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    • 专利摘要:
    The present invention relates to a method for obtaining CSM derived cells having improved transplantation properties from CSM, the method comprising a cell size reduction step, wherein said cell size reduction step is characterized by contacting CSM or cells derived from CSM in vitro or ex vivo with heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU / ml. The invention further relates to a method for obtaining cells derived from mesenchymal stem cells from mesenchymal stem cells (MSCs) comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-I3 and at least 0.01 IU / ml heparin or a derivative or analogue thereof. The invention further relates to cells and cell populations thus obtained, as well as additional products comprising them and uses thereof.
    公开号:BE1025935B1
    申请号:E2018/5662
    申请日:2018-09-25
    公开日:2019-10-18
    发明作者:Enrico Bastianelli；Xuan Mai Nguyen；Isabelle Tytgat；Sandra Pietri；Sabrina Ena；Pierre-Yves Laruelle
    申请人:Bone Therapeutics Sa；
    IPC主号:
    专利说明:

    BE2018 / 5662
    METHODS OF DIFFERENTIATION OF MESENCHYMAL STEM CELLS FIELD OF THE INVENTION
    The present invention relates to methods of expanding and / or differentiating mesenchymal stem cells (MSCs) into cells and cell populations derived from MSCs, and products comprising such cells and cell populations, methods and uses.
    CONTEXT
    The transplantation of stem cells capable of undergoing osteogenic differentiation, of cells which are programmed for osteogenic differentiation or of cells having osteoforming capacity is a promising route for the treatment of bone diseases, in particular when the treatment requires the production of new tissues. bony.
    Mesenchymal stem cells (MSCs) have previously been used to treat bone disorders (Gangji et al., 2005 Expert Opin Biol Ther 5: 437-42). However, although such relatively undifferentiated stem cells can be transplanted, they are not associated with an osteoblastic line and, therefore, a considerable proportion of such transplanted stem cells cannot ultimately contribute to the formation of the desired bone tissue. In addition, the quantity of such stem cells is often unsatisfactory.
    WO 2007/093431 relates to a method for the in vitro expansion of isolated MSCs, which produces cells with an osteoblastic phenotype. In said method, human MSCs were grown in the presence of serum or plasma and basic fibroblast growth factor (FGF-2).
    WO 2009/087213 relates to a process for obtaining osteoprogenitors, osteoblasts or cells exhibiting an osteoblast phenotype from human CSM in vitro or ex vivo, comprising contacting said CSM with human plasma or serum , FGF-2 and the transforming growth factor beta (TGF-β).
    There is a continuing need for new cells and cell populations useful in therapy, among others, such as new cells and cell populations derived from MSCs, and methods of producing them.
    ABSTRACT
    As corroborated by the experimental section, which illustrates certain embodiments representative of the invention, the inventors have realized that the transplantation potential of cells derived from mesenchymal stem cells (MSC) can be considerably increased when said cells are obtained by contacting
    CSM or cells derived from CSM in vitro or ex vivo with heparin or an E or 18/5662 derivative analog thereof. More particularly, the inventors have discovered that bringing CSM or cells derived from CSM into contact with heparin or a derivative or analog thereof, preferably heparin or a derivative or analog thereof, to a concentration of at least 0.01 IU / ml, leads to new populations of cells derived from MSC cells whose cells have a normalized, homogeneous and comparatively small cell size. Such cells derived from CSM cells have improved transplant properties, such as (i) improved compliance for parenteral administration (e.g., intravascular, including intravenous), (ii) the ability to administer a concentration of cells in vivo adjustable and high with a limited volume, (iii) a satisfactory in vivo safety profile and / or (iv) good syringability when administered parenterally.
    In addition, the present cells derived from MSCs are capable of inducing bone formation. In addition to the osteoinductive properties, the present inventors have discovered that the present cells derived from MSC also exhibit high osteogenic activity. Osteogenic activity advantageously leads to the occurrence of mineralized nodules produced by endochondral ossification.
    Therefore, in one aspect, the present invention relates to a method for obtaining CSM-derived cells having improved transplant properties from CSM, the method comprising a size reduction step, wherein said size reduction step is characterized by contacting CSM or cells derived from CSM in vitro or ex vivo with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    In another aspect the invention relates to a method for obtaining cells derived from CSM from CSM comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-ß and heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    In another aspect, the invention relates to a method for obtaining cells derived from CSM from CSM comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-ß and heparin or a derivative or analog thereof, in which at least 60% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm) and in which at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm.
    In another aspect, the invention relates to a population of cells derived from MSCs obtainable by in vitro or ex vivo expansion of MSCs, in which at least 60% of the cells derived from MSCs in suspension have a diameter equal to or less than 25 μm (D 60
    BE2018 / 5662 <25 μm) and in which at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm.
    In another aspect, the invention relates to a pharmaceutical composition comprising the population of cells derived from MSC as presently described.
    In another aspect, the invention relates to the population of cells derived from MSC or the pharmaceutical composition as presently described for use as a medicament.
    These and other preferred aspects and embodiments of the invention are described in the sections and in the appended claims. The subject matter of the appended claims is specifically incorporated into this specification.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1 illustrates the cell size of cells derived from MSC, in particular osteoforming cells derived from MSC, generated with fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-P1) (A) and osteoforming cells derived from CSM generated with FGF-2, TGF-P1 and heparin (B).
    FIG. 2 illustrates the generation of cells derived from MSC, in particular osteoforming cells derived from MSC, with different heparinoids: unfractionated heparin (HNF), dalteparin, danaparoid and heparan sulfate; used at 0.1 IU / ml.
    Figure 3 illustrates a culture of MSC with heparin compared to other anticoagulants: EDTA and Actilyse® (alteplase, tissue activator of recombinant plasminogen). After 36 h of culture, the cells brought into contact with EDTA at 2 mg / ml and Actilyse® at 0.1 mg / ml are in suspension, while the cells brought into contact with heparin at 1 or 100 IU / ml grow and are adherent.
    FIG. 4 illustrates cells derived from MSC, in particular osteoforming cells derived from MSC, cultured according to an embodiment of the method of the invention in which the plasma treated with solvent / detergent (S / D) (5% v / v) has been replaced by serum (5% v / v).
    FIG. 5 illustrates the in vitro mineralization of cells derived from MSC, assayed by alizarin red staining (ARS). Osteoforming B cells derived from MSC (generated with FGF-2, TGF-P1 and heparin) were cultured under osteogenic conditions. ARS was performed after 21 days (A) and 28 days (B) of culture under osteogenic conditions to color the calcium and phosphate deposits (magnification 100x).
    FIG. 6 illustrates the staining with hematoxylin and eosin and the staining of the cartilaginous extracellular matrix (proteoglycans and collagen) of sections of cell aggregates. Osteoforming B cells derived from MSCs (generated with FGF-2, TGF-P1 and heparin) are centrifuged to form cell aggregates, and cell aggregates are cultured for 21 days under chondrogenic or control conditions. Toluidine blue stains the proteoglycan of the cartilage extracellular matrix and cell nuclei. The orange saffron colors the proteoglycan of the cartilaginous extracellular matrix, and the red sirius colors the collagen.
    FIG. 7 illustrates the staining with hematoxylin and eosin of histological sections of pulmonary parenchyma (magnification 200 ×). (A) Animal after injection of osteoforming B cells derived from MSC: normal pulmonary parenchyma; (B) Animal after injection of osteoforming cells A: pulmonary parenchyma with numerous disseminated groups of cells injected into the alveolar capillaries (arrows).
    FIG. 8 illustrates the bone neoformation on a coronal section of the murine cranial vault demonstrated by murine and human fluorochromes binding to calcium, 2 weeks after the administration of excipient alone (control condition), of osteoforming A cells derived from CSM (generated with FGF-2 and TGF-ß1) or osteoforming B cells derived from CSM (generated with FGF-2, TGF-ß1 and heparin).
    FIG. 9 illustrates the quantification of bone formation (%) carried out on coronal sections of the murine cranial vault, 2 weeks after the administration of the excipient alone (control condition), of osteoforming A cells derived from MSC (generated with FGF-2 and TGF-ß1) or osteoforming B cells derived from CSM (generated with FGF-2, TGF-ß1 and heparin).
    FIG. 10 illustrates a double immunostaining (immunofluorescence) anti-collagen type I human and murine performed on coronal sections of the murine cranial vault, 2 weeks after the administration of osteoforming B cells derived from MSC (generated with FGF-2, TGF -ß1 and heparin). FIG. 10a illustrates a double anti-collagen type I human and murine immunostaining (fusion) while FIGS. 10b and 10c represent an anti-human and anti-murine type I collagen immunostaining, respectively.
    Figure 11 illustrates the cell size of (A) CSM, (B) osteoforming A cells derived from CSM (generated with FGF-2 and TGF-ß1), and (C) osteoforming B cells derived from CSM (generated with FGF- 2, TGF-ß1 and heparin).
    FIG. 12 illustrates the histological staining of coronal sections of the murine cranial vault, 2 weeks after the administration of excipient alone, of MSC, of osteoforming A cells derived from MSC generated with FGF-2 and TGF-β1 (cells of A) or osteoforming B cells derived from MSCs generated with FGF-2, TGF-ß1 and heparin (cells of B). (A) calcium binding fluorochromes are sequentially injected intraperitoneally (alizarin red from calcein blue from calcein blue from tetracycline) to demonstrate bone neoformation (arrows) and assess the dynamics of bone formation; (B) human + murine type I collagen immunofluorescence (IF); (C) murine type I collagen IF; (D) IF of human type I collagen. The dOu ^ § 18/5662 immunofluorescence of collagen type I anti-human and anti-murine is carried out to allow the detection of collagen type I human and murine secreted by the bone matrix;
    (E) ALP + Goldner staining: ALP: detection of the osteoblast activity in black (solid lines and areas), Masson-Goldner trichrome: detection of the osteoid (non-mineralized bone tissue) in black dotted line, mineralized bone in dark gray line; (F) tartrate resistant acid phosphatase (TRAP): detection of the activity of osteoclasts in dark gray / black.
    FIG. 13 represents photographs illustrating new bone formation on coronal sections of murine cranial vault, 2 weeks after the administration of excipient alone; from CSM; osteoforming A cells derived from MSCs generated with FGF-2 and TGF-ß1 (o-f A cells); or osteoforming B cells derived from MSCs generated with FGF-2, TGF-ß1 and heparin (o-f B cells). Bone neoformation is highlighted by fluorescence (marked by the sequential integration of different fluorochromes: alizarin red from calcein blue from calcein yellow from tetracycline). A red, green and blue coloration appears in light gray and the thickness of new bone formation is indicated with double arrows. The yellow coloration was surrounded by dotted lines.
    FIG. 14 represents a graph illustrating the total area of newly formed bone (mean ± SEM, * p <0.05) measured on sections of the murine cranial arch, 2 weeks after the administration of MSC (dark gray) or of cells osteoformers B (light gray).
    FIG. 15 illustrates the staining with orange safranine of the cartilaginous matrix (surrounded by dotted lines) of mineralized nodules performed on sagittal sections of murine cranial vault, one day after the administration of osteoforming B cells (J1) and during time (D7, D14, D21) up to 28 days (D28) after administration.
    Figure 16 illustrates the effect of MSC-derived cells in a segmental femoral subcritical size defect model. (A) represents a graph illustrating the measurement of the size of the defect on radiographs taken on the day of the surgical intervention / product administration (D0) and over time (1, 2, 3, 4, 5 weeks) up to '' at 6 weeks (6S) after the administration of the excipient alone, of osteoforming cells A (cells of A) or of osteoforming cells B (cells of B); mean ± SEM, ** p <0.01, *** p <0.001; (B) represents radiographs representative of segmental femoral defects on D0 and 6S after the administration of the excipient alone or of osteoforming B cells (o-f B cells); (C) represents a graph illustrating the measurement of bone repair volume by microtomodensitometry (micro-CT) analyzes at 6S after the administration of the excipient alone (n = 7) and of osteoforming B cells (n = 8) ; mean ± SEM, * p <0.05.
    DETAILED DESCRIPTION
    BE2018 / 5662
    In the present context, the singular forms "a", "an", and "the" include both references to the singular and plural, unless this is clearly contraindicated in the context.
