
[0054] SEQ ID NO: 6
[0055]
[0056]
[0057]
[0058] In the context of the present invention, any isoform of the BMP1 protein can be used, including additional members of the BMP1 / toloid-like family, such as toloid-1 (TLL1) or toloid-2-like (TLL2). ), which show a very high degree of homology with BMP1 at its catalytic N-terminus. There are four TLL1 protein isoforms created by alternative splicing, and one of TLL2: X1 isoform of toloid-like protein 1 (NCBI Reference Sequence: XP_016864059.1 (SEQ ID NO: 7)), X2 isoform of similar protein to toloid 1 (NCBI Reference Sequence: XP_011530516.1 (SEQ ID NO: 8)), protein isoform 2 similar to toloid 1 (NCBI Reference Sequence: NP_001191689.1 (SEQ ID NO: 9)), isoform 1 of protein similar to toloid 1 (Reference Sequence of NCBI: NP_036596.3 (SEQ ID NO: 10)), protein precursor similar to toloid 2 (NCBI Reference Sequence: NP_036597 (SEQ ID NO: 11)).
[0059] SEQ ID NO: 7
[0060]
[0061]
[0062] SEQ ID NO: 8
[0063]
[0064] SEQ ID NO: 9
[0065]
[0066] SEQ ID NO: 10
[0067]
[0068] SEQ ID NO: 11
[0069]
[0070]
[0071]
[0072]
[0073] In a particular embodiment of the ECM of the invention, the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 2.
[0074]
[0075] The term "identity" has been defined in the foregoing paragraphs, therefore variants of the BMP1 protein of SEQ ID NO: 2 are included within the context of the present invention. Apart from humans, other sources from which can obtain variants of the BMP1 protein include, but are not limited to, non-human animals such as non-human primates, pigs, mice and rats etc. In a more particular embodiment, the BMP1 protein comprises the sequence SEQ ID NO: 2. The present invention also includes fragments of the BMP1 protein, said fragments being considered variants of the BMP1 protein. As used herein, the term "BMP1 protein fragment" or "BMP1 fragment" refers to a polypeptide having one or more (several) amino acids deleted from the amino and / or carboxyl terminal end of the BMP1 protein, or a homologous sequence thereof, wherein the fragment has BMP1 activity. therefore, the present invention also includes variants of the BMP1 protein that show the same function as the BMP1 protein, ie, cleavage of the C-terminal propeptides of procollagen I, II, and III. Assays to evaluate the activity of BMP1 of a given protein are widely known in the state of the art. Examples of trials for measuring the activity of BMP1 include, but are not limited to, procollagen assay (Hartigan, N., Garrigue-Antar, L. and Kadler, KE 2003. The Journal of Biochemical Chemistry, 278 (20): 18045-18049; pro-lysyl oxidase processing assay (Uzel MI, Scott IC, Babakhanlou-Chase H, Palamakumbura AH, Pappano WN, Hong HH, Greenspan DS, Trackman PC J Biol Chem. 2001; 276 (25): 22537-43) In addition, commercial substrates can also be used to measure the activity of BMP1, such as the Recombinant Human BMP-1 / PCP Assay from R & D Systems.
[0076]
[0077] In a particular embodiment, the ECM of the invention comprises ECM producing cells. There are many types of cells that contribute to the development of the various types of extracellular matrix. Fibroblasts are the most common type of cell in connective tissue ECM, in which they synthesize, maintain and provide a structural framework; the fibroblasts secrete the precursor components of the ECM, including the ground substance. Chondrocytes are found in the cartilage and produce the cartilaginous matrix. Osteoblasts are responsible for bone formation. Therefore, in a particular embodiment of the ECM of the invention, the ECM producing cells are fibroblasts, keratinocytes, tenocytes, chondrocytes and / or any combination thereof. The cells are preferably obtained from a living human being; although, as an alternative, human donors or recently deceased animals can be used. In another particular embodiment, the ECM producing cells are fibroblasts.
[0078]
[0079] Fibroblasts can be obtained from a patient's tendon. For example, a longus palms tendon may be removed from the patient's arm. But for the addition of the ECM material, the obtained fibroblasts are isolated and cultured using conventional techniques, for example, the collected cells can be cultured in Hamm's F-12 culture medium, 10% fetal calf serum, L -glutamine (292 mu.g / cc), penicillin (100 u / cc), streptomycin (100.mu.g / cc), and ascorbic acid (5 .mu.g / cc) at 37 ° C.
[0080]
[0081] The ECM-producing cells can be genetically modified to produce the LOX enzyme, or a fragment thereof, and the BMP1 protein, or a fragment thereof. In the state of the art methods for genetically modifying cells are widely known. Therefore, in a particular embodiment of the ECM of the invention, the ECM-producing cells are genetically modified to produce the recombinant LOX enzyme and / or recombinant BMP1. This means that, in a particular embodiment, ECM producing cells comprise overexpression of the nucleotide sequences encoding the LOX enzyme and the BMP1 protein, i.e., the amount of LOX enzyme and BMP1 produced by the cell increases. The amino acid sequences encoding the enzyme LOX and BMP1 are described below. As the expert in the In other words, overexpression of a gene can be achieved, for example, by increasing the number of copies of the genes encoding the LOX enzyme and the BMP1 protein, or by expressing these genes under the appropriate regulatory sequences such as a promoter. In the state of the art, the nucleotide sequences encoding the LOX enzyme and the BMP1 protein are known and can be recovered from public databases.
[0082]
[0083] In another particular embodiment, the ECM may additionally comprise other cells, for example stem cells, apart from those that commonly contribute to the development of the extracellular matrix. The ECM can be used as a substrate to grow cells, such as, but not limited to, mesenchymal stem cells, smooth muscle cells and cardiomyocytes. Therefore, in a particular embodiment of the ECM of the invention, the ECM additionally comprises mesenchymal stem cells.
