• 专利附录
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    • 专利摘要:
    The invention relates to a new type of mesenchymal stem cell (MSC) which co-expresses at least one mesenchymal marker, preferably at least CD90 or CD105, and CD34. The invention also provides osteogenic cells having a similar phenotype. The invention also provides cells and cell populations, as well as other products comprising them, and their use in bone therapy.
    公开号:BE1018748A3
    申请号:E2009/0286
    申请日:2009-05-07
    公开日:2011-08-02
    发明作者:Cindy Badoer；Enrico Bastianelli；Xavier Pesesse
    申请人:Bone Therapeutics Sa；
    IPC主号:
    专利说明:

    NOVEL MESENCHYMAL STEM CELLS AND OSTEOGENIC CELLS
    The invention relates generally to the field of cellular phenotypes and cell differentiation, and uses of cells in medicine. More particularly, the invention relates to a novel type of mesenchymal stem cells (MSC) and relates to the osteogenic differentiation of said MSCs, and the therapeutic and prophylactic applications, in bone diseases, of said MSCs and osteogenic cells derived therefrom.
    The inventors of the present invention have found that a small subset of isolated and optionally amplified MSC cells co-express at least one mesenchymal marker, such as for example CD90 or CD105, with CD34, a marker that is usually considered to represent immature blood and endothelial cell character.
    Compared with general populations of MSC cells, this new cell type expresses significant levels of alkaline phosphatase (ALP), a marker of osteocytes, and can synthesize and mineralize a new bone matrix. As a result, the cells have a relatively high potential for production of osteogenic cells useful for bone reconstruction. The inventors have similarly observed that cells can be greatly amplified, whereby appropriate amounts of osteogenic cells can be produced for cell-based therapies, particularly for bone disorders. The inventors of the present invention have succeeded in enriching MSC cells in the above cellular subtype, which will then retain its phenotypic and biological properties.
    One aspect of the invention provides an isolated mesenchymal stem cell (MSC) characterized in that it co-expresses at least one mesenchymal marker and CD34.
    Another aspect provides a method for obtaining MSC cells co-expressing at least one mesenchymal marker and CD34, comprising (a) isolating MSC cells from a sample of a subject; (b) optionally, expanding the MSC cells of (a); and (c) isolating, from the MSC cells of (a) or (b), a subset of cells that co-express at least one mesenchymal marker and CD34.
    Another aspect relates to MSC cells obtainable by steps comprising (a) isolating MSC cells from a sample of a subject; (b) optionally, expanding the MSC cells of (a); and (c) isolating, from the MSC cells of (a) or (b), a subset of cells that co-express at least one mesenchymal marker and CD34.
    The invention also provides a cell population comprising isolated mesenchymal stem cells (MSCs), co-expressing at least one mesenchymal marker and CD34, as defined in any of the above aspects.
    Another aspect provides a method for the in vitro expansion of isolated mesenchymal stem cells (MSCs), preferably isolated MSCs that express at least one mesenchymal marker and CD34, as taught in the present invention, including the said hematopoietic growth factor MSC cells and / or an angiogenic growth factor, preferably a hematopoietic growth factor and an angiogenic growth factor.
    This method can also be adapted for enrichment, by means of appropriate culture conditions, of a population of MSC (i.e., a relatively more heterogeneous MSC population) into MSC cells that co- express at least one mesenchymal marker and CD34. This results in the exposure of said relatively more heterogeneous MSC population (eg, MSC cells isolated from a subject's sample) to a hematopoietic growth factor and / or an angiogenic growth factor, preferably at a specific factor. hematopoietic growth and an angiogenic growth factor.