    The terms "comprising", "comprises" and "consistingen", in this context, are synonymous with "comprising", "comprises" or "containing", "contains", and are inclusive or open and do not exclude members , additional elements or process steps, not mentioned. The terms further cover "consisting of" and "essentially consisting of", which have well-defined meanings in patent terminology.
    The quotation of numerical ranges by limits includes all the numbers and fractions included in the respective ranges as well as the limits mentioned.
    The terms "approximately" or "approximately", in the present context, with reference to a measurable value such as a parameter, a quantity, a time duration, and the like, are intended to cover variations of the specified value and relative to it, such as variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and even more preferably +/- 0.1% or less than and relative to the specified value, as long as such variations are suitable for application in the present invention. It should be understood that the value to which the modifier "about" refers is itself specifically and preferably described.
    Although the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, is clear as such, by further exemplification, the term covers inter alia a reference to any one of said members, or to two or more of any two of said members, such as, for example,> 3,> 4,> 5,> 6 or> 7 any, etc. , of said members, and up to all said members. In another example, "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7 or more.
    The discussion of the context of the invention is presently included to explain the context of the invention. This should not be taken as an admission that any part of the material has been published, known, or is part of general knowledge common in any country on the priority date of any of the claims.
    Throughout this description, various publications, patents and patent specifications published are referenced by an identified citation. All documents cited in this specification are currently incorporated by reference in their entirety. In particular, the lessons or sections of these documents specifically referenced herein are incorporated by reference.
    Unless otherwise indicated, all the terms used in the description of the invention, including technical and scientific terms, have their meanings commonly known to those skilled in the art to which this invention belongs. As a further indication, definitions of terms are included to better understand the teaching of the invention. When specific terms are defined in the context of a particular aspect of the invention or of a particular embodiment of the invention, such a connotation is intended to apply to the whole of this specification, it that is, also in the context of other aspects or embodiments of the invention, unless otherwise indicated.
    In the following sections, various aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment thus defined can be combined with one or more other aspect (s) or embodiment (s), unless clearly indicated to the contrary. In particular, a characteristic indicated as being preferred or advantageous can be combined with another characteristic or other characteristics indicated as being preferred or advantageous.
    Reference throughout this specification to "an embodiment" means that a particular element, structure or feature described in the context of the embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of "in one embodiment" in different places throughout this specification do not necessarily refer to the same embodiment, but it is possible. In addition, the particular elements, structures or characteristics can be combined in any suitable way, as will be apparent to those skilled in the art from the present description, in one or more embodiments. In addition, although some embodiments presently described include some, but not other elements included in other embodiments, combinations of elements of different embodiments are intended to be within the scope of the invention. , and constitute different embodiments, as will be apparent to those skilled in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
    As corroborated by the experimental section, which illustrates certain embodiments representative of the invention, the inventors have identified a method for obtaining cells derived from CSM or a population of cells derived from CSM having a transplant potential. increased. More particularly, the inventors have unexpectedly discovered that, when contacting CSM or cells derived from CSM with heparin or a derivative or analog thereof, preferably heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml, a new population of cells derived from MSC having a standardized, homogeneous and small size can be obtained. In certain embodiments, MSCs or cells derived from MSCs are contacted with a combination of FGF-2, TGF-β and heparin or a derivative or analogd® ^ 18/5662 thereof, preferably heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml. Such a normalized, homogeneous and small size represents improved transplantation properties such as (i) the potential for parenteral (for example, intravascular, including intravenous) administration of said cells derived from MSC, (ii) the possibility of administering in in vivo an adjustable and high cell concentration with a limited volume, (iii) a satisfactory in vivo safety profile and / or (iv) good syringability when administered parenterally. Consequently, a first aspect relates to a method for obtaining cells derived from CSM from CSM comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or analog thereof to a concentration of at least 0.01 IU / ml.
    The term “mesenchymal stem cell” or “MSC”, in the present context, designates a stem cell derived from adult mesoderm which is capable of generating cells from mesenchymal lines, typically from two or more mesenchymal lines, more typically three or mesenchymal lines plus, for example, an osteochondroblastic (bone and cartilage), osteoblastic (bone), chondroblastic (cartilage), myocytic (muscle), tenocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) and stromogenic (spinal cord) lineage . MSCs can be isolated from a biological sample, preferably a biological sample from a human subject, for example, bone marrow, trabecular bone, blood, umbilical cord, placenta, fetal yolk sac , skin (dermis), specifically fetal and adolescent skin, periosteum, dental pulp, tendon and adipose tissue. The term "biological sample" or "sample" in the present context means a sample obtained from a biological source, for example, from an organism, such as an animal or human subject, a culture of cells, tissue sample, etc. A biological sample from an animal or human subject means a sample taken from an animal or human subject and comprising cells thereof. The biological sample from an animal or human subject can comprise one or more types of tissue and can comprise cells from one or more types of tissue. Methods of obtaining biological samples from an animal or human subject are known in the art, for example, a tissue biopsy or a blood sample. Human MSCs, their isolation, in vitro expansion and differentiation, have been described in, for example, U.S. Patent No. 5,486,359; U.S. Patent No. 5,811,094; U.S. Patent No. 5,736,396; U.S. Patent No. 5,837,539; or U.S. Patent No. 5,827,740. The MSCs described in the art and isolated by any method described in the art can be adapted in the present method. In particular, MSCs can be defined as having a capacity for differentiating the trenchline mesenchymal in vitro into osteoblasts, adipocytes, and chondroblasts (Dominici et al., 2006, vol. 8, 315).
    The term "CSM" further covers the descendants of CSM, for example, a descend§n8ê 18/5662 obtained by proliferation (propagation / expansion) in vitro or ex vivo of CSM obtained from a biological sample of a subject animal or human.
    The term “stem cell” generally designates a non-specialized or relatively less specialized cell competent for proliferation, which is capable of self-renewal, that is to say that it can proliferate without differentiation, and which leads, or whose offspring can lead to at least one relatively more specialized type of cell. The term covers stem cells capable of substantially unlimited self-renewal, that is to say that the progeny of a stem cell or at least part of it substantially retains the non-specialized or relatively less specialized phenotype, the potential of differentiation and the proliferation capacity of the mother stem cell, as well as of stem cells which exhibit limited self-renewal, that is to say that the capacity of the progeny of part of it for proliferation and / or subsequent differentiation is demonstrably reduced compared to the parent cell. By way of example and without limitation, a stem cell can lead to descendants which can differentiate in one or more lines to produce more and more relatively more specialized cells, such descendants and / or cells more and more relatively Specialized can themselves be stem cells as presently defined, or even produce terminally differentiated cells, i.e., fully specialized cells, which can be post-mitotic.
    The term "adult stem cell" in the present context means a stem cell present in, or obtained from (for example, isolated from) an organism in the fetal stage, preferably after birth (for example, in particular but without limitation for a human organism, at the age of at least one month after birth, for example, at the age of at least 2 months, at least 3 months, for example, at least 4 months , at least 5 months, for example, at least 6 months after birth, such as, for example, at the age of 1 year or more, 5 years or more, at least 10 years or more, 15 years or more, 20 years or more, or 25 years or more after birth), such as, for example, after reaching adulthood. For example, adult stem cells can be obtained from human subjects which would otherwise be described by the conventional terms "infant", "child", "young", "adolescent" or "adult".
    Preferable MSCs have the potential to generate cells of at least the osteochondroblastic line, such as, for example, osteoblastic line cells, such as, for example, osteochondroprogenitor and / or osteoprogenitor and / or preostoblast and / or osteoblasts and / or osteocytes, and / or of chondroblastic line, such as, for example, osteochondroprogenitors and / or chondroprogéniteurs and / or prechondroblasts and / or chondroblasts and / or chondrocytes.
    Other preferable MSCs have the potential to generate cells from at least the osteoblastic line 18/5662 (bone), such as, for example, osteochondroprogenitors and / or osteoprogenitors and / or preosteoblasts and / or osteoblasts and / or osteocytes, etc. ; or at least the chondroblastic line (cartilage), such as, for example, osteochondroprogenitors and / or chondroprogenitors and / or prechondroblasts and / or chondroblasts and / or chondrocytes; fibroblastic line (connective tissue), such as, for example, fibroblasts, fibrocytes; or at least synoviocytes (synovial fluid); or tenocytes, etc.
    Unless otherwise indicated, "subject" or "patient" is used interchangeably and means animals, preferably vertebrates, more preferably mammals, and specifically includes human patients and non-human mammals. Preferred patients are human subjects. Animal subjects include prenatal forms of animals, such as, for example, fetuses. Human subjects can include fetuses, but not embryos.
    In one embodiment, CSMs can be obtained from a healthy subject, which can help to ensure the functionality of cells derived from CSMs obtained from said CSMs.
    In another embodiment, MSCs are obtained from a human subject who requires a cell transplant derived from MSCs.
    In certain embodiments of the products or methods as presently described, the MSCs or cells derived from MSCs may be allogenic for the subject to be treated. The terms "allogeneic" or "homologous" with reference to MSCs or cells derived from MSCs mean that the MSCs or cells derived from MSCs are obtained from one or more (grouped) subjects other than the subject to be brought into focus. contact or treated with cells derived from CSM.
    In certain embodiments of the products or methods as presently described, the MSCs or cells derived from MSCs may be autologous for the subject to be treated. The term "autologous", in reference to MSCs or cells derived from MSCs, means that the MSCs or cells derived from MSCs are obtained from the same subject to be contacted or treated with cells derived from MSCs.
    In certain embodiments of the products or methods as presently described, the MSCs or cells derived from MSCs may comprise a mixture of MSCs or cells derived from autologous and allogenic (i.e., homologous) MSCs as defined above. Preferably, the CSM or cells derived from CSM are allogenic for the subject to be treated.
    The term “cells derived from mesenchymal stem cells” or “cells derived from MSC 18/5662 ”, in the present context, designates cells of mesenchymal line (for example of osteochondroblastic line (bone and cartilage), osteoblastic line (bone), chondroblastic (cartilage), myocytic (muscle), tenocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic (medullary stroma)) obtained by differentiation of MSC, in particular obtained by differentiation of MSC in vitro (including ex vivo).
    The differentiation of CSM can implement the culture of CSM under conditions which can induce the differentiation of CSM towards the type of cells desired, more typically the culture of CSM in a medium comprising one or more agents (for example, growth factors ) which can induce the differentiation of CSM into a desired cell type. Protocols for CSM differentiation are known as such (see, inter alia, WO 2007/093431; as well as REGER, RL et al. "Differentiation and Characterization of Human MSCs", in: Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology), edited by DJ Prockop et al. Humana Press, 2008, vol. 449, p. 93-107; VERMURI, MC et al. (Ed.). Mesenchymal Stem Cell Assays and Applications (Methods in Molecular Biology). Humana Press, 2011, vol. 698, in particular pages 201 to 352).
    The term "growth factor" in this context means a biologically active substance which influences the proliferation, growth, differentiation, survival and / or migration of different types of cells, and can cause developmental changes , morphological and functional in an organism, alone or when it is modulated by other substances. A growth factor can typically act by binding, as a ligand, to a receptor (eg, a surface or intracellular receptor) present in cells sensitive to growth factor. A growth factor can presently be, in particular, a protein entity comprising one or more polypeptide chains. By way of example and not limitation, the term “growth factor” covers members of the family of fibroblast growth factors (FGF), the family of bone morphogenetic proteins (BMP), the family of derived growth factors platelets (PDGF), the family of transforming beta growth factors (TGF-β), the family of nerve growth factors (NGF), the family of epidermal growth factors (EGF), the family of insulinomimetic growth factors ( IGF), the family of growth differentiating factors (GDF), the family of hepatocyte growth factors (HGF), hematopoietic growth factors (HeGF), platelet-derived endothelial cell growth factor (PD-ECGF) ), angiopoietin, the family of vascular endothelium growth factors (VEGF), glucocorticoids, and the like. It will be apparent to those skilled in the art that the growth factor or combination of growth factors can be a growth factor or any combination of growth factors known to be capable of inducing the differentiation of CSM into E 18 / 5662 desired cell type. It will be apparent to those skilled in the art that in vitro methods for inducing the differentiation of MSC into a desired cell type (for example, in cells of osteochondroblastic, osteoblastic or chondroblastic lineage) can lead to a substantially pure population (this is that is, primarily made up) of cells of the desired cell type. Without limitation, a cell population obtained in this way may contain at least 90% (by number) of the desired cell type, such as, for example,>91%,>92%,>93%,>94%,> 95% ,>96%,>97%,>98%,> 99%, or 100% of the desired cell type.
