Abstract
The present invention relates to an extracellular matrix comprising ECM producer cells, a lysyloxidase (LOX), and bone morphogenetic protein-1 (BMP1), and its use for regulating the differentiation of mesenchymal stem cells and increasing the synthesis and/or deposit 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.
Claims
1. An extracellular matrix (ECM) comprising a lysyl oxidase (LOX), or a fragment thereof, and bone morphogenetic protein-1 (BMP 1), or a fragment thereof.
2. An extracellular matrix according to claim 1, wherein LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with SEQ ID NO: 1.
3. An extracellular matrix according to claim 1, wherein BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with SEQ ID NO: 2.
4. An extracellular matrix according to claim 1, wherein the ECM further comprises ECM producer cells.
5. An extracellular matrix according to claim 1, wherein the ECM producer cells are fibroblasts, keratinocytes, tenocytes, chondrocytes or any combination thereof.
6. An extracellular matrix according to claim 4, wherein the ECM producer cells are genetically modified for producing the LOX enzyme, or a fragment thereof, and/or BMP1 or a fragment thereof.
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15. An in vitro method for obtaining an extracellular matrix (ECM) according to claim 1 comprising incubating ECM producer 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 LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with SEQ ID NO: 1.
17. The method according to claim 15, wherein BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with SEQ ID NO: 2.
18. The method according to claim 15, wherein the ECM producer cells are fibroblasts, keratinocytes, tenocytes, chondrocytes or any combination thereof.
19. The method according to claim 15, wherein the ECM producer cells are genetically modified for producing the LOX enzyme, or a fragment thereof, and/or BMP1 or a fragment thereof.
20. An in vitro method for regulating the differentiation of mesenchymal stem cells comprising culturing the mesenchymal stem cells (ECM) in an extracellular matrix according to claim 1, wherein the ECM is decellularized.
21. A method for increasing the deposit of collagen in an extracellular matrix (ECM) comprising cultivating ECM producer cells in the presence of a composition comprising a lysyl oxidase (LOX) and bone morphogenetic protein-1 (BMP1), or culturing ECM producer cells genetically modified for producing the LOX enzyme and/or BMP 1.
22. The method according to claim 21, wherein LOX protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with SEQ ID NO: 1.
23. The method according to claim 21, wherein BMP1 protein comprises an amino acid sequence with an identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with SEQ ID NO: 2.
24. The method according to claim 21, wherein the ECM producer cells are fibroblasts, keratinocytes, tenocytes, chondrocytes or any combination thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1. Time-dependent stimulation of collagen synthesis and deposition in fibroblasts incubated with and without TGF-1. A) Soluble collagen in the supernatant. B) Pepsin-solubilized collagen fraction associated to cell monolayer. C) Insoluble collagen deposited into the matrix. Collagen fractions were determined from cells incubated for 1 to 4 days in the absence (white bars) or presence of 5 ng/ml TGF-I1 (black bars) as described under Materials and Methods. Values are represented as g collagen per million of cells (meanSEM, n=6; *P<0.05 or **P<0.01 vs one day in the absence of TGF-I1, and #P<0.05 vs the corresponding time value in the absence of TGF-1).
[0062] FIG. 2. Generation of HEK293 cells overexpressing secreted and active forms of LOX and BMP1 proteins. Induction of LOX (A) and BMP1 (B) proteins in HEK293 cells upon incubation with the tetracycline analog, doxycycline, at 10 M as assessed by western blotting using total extracts (left panel) or Amicon-concentrated aliquots of the cell supernatant (right panel). C) Combination of cell supernatants containing LOX and BMP1 proteins gives rise to the proteolytic activation of LOX as assessed by western blotting. The blots shown correspond to representative experiments performed twice with two independent preparations. LOX-immunoreactive bands from results shown in panel C were quantified and expressed as percentage of total: 50 KDa precursor (open circle), 30 KDa active form (closed circle), and 25 KDa unknown band (open squares). D) LOX enzymatic activity as measured using Amplex red assay in cell supernatants from uninduced cells (Basal, white bar), induced and without BMP1 (Only LOX, closed bar), or induced and combined with BMP1 supernatants for 5-60 minutes (LOX+BMP1, closed bars). Values are represented as arbitrary fluorescent units (meanSEM, n=6; *P<0.05, **P<0.01).