[0084]
[0085] The NDE can also comprise a "bioactive agent" or a "bioactive compound". In the present document, these terms are used to refer to a compound or entity that alters, inhibits, activates or otherwise influences biological or chemical processes. Examples of bioactive agents may include, but are not limited to, osteogenic or chondrogenic proteins or peptides, anticancer substances, antibiotics, immunosuppressants, antiviral substances, enzyme inhibitors, hormones, neurotoxins, opioids, hypnotic substances, antihistamines, lubricants, antispasmodic agents and agents of muscle contraction including channel blockers, miotics and anticholinergic agents, antiparasitic and / or antiprotozoal compounds, modulators of cell-extracellular matrix interactions including inhibitors of cell growth and anti-adhesion molecules, vasodilator agents, inhibitors of DNA, RNA or protein synthesis , antihypertensive agents, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, angiogenic factors, antisecretory factors, anticoagulant and / or antithrombotic agents, local anesthetics, prostaglandins and antidepressant people. In certain embodiments, the bioactive agent is a drug. In certain embodiments, the bioactive agent is a small molecule. Additional bioactive agents include RNA, such as siRNA, and osteoclast stimulating factors. In some embodiments, the bioactive agent may be a factor that stops, sequesters, or reduces the activity of bone growth inhibitors. In some embodiments, the bioactive agent is a growth factor, cytokine, extracellular matrix molecule or a fragment or derivative thereof, for example, a cell binding sequence such as RGD. In a particular embodiment, the bioactive agent is selected from transforming growth factor beta (TGF-beta), dextran sulfate, ascorbate and combinations thereof. The NDE may comprise more than one bioactive agent. By therefore, in another particular embodiment, the ECM additionally comprises a growth factor and extracellular matrix molecules. In a more particular embodiment, the ECM further comprises TGF-beta, dextran sulfate and ascorbate, these bioactive agents being able to stimulate the synthesis of extracellular matrix components. Other bioactive agents capable of stimulating the synthesis of extracellular matrix components also include profibrotic cytokines such as tumor necrosis factor alpha (TNF-alpha), bioactive peptides such as angiotensin II or proteins such as connective tissue growth factor (CTGF). . Molecules capable of increasing the density of the extracellular medium such as Ficoll ™ may also be understood as bioactive agents.
[0086]
[0087] Additionally, the ECM of the invention can be combined with the bone marrow aspirate from an autograft, autograft bone, selected autograft cell preparations, autograft cells containing bone stimulation genes prior to implantation at a defective site.
[0088]
[0089] In a particular embodiment, the extracellular matrix of the invention comprises an amount of insoluble collagen deposited in the matrix of at least 3 pg / 106 cells, more than four times the amount accumulated in the absence of LOX and BMP1 as can be seen in Figure 4
[0090]
[0091] As explained at the beginning of the present description, the inventors have discovered that performing cultures of fibroblasts with supernatants enriched in LOX and BMP1 from stable HEK293 cell lines greatly increased the deposition of collagen on the insoluble matrix at expense of the soluble fraction in the extracellular medium. Therefore, in a particular embodiment, the ECM of the invention is decellularized.
[0092]
[0093] Additionally, in another aspect, the present invention also includes a decellularized ECM comprising an increase in the amount of collagen compared to an ECM that has not been produced by culturing fibroblasts with LOX and BMP1. Therefore, the present invention relates to a decellularized extracellular matrix comprising the amount of insoluble collagen deposited in the matrix of at least 3 pg / 106 cells, more than four times the amount accumulated in the absence of LOX and BMP1.
[0094]
[0095] Uses of the invention
[0096]
[0097] Additionally, the authors of the present invention have also discovered that the carrying out cultures of fibroblasts with supernatants enriched in LOX and BMP1 greatly increases the deposition of collagen on the insoluble matrix at the expense of the fraction soluble in the extracellular medium (Example 1). These results support a convenient protocol to increase the ability of in vitro cell cultures to deposit collagen in the extracellular matrix, which represents a promising approach for its application in tissue engineering.
[0098]
[0099] Therefore, in another aspect, the present invention relates to an in vitro use of a composition comprising a lysyl oxidase (LOX), or a fragment thereof, and bone morphogenetic protein 1 (BMP1), or a fragment of the same, hereinafter "first use of the invention", to increase the synthesis and / or deposition of collagen in an extracellular matrix.
[0100]
[0101] The terms "Lisil oxidase" and "bone morphogenetic protein 1" and fragments thereof have been defined in previous paragraphs. As explained above, in a particular embodiment, the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 1. In another particular embodiment, the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 4. The term "identity" has been defined above.
[0102]
[0103] As can be seen in the examples of the present disclosure, a composition comprising the LOX, or a fragment thereof, and BMP1, or a fragment thereof, hereinafter "composition of the invention", can increase the synthesis and / or deposition of collagen in an extracellular matrix.The present composition, in addition to comprising the LOX and BMP1 proteins, can comprise another type of compounds that favor the activity of LOX and BMP1. Examples of these compounds are any of The bioactive agents mentioned above, therefore, in a particular embodiment, the bioactive agent is selected from transforming growth factor beta (TGF-beta), dextran sulfate, ascorbate and combinations thereof. a bioactive agent Therefore, in another particular embodiment, the ECM additionally comprises a growth factor and molecules of the extracellular matrix. More particular ion, the ECM further comprises TGF-beta, dextran sulfate and ascorbate.
[0104]
[0105] To increase the synthesis and / or deposition of collagen in an extracellular matrix, the composition of the invention can be contacted with the ECM-producing cells and then these cells can be deposited on a surface for cultivation or, alternatively, the composition of the invention can be contacted directly with an ECM or a surface such as those described above which already comprise ECM producing cells.
[0106]
[0107] Examples of ECM-producing cells, which may be cultured together with the composition of the invention or which may be present in the ECM, include, but are not limited to, fibroblast cells, keratinocyte cells, tenocyte cells, chondrocyte and / or any combination thereof. Therefore, in a particular embodiment, the extracellular matrix comprises fibroblast cells, keratinocyte cells, tenocyte cells, chondrocyte cells and / or any combination thereof. The medium and the conditions (pH, medium, temperature, etc.) for cultivating these cells are widely known in the state of the art.
[0108]
[0109] As explained above in the present invention, the inventors have discovered that matrices obtained from fibroblasts regulate the adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSCs), and that this effect was modulated with LOX and BMP1 (cf. Example 2). These results provided evidence that matrices obtained from fibroblasts are capable of regulating the adipogenic and osteogenic differentiation of human MSC, a powerful cellular tool in regenerative medicine.
[0110]
[0111] Therefore, in another aspect, the present invention relates to an in vitro use of the extracellular matrix of the invention, hereinafter "second use of the invention", for regulating the differentiation of stem cells, preferably mesenchymal stem cells, in which the ECM is decellularized.