    In particular embodiments, the cells derived from MSCs are of osteochondroblastic line (bone and cartilage), osteoblastic line (bone), such as, for example, osteochondroprogenitors and / or osteoprogenitors and / or preosteoblasts and / or osteoblasts and / or osteocytes, etc. ; of chondroblastic line (cartilage), such as, for example, osteochondroprogenitors and / or chondroprogenitors and / or prechondroblasts and / or chondroblasts and / or chondrocytes; adipogenic line (fat); myogenic (muscle); tenogenic (tenocytes); fibroblastic line (connective tissue), such as, for example, fibroblasts, fibrocytes; or synovial line (synovial fluid).
    In particular embodiments, the cells derived from MSC are of osteochondroblastic line. The term "cells derived from MSC of osteochondroblastic line", in the present context, can designate progenitor cells which have the capacity to differentiate into cells of osteoblastic line, such as osteochondroprogenitors, osteoprogenitors and / or preosteoblasts and / or osteoblasts and / or osteocytes, etc., or in cells of a chondroblastic line, such as osteochondroprogenitors, chondroprogenitors and / or prechondroblasts and / or chondroblasts and / or chondrocytes. It will be apparent to a person skilled in the art that the progenitor cells will differentiate into cells of osteoblastic line (for example, preosteoblasts or osteoblasts), or into cells of chondroblastic line (for example, prechondroblasts or chondroblasts) they are exposed, such as physical factors and / or chemical and biological components, such as growth factors.
    In particular embodiments, the cells derived from CSM are cells derived from CSM of osteoblastic or chondroblastic line. In preferred embodiments, the cells derived from CSM are cells derived from CSM of osteoblastic line. In more preferred embodiments, the cells derived from MSCs are osteoprogenitors, preosteoblasts, osteoblasts or osteocytes.
    In certain particularly preferred embodiments, the expressions “cells derived from MSC of osteoblastic line” or “osteoforming cells derived from MSC” can both designate types of cells having an osteoblastic phenotype, and which can contribute to, or are capable of, develop into cells that can contribute to the formation of bone material or bone matrix, such as osteochondroprogenitors, osteoprogenitors, preosteoblasts, osteoblasts or osteocytes, or mixtures thereof. In the present context, "osteoprogenitors" can include, in particular, early and late osteoprogenitors. Even more preferably, "cells derived from MSC of osteoblastic line" or "osteoforming cells derived from MSC" may also designate osteochondroprogenitors, osteoprogenitors, preosteoblasts or osteoblasts, or mixtures thereof, even more preferably, the term may refer to osteochondroprogenitors or preosteoblasts or osteoblasts, or mixtures thereof, so that, in some examples, the term may refer to preosteoblasts, or in certain other examples, the term may refer to osteoblasts. All of these terms are known as such.
    By way of additional indication and without limitation, osteoprogenitors, preosteoblasts and osteoblasts, as well as populations of cells comprising osteoprogenitors, preosteoblasts and / or osteoblasts may have the following characteristics:
    a) the cells exhibit the expression of the transcription factor linked to Runt 2 (Runx2), a multifunctional transcription factor which regulates the differentiation of osteoblasts and the expression of a large number of extracellular matrix protein genes during differentiation osteoblasts;
    b) the cells have the expression of at least one of the following: alkaline phosphatase (ALP), more specifically bone-liver-kidney ALP; and, more preferably, further comprise the expression of one or more additional bone markers such as osteocalcin (OCN, BGLAP), the amino-terminal propeptide of procollagen type 1 (P1NP), osteonectin (ON, SPARC), osteopontin (OPST, SPP1, OPN) and / or bone sialoprotein (BSP), and / or one or more additional bone matrix proteins such as decorin and / or osteoprotegerin (OPG);
    c) cells do not substantially express CD45 (for example, less than about 10%, preferably less than about 5%, more preferably less than about 2% of cells can express CD45);
    d) the cells show signs of being able to mineralize the external environment, or to synthesize an extracellular matrix containing calcium (for example, when they are
    RP9Q18 / 5662 exposed to an osteogenic medium; see Jaiswal et al. J Cell Biochem, 1997, vol. 64, 295-3T2). The accumulation of calcium inside cells and its deposition in matrix proteins can be conventionally measured, for example, by culture in 45 Ca 2+ , washing and reculture, then determination of the radioactivity present inside the cell or deposited in the extracellular matrix (US 5,972,703), or by means of a mineralization assay based on alizarin red (see, for example, Gregory et al. Analytical Biochemistry, 2004, vol. 329, 77 -84);
    e) the cells do not substantially differentiate into cells of adipocyte line (for example, adipocytes) or of chondroblastic line (for example, chondroblasts, chondrocytes). The lack of differentiation in these cell lines can be assessed using differentiation induction conditions (eg, see Pittenger et al. Science, 1999, vol. 284, 143-7), and established standard assay methods in the art (for example, when induced, adipocytes are typically stained with Oil Red O, indicating an accumulation of lipids; chondrocytes are typically stained with alkian blue or orange saffron). The substantial absence of propensity for adipogenic and / or chondrogenic differentiation can typically mean that less than 20%, or less than 10%, or less than 5%, or less than 1% of the cells tested would show signs of adipogenic differentiation or chondrogenic when applied in the respective test.
    By way of example, but without limitation, cell surface markers suitable for assessing the cellular identity of cells derived from MSCs of osteochondroblastic or osteoblastic lineage can include CD105, CD90, CD73, CD45 and ALP, in particular bone type ALP. -Liver-kidney. These cell surface markers can, for example, be detected by commercial monoclonal antibodies, such as monoclonal antibodies labeled with a fluorochrome allowing the detection of cells by flow cytometry. In particular, CD105, CD90, and CD73 are mesenchymal markers, and are typically strongly expressed by cells derived from CSM of osteoblastic line; CD45 is a hematopoietic marker, and is typically substantially absent from cells derived from MSC of the osteoblastic line; ALP is a marker for preosteoblasts and osteoblasts, and is typically expressed by a substantial fraction of cells derived from MSCs of the osteochondroblastic or osteoblastic line.
    In some embodiments, cells derived from MSCs of the osteochondroblastic or osteoblastic line may exhibit osteoinductive properties.
    The terms “osteoinductive properties”, “osteoinductive potential” or “osteoinductive activity”, in the present context, designates the capacity of cells to attract other cells secreting bone matrix and / or to induce (trans) differentiation other cells into bone matrix secreting cells.
    For example, the cellular activity of cells derived from MSCs of osteochondroblasBqUê 18/5662 or osteoblastic line can be determined by measuring the osteoforming properties of these cells. The capacity of cells derived from MSC of osteochondroblastic or osteoblastic line to induce bone formation can be measured in vivo, for example by evaluation of the thickness of newly mineralized bone after administration of the cells to mice by subcutaneous injection. on the cranial vault. The ability of cells derived from MSCs of osteochondroblastic or osteoblastic lineage to induce bone formation can also be measured, for example, by evaluation of alkaline phosphatase (ALP) activity by ALP substrate staining.
    In some embodiments, cells derived from MSCs of the osteochondroblastic or osteoblastic line may exhibit osteogenic properties.
    For example, the cellular activity of cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be determined by measuring the osteogenic activity of these cells. The osteogenic activity of cells derived from human CSM of osteochondroblastic or osteoblastic line can be measured in vivo, for example, by determining the presence of at least one mineralized nodule of human origin after administration of the cells to mice by injection under -cutaneous on the cranial vault. The osteogenic activity of cells derived from human MSCs of osteochondroblastic or osteoblastic line can be measured in vivo, for example, by evaluation of the thickness of newly mineralized nodules of human origin after administration of the cells to mice by subcutaneous injection into the cranial vault.
    For example, cells derived from human MSCs of osteochondroblastic or osteoblastic line, such as 2.5 x 10 6 cells formulated in 100 µl of excipient, can be administered to nude mice by single subcutaneous administration on the bone of the cranial vault. To mark new bone formation over time, calcium binding fluorochromes such as alizarin red (red), calcein (green and blue) and tetracycline (yellow) can be administered sequentially to mice by injection. intraperitoneally 3 days before and 4, 8, and 12 days after administration of MSC-derived cells, respectively. Mice can be euthanized 2 weeks after cell administration and the cranial vault of each mouse can be collected to assess bone formation properties by histomorphometry (eg, quantification of bone formation). The initial and final thicknesses of the skull can be used to calculate the percentage of new bone formation after cell administration. In addition, bone formation properties can also be assessed by immunofluorescence (e.g., murine or human origin of bone formation). The osteoblastic activity can be evaluated on cranial vaults using the enzymatic activity detection method of ALP. The osteoclastic activity can be evaluated on cranial vaults using the methods for detecting enzymatic activity ^ Sê 18/5662 TRAP. The mineralization status of newly formed bone can be assessed using Masson-Goldner trichrome staining on cranial vault sections stained with ALP, for example, using commercially available kits (eg Bio-Optica®). Cartilage formation can be assessed using orange saffron staining on sagittal paraffin sections of the cranial vault.
    The term "osteogenic potential" in this context means the ability of cells to (trans) differentiate into cells that secrete bone matrix or the ability of cells to secrete bone matrix (i.e., without require a (trans) differentiation step), in vivo, and optionally in vitro. The term covers the ability of cells to form bone tissue by intramembrane ossification or endochondral ossification. The ability of cells to form bone tissue by intramembrane ossification typically represents the ability of cells to form bone tissue without requiring a cartilage matrix classified as a model. The capacity of cells to form bone tissue by endochondral ossification typically represents the capacity of cells to form bone tissue, firstly, by forming a matrix of calcified cartilage and then, using said matrix of calcified cartilage as that model for the formation of bone tissue. The term does not cover the osteoinductive potential of cells, which represents the ability of cells to attract other cells that secrete bone matrix and / or induce the (trans) differentiation of other cells into cells that secrete bone matrix. It will be apparent to those skilled in the art that cells derived from MSCs of the osteochondroblastic or osteoblastic line as presently described can have both osteogenic and osteoinductive potential.
    In some embodiments, cells derived from MSCs of the osteochondroblastic or osteoblastic line may have both osteoinductive and osteogenic properties. Advantageously, the cells derived from MSC of osteochondroblastic or osteoblastic line as presently described, after a transplant in a subject in need thereof, allow a bone neoformation which exceeds the bone neoformation obtained with a graft of CSM or cells derived from MSC obtained by prior art methods.
    By way of example, but without limitation, cell surface markers suitable for assessing the cellular identity of cells derived from MSCs of osteochondroblastic or osteoblastic line can include CD73, CD105, CD10, and CD44. These cell surface markers can, for example, be detected by commercial monoclonal antibodies, such as monoclonal antibodies labeled with a fluorochrome allowing the detection of cells by flow cytometry. In particular, CD73 and CD105 are mesenchymal markers; CD44 is an adhesion marker; and CD10 is an osteochondroblastic marker which are typically expressed by a high fraction of celBUes 18/5662 derived from MSC of osteochondroblastic or osteoblastic line. The amount of CD73 on the cell surface of cells derived from MSCs of osteochondroblastic or osteoblastic lineage is typically high; the amount of CD105 on the cell surface of cells derived from MSCs of osteochondroblastic or osteoblastic line is typically low; and the amount of CD44 on the cell surface of cells derived from MSCs of osteochondroblastic or osteoblastic lineage is typically high.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for CD73, CD63 and CD166; substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%, > 98%,> 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line are negative for CD45.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the methods of obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from MSC are positive for CD90, CD105, CD73, CD63 and CD166.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the methods of obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from MSC are positive for CD90, CD105, CD73, CD63 and CD166; substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%, > 98%,> 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line are negative for CD45, CD14 and CD19.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line are negative for CD45, CD14 and CD19.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,>91%,>92%,>93%,>94%,>95%,>96%,>97%,>98%,> 99%, or 100%) of the cells derived from MSC of line 18/5662 osteochondroblastic or osteoblastic are negative for CD45, CD34 and CD3.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line are negative for CD45, CD34, CD3, CD14 and CD19.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the methods of obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from MSC are positive for any one or more, for example one, two, three or all, of CD73, CD105, CD10 or CD44 (i.e., express any one or more, such as , two, three or all of CD73, CD105, CD10 or CD44 on the cell surface). Preferably substantially all (for example, at least 90% (by number), such as, for example,> 91%,> 92%,> 93%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the methods of obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from CSM are positive for CD73 , CD105, CD10 and CD44 (i.e., express CD73, CD105, CD10 and CD44 on the cell surface).