    Advantageously, the method can enrich a population of initial MSC or starter MSC cells comprising the expression of CD34 and one or more mesenchymal markers, such as preferably CD105, CD90 and / or CD73. Advantageously, the MSC cells in which said population has been enriched may not have expression of hematopoietic and / or endothelial markers, such as, for example, CD45, CD133, CD31, CD14 and / or CD19. Accordingly, in one embodiment, MSC cells in which the cell population has been enriched may comprise the expression of CD34 and one or more, preferably all, CD105, CD90 and CD73 markers, and may not not contain the expression of one or more, or preferably all, markers CD45, CD133, CD31, CD14 and CD19. Advantageously, the method of the present invention achieves an increase in the proportion of MSC cells comprising co-expression of CD34 and one or more mesenchymal markers in an initial MSC population during application of the method (e.g. in a non-limiting experiment, the proportion of CD105-positive and CD34-positive MSC cells increased, respectively from 15 to 95% and from 1 to 59.4% during the culture).
    The invention also provides MSC cells obtainable, or which are directly obtained by said methods of expanding or enriching MSC cells or cell populations by using a hematopoietic growth factor and / or an angiogenic growth factor.
    Another aspect relates to methods for differentiating in vitro mesenchymal stem cells (MSC) coexpressing at least one mesenchymal marker and CD34 as defined in the foregoing aspects into osteogenic cells, such as, for example, osteoblasts or cells. osteoprogenitor.
    Another aspect provides osteogenic cells, such as for example osteoblasts or osteoprogenitor cells, obtainable by in vitro differentiation of mesenchymal stem cells (MSC) co-expressing at least one mesenchymal marker and CD34 as defined in the previous aspects, into osteogenic cells.
    Since at least some of these osteogenic cells can retain the appropriate expression profile of the original MSC cells, another aspect provides isolated osteogenic cells, such as, for example, osteoblasts or osteoprogenitor cells, characterized in that that they co-express at least one mesenchymal marker and the CD34.
    The invention also discloses a cell population comprising isolated osteogenic cells co-expressing at least one mesenchymal marker and CD34.
    The invention also discloses a method for obtaining osteogenic cells co-expressing at least one mesenchymal marker and CD34, comprising differentiating, into osteogenic cells, MSC cells coexpressing said at least one mesenchymal marker and CD34.
    These osteogenic cells co-expressing at least one mesenchymal marker and CD34 can also be obtained by other methods. Thus, the invention also discloses a method for obtaining osteogenic cells co-expressing at least one mesenchymal marker and CD34, comprising (a) isolating osteogenic cells from a sample of a subject, or differentiating osteogenic cells from MSC cells isolated from a sample of a subject; (b) optionally, the expansion of osteogenic cells of (a); and (c) isolating, from the osteogenic cells of (a) or (b), a subset of cells that co-express at least one mesenchymal marker and CD34.
    Another aspect provides a method for in vitro expansion of isolated osteogenic cells, preferably isolated osteogenic cells that co-express at least one mesenchymal marker and CD34 as taught in the present invention, comprising exposing said cells osteogenic to a hematopoietic growth factor and / or an angiogenic growth factor, preferably to a hematopoietic growth factor and an angiogenic growth factor.
    This method can also be adapted for enrichment, by means of appropriate culture conditions, of a population of osteogenic cells (i.e., a relatively more homogeneous osteogenic cell population) into osteogenic cells which co-express at least one mesenchymal marker and CD34. This results in the exposure of said relatively more heterogeneous population of osteogenic cells (i.e. osteogenic cells isolated from a sample of a subject or differentiated from MSC cells isolated from a sample of a patient) to a hematopoietic growth factor and / or an angiogenic growth factor, preferably a hematopoietic growth factor and an angiogenic growth factor.
    Advantageously, the method can enrich a population of osteogenic initial cells or osteogenic cells comprising the expression of CD34 and one or more mesenchymal markers, such as for example CD105, CD90 and / or CD73. Advantageously, the osteogenic cells in which the population has been enriched may not include the expression of hematopoietic and / or endothelial markers, such as, for example, CD45, CD133, CD31, CD14 and / or CD19. Accordingly, in one embodiment, the osteogenic cells in which the population has been enriched may comprise the expression of CD34 and one or more, or preferably all, of the CD105, CD90 and CD73 markers, and may not not contain the expression of one or more, or preferably all, markers CD45, CD133, CD31, CD14 and CD19. Advantageously, the method of the present invention achieves an increase in the proportion of osteogenic cells comprising co-expression of CD34 and one or more mesenchymal markers in an initial osteogenic cell population during application of the method.