[0063] FIG. 3. LOX immunoreactivity in the supernatants of fibroblast cultures supplemented with LOX- and BMP1-containing conditioned media. LOX, BMP1 or both LOX/BMP1 supernatants were added to fibroblast cultures in the presence (T) or absence (basal, B) of TGF-1 and LOX immunoreactivity assessed by western blotting at the beginning of the experiment (A, one day) or at the end (B, four days). The blots shown correspond to representative experiments performed twice with two independent preparations.
[0064] FIG. 4. Effect of the supplementation with LOX/BMP1 supernatants on collagen deposition from fibroblast cultures. Collagen fractions as measured in FIG. 2 were analyzed in fibroblasts exposed to conditioned media from control or LOX- and BMP1-overexpressing cells and incubated with and without TGF-1 for 4 days. A) Soluble collagen in the supernatant. B) Pepsin-solubilized collagen fraction associated to cell monolayer. C) Insoluble collagen deposited into the matrix. D) LOX-derived pyridinoline (PYD) cross-link levels in the deposited matrix from fibroblast cultures exposed to conditioned media as assessed by specific ELISA. Values are represented as g collagen or concentration of PYD per million of cells (meanSEM, n=6; *P<0.05 vs the corresponding control values with TGF-1).
[0065] FIG. 5. Collagen type I immunoreactivity in the supernatants of fibroblast cultures supplemented with LOX- and BMP1-containing conditioned media. Fibroblast cultures were exposed to control or LOX/BMP1 supernatants in the presence (T) or absence (B) of TGF-1 for 4 days and collagen type I immunoreactivity assessed by western blotting as described under Materials and Methods. Specific collagen type I immunoreactivity was detected as a TGF-1-induced band of approximately 150 KDa. The blots shown correspond to representative experiments performed twice with two independent preparations.
[0066] FIG. 6. Immunofluorescence analysis of collagen type I deposition from fibroblast cultures exposed to LOX/BMP1 supernatants. Fibroblasts exposed to control or LOX/BMP1 supernatants and incubated in the presence of TGF-1 for 4 days were processed for immunofluorescence analysis of collagen type I as described under Materials and Methods. Micrographs shown correspond to representative results of staining for collagen type I (left column) and nuclei using DAPI (right column) performed twice with two independent preparations.
[0067] FIG. 7. Immunofluorescence detection of deposited collagen I in decellularized matrices from fibroblasts exposed to LOX/BMP1 supernatants. Fibroblast monolayers exposed to control or LOX/BMP1 supernatants in the presence of TGF-1 for 4 days were decellularized before processing for immunofluorescence analysis of collagen type I as described under Materials and Methods. Micrographs shown correspond to representative results of staining for collagen type I (left column) performed twice with two independent preparations. The absence of DAPI staining confirmed the effectiveness of the decellularization procedure.
[0068] FIG. 8. Adipogenic differentiation of human MSC seeded on decellularized matrices from fibroblasts exposed to LOX/BMP1 supernatants. Adipogenic capacity was evaluated by microscopic examination (A) and quantified by spectrophotometric analysis (B) using Oil Red O staining in human MSC seeded without matrix, with matrix from TGF--stimulated fibroblasts exposed to control medium or with LOX/BMP1. Micrographs shown correspond to representative results of staining performed twice with two independent preparations. Values are represented as absorbance at 540 nm (meanSEM, n=6; *P<0.05 vs no matrix, and #P<0.05 vs matrix fibroblast-derived matrix under control medium).