[0112]
[0113] As used herein, the term "regulating" or "regulates" refers to the control of the ability of stem cells to differentiate into a more specialized cell type. In developmental biology, cell differentiation is the process by which a cell changes from one cell type to another. As the person skilled in the art knows, stem cells are a class of undifferentiated cells that are capable of differentiating into specialized cell types. In a particular embodiment of the present invention, the stem cell whose differentiation is regulated by the ECM of the invention is a mesenchymal stem cell. Mesenchymal or stromal stem cells are a population of stromal cells, present in the bone marrow, adipose tissue and most of the connective tissues, capable of differentiation in mesenchymal tissues such as adipose tissue (adipogenesis), bone tissue (osteogenesis) and cartilage (chondrogenesis). The NDE of the invention can be used to culture MSCs and regulate their differentiation into adipose, bone and cartilaginous tissue. In a particular embodiment of the second use of the invention, the adipogenic and / or osteogenic differentiation of mesenchymal stem cells is regulated by the ECM of the invention, in which the ECM is decellularized. In a more particular embodiment, the differentiation of mesenchymal stem cells is reduced or inhibited with the ECM of the invention, in which the ECM is decellularized.
[0114]
[0115] In the present description, it is considered that "the differentiation of mesenchymal stem cells is reduced" or "the differentiation of mesenchymal stem cells is inhibited" when more than 50%, preferably more than 60% of the whole cell population of stem cells Mesenchymal does not differ in mesenchymal tissues.
[0116]
[0117] As used herein, the term "decellularized" refers to the process used in biomedical engineering to isolate the ECM from its resident cells, leaving an ECM framework. The person skilled in the art is able to eliminate the cells within the ECM without damaging the extracellular components. To remove the cells from an ECM, physical, chemical and enzymatic methods can be used. The most common physical methods used to lyse, remove, and remove cells from the matrix of a tissue are the use, for example, of temperature, force and pressure, or electrical stimulation. As an alternative to physical methods, an appropriate combination of chemical agents can be selected for decellularization depending on the thickness, composition of the extracellular matrix, and the use for which the ECM is intended. The chemical agents used to remove and remove the cells include, but are not limited to, acids, alkaline treatments, ionic detergents, non-ionic detergents, and zwitterionic detergents. Sodium dodecyl sulfate (SDS), the ionic detergent, is commonly used due to its high efficiency to lyse cells without significantly damaging the ECM. The detergents act efficiently to lyse the cell membrane and expose the contents to further degradation. After the SDS smooths the cell membrane, endonucleases and exonucleases degrade the genetic contents, while other components of the cell are solubilized and removed from the matrix by washing. Alkaline and acid treatments can be effective partners with SDS treatment due to their ability to degrade nucleic acids and solubilize cytoplasmic inclusions. The best-known non-ionic detergent is Triton X-100, which is popular because of its ability to alter the interactions between lipids and between lipids and proteins. Triton X-100 does not alter protein-protein interactions, which are beneficial in maintaining the ECM intact. EDTA is a chelating agent that binds to calcium, which is a necessary component for proteins to interact with each other. By preventing the availability of calcium, EDTA prevents proteins that act as bridges between cells from binding to each other. EDTA is often used with trypsin, an enzyme that acts as a protease to break existing bonds between Integral proteins of neighboring cells within a tissue. Taken together, the EDTA-trypsin combination forms a good equipment for the decellularization of the ECM. Enzymes used in decellularization treatments are used to break down the bonds and interactions between nucleic acids, cells that interact through neighboring proteins, and other cellular components. Lipases, thermolysin, galactosidase, nucleases, and trypsin have all been used in the removal of cells. After lysing a cell with a detergent, acid, physical pressure, etc., endonucleases and exonucleases can start the degradation of the genetic material.
[0118]
[0119] Within the context of the present invention, the use of the ECM of the invention to select drugs in the treatment of diseases is also included, as well as the use of the ECM of the invention as a support material for regenerating a biological tissue.
[0120]
[0121] Methods of the invention
[0122]
[0123] In another aspect, the invention relates to an in vitro method for obtaining an extracellular matrix of the invention, hereinafter "first method of the invention", which comprises incubating the ECM-producing cells in the presence of a composition comprising a lysyl oxidase (LOX), or a fragment thereof, and bone morphogenetic protein 1 (BMP1), or a fragment thereof, or culturing ECM-producing cells genetically modified to produce a LOX enzyme, or a fragment of the same, and / or BMP1, or a fragment thereof.
[0124]
[0125] The first method of the invention comprises the incubation of cells in the presence of a composition comprising LOX and BMP1 proteins or a fragment thereof. Methods and means for incubating cells are widely known in the state of the art. An example of these methods can be found in the illustrative examples of the present invention. Briefly, the ECM-producing cells are incubated in DMEM medium (pH 7.4) without serum or phenol red, and then dextran sulphate and ascorbate are added in the presence or absence of TGF-beta for 4 days at 37 ° C.
[0126]
[0127] In addition, the composition further comprising LOX and BMP1 proteins may also comprise a "bioactive agent" or a "bioactive compound". In the present document, these terms are used to refer to a compound or entity that alters, inhibits, activates or otherwise influences biological or chemical processes. Examples of bioactive agent have been mentioned above in the present description.
[0128] In a particular embodiment of the first method of the invention, the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 1. In a particular embodiment of the first method of the invention, the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or a 99% with SEQ ID NO: 2. These particular embodiments, as well as the terms and expressions used, have been explained and defined in previous inventive aspects of the present invention.
[0129]
[0130] In another particular embodiment, the ECM producing cells are fibroblasts, keratinocytes, tenocytes, chondrocytes and / or any combination thereof. In another particular embodiment, the ECM-producing cells are genetically modified to produce the LOX enzyme, or a fragment thereof, and / or BMP1 or a fragment thereof.
[0131]
[0132] After having incubated the cells with the composition comprising LOX and BMP1 proteins, they can be allowed to form an ECM in tissue culture or, after sowing on a scaffold. In the first method of the invention to produce the ECM, the cells can be exposed to various factors or compounds to stimulate the production of said ECM. Once the NDE occurs, it can be isolated by techniques well known in the state of the art. The NDE obtained in this way shows an increase in collagen deposition with respect to another ECM whose cells have not been incubated with a combination of LOX and BMP1 proteins.
[0133]
[0134] Alternatively, the first method of the invention may comprise culturing ECM producing cells genetically modified to produce the enzyme LOX and / or BMP1. In the present description, examples of methods for genetically modifying cells have been previously described.
[0135]
[0136] As a consequence of the implementation of the first method of the invention, an ECM is obtained. Therefore, in another aspect, the present invention relates to an ECM obtained with the first method of the invention.
[0137]
[0138] In another aspect, the invention relates to an in vitro method for regulating the differentiation of stem cells, preferably mesenchymal stem cells, hereinafter "second method of the invention", which comprises culturing the stem cells, preferably cells mesenchymal stem (MSC), in the extracellular matrix of the invention, in which the ECM is decellularized The term "decellularized" has been described in previous paragraphs.