    Advantageously, the osteogenic cells amplified and / or enriched by exposure to hematopoietic and / or angiogenic growth factors, as taught in the present invention, may have both appropriate osteogenic properties (for example as evidenced by an expression and staining of ALP) than advantageous pro-angiogenic properties (for example as evidenced by the expression of vWF and VEGF and / or by formation, in an appropriate test, of structures similar to capillaries).
    The invention also provides osteogenic cells obtainable, or obtained directly, by said methods of expanding or enriching osteogenic cells or cell populations, using a hematopoietic growth factor and or an angiogenic growth factor.
    In one embodiment, the hematopoietic growth factor, as provided throughout the present disclosure, is selected from a group consisting of or consisting of colony stimulating factor 2 (CSF2), CSF3, macrophage CSF. (M-CSF), CSF of granulocytes and monocytes (GM-CSF), interferon (IFN), including inter alia IFN-gamma, tumor necrosis factor (TNF) and cytokines with hematopoietic activity , such as inter alia interleukin 2 (IL2). Preferably, the hematopoietic growth factor may be selected from GM-CSF and IFN-gamma, and more particularly it may be IFN-gamma.
    In one embodiment, the angiogenic growth factor, as provided throughout the present disclosure, is selected from a group consisting of or consisting of platelet derived growth factor (PDGF), vascular endothelial growth factors ( VEGF1 or 2), von Willebrand factor (vWF), angiopoietin 2, fibroblast growth factors (FGF1, FGF-3 or other FGF factors, or FGF factor other than FGF1) and erythropoietin (EPO). In another embodiment, the angiogenic growth factor may be selected from a group consisting of or consisting of PDGF, VEGF, vWF, angiopoietin 2 and ΓΕΡΟ. Preferably, the angiogenic growth factor may be selected from PDGF, FGF-1 and FGF-3, it may be more particularly PDGF or FGF-3 and even more preferably PDGF.
    Preferably, besides said hematopoietic growth factor and / or said angiogenic growth factor, MSC or osteogenic cells can be exposed, preferably simultaneously exposed, to FGF-2 to further stimulate maintenance, expansion, enrichment. and / or osteogenic differentiation of said cells.
    Thus, in a preferred embodiment, the expansion of MSC or osteogenic cells, such as for example MSC or osteogenic cells comprising the expression of CD34 and one or more mesenchymal markers, as taught in the invention, can understanding the exposure of said MSC or osteogenic cells to one or more growth factors selected from GM-CSF, IFN-gamma, PDGF, FGF-1 and FGF-3; for example one or more GM-CSF and IFN-gamma growth factors and / or one or more PDGF, FGF-1 and FGF-3 growth factors; for example one or more GM-CSF and IFN-gamma growth factors and one or more PDGF, FGF-1 and FGF-3 growth factors. Optionally, in these embodiments, the cells may also be exposed, preferably simultaneously exposed, to FGF-2. In a particularly preferred embodiment, the expansion of MSC or osteogenic cells, such as for example MSC or osteogenic cells comprising the expression of CD34 and one or more mesenchymal markers, as taught in the invention, may comprise exposing said MSC or osteogenic cells to IFN-gamma. This treatment considerably strengthens the osteogenic and pro-angiogenic characteristics of the cells thus treated.
    In another preferred embodiment, enrichment into MSC or osteogenic cells comprising expression of CD34 and one or more mesenchymal markers as taught in the invention, from an initial population of MSC or osteogenic cells. may include exposing said initial population of MSC or osteogenic cells to one or more growth factors selected from GM-CSF, IFN-gamma, PDGF, FGF-1 and FGF-3; for example one or more GM-CSF and IFN-gamma growth factors and / or one or more PDGF, FGF-1 and FGF-3 growth factors; for example one or more GM-CSF and IFN-gamma growth factors and one or more PDGF, FGF-1 and FGF-3 growth factors. Optionally, in these embodiments, the cells may also be exposed, preferably simultaneously exposed, to FGF-2. In a particularly preferred embodiment, enrichment into MSC or osteogenic cells comprising expression of CD34 and one or more mesenchymal markers, as taught in the invention, from an initial population of MSC cells or osteogenic, may include exposure of said MSC or osteogenic cells to IFN-gamma. This treatment considerably strengthens the osteogenic and pro-angiogenic characteristics of the cells thus treated.