[0069] FIG. 9. Osteogenic differentiation of human MSC seeded on decellularized matrices from fibroblasts exposed to LOX/BMP1 supernatants. The capacity of human MSC to differentiate into osteoblasts in substrates without matrix, with matrix from TGF-1 stimulated fibroblasts exposed to control medium or with LOX/BMP1was assessed by microscopic examination (A) and quantified by spectrophotometry (B) using Alizarin Red S staining. Micrographs shown correspond to representative results of staining performed twice with two independent preparations. Values are represented as absorbance at 405 nm (meanSEM, n=6; *P<0.05 vs no matrix, and #P<0.05 vs matrix fibroblast-derived matrix under control medium).
EXAMPLES
I. Material and Methods
Fibroblast Cell Culture
[0070] The human fibroblast cell line CCD-19Lu (ATCC) was maintained in culture medium as already described (Puig et al., 2015, Molecular Cancer Research 13, 161-173). For collagen analysis, fibroblasts were seeded on 100-mm dishes in culture medium without serum and phenol red but containing 100 g/ml 500 KDa dextran sulfate (DxS) and 29 g/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, Mo.), for up to four days in the absence or presence of 5 ng/ml TGF-1 (R&D Systems, Minneapolis, Minn.).
Collagen Analysis
[0071] At the end of the experimental time, culture media were collected and soluble collagen measured upon concentration with Sircol Soluble Collagen Assay (Biocolor, Carrickfergus, United Kingdom) following manufacturer's instructions. Cell layers were scrapped, extracted overnight with acid-based buffer (0.5 M acetic acid), and resulting pellets digested with 0.5 mg/ml pepsin (Sigma-Aldrich) in 10 mM HCl. Corresponding solubilized fractions were analyzed for collagen with Sircol. Insoluble collagen after pepsin digestion was hydrolyzed at 100 C. for 16 hours with 12 M HCl, neutralized with NaOH and analyzed by hydroxyproline assay using hydrolyzed type I collagen as standard (Kesava Reddy and Enwemeka, 1996, Clinical Biochemistry 29, 225-229.). Hydrolyzed fractions were also assayed for the content of the pyridinoline cross-links (PYD) using a commercially available ELISA kit (Quidel, Athens, Ohio).
[0072] Soluble collagen in the supernatant was also analyzed by western blotting using an specific anti-collagen al type I antibody (sc-8784, Santa Cruz, Dallas, Tex.) upon protein fractionation in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following protocols previously described (Busnadiego et al., 2013, Molecular and Cellular Biology 33, 2388-2401).
Generation of HEK293 Cell Clones Overexpressing LOX and BMP1
[0073] 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 Victor L. Ruiz-Prez (Instituto de Investigaciones Biomdicas Alberto Sols, Madrid, Spain) (Martnez-Glez et al., 2012, Human Mutation 33, 343-350). Both constructs were cloned into the vector pcDNA5/FRT/TO (Invitrogen, Carlsbad, Calif.), to obtain the corresponding pcDNA5/FRT/TO-LOX and -BMP1 plasmids. These constructs were then co-transfected with the Flp recombinase expression plasmid pOG44 into the Flp-In T-REx 293 cell line using Lipofectamine 2000 (Invitrogen). These cells stably express the Tet repressor and contain a single integrated FRT (Flp recombination target) site. Flp recombinase expression from the pOG44 vector mediates insertion of the cDNA cassettes into the genome at the integrated FRT site through site-specific DNA recombination. After 48 hours, cells were selected for hygromycin B resistance (Roche Diagnostics, Barcelona, Spain), and clones appeared after 10-15 days. Isogenic pooled clones were expanded and checked for transgene expression after 48 hours of incubation in the absence or presence of the tetracycline analog, doxycycline at 10 M. Culture media were concentrated using Amicon Ultra-4 centrifugal filter units (Ultra-Cel 10K, Millipore, Cork, Ireland). LOX and BMP1 protein levels in cell layers or concentrated supernatants were detected by western blotting using specific antibodies against LOX (ab31238, Abcam, Cambridge, United Kingdom) and BMP1 (AF1927, R&D Systems). LOX enzymatic activity was determined using a commercially available assay from Abcam.