    In particular embodiments, the cells derived from CSM of osteochondroblastic or osteoblastic line obtained by the methods of obtaining cells derived from CSM of osteochondroblastic or osteoblastic line from CSM have any one or more among a normalized median of fluorescence intensity (nMFI) for CD73 (nMFI CD73 ) of at least 500, an nMFI for CD44 (nMFI CD44 ) of at least 100 or an nMFI for CD105 (nMFI C D 105 ) of at most 150. By example, cells derived from MSC of osteochondroblastic or osteoblastic line have any one or more of an nMFI C D 73 of at least 550, at least 600, at least 650, at least 700, at least 750, at least 800 , at least 850 or at least 900; an nMFI C D 44 of at least 110, at least 120, at least 130, at least 140, at least 150, at least 200, at least 250, at least 300 or at least 350; or CD105 nMFI at most 180, at most 170, at most 160, at most 150, at most 140, at most 130, at most 120, at most 110 or at most 100. Preferably, cells derived MSCs line ostéochondroblastique or osteoblast obtained by the methods for obtaining cells derived from MSCs ostéochondroblastique or osteoblastic lineage from CSM have nMFI CD73 of at least 500, a nMFI CD44 of at least 100, and CD105 of nMFI '' at most 150.
    In particular embodiments, substantially all (for example, at 18/ 5662 % (by number), such as, for example,>91%,>92%,>93%,>94%,> 95%, >96%,>97%,>98%,> 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the processes for obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from CSMs are positive for any one or more, for example one, two, three or all, of CD73, CD105, CD10 or CD44 (i.e., express any one or more, such as one, two, three or all of CD73, CD105, CD10 or CD44 on the cell surface), and cells derived from CSM of osteochondroblastic or osteoblastic line obtained by the methods of obtaining cells derived from CSM of osteochondroblastic line or osteoblastic from CSM have any one or more of an nMFI C D 73 of at least ins 500, a nMFI C D 44 of at least 100 or a nMFI CD105 at most 150. Preferably, substantially all (e.g., at least 90% (in number), such as, for example,> 91 %,>92%,>93%,>94%,>95%,>96%,>97%,>98%,> 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the methods for obtaining cells derived from CSM of osteochondroblastic or osteoblastic line from CSM are positive for CD73, CD105, CD10 and CD44 (i.e., express CD73, CD105, CD10 and CD44 on the cell surface ), and the cells derived from CSM of osteochondroblastic or osteoblastic line obtained by the methods for obtaining cells derived from CSM of osteochondroblastic or osteoblastic line from CSM have an nMFI C D 73 of at least 500, an nMFI C D 44 of at least 100, and an nMFI C D 105 of at most 150.
    The “normalized fluorescence intensity median” or “nMFI”, in the present context, denotes the ratio of the MFI of the total population of labeled cells analyzed with one or more antibodies conjugated to fluorochromes (MFI channel _ mark u r ) to the MFI of the population of cells labeled with one or more isotype control antibodies conjugated to fluorochromes (MFI channel j so t ype ), such as an immunoglobulin G (IgG) control conjugated with a fluorochrome such as fluorescein isothiocyanate (FITC), allophycocyanin (APC) or phycoerythrin (PE). The results of nMFI are proportional to the quantity of markers present on the cell surface of a population of interest. The (n) MFI is typically related to the wavelength at which the emission of the fluorescent signal is measured.
    The expressions “an nMFI for CD73” or “nMFI CD73 ”, in the present context, denote the ratio of the MFI of the total population of analyzed cells labeled with an antibody against CD73 conjugated to APC (for example, BD Biosciences®, ref No. 560847) to the MFI of the population of cells labeled with a control IgG conjugated to APC (for example, BD Biosciences®, ref. No. 555751). Preferably, the nMFI C D 73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC.
    The expressions “an nMFI for CD44” or “nMFI C D44”, in the present context, denote the ratio of the MFI of the total population of analyzed cells labeled with an antibody against CD44 conjugated to PE (for example, BD Biosciences®, no. 550989) to the MFI of the population of cells labeled with a control IgG conjugated to PE (for example, BD Biosciences®, ref. no 556650). Preferably, nMFI CD44 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE.
    The terms "a nMFI for CD105" or "nMFI CD105", as used herein, refer to the ratio of the MFI of the total population analyzed labeled cells with an antibody against CD105 conjugated to APC (e.g., BD Biosciences®, ref No. 562408) to the MFI of the population of cells labeled with a control IgG conjugated to APC (for example, BD Biosciences®, ref. No. 555751). Preferably, the nMFI CD105 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC.
    In certain embodiments, the cells derived from MSC of osteochondroblastic or osteoblastic line can comprise one or more of:
    - increased expression of a gene coding for an osteochondroblastic marker chosen from the group consisting of RUNX2, SOX9, ZNF521, ALPL, BMP2, OPG, POSTN, CHI3L1, MMP13, CADM1, CX43, CD10, and WISP1;
    - increased expression of a gene encoding a bone or cartilage matrix protein chosen from DCN or SPON1;
    - a reduced expression of the DKK1 gene coding for an osteochondrogenesis inhibitor; and or
    a reduced expression of a gene coding for a proliferation marker chosen from KI67 or PCNA, compared to the expression of the respective gene in MSCs.
    In certain embodiments, the expression of a gene coding for a marker linked to apoptosis chosen from BCL2 or BAX can be similar for cells derived from CSM of osteochondroblastic or osteoblastic line and of CSM.
    In certain embodiments, the cells derived from MSC of osteochondroblastic or osteoblastic line comprise one or more of:
    - increased expression of the PPARG gene coding for a protein involved in adipogenesis;
    - increased expression of a gene coding for an osteochondroblastic protein chosen from CD73 or BMP2; and or ,, .. ,, - , . .,. .,,, .. ..BE2018 / 5662 a reduced expression of a gene coding for an osteochondroblastic protein chosen from the group consisting of COL1A1, BGN, SPARC, ALPL and BCL2, compared to the expression of the respective gene in osteoforming cells generated by a method which is substantially identical for substantially all of the parameters to the method as presently described for obtaining cells derived from CSM from CSM, except for the presence vs. the absence of heparin or its analog or derivative.
    In certain embodiments, the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be increased (i.e., amplified) by at least about 1% by relative to (i.e., compared to) (i.e., the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be at least approximately 1.01 times) the expression of said gene in a corresponding control cell as presently defined. For example, the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be increased (i.e., amplified) by at least about 2% (this is i.e., 1.02 times), at least about 5% (i.e., 1.05 times), at least about 10% (i.e., 1.10 times) , at least about 15% (i.e., 1.15 times), at least about 20% (i.e., 1.20 times), at least about 25% (that's i.e., 1.25 times), at least about 30% (i.e., 1.30 times), at least about 35% (i.e., 1.35 times), at least about 40% (i.e., 1.40 times), at least about 45% (i.e., 1.45 times), at least about 50% (i.e. - say, 1.50 times), at least about 55% (i.e., 1.55 times), at least about 60% (i.e., 1.60 times), at least about 65% (i.e., 1.65 times), at least about 70% (i.e., 1.70 times), at least about 75% (i.e., 1.75 times), at least ins about 80% (i.e., 1.80 times), at least about 85% (i.e., 1.85 times), at least about 90% (i.e. say, 1.90 times), at least about 95% (i.e., 1.95 times), or at least about 100% compared to (i.e., compared to) (c (ie, 2 times) the expression of said gene in a corresponding control cell as presently defined.
    In some embodiments, the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1000 times, at least about 2000 times, at least about 3000 times or at least about 5000 times, the expression of a control gene in a corresponding control cell as presently defined.
    In certain embodiments, the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be decreased (i.e., reduced) by at least about 1% per relative to (i.e., compared to) (i.e., the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be at least about 0.99 times) the expression of said gene in a corresponding control cell as presently defined. For example, the expression of a gene encoding a protein in cells derived from MSCs of osteochondroblastic or osteoblastic lineage can be decreased (i.e., reduced) by at least about 2% (this is i.e., 0.98 times), at least about 5% (i.e., 0.95 times), at least about 10% (i.e., 0.90 times) , at least about 15% (i.e.,
    0.85 times), at least about 20% (i.e., 0.80 times), at least about 25% (i.e.,
    0.75 times), at least about 30% (i.e., 0.70 times), at least about 35% (i.e.,
    0.65 times), at least about 40% (i.e., 0.60 times), at least about 45% (i.e.,
    0.55 times), at least about 50% (i.e., 0.50 times), at least about 55% (i.e.,
    0.45 times), at least about 60% (i.e., 0.40 times), at least about 65% (i.e.,
    0.35 times), at least about 70% (that is, 0.30 times), at least about 75% (that is,
    0.25 times), at least about 80% (i.e., 0.20 times), at least about 85% (i.e.,
    0.15 times), at least about 90% (i.e., 0.10 times), at least about 95% (i.e.,
    0.05 times), or at least about 99% relative to (i.e., compared to) (i.e., 0.01 times) the expression of said gene in a control cell corresponding as currently defined.
    In certain embodiments, the expression of a gene coding for a protein in cells derived from MSC of osteochondroblastic or osteoblastic line can be at least approximately 0.005 times, at least approximately 0.001 times, at least approximately 0.0005 times, or at least about 0.0001 times, the expression of a control gene in a corresponding control cell as presently defined.
    In some embodiments, the control cells as presently described may be MSCs or may be osteoforming cells generated by a method which is substantially identical for substantially all parameters to the method as presently described for obtaining cells derived from MSCs from CSM, except presence vs. the absence of heparin or its analogue or derivative.
    In some embodiments, cells derived from MSCs of osteochondroblastic or osteoblastic lineage secrete higher amounts of proteins involved in osteochondrogenesis selected from CHI3L1 or MMP13, compared to MSCs or osteoforming cells generated by a process which is substantially identical for substantially all the parameters to the process as presently described for obtaining cells derived from CSM from CSM, except for the presence vs. the absence of heparin or its analogue or derivative. In some embodiments, cells derived from MSCs of the osteochondroblastic or osteoblastic line secrete lower amounts of protein
    DKK1 involved in the inhibition of osteogenesis compared to MSCs or osteoforming ceiTufes generated by a method which is substantially identical for substantially all the parameters to the method as presently described for obtaining cells derived from MSCs from MSCs , apart from the presence vs. the absence of heparin or its analogue or derivative.
    As previously described, the methods detailed above can produce cells derived from MSC of osteochondroblastic or osteoblastic line, or populations of such cells derived from MSC, having superior characteristics, such as, in particular, (i) high expression ALP, which represents the orientation of the cell towards the osteochondroblastic or osteoblastic line, and (ii) a low expression of HLA-DR, which represents the limited immunogenicity of cells derived from MSCs of osteochondroblastic or osteoblastic line, which indicates that the cells are more suitable for transplanting cells, for example to allogeneic subjects.
    Consequently, in particular embodiments, at least 70% (by number) of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for alkaline phosphatase (ALP); and less than 10% (by number) of cells derived from MSCs of osteochondroblastic or osteoblastic line are positive for HLA-DR.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example, s 91%, s 92%, s 93%, s 94%, s 95%, s 96%,> 97%, s 98%, s 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the processes for obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from MSC are positive for CD73, CD63 and CD166; substantially all (for example, at least 90% (by number), such as, for example, s 91%, s 92%, s 93%, s 94%, s 95%, s 96%, s 97%, s 98%, s 99%, or 100%) of the cells derived from MSC of osteochondroblastic or osteoblastic line are negative for CD45; at least 70% of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for alkaline phosphatase (ALP); and less than 10% of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for HLA-DR.