    Another aspect provides a pharmaceutical composition comprising mesenchymal stem cells (MSC) co-expressing at least one mesenchymal marker and CD34 as defined in the foregoing aspects, or osteogenic cells as defined in the foregoing aspects, such as osteogenic cells co-expressing at least one mesenchymal marker and CD34, and one or more pharmaceutically acceptable carriers / excipients. The pharmaceutical composition may further comprise a cell population comprising said MSC or osteogenic cells, and one or more pharmaceutically acceptable carriers / excipients.
    A process for preparing the pharmaceutical formulation is also provided, and comprises mixing the mesenchymal stem cells (MSC) co-expressing at least one mesenchymal marker and CD34 as defined in the foregoing aspects, or osteogenic cells such as defined in the foregoing aspects, such as osteogenic cells co-expressing at least one mesenchymal marker and CD34, or a cell population comprising said MSC or osteogenic cells, with said pharmaceutically acceptable carrier / excipient.
    The cellular products of the invention are particularly suitable for prophylactic and therapeutic treatments of bone disorders. Thus, other aspects relate to: mesenchymal stem cells (MSC) co-expressing at least one mesenchymal marker and CD34 as defined in the foregoing aspects, or osteogenic cells as defined in the foregoing aspects, such as cells osteogenic co-expressing at least one mesenchymal marker and CD34, or cell populations or pharmaceutical compositions comprising the cells, as defined above, for use in the treatment of a bone disorder; the use of mesenchymal stem cells (MSC) co-expressing at least one mesenchymal marker and CD34 as defined in the foregoing aspects, or osteogenic cells as defined in the foregoing aspects, such as osteogenic cells coexpressing at minus a mesenchymal marker and CD34, or cell populations comprising the cells as defined above, for the manufacture of a medicament for the treatment of bone disorders; a method of treating a bone disorder in a subject in need of such treatment, comprising administering to said subject a therapeutically or prophylactically effective amount of the mesenchymal stem cells (MSC) coexpressing at least one mesenchymal marker and CD34 as defined in the foregoing aspects, or osteogenic cells as defined in the foregoing aspects, such as osteogenic cells co-expressing at least one mesenchymal marker and CD34, or cell populations or pharmaceutical compositions comprising cells, as defined above.
    The invention also provides an arrangement comprising a surgical instrument adapted for administering a composition to a subject, for example systemically, or at the site of a bone lesion, and further comprising any one or more cells, cell populations or pharmaceutical compositions defined above.
    In some embodiments of any of the above aspects, the cells (MSC cells, osteogenic cells) are of human origin. Preferably, the MSCs can come from the bone marrow.
    In a preferred embodiment, MSCs or MSC populations, as used in the invention, may be from the bone marrow, and may for example be isolated and optionally amplified from a bone marrow sample. . MSCs and bone marrow MSC populations may have characteristics (eg, marker profile, function, expansion, differentiation, etc.) different from, and / or more interesting than, MSCs from other tissues. latter, and in particular, but without limitation, can differentiate in a more efficient and / or more regulatable manner into osteogenic cells.
    MSC cells exhibiting the above marker profile may also represent other attributes of mesenchymal cells. For example, MSC cells may further express one, more, or all of the mesenchymal markers selected from CD106 (VCAM), CD166 (ALCAM), CD29, CD44, CD54, and GATA-4. MSC cells may further exhibit certain morphological characteristics, such as one or more adhesions to the plastic of the tissue culture; monolayer growth; and an ovoid, stellate or fusiform form of mononuclear cells, with circular oval nuclei, with prominent nucleoli.