Immunofluorescence Studies
[0074] Fluorescence microscopy was performed as previously described (Lagares et al., 2012). Briefly, cells were seeded onto 10 mm glass diameter coverslips (No. 1.5) in 35 mm culture dishes (Mattek, Ashland, Mass.). After the corresponding treatment, 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-collagen al type I antibody (Santa Cruz), followed by the corresponding fluorescent secondary antibodies. Cell fluorescence was visualized by microscopy with a Nikon Eclipse T2000U (Nikon, Amstelveen, The Netherlands).
[0075] For analysis of the matrix deposited from cells, decellularization was performed by incubation with an extraction buffer containing 0.5% (v/v) Triton X-100 and 20 mM NH.sub.4OH in PBS for 3-5 minutes as previously described (Cukierman, 2001, Preparation of Extracellular Matrices Produced by Cultured Fibroblasts, Current Protocols in Cell Biology. John Wiley & Sons, Inc.).
Analysis of Adipogenic and Osteogenic Differentiation of Human Mesenchymal Stem Cells
[0076] Human mesenchymal stem cells (MSC) (Promocell, Heidelberg, Germany) were maintained in culture under basal medium (Promocell) and then induced for adipogenesis and osteogenesis with corresponding differentiation media (Promocell) for 21 days with medium change every 2-3 days. Phenotypic changes induced by lineage differentiation, i.e. the formation of lipid vesicles for adipogenesis and the extracellular deposition of calcium phosphate for osteogenesis, were monitored by staining with Oil Red O and Alizarin Red S (Santa Cruz), respectively, as described previously (Bruedigam et al., 2007, Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses, Current Protocols in Stem Cell Biology. John Wiley & Sons, Inc.). Differentiation was assessed by microscopic examination and quantitatively determined by spectrophotometric analysis upon dye solubilization.
Statistical Analysis
[0077] Experimental data were analyzed using the unpaired Student t test in the case of normal distribution of data or using nonparametric tests as appropriate. The P values obtained are indicated in the figure legends when statistically significant (P<0.05).
II. Results
Example 1: Composition Comprising Lysyl Oxidase (LOX) and Bone Morphogenetic Protein-1 (BMP1) Strongly Increases Collagen Deposition In Vitro
In Vitro Collagen Deposition is Slow and has Low Efficiency
[0078] We have studied the synthesis and deposition of collagen in cultures of human lung fibroblast (CCD19-Lu) cells under basal conditions or incubated with the profibrotic cytokine transforming growth factor (TGF)-1 for time periods ranging from one to four days (FIG. 1). Several fractions of collagen can be extracted from the cultures representing the sequential steps in the biosynthetic process. Cell supernatants were assayed for the soluble form of secreted collagen. Acid-based buffer was used to extract recently deposited, non-crosslinked collagen in cell monolayers. Pepsin treatment was then used to proteolytically digest the non-collagenous telopeptide segments, and thereby, solubilize recently cross-linked collagen. A Sirius-based colorimetric assay was used to determine collagen made soluble by these procedures. Insoluble collagen in the cell pellets was finally hydrolyzed with strong acid and heat and hydroxyproline measured as an estimation of heavily cross-linked collagen. As shown in FIG. 1A, soluble collagen progressively accumulated in cell supernatants from fibroblasts incubated under basal conditions, and this effect was further augmented in cells stimulated with TGF-131. In spite of this rate of synthesis and accumulation of soluble collagen, deposition into the matrix as pepsin-soluble or insoluble forms only modestly increased in cells incubated for four days with TGF-1 (FIG. 1B and FIG. 1C). Acid-based buffer solubilized negligible amounts of collagen, indicating this pool is not stable in our experimental conditions (data not shown). Overall, these results indicate that, despite an active production and secretion of collagen precursors, in vitro deposition is an unfavoured process, even in conditions of macromolecular crowding, such as those used in our study.