[0139] Substrates for conventional cell culture research include plastic, glass, and microporous filters (e.g., cellulosics, nylon, fiberglass, polystyrene, polyester, and polycarbonate). Substrates for bioreactors used in discontinuous or continuous cell culture or in genetic engineering include hollow fiber tubes or microparticles. In some embodiments, the substrate / container may be made of any suitable material capable of allowing the components of the extracellular matrix to adsorb or bind to at least one surface of the substrate or container. Materials of this type may include the following: cellulose, polystyrene, polycarbonate, polytetrafluoroethylene, nylon, glass, polyethylene terephthalate, polymethylpentane, polypropylene, polyethylene, and combinations thereof. Other materials that can be used include Permanox®, polyester, polyamide, polyimide, and silica-based materials, including glass containers and the like. Combinations of any of the materials mentioned above may also be used. These materials can be porous or non-porous
[0140] The medium used to culture the stem cells, preferably mesenchymal stem cells, is a conditioned or defined cell culture medium. In one embodiment, the medium is MEF conditioned medium supplemented with basic fibroblast growth factor (bFGF). The bFGF may be present in an amount of about 4 to about 20 ng / ml in the medium. However, it is noted that the culture method is not limited to this culture medium. It has been reported that a large number of media are compatible with the cultivation of MSC cells.
[0141]
[0142] By way of illustration, the conditioned medium can be prepared by culturing primary mouse embryonic fibroblasts irradiated or inactivated with mitomycin C in a serum replacement medium such as, for example, DMEM, K / O DMEM, or DMEM / T12 containing 4 ng / ml of basic fibroblast growth factor (bFGF). Usually, the culture supernatant is collected after 1 day at 37 ° C, and supplemented with additional growth factors, including bFGF. More specifically, from the following components, culture media can be prepared without a feeder, with a suitable base: Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified Eagle's medium for genetic deactivation (KG DMEM), basal medium of Ham F12 / 50% DMEM (Ham's F12 / 50% DMEM basal medium ); 200 mM L-glutamine, non-essential amino acid solution, p-mercaptoethanol, human recombinant basic fibroblast growth factor (bFGF).
[0143]
[0144] Next, the medium / media is combined with the cells used for conditioning in an environment that allows the cells to release the components that maintain stem cells in the medium. Optionally, the cells can be inactivated (ie, rendering it incapable of substantial replication) by radiation (eg, about 4,000 rads), treatment with a chemical inactivator such as mitomycin C, or by any other effective method. Inactivation of the cells is not necessary in cases in which the medium is separated from the conditioned cells before its use in stem cell cultures. The cells are cultured in the medium for a period of time sufficient to allow an adequate concentration of the released factors (or consumption of components of the medium) to produce a medium that facilitates the culture of embryonic stem cells without differentiation. In general, the medium conditioned by culture for 24 h at 37 ° C produces a medium that allows the cultivation of stem cells for 24 hours. However, the cultivation period can be adjusted upwards or downwards, determining empirically (or by testing the concentration of essential factors) what constitutes an adequate period.
[0145]
[0146] The stem cells, preferably mesenchymal stem cells, can be plated on the ECM of the invention in an appropriate distribution and in the presence of the conditioned medium.
[0147]
[0148] A convenient way to determine if MSCs are differentiating is to follow the morphological characteristics of the colonies. For example, the distinctive morphological features characteristic of undifferentiated MSCs are known to those skilled in the art, and include a high nuclear / cytoplasmic ratio, prominent nucleoli, and formation of compact colonies with poorly discernible cell junctions. During the passage, some cells can be differentiated (in particular when they are planted as individual cells, or when large groups are allowed to form). However, crops generally re-establish a larger proportion of undifferentiated cells during the culture period. Ideally, the propagated cells will have a doubling time of no more than about 20-40 hours.
[0149]
[0150] The present invention also relates to a method for maintaining and expanding stem cells, preferably mesenchymal stem cells, in culture in an undifferentiated state, which method comprises culturing the mesenchymal stem cells in the extracellular matrix of the invention in which the ECM is decellularized
[0151]
[0152] In another aspect, the present invention relates to a method for increasing the deposition of collagen in an extracellular matrix, hereinafter "third method of the invention", which comprises culturing cells in the presence of a composition comprising a lysyl oxidase (LOX) and bone morphogenetic protein 1 (BMP1), or cultivate genetically modified ECM producing cells to produce the LOX enzyme and / or BMP1.
[0153]
[0154] In a particular embodiment of the third method of the invention, the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 1.
[0155]
[0156] In a particular embodiment of the third method of the invention, the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEQ ID NO: 2.
[0157]
[0158] In a particular embodiment, the ECM producing cells are fibroblasts, keratinocytes, tenocytes, chondrocytes and / or any combination thereof.
[0159]
[0160] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the present invention pertains. In the practice of the present invention methods and materials similar or equivalent to those described herein can be used. Through the description and claims, the term "understand" and its variations are not intended to exclude other features, additives, components, or steps. The objects, advantages and additional features of the invention will become apparent to those skilled in the art upon examination of the description or can be learned with the practice of the invention. The examples and figures that follow are provided by way of illustration and are not intended to limit the present invention.
[0161]
[0162] BRIEF DESCRIPTION OF THE FIGURES
[0163]
[0164] Figure 1. Stimulation of time-dependent collagen synthesis and deposition in fibroblasts incubated with and without TGF-P1. A) Soluble collagen in the supernatant. B) Fraction of collagen solubilized by pepsin associated with the cell monolayer. C) Insoluble collagen deposited in the matrix. The collagen fractions were determined from cells incubated from 1 to 4 days in the absence (white bars) or the presence of 5 ng / ml of TGF-P1 (black bars) as described in Materials and Methods. Values are represented as ^ g of collagen per million cells (mean value ± SEM, n = 6; * P <0.05 or ** P <0.01 with respect to a day in the absence of TGF-P1, and # P <0.05 with respect to the corresponding time value in the absence of TGF-P1).
[0165] Figure 2. Generation of HEK293 cells that overexpress secreted and active forms of LOX and BMP1 proteins. Induction of LOX (A) and BMP1 (B) proteins in HEK293 cells after incubation with the tetracycline analog, doxycycline, at 10 pM as assessed by immunoblotting using total extracts (left panel) or aliquots of the cell supernatant concentrated with Amicon (right panel). C) The combination of cellular supernatants containing LOX and BMP1 proteins results in the proteolytic activation of LOX as assessed by immunoblotting. The transfers shown correspond to representative experiments performed twice with two independent preparations. LOX immunoreactive bands were quantified from the results shown in panel C and expressed as a percentage of the total: 50 kDa precursor (open circle), 30 kDa active form (closed circle), and unknown band of 25 KDa (open squares). D) LOX enzymatic activity as measured using an Amplex red assay in cell supernatants from non-induced cells (Basal, white bar), induced and without BMP1 (LOX only, closed bar), or induced and combined with supernatants of BMP1 for 5-60 minutes (LOX + BMP1, closed bars). The values are represented as arbitrary units of fluorescence (mean value ± SEM, n = 6; * P <0.05, ** P <0.01).