    The term "osteogenic cells" as used in the invention generally refers to cells capable of contributing to the formation of bone material and / or bone matrix, and in particular refers to isolated cells or cell populations that have wholly or partly progressed along an osteogenic differentiation pathway. Without limitation, osteogenic cells include, in particular, osteoprogenitor cells, osteoblasts, osteocytes and other cell types of the osteogenic lineage, as is known in the art.
    Those skilled in the art thus generally recognize the limits of the expression "osteogenic cells" as provided for in the invention. Nevertheless, as a further guide and not a limitation, the osteogenic cells of the present invention may have any one or more or all of the following characteristics: a) the cells comprise the expression of alkaline phosphatase (ALP) ), more particularly ALP of the bone, hepatic and renal types; b) the cells comprise the expression of one or more of an aminoterminal propeptide of procollagen type 1 (P1NP), osteonectin (ON), osteopontin (OP), osteocalcin (OCN) and bone sialoprotein (BSP); c) the cells exhibit evidence of their ability to mineralize their external environment, or to synthesize the extracellular matrix containing calcium (e.g., when exposed to osteogenic media, see Jaiswal et al., 1997. J. Cell Biochem. 64: 295-312). The accumulation of calcium inside the cells, and its deposition in the proteins of the matrix, can be conventionally measured for example by culture in 45Ca2 +, washing and new culture, then by determining a possible radioactivity present in the cell interior or deposited in the extracellular matrix (US 5,972,703), or using an alizarin red mineralization assay (see, eg, Gregory et al., 2004. Analytical Biochemistry 329: 77 -84); d) the cells do not differ considerably in any of the cells of the adipocytic line (for example adipocytes) or the chondrocytic line (for example the chondrocytes), and preferably do not differ in any of these. The lack of differentiation in these cell lines can be tested by using standard differentiation-inducing conditions, well established in the art (see, for example, Pittenger et al., 1999, Science 284: 143-7) and methods of differentiation. analysis (for example, after induction, the adipocytes are representative of staining with oil red O, which shows an accumulation of lipids, the chondrocytes are usually stained alcian blue or safranin O). The almost complete absence of a tendency to adipogenic and / or chondrogenic differentiation can usually mean that less than 50%, or less than 30%, or less than 5%, or less than 1% of the cells studied should show signs adipogenic or chondrogenic differentiation when applied to the test under consideration.
    In one embodiment, the osteogenic cells may have all the characteristics mentioned in a), c) and d) above.
    Preferably, the MSC cells or the osteogenic cells, as described in the invention, or the cell populations which comprise them, are of animal origin, more particularly come from a non-human mammal or from a human, and from an even more preferred way are of human origin.
    MSC cells coexpressing at least one mesenchymal marker and CD34, and optionally any additional marker of interest, as described herein, can be obtained by methods that are known per se.
    For example, conventional methods make it possible to isolate and optionally amplify MSC or B MSC cells from biological samples of subjects.
    It has been described that MSCs or BMSCs can be isolated and amplified from bone marrow or other sources by selecting and culturing, from them, cells that can adhere to a substrate surface, by particularly a tissue culture plastic surface. Protocols based on this principle are described in detail in Pittenger et al., 1999 (see above) and in related patent documents mentioned elsewhere in this specification, and are discussed in detail. together in Alhadlaq & Mao 2004 (Stem Cells Dev. 13: 436-48). These protocols are applicable in the present invention.
    Osteogenic cells can be obtained by differentiation from isolated MSC cells, as known in the art. In one example, the method of WO 2007/093431 can be used, wherein isolated MSC cells are cultured in the presence of serum or plasma and a basic fibroblast growth factor (FGF-2). In another example, cells of the osteogenic line can be obtained by differentiating MSC cells in an osteogenic medium, as described by Pittenger et al., 1999 (see above) and Jaiswal et al., 1997 (see below). above). Optionally, a traditional osteogenic medium may be supplemented with FGF-2. It is likewise possible to isolate and cultivate osteogenic cells directly from the trabecular bone, as described by Skjodt et al., 1985 (J. Endocrinol., 105: 391-6).