    In particular embodiments, substantially all (for example, at least 90% (by number), such as, for example, s 91%, s 92%, s 93%, s 94%, s 95%, s 96%, s 97%, s 98%, s 99%, or 100%) of cells derived from MSC of osteochondroblastic or osteoblastic line obtained by the processes for obtaining cells derived from MSC of osteochondroblastic or osteoblastic line from MSC are positive for CD90, CD105, CD73, CD63 and CD166; substantially all (for example, at least 90% (by number), such as, for example, s 91%, s 92%, s 93%, s 94%, s 95%, s 96%, s 97%, s 98%, s 99%, or 100%) of cells derived from osteochondroblastic or osteoblastic MSCs are negative for CD45, CD14 and CD19; at least
    BE2018 / 5662 70% of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for alkaline phosphatase (ALP); and less than 10% of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for HLA-DR.
    In certain particularly preferred embodiments, the expression "cells derived from MSC of chondroblastic line (cartilage)" can designate types of cells having a chondroblastic phenotype, and which can contribute to, or are capable of developing into cells which can contribute to, the formation of cartilage or cartilage matrix. In the present context, "chondroprogenitors" can include, in particular, early and late chondroprogenitors. Even more preferably, "cells derived from MSCs of the chondroblastic line (cartilage)" may denote osteochondroprogenitors, chondroprogenitors, prechondroblasts or chondroblasts, or mixtures thereof, even more preferably, the expression may denote prechondroblasts or chondroblasts, or mixtures thereof, in particular, in some examples, the expression may denote prechondroblasts or, in certain other examples, the expression may denote chondroblasts. All of these terms are known as such.
    By way of additional indication and without limitation, the cells of osteochondroblastic and / or chondroblastic lineage, such as osteochondroprogenitors, chondroprogenitors, prechondroblasts and chondroblasts, as well as populations of cells including osteochondroprogenitors, chondroprogenitors and prechondroblasts chondroblasts, can have the following characteristics:
    a) the cells exhibit the expression of SOX9, a transcription factor which plays a central role during the differentiation of the chondroblasts and the formation of cartilage;
    b) the cells exhibit the expression of at least one of the following: aggrecan (ACAN), type II collagen or CD90;
    c) the cells do not substantially express CD45 (for example, less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45);
    d) cells show signs of being able to produce high levels of types II, IX, and XI collagen and proteoglycans, the main constituents of the extracellular matrix (ECM) of hyaline in situ. Cartilage formation can be conventionally measured, for example, using a solid orange / green safranin assay to stain proteoglycans and non-collagen proteins, respectively (see, for example, Lee et al. Tissue Engineering, 2011, vol. 18, 484-98);
    e) human articular chondrocytes can exhibit cell expression characteristics as summarized in Diaz-Romero et al. 2005 (J Cell Physiol, vol. 202 (3), 731-
    42), for example, they can express integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51 / 61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99 ). During monolayer culture, chondrocytes can upregulate certain markers considered to be distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166). Therefore, such markers can also be expressed by less mature prechondroblasts or chondroblasts.
    f) the cells do not substantially differentiate into cells of adipocyte line (for example, adipocytes) or of osteoblastic line (for example, osteoblasts, chondrocytes). The lack of differentiation in these cell lines can be assessed using differentiation induction conditions (eg, see Pittenger et al. Science, 1999, vol. 284, 143-7), and established standard assay methods in the art (for example, when induced, adipocytes are typically stained with Oil Red O, indicating an accumulation of lipids; preostoblasts and osteoblasts are typically stained for ALP). The substantial absence of propensity for adipogenic and / or osteoblastic differentiation can typically mean that less than 20%, or less than 10%, or less than 5%, or less than 1% of the cells tested would show signs of adipogenic differentiation or osteoblastic when applied in the respective test.
    As is known in the art, cells of fibroblastic line can contribute to, or are capable of developing into cells which can contribute to, the formation of connective tissue.
    As an additional indication and without limitation, fibroblastic cells may have the following characteristics:
    a) the cells exhibit the expression of FSP1 (protein specific to fibroblasts 1);
    b) the cells have the expression of at least one of the following: collagen, vimentin, desmin or CD90;
    c) the cells do not substantially express CD45 (for example, less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45);
    d) the cells show signs of capacity to produce collagen, glycosaminoglycan, reticular and elastic fibers, glycoproteins to form the extracellular matrix of connective tissues. Fibroblasts contribute to the structural integrity of ligaments and tendons and have a tissue repair function. Collagen deposition can be visualized using trichrome staining (Li et al. World J Gastroenterol, 2014 vol. 20 (16), 4648-61). Collagen type I (Chondres, Redmond, WA) and tenascin C B (Tn- 18/5662 C; IBL-America, Minneapolis, MN) are two markers of ligament fibroblasts, and can be determined by ELISA (Brissett and al. Arthritis Rheum, 2012, vol. 64 (1), 272-80).
    As is known in the art, cells of the tendinocyte line can contribute to the formation of tendon material or tendon matrix. A tendon is made up of large bundles of fibers that include a network of collagen fibrils and different types of cells, including synovial cells, endothelial cells, tenoblasts, and tenocytes arranged longitudinally in rows in collagen molecules. Tenoblasts are an immature form of tendon cells that differentiate into tenocytes when they age with reduced metabolic activity.
    As additional information and without limitation, tenocytes may have the following characteristics:
    a) the cells comprise the expression of Scleraxis (SCX), a member of the basic helix-loop helix family of transcription factor involved in cell differentiation and organization of the extracellular matrix in tendons;
    b) the cells comprise the expression of at least one of the following: tenomodulin (TNMD) and tenascin C (Tn-C);
    c) cells express substantially CD44, CD73, CD90 and CD105, but do not express CD34, CD45, CD146, or stro-1;
    d) the cells show signs of being able to produce an extracellular component of tendon which consists of collagens of type I, III and V, proteoglycans, fibronectin, and elastic fibrils for the regeneration of tendon tissue (Güngörmüs et al. Connect Tissue Res , 2008, vol. 53 (6), 485-91);
    e) the cells do not substantially differentiate into cells of adipocyte line (for example, adipocytes), of chondroblastic line (for example, chondroblasts, chondrocytes) or of osteoblastic line (for example, osteoblasts, osteocytes).
    As is known in the art, cells of the synoviocyte line (synovial fluid) typically include type A or macrophage type synovial cells and type B or fibroblast (FLC) synoviocytes, which can contribute to the formation of the synovial membrane and synovial fluid. All of these terms are known as such. The term "synoviocyte", in the present context, thus designates any one, as well as, collectively, all of these types of cells.
    As an additional indication and without limitation, synoviocytes may have the following characteristics:
    a) the cells show signs of the ability to secrete proteoglycan 4 (PRG4) and 8Offl 18/5662 the main source of surfactant phospholipids (SAPL) as well as hyaluronan (HA) present in the synovial fluid (Tamer et al. Interdiscip Toxicol, 2013, vol. 6 (1), 111-125);
    b) synovial cells of type A or of macrophage type include the expression of markers of hematopoietic origin comprising CD11b, CD86, CD14, CD163, the DR antigen and the Fc receptor. Fibroblast or type B synoviocytes are mesenchymal cells that exhibit many fibroblast characteristics, including the expression of types IV and V collagens, vimentin and CD90. In addition, type B cells have unique properties in situ that distinguish them from many other fibroblast lines, including resident sub-border fibroblasts. For example, cadherin 11 (specific adhesion molecule playing a key role in the homotypic aggregation of FLS), CD55 (degradation acceleration factor), VCAM-1 (vascular adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) (Bartok et al. Immunol Rev, 2011, vol. 233 (1), 233-255);
    c) the cells do not substantially express CD45 (for example, less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45);
    d) the cells do not substantially differentiate into cells of adipocyte line (for example, adipocytes), of chondroblastic line (for example, chondroblasts, chondrocytes) or of osteoblastic line (for example, osteoblasts, osteocytes).
    When a cell is said to be positive (or expresses or understands the expression of) a particular marker, it means that those skilled in the art will conclude that there is a distinct signal, for example detection of antibodies or detection by polymerase chain reaction by reverse transcription, for this marker when carrying out the appropriate measurement, compared with suitable controls. When the method allows quantitative evaluation of the marker, positive cells can, on average, generate a signal which is significantly different from the control, for example, but without limitation, at least 1.5 times higher than such a generated signal. by control cells, for example, at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times higher or even higher.
    The expression of the above specific cell markers can be detected using any suitable immunological technique known in the art, such as immunohistochemistry or affinity adsorption, Western blot analysis, flow cytometry, ELISA, etc., or by any suitable biochemical assay of an enzymatic activity (for example, for ALP), or by any suitable technique of measuring the amount of marker mRNA, for example, transfer Northern, semi-quantitative or quantitative RT-PCR, etc. Sequence data for markers listed in the pre- 18-5662 description are known and can be obtained from public databases such as GenBank (http://www.ncbi.nlm.nih.gov/).
    In certain embodiments of the methods, as described herein, the MSCs or cells derived from MSCs can be animal cells, preferably warm-blooded animal cells, more preferably mammalian cells, such as human cells or non-human mammalian cells and, most preferably, human cells.
    The MSCs or cells derived from MSCs as presently described are preferably adherent, that is to say that they need a surface to grow and, typically, grow in the form of an adherent monolayer on said surface (i.e., a culture of adherent cells), rather than as cells floating freely in a culture medium (suspension culture). The adhesion of cells to a surface, such as the surface of a plastic tissue culture vessel, can be readily examined by visual inspection with an inverted microscope. Cells grown in adherent culture require periodic passage, in which cells can be removed from the surface enzymatically (e.g., using trypsin), suspended in culture medium and again spread in a / new culture container (s). In general, a surface or substrate which allows cell adhesion thereto can be any substantially hydrophilic substrate. As is known in the art, tissue culture vessels, for example culture flasks, well plates, dishes or the like, can generally consist of a wide variety of polymeric materials, surface treated suitably or coated after molding to provide hydrophilic substrate surfaces. The term "bringing into contact", in the present context, means bringing one or more molecules, components or materials into contact with one another, directly or indirectly, so as to promote interactions between them. Typically, one or more agents capable of inducing the expansion and / or differentiation of CSM or cells derived from CSM can be brought into contact with CSM or cells derived from CSM by means of their inclusion in the medium, in which the CSM or cells derived from CSM are cultured.
    The term "in vitro" in this context means outside, or outside, an animal or human body. The term "in vitro" in the present context should be understood to include "ex vivo". The term "ex vivo" typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, for example, in a culture vessel. The terms "fibroblast growth factor 2 (FGF-2)", "basic FGF", "FGF-b", "FGFB", "BFGF", "heparin-binding growth factor 2 (HBGF-2 ) ”Or“ prostatropin ”can be used interchangeably and refer to a known member of the fibroblast growth factor family. The inventors have observed that FGF-2 is particularly effective in the process of. BE2018 / 5662 present invention.
    The term "beta transforming growth factor (TGF-β)", "TGFB" or "TGFbeta", in the present context, designates a member of the family of beta transforming growth factor (TGF-β). The inventors have observed that TGF-β is particularly effective in the process of the present invention. In a further embodiment, said member of the TGF-β family is chosen from the group consisting of TGF-beta-1, TGF-beta-2, TGF-beta3, TGF-beta-4, GDF1 (differentiation factor of growth 1), GDF-2, GDF-3, GDF-5, GDF6, GDF-7, GDF-8, GDF-9, GDF-11, GDF-15, INHA (inhibin alpha chain), INHBA (chain inhibin beta A), INHBB (inhibin beta B chain), INHBC (inhibin beta C chain), INHBE (inhibin beta E chain), MIS (mueller inhibitor factor), and in addition members of the GDNF subfamily, comprising GDNF (neurotrophic factor derived from glial cell line), NRTN (neurturin), PSPN (persephine), and mixtures thereof.
    In a particular embodiment, TGF-β is chosen from the group consisting of TGF-β! TGF ^ 2, TGF ^ 3, and mixtures thereof. In a particular embodiment, TGF-β is TGF-βΤ For example, TGF ^ 1 can be used in the present methods as the only cytokine TGFB.