[0166]
[0167] Figure 3. LOX immunoreactivity in the supernatants of fibroblast cultures supplemented with conditioned media containing LOX and BMP1. LOX, BMP1 or both LOX / BMP1 supernatants were added to fibroblast cultures in the presence (T) or absence (basal, B) of TGF-P1, and LOX immunoreactivity was assessed by immunoblotting both at the start of the experiment (A , one day) as in the end (B, four days). The transfers shown correspond to representative experiments performed twice with two independent preparations.
[0168]
[0169] Figure 4. Effect of the supplement with LOX / BMP1 supernatants in the deposition of collagen from fibroblast cultures. The collagen fractions as measured in Figure 2 were analyzed in fibroblasts exposed to media conditioned from control cells or cells overexpressing LOX and BMP1 and incubated with and without TGF-P1 for 4 days. A) Soluble collagen in the supernatant. B) Fraction of collagen solubilized by pepsin associated with the cell monolayer. C) Insoluble collagen deposited in the matrix. D) Levels of crosslinking of pyridinoline obtained from LOX (PYD) in the matrix deposited by cultures of fibroblasts exposed to conditioned media as evaluated by specific ELISA. The values are represented as pg of collagen or concentration of PYD per million cells (mean ± SEM, n = 6; * P <0.05 with respect to the values of the corresponding control with TGF-P1).
[0170] Figure 5. Immunoreactivity of type I collagen in the supernatants of fibroblast cultures supplemented with conditioned media containing LOX and BMP1. Fibroblasts cultures were exposed to control or LOX / BMP1 supernatants in the presence (T) or absence (B) of TGF-P1 for 4 days and the immunoreactivity of type I collagen was evaluated by immunoblotting as described in Materials and Methods. The specific immunoreactivity of type I collagen was detected as a band induced by TGF-P1 of approximately 150 KDa. The transfers shown correspond to representative experiments performed twice with two independent preparations.
[0171]
[0172] Figure 6. Immunofluorescence analysis of deposition of type I collagen from cultures of fibroblasts exposed to LOX / BMP1 supernatants. Fibroblasts exposed to control or LOX / BMP1 supernatants and incubated in the presence of TGF-P1 for 4 days were processed for immunofluorescence analysis of type I collagen as described in Materials and Methods. The photomicrographs shown correspond to representative staining results for type I collagen (left column) and nuclei using DAPI (right column) performed twice with two independent preparations.
[0173]
[0174] Figure 7. Detection of collagen I immunofluorescence deposited in decellularized matrices of fibroblasts exposed to LOX / BMP1 supernatants. Monolayers of fibroblasts exposed to control or LOX / BMP1 supernatants in the presence of TGF-P1 for 4 days were decellularized before processing for immunofluorescence analysis of type I collagen as described in Materials and Methods. The photomicrographs shown correspond to representative staining results for type I collagen (left column) performed twice with two independent preparations. The absence of DAPI staining confirmed the efficacy of the decellularization procedure.
[0175]
[0176] Figure 8. Adipogenic differentiation of human MSCs seeded in decellularized matrices of fibroblasts exposed to LOX / BMP1 supernatants. The adipogenic capacity was evaluated by microscopic examination (A) and quantified by spectrophotometric analysis (B) using Red O Oil staining in human MSC seeded without matrix, with the matrix of fibroblasts stimulated with TGF-p exposed to control medium or with LOX / BMP1. The microphotographs shown correspond to representative staining results performed twice with two independent preparations. The values are represented as absorbance at 540 nm (mean ± SEM, n = 6; * P <0.05 with respect to experiments without matrix, and #P <0.05 with respect to matrix obtained from matrix fibroblasts in control medium).
[0177]
[0178] Figure 9. Osteogenic differentiation of human MSCs seeded in decellularized matrices of fibroblasts exposed to LOX / BMP1 supernatants. The ability of human MSCs to differentiate into osteoblasts on substrates without a matrix, with fibroblast matrix stimulated with TGF-P1 exposed to control medium or with LOX / BMP1 was evaluated by microscopic examination (A) and quantified by spectrophotometry (B) using staining with Alizarina Red S. The microphotographs shown correspond to representative staining results performed twice with two independent preparations. The values are represented as absorbance at 405 nm (mean ± SEM, n = 6; * P <0.05 with respect to experiments without matrix, and #P <0.05 with respect to a matrix obtained from fibroblasts under medium control).
[0179]
[0180] EXAMPLES
[0181]
[0182] I. MATERIAL AND METHODS
[0183]
[0184] Cell culture of fibroblasts
[0185] The CCD-19Lu human fibroblast cell line (ATCC) was maintained in culture medium as previously described (Puig et al., 2015, Molecular Cancer Research 13, 161-173). For collagen analysis, the fibroblasts were seeded in 100 mm plates in culture medium without serum or phenol red but containing 100 μg / ml 500 kDa dextran sulfate (DxS) and 29 ^ g / ml 2- L-ascorbic acid phosphate (Sigma-Aldrich, St. Louis, MO), up to four days in the absence or presence of 5 ng / ml of TGF-P1 (R & D Systems, Minneapolis, MN).
[0186]
[0187] Collagen analysis
[0188] At the end of the experimental time, the culture media was collected and the soluble collagen was measured after concentration with the Sircol Soluble Collagen Assay (Biocolor, Carrickfergus, UK) following the manufacturer's instructions. The cell layers were scraped, extracted overnight with acid-based buffer (0.5 M acetic acid), and the resulting pellets were digested with 0.5 mg / ml pepsin (Sigma-Aldrich) in 10 mM HCl. . The corresponding solubilized fractions were analyzed for collagen with Sircol. The insoluble collagen after digestion with pepsin was hydrolyzed at 100 ° C for 16 hours with 12 M HCl, neutralized with NaOH and analyzed with the hydroxyproline assay using type I collagen hydrolyzed as standard (Kesava Reddy and Enwemeka, 1996, Clinical Biochemistry 29, 225-229.). The hydrolyzed fractions were also evaluated for the crosslinking content of pyridinoline (PYD) using a kit of Commercially available ELISA (Quidel, Athens, OH).