    In the context of the various aspects of the invention, the term "bone disorder" refers to any type of bone disease whose treatment may benefit from the administration of the osteogenic line or osteogenic cells, for example osteoprogenitor cells, osteoblasts or osteocytes, to a subject with this disorder. In particular, these disorders can be characterized for example by a decrease in bone formation or by excessive bone resorption, by a decrease in the number, viability or function of osteoblasts or osteocytes present in the bone, by a decreased bone mass in a subject, thinning of the bone, decreased strength or elasticity of bone, etc.
    By way of example and not limitation, the term encompasses local and systemic disorders, such as any type of osteoporosis or osteopenia, for example primary, postmenopausal, senile, due to corticosteroids, any secondary osteonecrosis , monositis or multisite, any type of fracture, for example pseudarthrosis, malunion, fracture consolidation delay or compression, conditions requiring bone fusion (eg spondylodesis and reconstruction), maxillofacial fractures bone reconstruction, for example after traumatic injury or surgery in oncology, reconstruction of cranial-facial bones, osteogenesis imperfecta, osteolytic bone cancer, Paget's disease, endocrinological disorders, hypophosphatemia, hypocalcemia, renal osteodystrophy, osteomalacia, adynamic bone diseases, rheumatoid arthritis, hyperparathyroidism, hyperpa primary rathyroidism, secondary hyperparathyroidism, periodontal disease, Gorham-Stout disease and McCune-Albright syndrome.
    MSC, BMSC and osteogenic cells, and populations comprising cells of this type as described in the invention, find various uses, particularly in the fields of research, prevention and treatment of bone disorders.
    In one aspect, cells or cell populations can be used to produce bone matrix in vitro. This bone matrix can be used, for example, in conjunction with or without cells or cell populations in the treatment of bone diseases.
    The treatment can use cells or populations of MSC, BMSC or osteogenic cells, as defined in the present invention, autologous (i.e., cells derived from the subject to be treated), allogeneic (ie that is, cells derived from one or more subjects other than the subject to be treated, but belonging to the same species) or xenogeneic (that is, cells derived from one or more subjects belonging to a species other than that of the subject to be treated).
    Particularly contemplated are the treatments of human subjects using cells or populations of MSC, BMSC or osteogenic cells, as obtained in the invention, autologous or allogeneic.
    Advantageously, the cells and populations of MSC, BMSC or osteogenic cells obtained in the present invention can be formulated into pharmaceutical compositions and administered in the form of pharmaceutical compositions.
    The pharmaceutical compositions will usually comprise the cells or populations of MSC, BMSC or osteogenic cells of the invention as active ingredient, and one or more pharmaceutically acceptable carriers / excipients.
    In another aspect, the invention relates to an arrangement comprising a surgical instrument for administering a composition to a subject, such as, for example, systemically, topically or at the site of a bone lesion, and further comprising the cells or cell populations of the invention, or a pharmaceutical composition comprising said cells or cell populations, the arrangement being adapted to the administration of the pharmaceutical composition for example systemically, topically or at the site of the bone lesion. For example, a suitable surgical instrument may be capable of injecting a liquid composition comprising cells of the present invention, especially systemically or at the site of the bone lesion.
    The cells or cell populations may be administered in a manner that allows them to graft or migrate to the intended tissue site, by reconstituting or regenerating the functionally deficient area. Administration of the composition will depend on the musculoskeletal site being repaired. For example, osteogenesis may be facilitated in accordance with a surgical procedure to reshape the tissue or insert a fissure, or a prosthetic device such as a hip prosthesis.
    In other circumstances, invasive surgery will not be required, and the composition may be administered by injection (for example for spinal repair), using a steerable endoscope.
    In one embodiment, the pharmaceutical cellular composition as defined above may be administered in the form of a liquid composition. In embodiments, the cells or pharmaceutical composition comprising the same may be administered systemically, topically, or at the site of injury.