    In a further embodiment, the CSMs or cells derived from CSM can be, in addition to FGF-2 and TGF-β, contacted with one or more additional growth factors, exogenously added, other than FGF- 2 and TGF-β. In another embodiment, FGF-2 and TGF-β may be the only exogenous growth factors with which the MSCs or cells derived from MSCs are contacted.
    In a preferred embodiment, the growth factor used in the present method is a human growth factor. In the present context, the term “human growth factor” designates a growth factor substantially identical to a human growth factor of natural origin. For example, when the growth factor is a protein entity, the peptide constituent (s) or polypeptide (s) thereof may have a primary amino acid sequence identical to a human growth factor of natural origin. The use of human growth factors in the present method is preferable, since such growth factors are expected to induce a desirable effect on cell function.
    The term "naturally occurring" is used to describe an object or entity that is found in nature, as opposed to its artificial production by humans. For example, a polypeptide sequence present in an organism, which can be isolated from a natural source and which has not been intentionally modified in the laboratory by humans, is natural. When reference is made to a particular entity, for example a polypeptide or a protein, the term covers all of the forms and variants thereof which are present 18/5662 in nature, for example due to normal variation between individuals. For example, when referring to a protein growth factor, the term "naturally occurring" covers growth factors with differences in the primary sequence of their peptide constituent (s) or polypeptide (s) due to normal allelic variation between individuals.
    The present method can use a biologically active variant or fragment of a growth factor. In the method of the invention, the "biologically active" variants or fragments of a growth factor produce at least about the same degree of obtaining cells derived from CSM from CSM as the respective growth factor, when the other conditions are much the same.
    A "variant" of a polypeptide has an amino acid sequence that is substantially identical (that is, largely, but not completely, identical) to the amino acid sequence of the polypeptide. At present, the term “substantially identical” designates at least 85% identical, for example at least 90%, preferably at least 95%, and, for example, at least 99%. Sequence differences may result from the insertion (addition), deletion and / or substitution of one or more amino acids.
    In another embodiment, the growth factors used in the present method, namely at least FGF-2 and TGF-β, can be growth factors from non-human animals, and in particular mammalian growth factors non-human, or biologically active variants or derivatives thereof. In the present context, the terms "non-human animal growth factor" and "non-human mammalian growth factor" mean a growth factor practically identical to, respectively, a non-human animal or mammalian growth factor non-human of natural origin. For example, when the growth factor is a protein entity, the constituent (s) peptide (s) or polypeptide (s) thereof can have a primary amino acid sequence identical to a growth factor d non-human animal or non-human mammal of natural origin. It will be apparent to those skilled in the art that non-human animal or non-human mammalian growth factors may be applicable in the present process, but to a lesser degree than human growth factors, since these are of the same origin as CSM cells. In particular, non-human animal or non-human mammal growth factors can be used if they induce the desired effect, for example an effect similar to a human growth factor (analog).
    In a preferred embodiment, the growth factors or biologically active variants or derivatives thereof are recombinant, i.e. produced by a host organism by expression of a recombinant nucleic acid molecule , which was introduced into the host organism or one of its ancestors, and which comprises a sequence coding for the said
    BE2018 / 5662 polypeptide. The term "recombinant nucleic acid molecule" in the present context means a nucleic acid molecule (for example, a DNA or cDNA molecule) which is made up of segments assembled together using d technology Recombinant DNA.
    In particular embodiments, the MSCs or cells derived from MSCs are also brought into contact with, for example when the medium also comprises, one or more elements among the plasma, serum or a substitute thereof.
    The term "plasma" is as conventionally defined and includes fresh plasma, frozen thawed plasma, solvent / detergent treated plasma, processed plasma (eg PRP), or a mixture of two or more thereof . Plasma is usually obtained from a whole blood sample, to which is added or which is brought into contact with an anticoagulant (for example, heparin (at very low concentrations, typically about 15 x 10 -5 IU / ml, citrate, oxalate or EDTA). Next, the cellular components of the blood sample are separated from the liquid component (plasma) by an appropriate technique, typically by centrifugation. As a specific example, but without limitation, to obtain plasma suitable for use in the present invention, a blood sample can be drawn from a vacutainer tube containing the EDTA (ethylenediaminetetraacetic acid) anticoagulant (e.g. BD Vacutainer plastic EDTA tube, 10 ml, 1.8 mg / The sample is gently stirred and then centrifuged for 10 min at room temperature between 1000 and 2000 g to separate the plasma from the red blood cells. The supernatant (plasma) is collected, optional. grouped (if several blood samples are used), and aliquoted in Cryovials, which are stored at -80 ° C until used. The term "plasma" denotes a composition that is not part of a human or animal body. The term “plasma” may, in certain embodiments, specifically include transformed plasma, that is to say plasma subjected, after its separation from whole blood, to one or more processing steps which modify its composition, specifically its chemical, biochemical or cellular composition. Accordingly, the term "plasma" in the present context may include platelet-rich plasma (PRP), that is, plasma which has been enriched in platelets. Typically, a PRP can contain about 1.0x10 6 platelets / µl, while the platelet concentration in whole blood can be from about 1.5x10 5 to 3.5x10 5 / µl.
    The plasma can be treated with a solvent or detergent. The terms “plasma treated by solvent / detergent”, “plasma treated by S / D” or “plasma S / D” generally denote a decellularized plasma which can be obtained or obtained by a process comprising the steps of: (a) treatment of the plasma with solvent and detergent and (b) filtration of the solvent / detergent treated plasma. Solvents suitable for such treatment are solvents such as dialkyl or trialkyl phosphates and detergents which are described in the document U.S.
    No. 4 764 369. The detergent used to prepare the plasma S / D is preferably a detergent nontoxic 18/5662 (e.g., Tween® 20 or Tween® 80).
    The term "serum" is as conventionally defined and includes fresh serum, frozen thawed serum or serum prepared from plasma, or a mixture of any two or more of these. Serum can usually be obtained from a whole blood sample by first allowing coagulation to occur in the sample, then separating the clot thus formed and the cellular components of the blood sample from the liquid component. (serum) by an appropriate technique, usually by centrifugation. Coagulation can be facilitated by an inert catalyst, for example beads or glass powder. Alternatively, serum can be obtained from plasma by eliminating the anticoagulant and fibrin. By way of a specific example, but without limitation, to obtain a serum suitable for use in the present invention, a blood sample can be taken in a vacutainer tube not containing an anticoagulant (for example, a BD Vacutainer plastic tube Plus, 10 ml) and incubated for 30 to 45 min at room temperature to allow coagulation. The tube is then centrifuged for 15 min at room temperature between 1000 and 2000 g to separate the serum from the red blood cells. The supernatant (serum) is collected, optionally pooled (if multiple blood samples are used), and aliquoted in Cryovials, which are stored at -80 ° C until used. Therefore, the term "serum" refers to an acellular composition which is not part of a human or animal body. The serum as presently described is human serum, that is to say obtained from a single human subject or from a plurality of human subjects (for example, a pool of mixed serum). The serum can be untransformed serum, that is to say a serum derived by separation from whole blood and not subjected to downstream treatment steps modifying its chemical, biochemical or cellular composition, other than inactivation by optional heat, storage (cryogenic or non-cryogenic), sterilization, freeze-drying and / or filtration. In some embodiments, the serum can be obtained from solvent / detergent treated plasma.
    The isolated plasma, isolated serum or a substitute thereof can be used directly in the process of the present invention. They can also be stored appropriately for later use (for example, for shorter periods, for example up to about 1 to 2 weeks, at a temperature above the respective freezing points of plasma, serum or a substitute for these, but lower than room temperature, this temperature will generally be approximately 4 ° C to 5 ° C or longer, by storage by freezing, between approximately -70 ° C and approximately -80 ° C).
    Isolated plasma, serum, or a substitute for them can be heat inactivated as is known in the art, particularly to remove complement. When the present method uses autologous plasma, serum or a substitute thereof for cells grown in the presence thereof, it may not be necessary to heat inactivate plasma, serum or a substitute them. When the plasma, serum or a substitute thereof is at least partially allogenic for the cultured cells, it may be advantageous to heat inactivate the plasma, serum or a substitute thereof. Optionally, plasma, serum or a substitute thereof can also be sterilized before storage or use, using conventional microbiological filters, preferably having a pore size of 0.2 μm or less.
    In one embodiment, the present method may use human plasma, serum or a substitute thereof, which is autologous for MSCs or cells derived from human MSCs contacted therewith. The term "autologous", with reference to plasma, serum or a substitute thereof, means that plasma, serum or a substitute thereof is obtained from the same subject as MSCs or cells derived from CSM to be brought into contact with said plasma, serum or substitute thereof. The use of autologous plasma, serum or substitute therefor can ensure optimal acceptance of cells by the subject and / or avoid accidental transmission of infectious agents from, for example, other sera.
    In another embodiment, the method can use human plasma, human serum or a substitute thereof, which is "homologous" or "allogenic" for MSCs or cells derived from human MSCs contacted therewith. ci, i.e. obtained from one or more (grouped) human subjects other than the subject from which MSCs are obtained.
    In another embodiment, the method can use a mixture of autologous and allogeneic (i.e., homologous) plasma, sera or substitutes thereof as defined above. The expression “serum or plasma substitute”, in the present context, designates a non-toxic natural or artificial composition having one or more of the functions of plasma and / or serum, such as compositions which can induce growth and / or the expansion of CSM or cells derived from CSM. Nonlimiting examples of serum or plasma substitutes include a platelet lysate and compositions for cell culture comprising one or more fractionated components of plasma or serum, such as human serum albumin. It will be apparent to those skilled in the art that human plasma, human serum and substitutes thereof are complex biological compositions which may include one or more growth factors, cytokines or hormones.
    The growth factors FGF-2 and TGF-β or their respective biologically active variants or derivatives are expected to be provided in addition to, i.e., exogenously or in addition to, one or more elements from plasma, serum or a substitute thereof.
    The term "heparin" in the present context means a polymer from the glucosaminoglycan carbohydrate family having a molecular weight in the range of 3 to 30 kDa characterized by its anticoagulant effects. The activity of heparin or its derivatives or analogues can be determined in vitro by a biological assay in which the concentration of heparin necessary to prevent coagulation of sheep, goat or human plasma is compared to the concentration of an internationally recognized reference standard based on units of heparin activity per milligram. One mg of heparin is typically 140 to 180 international units (IU).
    The term "IU" or "international units" is a standard measure of the quantity of a biological substance expressed in biological activity or effect of said biological substance. For each substance to which this unit is assigned, an internationally accepted biological activity or effect is expected with a dose of 1 IU when this is assessed according to an internationally accepted biological procedure.
    In particular embodiments, heparin or heparin derivative or analog is selected from the group consisting of unfractionated heparin (HNF); low molecular weight heparin (LMWH), such as enoxaparin, dalteparin, nadroparin, tinzaparin, certoparin, reviparin, ardeparin, parnaparin, bemiparin, or mixtures thereof; a heparinoid, such as heparan sulfate, dermatan sulfate, chondroitin sulfate, acharan sulfate, keratan sulfate, or mixtures thereof, such as danaparoid; a heparin salt; a heparinoid salt; a heparin fragment; a heparinoid fragment; and mixtures thereof. Preferably, heparin or heparin derivative or analog is chosen from the group consisting of HNF, dalteparin, danaparoid and heparan sulfate.
    In particular embodiments, said FGF-2, said TGF-β, said heparin or a derivative or analog thereof, and optionally one or more of plasma, serum or a substitute thereof , are included in a medium, generally a liquid cell culture medium. Typically, the medium will include a base medium formulation known in the art. Many basic medium formulations (available, for example, in the American Type Culture Collection, ATCC; or from Invitrogen, Carlsbad, California) can be used to grow the cells of the invention, including, but not limited to to, the minimum essential medium of Eagle (MEM), the medium of Eagle modified by Dulbecco (DMEM), the minimum essential medium modified alpha (alpha-MEM), the basic essential medium (BME), BGJb, the mixture nutrients F-12 (Ham), Dulbecco's modified medium from Iscove (IMDM), or serum-free medium X-VIVO TM (clinical quality), available from Invitrogen or Cambrex (New Jersey), and modifications and / or combinations thereof. The compositions of the above basic media are generally known in the art and those skilled in the art will be able to modify or modulate the concentrations of medium and / or medium supplements, as required for the cultured cells. Such base medium formulations contain components necessary for the development of mammalian cells, which are known
    BE2018 / 5662 as such. By way of illustration and not by way of limitation, these components may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and optionally Cu, Fe, Se and Zn), physiological buffers (for example example, HEPES, bicarbonate), nucleotides, nucleosides and / or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g. glutathione) and carbon sources ( for example, glucose, sodium pyruvate, sodium acetate), etc.