[0189]
[0190] Soluble collagen in the supernatant was also analyzed by immunoblotting using a specific anti-collagen type 1 a1 antibody (sc-8784, Santa Cruz, Dallas, Texas) after fractionation of proteins in sodium dodecyl sulfate-polyacrylamide electrophoresis gels ( SDS-PAGE) following protocols that have been previously described (Busnadiego et al., 2013, Molecular and Cellular Biology 33, 2388-2401).
[0191]
[0192] Generation of HEK293 cell clones that overexpress LOX and BMP1
[0193] A full-length human LOX construct in pYX-Asc vector was obtained from Imagenes GmbH (Berlin, Germany). A full length human BMP1 construct in pBabe vector was kindly provided by Víctor L. Ruiz-Pérez (Biomedical Research Institute "Alberto Sols", Madrid, Spain) (Martínez-Glez et al., 2012, Human Mutation 33, 343 -350) Both constructs were cloned into the vector pcDNA5 / FRT / TO (Invitrogen, Carlsbad, CA), to obtain the corresponding plasmids of pcDNA5 / FRT / TO-LOX and -BMP1, then these constructs were cotransfected with the plasmid pOG44 expression of Flp recombinase in the Flp-In T-REx 293 cell line using Lipofectamine 2000 (Invitrogen) These cells stably express the Tet repressor and contain a unique FRT (target recombination site of Flp). Expression of Flp recombinase from vector pOG44 promotes the insertion of cDNA cassettes into the FRT site of the genome through site-specific DNA recombination.After 48 hours, cells are selected on resistance to hygromycin B (Roche Diagnostics, Barcelona, Spain), and clones appeared after 10-15 days. The isogenic clones were expanded and the expression of the transgene was analyzed after 48 hours of incubation in the absence or presence of the tetracycline analogue, doxycycline at 10 pM. The culture media was concentrated using Amicon Ultra-4 filter units (Ultra-Cel 10K, Millipore, Cork, Ireland). LOX and BMP1 protein levels in cell layers or concentrated supernatants were detected by immunoblotting using specific antibodies against LOX (ab31238, Abcam, Cambridge, UK) and BMP1 (AF1927, R & D Systems). The enzymatic activity of LOX was determined using a commercially available Abcam assay.
[0194]
[0195] Immunofluorescence studies
[0196] Fluorescence microscopy was performed as described previously (Lagares et al., 2012). Briefly, the cells were seeded on 10 mm diameter glass coverslips (No. 1.5) in 35 mm culture dishes (Mattek, Ashland, MA).
[0197] After the corresponding treatment, the cells were fixed with cold methanol for 5 minutes, blocked with 1% BSA in phosphate buffered solution (PBS) for 1 h, and then incubated overnight at 4 ° C with anti-cancer antibody. collagen a1 type I (Santa Cruz), followed by the corresponding fluorescent secondary antibodies. Cell bloom was visualized by microscopy with a Nikon Eclipse T2000U (Nikon, Amstelveen, The Netherlands).
[0198]
[0199] For analysis of the matrix deposited from cells, decellularization was performed by incubation with an extraction buffer containing 0.5% Triton X-100 (v / v) and 20 mM NH4OH in PBS for 3-5 minutes as it has been previously described (Cukierman, 2001, Preparation of Extracellular Matrices Produced by Cultured Fibroblasts, Current Protocols in Cell Biology, John Wiley & Sons, Inc.).
[0200]
[0201] Analysis of adipogenic and osteogenic differentiation of human mesenchymal stem cells
[0202] Human mesenchymal stem cells (MSC) (Promocell, Heidelberg, Germany) were kept in culture in basal medium (Promocell) and then induced for adipogenesis and osteogenesis with the corresponding differentiation media (Promocell) for 21 days with medium change each 2-3 days The phenotypic changes induced by lineage differentiation, ie the formation of lipid vesicles for adipogenesis and the extracellular calcium phosphate deposition for osteogenesis, were monitored by staining with Red Oil O and Red Alizarin S (Santa Cruz), respectively, as previously described (Bruedigam et al., 2007, Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation in Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyzes, Current Protocols in Stem Cell Biology. John Wiley & Sons, Inc.). Differentiation was evaluated by microscopic examination and quantified by spectrophotometric analysis after solubilization of the dye.
[0203]
[0204] Statistic analysis
[0205] Experimental data were analyzed using the Student t- test for unrelated or independent samples in the case of normal data distribution or using nonparametric tests when appropriate. The P values obtained are indicated in the legends of the figures when they are statistically significant (P <0.05).
[0206]
[0207] Example 1: The composition comprising Lisil oxidase (LOX) and bone morphogenetic protein 1 (BMP1) produces a strong increase in collagen deposition in vitro.
[0208]
[0209] Collagen deposition in vitro is slow and has low efficiency .
[0210] The inventors have studied the synthesis and deposition of collagen in cultures of human lung fibroblast cells (CCD19-Lu) under basal conditions or incubated with the profibrotic cytokine transforming growth factor beta (TGF-pi) for periods of time ranging from one to four days (Figure 1). Several fractions of collagen can be extracted from the cultures that represent the sequential stages in the biosynthetic process. Cell supernatants were evaluated for the soluble form of secreted collagen. Acid-based buffer was used to extract the non-crosslinked collagen, recently deposited, in monolayers of cells. Next, a pepsin treatment was used to digest proteolyticly the telopeptide segments without collagen, and thereby, solubilize the recently cross-linked collagen. To determine the collagen that becomes soluble with these procedures, a colorimetric assay based on Sirius red (Sircol) was used. Finally, the insoluble collagen in the cell pellets was hydrolyzed with strong acid and heat, and the hydroxyproline was measured as an estimate of strongly crosslinked collagen. As shown in Figure 1A, soluble collagen was progressively accumulated in cellular supernatants of fibroblasts incubated under basal conditions, and this effect was further increased in cells stimulated with TGF-pi. Despite this rate of synthesis and accumulation of soluble collagen, deposition in the matrix as well as soluble or insoluble forms of pepsin only slightly increased in cells incubated for four days with TGF-P1 (Figure 1B and Figure 1C). The acid buffer solubilized insignificant amounts of collagen, indicating that this combination is not stable under the experimental conditions of the inventors (data not shown). In general, these results indicate that, in spite of an active production and secretion of collagen precursors, in vitro deposition is a process that is not favored, even in conditions of "macromolecular aggregation" (macromolecular crowding), such as studied in the study of the inventors.