Generation of HEK293 Cell Lines Overexpressing Lysyl Oxidase (LOX) and Bone Morphogenetic Protein-1 (BMP1)
[0079] Several evidences in the literature suggest that an incomplete conversion of procollagen by C-proteinase/bone morphogenetic protein 1 (BMP1) significantly limits collagen deposition in vitro. Among several matrix (and non-matrix) substrates, BMP1 also catalyzes the proteolytic conversion of the precursor of lysyl oxidase (LOX) to yield the active form, thereby promoting the initial step in the process of collagen cross-linking. We hypothesized that the addition of LOX and/or BMP1 may represent a strategy to boost in vitro deposition of collagen. For that purpose, we generated HEK293 cell clones stably expressing LOX and BMP1 constructs under tetracyclin-dependent control. As shown in FIG. 2A, LOX transfectants expressed and secreted to the extracellular medium several LOX immunoreactive bands including the precursor of about 50 KDa, and shorter bands of 25 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, likely representing precursor and processed forms (FIG. 2B). The presence in LOX-overexpressing cell of the 50 KDa band of LOX indicates a limited capacity of the cells to process and activate the enzyme. Interestingly, incubation of cell supernatants containing LOX with those with BMP1 promoted the proteolysis of the precursor pro-LOX to the active form of 30 KDa in a time-dependent manner (FIG. 2C and D). The shortest LOX form of 25 KDa was not modified by the action of BMP1. LOX enzymatic activity was assessed in a fluorometric assay using supernatants from basal and doxycycline-incubated cells. As shown in FIG. 2E, the induction of the expression of LOX promoted a strong increase in LOX enzymatic activity, that was further augmented upon incubation with BMP1 supernatants in a time-dependent manner. Taken together, we succeed in generating HEK293-based cell systems to produce supernatants enriched with LOX and BMP1 enzymes which, when combined together, recapitulated in vitro the proteolytic activation of LOX.
Addition of Recombinant Lysyl Oxidase (LOX) and Bone Morphogenetic Protein-1 (BMP1) Strongly Increases Collagen Deposition In Vitro
[0080] We have first checked the proteolytic activation of LOX in fibroblasts exposed to supernatants. As shown in FIG. 3A, fibroblasts incubated for one day with only LOX supernatants displayed a significant amount of the unprocessed LOX precursor, again indicating a limited cell capacity to in vitro process the proenzyme. In contrast, the combination of recombinant LOX and BMP1 resulted in complete proteolysis of the pro-LOX. The presence of processed forms of LOX in fibroblasts incubated with BMP1 alone indicated that the protease promoted the processing of endogenously produced LOX. No detectable LOX bands were observed in fibroblasts exposed to control media. After four days of incubation with supernatants, proteolytic conversion of pro-LOX enzyme was complete, even in the absence of added BMP1 (FIG. 3B). Interestingly, LOX immunoreactive signals were lower in supernatants from LOX/BMP1 than those from only LOX (at both one and four days), as well as in LOX (or LOX/BMP1) at one day compared to corresponding samples at four days, indicating that as soon as the processed forms of LOX are generated, they are either degraded or retained into the matrix. We have then studied the effect of these supernatants on collagen synthesis and deposition. As shown in FIG. 4A, as opposed to cells exposed to control media, the incubation of fibroblasts with cell supernatants containing either LOX, BMP1 or a mixture of both abrogated the accumulation of soluble collagen in the extracellular medium, both in the absence or presence of TGF-1, an effect that was further corroborated by immunoblotting using an anti-col11 antibody (FIG. 5). Concomitantly with this drastic reduction, both pepsin-soluble and -insoluble fractions from TGF-1-treated cells were found to significantly increase, being higher in cells incubated with the mixture of LOX/BMP1 than with either only LOX or BMP1, an observation that suggests a synergic action for the effect of both enzymes (FIG. 4B and C). LOX enzyme catalyzes the oxidative deamination of telopeptide lysine/hydrolysine residues to yield highly reactive aldehydes that further react to form immature and then mature permanent cross-links. The preferential use of hydroxylysine versus lysine in cross-linking reactions determines a distinctive pattern of maturational products, with higher levels of pyridinolines than of pyrroles, as is usually found in cartilage, bone or aorta. Hydrolyzed pepsin-insoluble pellets were assayed with a specific ELISA for the presence of pyridinoline cross-links (PYD). As shown in FIG. 4D, compared with control, the exposure of fibroblasts to LOX and/or BMP1 supernatants promoted the formation of PYD cross-links, indicating that a significant part of the deposited collagen is formed through this maturation pathway.