    In another embodiment, the cells or cell populations may be transferred to a suitable substrate and / or cultured on that substrate to provide implants. The substrate on which the cells can be applied and cultured may be a metal such as titanium, a cobalt / chromium alloy or stainless steel, a bioactive surface such as a calcium phosphate, polymeric surfaces such as polyethylene, and the like. Although it is less preferred, it is also possible to use as a substrate a siliceous material such as glass-ceramics. Metals such as titanium and calcium phosphates are particularly preferred, although calcium phosphate is not an indispensable component of the substrate. The substrate may be porous or non-porous.
    For example, cells that have proliferated, or are being differentiated into culture dishes, can if necessary be transferred to solid three-dimensional supports in order to be multiplied and / or to continue the process of differentiation, by incubation of the solid support in a liquid nutrient medium of the invention. The cells may be transferred to a solid three-dimensional support, for example by impregnation of said support with a liquid suspension containing said cells. The impregnated supports obtained in this way can be implanted in a human subject. These impregnated supports can also undergo a new culture by immersion in a liquid culture medium, before final implantation.
    The solid three-dimensional support must be biocompatible, so that it can be implanted in a human. It may have any suitable shape, such as a cylinder, sphere, plate, or arbitrary shape. Among the materials suitable for the biocompatible three-dimensional solid support, a particular mention may be made of calcium carbonate and in particular aragonite, in particular in the form of a coral skeleton, with porous ceramics based on alumina, zirconium, tricalcium phosphate and / or hydroxyapatite, imitation coral skeleton obtained by hydrothermal exchange, allowing the calcium carbonate to be converted to hydroxyapatite, or otherwise to a vitroceramic apatite-wollastonite, to a bioactive glass-ceramic such as Bioglass® glasses.
    权利要求:
    Claims (23)
    [1]
    1. Isolated mesenchymal stem cell (MSC), preferably from the bone marrow, characterized in that it co-expresses at least one mesenchymal marker and CD34.
    [2]
    An isolated MSC cell according to claim 1, wherein the at least one mesenchymal marker is selected from CD105, CD90 and CD73.
    [3]
    An isolated MSC cell according to claim 1 which coexpresses CD105, CD90, CD73 and CD34.
    [4]
    A process for obtaining MSC cells preferably from the bone marrow, co-expressing at least one mesenchymal marker, as defined in any one of claims 1 to 3 and CD34, comprising: (a) isolating MSC cells from a sample of a subject; (b) optionally, expanding the MSC cells of (a); and (c) isolating, from the MSC cells of (a) or (b), a subset of cells that co-express at least one mesenchymal marker as defined in any of the claims 1 to 3, and the CD34.
    [5]
    An in vitro method of expanding isolated MSC cells, preferably isolated MSC cells according to any one of claims 1 to 3, comprising exposing said MSC cells to a hematopoietic growth factor or an angiogenic growth factor. , or both to a hematopoietic growth factor and to an angiogenic growth factor.
    [6]
    A method of enriching a MSC population in MSC cells as defined in any one of claims 1 to 3, comprising exposing said MSC population to a hematopoietic growth factor or a growth factor. angiogenic or both to a hematopoietic growth factor and to an angiogenic growth factor.
    [7]
    The method of any of claims 5 or 6, wherein the hematopoietic growth factor is selected from a group consisting of colony stimulating factor 2 (CSF2), CSF3, macrophage CSF (M-CSF). , CSF of monocytes and granulocytes (GM-CSF), interferon (IFN), tumor necrosis factor (TNF) and hematopoietic cytokines, and angiogenic growth factor is selected from a group comprising the factor platelet derived growth (PDGF), vascular endothelial growth factor (VEGF), von Willebrand factor (vWF), angiopoietin 2, fibroblast growth factor, and erythropoietin (EPO).
    [8]
    The method according to any of claims 5 or 6, wherein the hematopoietic growth factor is GM-CSF or IFN-gamma, more particularly IFN-gamma, and the angiogenic growth factor is PDGF. , FGF-1 or FGF-3, more particularly PDGF or FGF-3.
    [9]
    9. Isolated MSC cells obtainable by the method of any one of claims 4 to 8.
    [10]
    10. Osteogenic cells obtainable by in vitro differentiation, in osteogenic cells, of MSC cells according to any one of claims 1 to 3.