[0211]
[0212] Generation of HEK293 cell lines overexpressing lysyl oxidase (LOX) and bone morphogenetic protein 1 (BMP1)
[0213] Several evidences in the literature suggest that an incomplete conversion of procollagen by the morphogenetic protein of C-proteinase / bone 1 (BMP1) significantly limits the deposition of collagen in vitro. Among various matrix substrates (and not matrix), BMP1 also catalyzes the proteolytic conversion of the lysyl oxidase (LOX) precursor to produce its active form, thereby stimulating the initial step in the collagen cross-linking process. The inventors hypothesized that the addition of LOX and / or BMP1 may represent a strategy to stimulate collagen deposition in vitro. For this purpose, the inventors generated clones of HEK293 cells that stably express LOX and BMP1 constructs under tetracycline-dependent control. As shown in Figure 2A, the LOX transfectants expressed and secreted into the extracellular medium several immunoreactive bands for LOX, including the precursor of approximately 50 KDa, and shorter bands of 25 KDa and 30 KDa. In a similar approach, BMP1 transfectants showed doxycycline sensitive expression and secretion of a complex mixture of BMP1 forms ranging from 60-100 KDa, which probably represents precursor and processed forms (Figure 2B). The presence in cells that overexpress LOX from the 50 KDa band of LOX indicates a limited capacity of the cells to process and activate the enzyme. Interestingly, the incubation of cellular supernatants containing LOX with those containing BMP1 stimulated the proteolysis of the pro-LOX precursor to the active form of 30 KDa in a time-dependent manner (Figure 2C and D). The shorter LOX form of 25 KDa was not modified by the action of BMP1. The enzymatic activity of LOX was evaluated in a fluorometric assay using basal cell supernatants and incubated with doxycycline. As shown in Figure 2E, the induction of LOX expression stimulated a strong increase in the enzymatic activity of LOX, which in turn increased further after incubation with BMP1 supernatants in a time-dependent manner. Taken together, the inventors succeeded in generating cellular systems based on HEK293 to produce supernatants enriched with LOX and BMP1 enzymes which, when combined, promoted in vitro the proteolytic activation of LOX.
[0214]
[0215] The addition of lysyl oxidase (LOX) and bone morphogenetic protein 1 (BMP1) produces a strong increase in collagen deposition in vitro
[0216] First, the inventors checked the proteolytic activation of LOX in fibroblasts exposed to supernatants. As shown in Figure 3A, fibroblasts incubated for one day with only LOX supernatants exhibited a significant amount of the unprocessed LOX precursor, again indicating a limited cellular capacity to process the proenzyme in vitro. In contrast, the combination of recombinant LOX and BMP1 resulted in complete proteolysis of pro-LOX. The presence of processed forms of LOX in fibroblasts incubated with BMP1 only indicated that the protease stimulated the processing of LOX produced endogenously. No detectable LOX bands were observed in fibroblasts exposed to control medium. After four days of incubation with supernatants, the proteolytic conversion of the enzyme proLOX was complete, even in the absence of added BMP1 (Figure 3B). Interestingly, the immunoreactivity of LOX was lower in LOX / BMP1 supernatants than those of LOX alone (both one day and four days), as well as in LOX (or LOX / BMP1) at one day compared to the corresponding samples after four days, which indicates that as soon as the processed forms of LOX are generated, they are degraded or retained in the matrix. Next, the inventors studied the effect of these supernatants on the synthesis and deposition of collagen. As shown in Figure 4A, unlike cells exposed to medium control, the incubation of fibroblasts with cell supernatants containing either LOX, BMP1 or a mixture of both abolished the accumulation of soluble collagen in the extracellular medium, both in the absence of in the presence of TGF-pi, an effect that was further confirmed by immunoblotting using an anti-coli1 antibody (Figure 5). Simultaneously with this drastic reduction, it was found that the fractions both soluble and insoluble by pepsin of TGF-pi treated cells increased significantly, being higher in cells incubated with the LOX / BMP1 mixture than with either LOX or BMP1 alone, an observation that suggests a synergistic action for the effect of both enzymes (Figure 4B and C). The LOX enzyme catalyzes the oxidative deamination of telopeptide lysine / hydrolysin residues to produce highly reactive aldehydes that additionally react to form immature and then mature permanent crosslinks. The preferred use of hydroxylysine with respect to lysine in crosslinking reactions determines a distinctive pattern in the maturation products, with higher levels of pyridinolines than pyrroles, as is normally found in cartilage, bone or aorta. The insoluble pellets in hydrolysed pepsin were tested with a specific ELISA for the presence of pyridinoline crosslinks (PYD). As shown in Figure 4D, in comparison with the control, the exposure of fibroblasts to LOX and / or BMP1 supernatants stimulated the formation of PYD crosslinks, indicating that a significant part of the deposited collagen is formed through this maturation route.
[0217]
[0218] In addition, the inventors analyzed the effect of supernatants containing LOX and BMP1 by immunofluorescence analysis using an anti-col1a1 antibody. As shown in Figure 6, fibroblasts exposed to control medium exhibited type I collagen immunoreactivity in the form of small and large aggregates. Although this aspect would not be significantly modified by the LOX supernatants, the cells exposed to BMP1 and in particular to the BMP1 and LOX mixture showed a more distinctive pattern of immunoreactivity that included the presence of fibrous material, probably consistent with its deposition to the matrix, instead of being associated with the cell layer. Furthermore this was confirmed in experiments in decellularized matrices. As shown in Figure 7, after removing the cell-associated material, the observed a more fibrous pattern in the matrix deposited by cells exposed to BMP1 and to the mixture of BMP1 and LOX. DAPI staining confirmed that the extraction procedure effectively removed the cell layer. Taken together, the results of the inventors show that the implementation of fibroblast cultures with supernatants enriched in LOX and BMP1 constitutes an effective approach to greatly increase the deposition of collagen on the insoluble matrix.