[0081] We have also analyzed the effect of LOX and BMP1-containing supernatants by immunofluorescence analysis using an anti-collal antibody. As shown in FIG. 6, fibroblasts exposed to control media displayed collagen type I immunoreactivity in the form of small and large aggregates. While this appearance was not significantly modified by LOX supernatants, cells exposed to BMP1 and particularly to the mixture of BMP1 and LOX showed a more distinctive pattern of immunoreactivity that includes the presence of fibrous material, likely consistent with their deposition to the matrix, rather than associated with the cell layer. This was further corroborated with experiments in decellularized matrices. As shown in FIG. 7, upon removal of the cell-associated material, a more fibrous pattern was observed in deposited matrix from cells exposed to BMP1 and the mixture of BMP1 and LOX. DAPI staining confirmed that the extraction procedure efficiently removed the cell layer. Taken together, our results show that the implementation of fibroblast cultures with supernatants enriched in LOX and BMP1 was an effective approach to strongly increase the deposition of collagen onto the insoluble matrix.
Example 2: Fibroblast-Derived Matrix Modified by Lysyl Oxidase (LOX) and Bone Morphogenetic Protein-1 (BMP1) Regulates the Differentiation of Human Mesenchymal Stem Cells (MSC)
[0082] Mesenchymal stem cells are a promising source for regenerative medicine due to its capacity to self-renew and to differentiate into various tissue lineages, such as adipocytes, osteoblasts, and chondrocytes. Since the ECM provides physical and chemical cues to regulate MSC activities, we investigated the effects of fibroblast-derived matrix modified by LOX/BMP1 on regulating MSC differentiation to adipogenic and osteogenic lineages. For that purpose, we exposed fibroblast cultures to control media or to LOX and BMP1-containing supernatants as described above, then cells were removed and deposited matrix used as a substrate to establish MSC cultures. Once these cultures reached confluence, they were induced into adipogenic and osteogenic lineages by incubation with the corresponding differentiation media. These cultures were then compared with equivalent MSC seeded without any matrix. As shown in FIG. 8, after 14 days under adipogenic differentiation medium MSC without matrix develop lipid droplets that can be visualized with Oil Red O. MSC cultured on matrices derived from fibroblasts exposed to control media showed a reduced capacity to differentiate to adipocytes, and this behavior was further exacerbated in matrices from fibroblasts incubated with LOX/BMP1. On the other hand, MSC differentiation into osteogenic lineage results in the formation of extracellular calcium deposits that can be specifically stained using Alizarin Red S, as shown in FIG. 9 for MSC without matrix. Osteogenic differentiation was strongly enhanced in MSC seeded on matrices from fibroblasts exposed to control media, this effect being attenuated in matrices from fibroblasts incubated with LOX/BMP1 supernatants. These results indicate that fibroblast-derived matrix is able to regulate adipogenic and osteogenic differentiation capacity of MSC, being the modification promoted by LOX/BMP1 capable to fine-tune this ability.