    [11]
    11. Isolated osteogenic cell, characterized in that it co-expresses at least one mesenchymal marker and CD34.
    [12]
    An isolated osteogenic cell according to claim 11, wherein the at least one mesenchymal marker is selected from CD105, CD90 and CD73.
    [13]
    An isolated osteogenic cell according to claim 11 which coexpresses CD105, CD90, CD73 and CD34.
    [14]
    14. Process for in vitro expansion of isolated osteogenic cells, preferably isolated osteogenic cells according to any one of claims 10 to 13, comprising exposing said osteogenic cells to a hematopoietic growth factor or to an angiogenic growth factor. or both to a hematopoietic growth factor and to an angiogenic growth factor.
    [15]
    The method of enriching, in osteogenic cells according to any of claims 10 to 13, an osteogenic cell population, comprising exposing said osteogenic cell population to a hematopoietic growth factor or a angiogenic growth or both to a hematopoietic growth factor and to an angiogenic growth factor.
    [16]
    The method of any one of claims 14 or 15, wherein the hematopoietic growth factor is selected from a group consisting of colony stimulating factor 2 (CSF2), CSF3, macrophage CSF (M-CSF). , CSF of monocytes and granulocytes (GM-CSF), interferon (IFN), tumor necrosis factor (TNF) and hematopoietic cytokines, and angiogenic growth factor is selected from a group comprising the factor platelet derived growth (PDGF), vascular endothelial growth factor (VEGF), von Willebrand factor (vWF), angiopoietin 2, fibroblast growth factor, and erythropoietin (EPO).
    [17]
    The method according to any one of claims 14 or 15, wherein the hematopoietic growth factor is GM-CSF or NFN-gamma, more particularly IFN-gamma, and the angiogenic growth factor is PDGF, FGF-1 or FGF-3, more particularly PDGF or FGF-3.
    [18]
    18. Isolated osteogenic cells obtainable by the method according to any of claims 14 to 17.
    [19]
    19. Isolated MSC cells according to any one of claims 1 to 3 or 9, or isolated osteogenic cells according to any one of claims 10 to 13 or 18, which are of human origin.
    [20]
    A cell population comprising isolated MSC cells according to any one of claims 1 to 3, 9 or 19 or isolated osteogenic cells according to any one of claims 10 to 13, 18 or 19.
    [21]
    21. Isolated MSC cells according to any one of claims 1 to 3, 9 or 19, or isolated osteogenic cells according to any one of claims 10 to 13, 18 or 19, or cell population according to claim 20, for use in the treatment of a bone disorder.
    [22]
    22. A pharmaceutical composition comprising the isolated MSC cells according to any one of claims 1 to 3, 9 or 19, or the isolated osteogenic cells according to any one of claims 10 to 13, 18 or 19, or the cell population according to claim 20, and one or more pharmaceutically acceptable carriers / excipients.
    [23]
    A process for preparing the pharmaceutical formulation according to claim 22, comprising mixing, with one or more pharmaceutically acceptable carriers / excipients, isolated MSC cells according to any one of claims 1 to 3, 9 or 19, or isolated osteogenic cells according to any one of claims 10 to 13, 18 or 19, or the cell population of claim 20.
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    同族专利:
    公开号 | 公开日
    AU2009245751B2|2014-09-18|
    JP6097479B2|2017-03-15|
    EP2291516A2|2011-03-09|
    EP2291516B1|2019-01-02|
    KR20110013450A|2011-02-09|
    WO2009135905A2|2009-11-12|
    JP2017099386A|2017-06-08|
    US20110262404A1|2011-10-27|
    US9371515B2|2016-06-21|
    AU2009245751A1|2009-11-12|
    JP2011520434A|2011-07-21|
    WO2009135905A3|2010-02-25|
    CN102016008A|2011-04-13|
    US20170290859A1|2017-10-12|
    CA2722625A1|2009-11-12|
    US20140093480A1|2014-04-03|
    KR20160065213A|2016-06-08|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    EP08155764|2008-05-07|
    EP08155764|2008-05-07|
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