[0219]
[0220] Example 2: The matrix obtained from fibroblasts modified by lysyl oxidase (LOX) and bone morphogenetic protein 1 (BMP1) regulates the differentiation of human mesenchymal stem cells (MSCs)
[0221] Mesenchymal stem cells are a promising source for regenerative medicine due to their capacity for self-renewal and differentiation in various tissue lineages, such as adipocytes, osteoblasts, and chondrocytes. Since the NDE provides physical and chemical signals to regulate the activity of the MSCs, the inventors investigated the effects of the matrix obtained from fibroblasts modified by LOX / BMP1 on the regulation of the adipogenic and osteogenic differentiation of MSCs. For that purpose, the inventors exposed cultures of fibroblasts to control media or to supernatants containing LOX and BMP1 as described above, and then the cells were removed and the deposited matrix was used as a substrate to establish MSC cultures. Once these cultures reached confluence, they were induced in adipogenic and osteogenic lineages by incubation with the corresponding differentiation media. Then, these cultures were compared with equivalent MSCs seeded without matrix. As shown in Figure 8, after 14 days in MSC medium of adipogenic differentiation without matrix, lipid droplets develop that can be visualized with O-Red Oil. MSC cells cultured in matrices obtained from fibroblasts exposed to control means showed a reduction in the ability to differentiate into adipocytes, and this behavior was further exacerbated in fibroblast matrices incubated with LOX / BMP1. On the other hand, differentiation of MSC into osteogenic lineage results in the formation of extracellular calcium deposits that can be stained specifically using Alizarin Red S, as shown in Figure 9 for MSC cells seeded without matrix. Osteogenic differentiation increased greatly in MSCs seeded on matrices of fibroblasts exposed to control media, this effect being attenuated in matrices of fibroblasts incubated with LOX / BMP1 supernatants. These results indicate that the matrix obtained from fibroblasts is able to regulate the capacity of adipogenic and osteogenic differentiation of MSCs, the modification being stimulated by LOX / BMP1 able to modulate this effect.

权利要求:
Claims (24)
[1]
An extracellular matrix comprising a lysyl oxidase (LOX), or a fragment thereof, and bone morphogenetic protein 1 (BMP1), or a fragment thereof.
[2]
2. An extracellular matrix according to claim 1, wherein the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 % with SEQ ID NO: 1.
[3]
An extracellular matrix according to claim 1 or 2, wherein the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with SEQ ID NO: 2.
[4]
4. An extracellular matrix according to any one of claims 1 to 3, wherein the ECM further comprises ECM-producing cells.
[5]
5. Extracellular matrix according to any one of claims 1 to 4, wherein the ECM-producing cells are fibroblasts, keratinocytes, tenocytes, chondrocytes and / or any combination thereof.
[6]
6. Extracellular matrix according to any one of claims 1 to 5, wherein the ECM-producing cells are genetically modified to produce the LOX enzyme, or a fragment thereof, and / or BMP1 or a fragment thereof .
[7]
7. An in vitro use of an extracellular matrix according to any one of claims 1 to 6 for regulating the differentiation of mesenchymal stem cells, wherein the ECM is decellularized.
[8]
8. An in vitro use of the composition according to claim 7, wherein the differentiation of mesenchymal stem cells is an adipogenic and / or osteogenic differentiation.
[9]
9. An in vitro use according to claim 7 or 8, wherein the differentiation of mesenchymal stem cells is reduced or inhibited.
[10]
10. An in vitro use of a composition comprising a lysyl oxidase (LOX), or a fragment thereof, and bone morphogenetic protein 1 (BMP1), or a fragment thereof, to increase the synthesis and / or deposit of collagen in an extracellular matrix.
[11]
11. An in vitro use according to claim 10, wherein the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or a 99% with SEQ ID NO: 1.
[12]
12. An in vitro use according to claim 10 or 11, wherein the BMP1 protein comprises an amino acid sequence with an identity of at least one 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with SEQ ID NO: 2.
[13]
13. An in vitro use according to any one of claims 10 to 12, wherein the composition additionally comprises ECM producing cells, preferably, the ECM producing cells are fibroblast cells, keratinocyte cells, tenocyte cells, chondrocyte cells and / or any combination thereof.
[14]
14. An in vitro use according to claim 13, wherein the ECM-producing cells are genetically modified to produce the LOX enzyme, or a fragment thereof, and / or BMP1 or a fragment thereof.
[15]
15. An in vitro method for obtaining an extracellular matrix according to any one of claims 1 to 6 comprising incubating the ECM-producing cells in the presence of a composition comprising a lysyl oxidase (LOX), or a fragment thereof. , and bone morphogenetic protein 1 (BMP1), or a fragment thereof.
[16]
The method according to claim 15, wherein the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with SEQ ID NO: 1.
[17]
17. Method according to claim 15 or 16, wherein the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 % with SEQ ID NO: 2.
[18]
18. Method according to any one of claims 15 to 17, wherein the ECM-producing cells are fibroblasts, keratinocytes, tenocytes, chondrocytes and / or any combination thereof.
[19]
19. Method according to any one of claims 15 to 18, wherein the ECM-producing cells are genetically modified to produce the LOX enzyme, or a fragment thereof, and / or BMP1 or a fragment thereof.
[20]
20. An in vitro method for regulating the differentiation of mesenchymal stem cells comprising culturing the mesenchymal stem cells in an extracellular matrix according to any one of claims 1 to 5, wherein the ECM is decellularized.
[21]
21. A method for increasing the deposition of collagen in an extracellular matrix comprising culturing ECM-producing cells in the presence of a composition comprising a lysyl oxidase (LOX) and bone morphogenetic protein 1 (BMP1), or culturing modified ECM-producing cells genetically to produce the enzyme LOX and / or BMP1.
[22]
22. A method according to claim 21, wherein the LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% with SEQ ID NO: 1.
[23]
23. A method according to claim 21 or 22, wherein the BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or a 99% with SEQ ID NO: 2.
[24]
24. A method according to any one of claims 21 to 23, wherein the ECM-producing cells are fibroblasts, keratinocytes, tenocytes, chondrocytes and / or any combination thereof.

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同族专利:

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公开号 | 公开日

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WO2019121768A1|2019-06-27|

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ES2717374B2|2020-02-20|

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US20200345898A1|2020-11-05|

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EP3728558A1|2020-10-28|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2012079086A2|2010-12-10|2012-06-14|Florida State University Research Foundation, Inc.|Mesenchymal stem cells expansion methods and materials|

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ES201731441A|ES2717374B2|2017-12-20|2017-12-20|Extracellular matrix and its use to regulate the differentiation of mesenchymal stem cells|ES201731441A| ES2717374B2|2017-12-20|2017-12-20|Extracellular matrix and its use to regulate the differentiation of mesenchymal stem cells|

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US16/955,441| US20200345898A1|2017-12-20|2018-12-18|Extracellular matrix and its use for regulating the differentiation of mesenchymal stem cells|

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EP18825996.4A| EP3728558A1|2017-12-20|2018-12-18|Extracellular matrix and its use for regulating the differentiation of mesenchymal stem cells|

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PCT/EP2018/085634| WO2019121768A1|2017-12-20|2018-12-18|Extracellular matrix and its use for regulating the differentiation of mesenchymal stem cells|