    For use in culture, one or more other components can be added to the base medium. For example, additional supplements can be used to provide cells with the trace elements and substances necessary for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a saline solution such as, without limitation, Hanks Balanced Saline Solution (HBSS), Earle's saline solution. Additional antioxidant supplements can be added, for example, β-mercaptoethanol. Although many basic media already contain amino acids, some amino acids can be supplemented later, for example L-glutamine, which is known to be less stable in solution. A medium can also be supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, by way of example but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin , tetracycline, tylosin and zeocin. Lipids and lipid carriers can also be used to supplement cell culture media. These lipids and carriers can include, without limitation, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid oleic acid unconjugated and conjugated to albumin, among others. Albumin can be similarly used in formulations without fatty acids.
    In particular embodiments, one or more elements among human plasma, serum or a substitute thereof can be contained in said medium in a proportion (volume of one or more among human plasma, the serum or a substitute thereof / volume of medium) of between approximately 0.5% and approximately 30%, preferably between approximately 1% and approximately 15%, more preferably between 2% and 10%. The present methods can exhibit satisfactory performance with relatively small amounts of one or more of plasma, serum or a substitute thereof, for example about 5 or 10% by volume or less, for example about 1, approximately 2, approximately 3 or approximately 4% by volume, which makes it possible to decrease the volume of one or more among the plasma, the serum or a su
    18/5662 of these which must be obtained in order to cultivate the MSCs or cells derived from MSCs.
    In other additional embodiments, one or more of concentrated plasma products (e.g., plasma concentrates such as frozen plasma concentrates), concentrated serum products or plasma substitute products or concentrated serum can be used. Such concentrated products can be included in the composition at a concentration lower than the desired concentration of one or more of plasma, serum or a substitute thereof, so as to displace (counterbalance, compensate) the factor concentration.
    In particular embodiments, combinations or mixtures of any two or more of human plasma, human serum and / or a substitute thereof can be used.
    In particular embodiments, FGF-2 and TGF-β are included in said medium at concentrations sufficient to induce differentiation towards a desired cell type.
    In particular embodiments, FGF-2 and TGF-β are included in said medium at concentrations sufficient to induce differentiation of CSM into cells derived from CSM of osteochondroblastic or osteoblastic line. Typically, FGF-2 or a biologically active variant or fragment thereof can be included in the medium at a concentration of between 0.1 and 100 ng / ml, preferably between 0.5 and 20 ng / ml, for example about 19,
    18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 ng / ml, or about 5 ng / ml or less, for example about 4, 3, 2 , 1 or 0.5 ng / ml. Typically, TGF-β, such as TGF-β! or a biologically active variant or fragment thereof can be included in the medium at a concentration of between 0.1 and 100 ng / ml, preferably between 0.25 and 20 ng / ml, for example of approximately
    19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 ng / ml, or about 5 ng / ml or less, for example about 4, 3 , 2, 1 or 0.5 ng / ml. Said values are intended to designate concentrations of the respective growth factors or of biologically active variants or fragments thereof, as supplemented exogenously in the medium.
    In particular embodiments, heparin or a derivative or analog thereof is included in said medium at a concentration of at least 0.01 IU / ml, at least 0.02 IU / ml, at least 0 , 03 IU / ml, at least 0.04 IU / ml, 0.05 IU / ml, at least 0.06 IU / ml, at least 0.07 IU / ml, at least 0.08 IU / ml, at at least 0.09 IU / ml, at least 0.1 IU / ml, at least 0.5 IU / ml, at least 1 IU / ml, at least 5 IU / ml, at least 10 IU / ml, at least 20 IU / ml, at least 30 IU / ml, at least 40 IU / ml, at least 50 IU / ml, at least 60 IU / ml, at least 70 IU / ml, at least 80 IU / ml, at least 90 IU / ml or at least 100 IU / ml. In particular embodiments, heparin or a derivative or analog thereof is included in said medium at a concentration of at least 0.10 IU / ml. In certain preferred embodiments, heparin or a derivative or
    BE2018 / 5662 analog thereof is included in said medium at a concentration of about 0.1 IU / ml:
    In certain embodiments, heparin or a derivative or analog thereof can be included in said medium at a concentration of approximately 0.10 IU / ml, 0.20 IU / ml, 0.30 IU / ml , 0.40 IU / ml, 0.50 IU / ml, 0.60 IU / ml, 0.70 IU / ml, 0.80 IU / ml, 0.90 IU / ml or 1.0 IU / ml.
    In particular embodiments, the concentration of heparin or a derivative or the like thereof is at least 0.05 IU / ml, preferably about 0.1 IU / ml.
    In one embodiment, the above concentrations may denote the total concentration of growth factors or biologically active variants or fragments thereof or of said heparin or a derivative or analog thereof in the medium, it i.e. the sum of the concentrations of said growth factors or biologically active variants or fragments thereof or of said heparin or derivative or analog thereof supplied by plasma, serum or a substitute thereof and as provided in addition to these.
    In another embodiment, the above concentrations may denote the concentration of said growth factors or biologically active variants or fragments thereof or of said heparin or derivative or analog thereof, as provided in addition to those already provided by plasma or serum. Of course, if the growth factors or heparin or a derivative or analog thereof to be added are not normally present (not detectable) in plasma, serum or a substitute thereof, the total concentration and added growth factors or heparin or a derivative or the like thereof will be (substantially) identical.
    In particular embodiments, the method for obtaining cells derived from CSM from CSM, as presently described, comprises the steps of:
    (a) culture of CSM recovered from a biological sample of a subject in a medium comprising FGF-2, TGF-β and heparin or a derivative or analog thereof at a concentration of at least 0.01 IU / ml;
    (b) elimination of the non-adherent material and continuation of the culture of the adherent cells in the medium comprising FGF-2, TGF-β and heparin or a derivative or analog thereof thereof at a concentration of at least 0 , 01 IU / ml, so as to obtain the cells derived from MSC. In a preferred embodiment, the MSCs recovered from a biological sample of a subject as defined elsewhere herein are grown in a culture vessel. The culture vessel can provide a plastic surface to allow cell adhesion. In another embodiment, the surface may be a glass surface. In another additional embodiment, the surface can be coated with a suitable material allowing adhesion and growth of cells, for example, Matrigel®, laminin or collagen.
    BE2018 / 5662 In particular embodiments, MSCs can be recovered from bone marrow (or other sources) by selecting cells (mononuclear) which can adhere to a substrate surface, for example a plastic surface.
    In particular embodiments, the cells are allowed to settle for about 1 to 8 days, more typically between about 2 and 6 days, more typically about 4 days before removing the non-adherent material in step (b). Otherwise, step (b) is carried out at most 8 days, at most 6 days, at most 4 days, preferably at most 4 days, after the start of step (a).
    In particular embodiments the cells can be cultured in steps (a) and (b) considered together for a period of between about 7 and about 35 days, generally between about 10 and about 28 days, and more preferably for about 12 at 21 days. Alternatively, cells can be grown in steps (a) and (b) taken together until their confluence reaches about 60% or more, or about 80% or more, or about 90% or more, or even up to '100 %.
    In one embodiment, after step (b), the method can comprise collecting the cells or the population of cells obtained in this way.
    In one embodiment, after step (b), the method can comprise detaching, subculturing and culturing cells derived from MSC in the medium comprising FGF-2, TGF-β and heparin or a derivative or the like, preferably at a concentration of at least 0.01 IU / ml.
    In one embodiment, after step (b), the method may include detaching, subculturing and culturing cells derived from MSC in an osteogenic or chondrogenic differentiation medium.
    Osteogenic and chondrogenic differentiation media are known in the art. Without limitation, osteogenic differentiation media can include basic media supplemented with ascorbic acid, ^ -glycerophosphate and dexamethasone. Without limitation, chondrogenic differentiation media can include basic media supplemented with insulin, transferrin, sodium selenite, ascorbic acid, TGF-β-! sodium pyruvate and dexamethasone.
    Detachment, subculturing and culturing of cells derived from MSC after step (b) can be carried out once or several times, for example once, twice, three times, four times, five times, six times, seven times , eight times, nine times or ten times. It will be apparent to those skilled in the art that this can generate cell cultures of passage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4 (P4), passage 5 (P5), passage 6 ( P6), passage 7 (P7), passage 8 (P8), passage 9 (P9) or passage 10 (P10), respectively. Passage 0 (P0) can denote MSCs or cells derived from MSCs that have not been detached and / or subcultured.
    BE2018 / 5662 Differentiation of CSM, for example, in particular, osteogenic differentiation of CSM, typically leads to cells derived from CSM which have a larger cell size than the CSM from which they are derived. The inventors have discovered that this increase in cell size does not occur or is reduced or minimized when cells derived from CSM are obtained from CSM by contacting CSM or cells derived from CSM in vitro or ex vivo with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml. Such smaller CSM-derived cells advantageously exhibit improved transplant properties, as described herein elsewhere.
    Accordingly, another aspect relates to a method of obtaining cells derived from CSM having improved transplant properties from CSM, the method comprising a size reduction step, wherein said size reduction step is characterized by contacting CSM or cells derived from CSM in vitro or ex vivo with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    The term "size reduction" in this context means (i) reduced physical dimensions or size of cells derived from MSCs (for example, as measured by average size, diameter or volume, or a variable indication of suitable cell size, such as D 60 , D 65 , D 70 , or D 75 ) obtained by a method comprising the step of reduction in size compared to cells derived from CSM obtained by an identical method by elsewhere not including the size reduction step. The reduction in size may be a decrease in average cell size of at least 30%, at least 25%, at least 20%, preferably at least 30%, of cells derived from MSCs obtained with the reduction step. size relative to the average cell size of MSC-derived cells obtained without the size reduction step.
    In particular embodiments, the method for obtaining cells derived from CSM having improved transplant properties from CSM as presently described comprises a step of culturing CSM in vitro or ex vivo in an appropriate supplemented culture medium. to achieve a high proliferation rate with early and late stage differentiation characteristics, said step being carried out simultaneously with or before the size reduction step.
    In particular embodiments, the method may include contacting CSM with one or more agents capable of inducing the expansion and / or differentiation of CSM simultaneously with or before contacting the cells with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    The term “agent” generally designates any chemical substance (for example, inorganic or organic), a substance, molecule or macromolecule (for example, a material or tissue and / or the
    BE2018 / 5662 biological macromolecule) biochemical or biological, a combination or a mixture thereof, a sample of indeterminate composition, or an extract consisting of biologicals such as bacteria, plants, fungi or animal cells. Nonlimiting examples of agents capable of inducing the differentiation expansion of CSM are growth factors, such as FGF-2 and TGF-β, and plasma or serum or a substitute for these. It will be apparent to those skilled in the art that the growth factor or combination of growth factors can be a growth factor or any combination of growth factors known to be capable of inducing the differentiation of MSC into a cell type wish.
    In particular embodiments, the method for obtaining cells derived from CSM from CSM having improved transplant properties as described herein further comprises a step of bringing CSM or cells derived from CSM into contact in vitro or ex vivo with FGF-2 and TGF-β. Contacting said MSCs or cells derived from MSCs in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or analog thereof at a concentration of at least 0.01 IU / ml is , preferably, performed simultaneously.
    In particular embodiments, the MSCs or cells derived from MSCs are also brought into contact with, for example when the medium also comprises, one or more elements among the plasma, serum or a substitute thereof.
    As described above, the methods detailed above produce cells derived from MSCs, or populations comprising them, having superior characteristics such as, in particular, a smaller and more homogeneous size than the cells derived from MSCs described above . The smaller and more homogeneous size of the MSC-derived cells obtainable by the methods as described herein makes the cells with improved transplant properties. More particularly, the smaller and more homogeneous size of the cells derived from MSC obtainable by the methods as presently described makes the cells suitable for all routes of administration and, in particular, intravascular administration, inter alia, by reduction or elimination of the risk of embolism and pulmonary infarction, by offering a satisfactory in vivo safety profile and / or syringability. In addition, cells derived from MSC obtainable by the methods as presently described allow an adjustable and high cell concentration to be administered at a site with a limited administered volume.
    Consequently, the process of the invention can be defined further by the size, characterized by the diameter and / or the volume of a cell, of the cells derived from CSM resulting from the contacting of CSM with FGF-2, TGF-β and heparin or a derivative or the like thereof.
    In particular embodiments, the average diameter of cells derived from CSM in suspension is less than 30 μm, less than 29 μm, less than 28 μm, less than 27 μm, less than 26 μm, less than 25 μm, or less at 24 μm. Preferably, the average diameter of the cells derived from CSM in suspension is less than 24 μm.
    The terms “suspension” and “cell suspension” generally designate cells derived from MSCs, in particular cells derived from viable MSCs, dispersed in a liquid phase.
    In particular embodiments, the average diameter of the cells derived from CSM in suspension is greater than 10 μm, greater than 11 μm, greater than 12 μm, greater than 13 μm, greater than 14 μm, greater than 15 μm, greater than 16 μm, greater than 17 μm or greater than 18 μm.
    In particular embodiments, the average diameter of the cells derived from CSM in suspension is between 16 μm and 26 μm, preferably between 20 μm and 25 μm.
    In particular embodiments, at least 60% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm), equal to or less than 24 μm (D 60 <24 μm), equal or less than 23 μm (D 60 <23 μm), equal to or less than 22 μm (D 60 <22 μm), equal to or less than 21 μm (D 60 <21 μm), or equal or less than 20 μm (D 60 <20 μm), preferably equal to or less than 25 μm (D 60 <25 μm).
    In particular embodiments, at least 65% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 65 <25 μm), equal to or less than 24 μm (D 65 <24 μm), equal or less than 23 μm (D 65 <23 μm), equal to or less than 22 μm (D 65 <22 μm), equal to or less than 21 μm (D 65 <21 μm), or equal or less than 20 μm (D 65 <20 μm), preferably equal to or less than 25 μm (D 65 <25 μm).
    In particular embodiments, at least 70% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 70 <25 μm), equal to or less than 24 μm (D 70 <24 μm), equal or less than 23 μm (D 70 <23 μm), equal to or less than 22 μm (D 70 <22 μm), equal or less than 21 μm (D 70 <21 μm), or equal or less than 20 μm (D 70 <20 μm), preferably equal to or less than 25 μm (D 70 <25 μm).
    In particular embodiments, at least 75% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 75 <25 μm), equal to or less than 24 μm (D 75 <24 μm), equal or less than 23 μm (D 75 <23 μm), equal to or less than 22 μm (D 75 <22 μm), equal to or less than 21 μm (D 75 <21 μm), or equal or less than 20 μm (D 75 <20 μm), preferably equal to or less than 25 μm (D 75 <25 μm).
    In particular embodiments, the cells derived from CSM in suspension have a D 60 equal to or less than 25 μm (D 60 <25 μm) and at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm . In particular embodiments, the
    BE2018 / 5662 cells derived from CSM in suspension have a D 65 equal to or less than 25 μm (D 65 <25 μm) and at most 5% of cells derived from CSM in suspension have a diameter greater than 35 μm.
    In particular embodiments, the cells derived from CSM in suspension have a D 70 equal to or less than 25 μm (D 70 <25 μm) and at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm .
    In particular embodiments, cells derived from suspended MSCs have a
    D 75 equal to or less than 25 μm (D 75 <25 μm) and at most 5% of the cells derived from MSC in suspension have a diameter greater than 35 μm.
    In particular embodiments, cells derived from suspended MSCs have a
    D 60 between approximately 25 μm and approximately 10 μm (10 μm <D 60 <25 μm), between approximately 24 μm and approximately 10 μm (10 μm <D 60 <24 μm), between approximately 23 μm and approximately 10 μm ( 10 μm <D 60 <23 μm), between approximately 22 μm and approximately 10 μm (10 μm <D 60 <22 μm), between approximately 21 μm and approximately 10 μm (10 μm <D 60 <21 μm) or between approximately 20 μm and approximately 10 μm (10 μm <D 60 <20 μm), preferably between approximately 25 μm and approximately 10 μm (10 μm <D 60 <25 μm).
    In particular embodiments, cells derived from suspended MSCs have a
    D 65 between approximately 25 μm and approximately 10 μm (10 μm <D 65 <25 μm), between approximately 24 μm and approximately 10 μm (10 μm <D 65 <24 μm), between approximately 23 μm and approximately 10 μm ( 10 μm <D 65 <23 μm), between approximately 22 μm and approximately 10 μm (10 μm <D 65 <22 μm), between approximately 21 μm and approximately 10 μm (10 μm <D 65 <21 μm) or between approximately 20 μm and approximately 10 μm (10 μm <D 65 <20 μm), preferably between approximately 25 μm and approximately 10 μm (10 μm <D 65 <25 μm).
    In particular embodiments, cells derived from suspended MSCs have a
    D 70 between approximately 25 μm and approximately 10 μm (10 μm <D 70 <25 μm), between approximately 24 μm and approximately 10 μm (10 μm <D 70 <24 μm), between approximately 23 μm and approximately 10 μm ( 10 μm <D 70 <23 μm), between approximately 22 μm and approximately 10 μm (10 μm <D 70 <22 μm), between approximately 21 μm and approximately 10 μm (10 μm <D 70 <21 μm) or between approximately 20 μm and approximately 10 μm (10 μm <D 70 <20 μm), preferably between approximately 25 μm and approximately 10 μm (10 μm <D 70 <25 μm).
    In particular embodiments, cells derived from suspended MSCs have a
    D 75 between approximately 25 μm and approximately 10 μm (10 μm <D 75 <25 μm), between approximately 24 μm and approximately 10 μm (10 μm <D 75 <24 μm), between approximately 23 μm and approximately 10 μm ( 10 μm <D 75 <23 μm), between approximately 22 μm and approximately 10 μm (10 μm <D 75 <22 μm), between approximately 21 μm and approximately 10 μm (10 μm <D 75 <21 μm) or between approximately 20 μm and approximately 10 μm (10 μm <D 75 <20 μm), preferably between approximately 25 μm and approximately 10 μm (10 μm <D 75 <25 μm).
    In particular embodiments, at least 90% of the cells derived from CSM in suspension have a diameter equal to or less than 30 μm (D 90 <30 μm), equal to or less than 29 μm (D 90 <29 μm), equal or less than 28 μm (D 90 <28 μm), equal to or less than 27 μm (D 90 <27 μm), equal or less than 26 μm (D 90 <26 μm), or equal or less than 25 μm (D 90 <25 μm), preferably equal to or less than 30 μm (D 90 <30 μm).
    In particular embodiments, the cells derived from CSM in suspension have a D 90 equal to or less than 30 μm (D 90 <30 μm) and at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm .
    In particular embodiments, the cells derived from CSM in suspension have a D 90 of between approximately 30 μm and approximately 10 μm (10 μm <D 90 <30 μm).
    In particular embodiments, the diameter of each cell derived from CSM in suspension is greater than 10 μm, greater than 11 μm, greater than 12 μm, greater than 13 μm, greater than 14 μm or greater than 15 μm, preferably greater than 10 μm.
    The diameter of a cell can be determined by any method known in the art, for example using a digital microscope and associated software for image analysis (for example, Motic Image Plus 2.02) . The mean cell diameter presently mentioned should be determined based on the diameter of the cells in a free, unfixed state, i.e., cells in suspension. The cells are preferably suspended in a solution comprising a transparent, non-toxic, isotonic buffer, such as PBS, and optionally a dye to differentiate living and dead cells, such as trypan blue. Preferably, at least one hundred cells should be measured to consider the analysis statistically significant.
    In particular embodiments, the diameter of each cell derived from CSM in suspension is less than 38 μm, less than 37 μm, less than 36 μm, less than 35 μm, preferably less than 35 μm.
    In particular embodiments, the standard deviation of the average diameter of the cells derived from CSM in suspension is less than 7.0 μm, less than 6.5 μm, less than 6.0 μm, less than 5.5 μm, less than 5 μm, less than 4.5 μm, less than 4 μm or less than 3.5 μm. Preferably, the standard deviation of the average diameter of cells derived from CSM in suspension is less than 4.0 μm, for example between 3.0 and 3.5 μm.
    The inventors have discovered that the cell size distribution of cells derived from MSCs obtained by the methods as presently described is stable. Consequently, the diameter and / or volume of the MSC-derived cells obtained by the methods as presently described can be determined at any time and at any confluence during an in vitro culture. In preferred embodiments, the diameter and / or volume of cells derived from MSC is determined when the cells reach a confluence of between 30% and 80%, preferably between 40% and 70%, such as 40%, 45 %, 50%, 55%, 60%, 65% or 70%.
    Another aspect relates to a method for obtaining cells derived from CSM from C SM 18/5662 comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative. or the like, in which the average diameter of the cells derived from CSM in suspension is less than 25 μm, for example less than 24 μm or, for example, between 20 μm and 25 μm.
    Another aspect relates to a method for obtaining cells derived from CSM from CSM comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or analog thereof. here, in which at least 60% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm), preferably at least 70% of the cells derived from CSM in suspension have an equal diameter or less than 25 μm (D 70 <25 μm), and at most 5% of the cell population have a diameter greater than 35 μm.
    It will be apparent to those skilled in the art that all the embodiments described above, including the embodiments relating to the average diameter of cells derived from CSM, maximum individual diameter of cells derived from CSM, average volume of cells derived from CSM, D 60 , D 65 , D 70 , D 75 , D 90 , concentration of heparin or derivative or analog thereof, cell line derived from CSM and agents capable of inducing the expansion and / or differentiation of CSM, apply to all the methods for obtaining cells derived from CSM as presently described.
    Another aspect relates to a population of cells derived from MSCs obtainable by expansion of MSCs in vitro or ex vivo, in which the average diameter of cells derived from MSCs in suspension is less than 30 μm, less than 29 μm, less than 28 μm, less than 27 μm, less than 26 μm, less than 25 μm or less than 24 μm. Preferably, the average diameter of the cells derived from CSM in suspension is less than 24 μm.
    Another aspect concerns a population of cells derived from MSCs obtainable by in vitro or ex vivo expansion of MSCs, in which at least 60% of the cells derived from MSCs in suspension have a diameter equal to or less than 25 μm (D 60 < 25 μm), preferably at least 70% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 70 <25 μm), and at most 5% of the cell population have a diameter greater than 35 .mu.m. The term "population", in the present context, denotes a substantially pure (that is to say, mainly composed of) and homogeneous group of cells of a type of cell derived from the desired MSC.
    In particular embodiments, the diameter of each cell derived from CSM in suspension is less than 38 μm, less than 37 μm, less than 36 μm, preferably less than 35 μm.
    In particular embodiments, the standard deviation of the average diameter of the cells derived from CSM in suspension is less than 6.0 μm, less than 5.5 μm, less than 5.0 μm, less than 4.5 μm, less than 4.0 μm or less than 3.5 μm. Preferably, the typ§ E difference dfl 18/5662 mean diameter of the cells derived from CSM in suspension is less than 4.0 μm, for example between 3.0 and 3.5 μm.
    In particular embodiments, the population of cells derived from CSM can be obtained by the methods of obtaining cells derived from CSM from CSM as presently described.
    It will be apparent to those skilled in the art that all the embodiments described above, including the embodiments relating to the average diameter of cells derived from CSM, maximum individual diameter of cells derived from CSM, average volume of cells derived from CSM, D 60 , D 65 , D 70 , D 75 , D 90 , concentration of heparin or derivative or analog thereof, cell line derived from CSM and agents capable of inducing the expansion and / or differentiation of CSM, apply to all methods of obtaining cells derived from CSM from CSM as described herein and, therefore, also to cells derived from CSM and to a population of cells derived from CSM obtainable by said methods such than currently described.
    Accordingly, another aspect relates to a composition comprising cells derived from CSM or the population of cells derived from CSM as presently defined. The invention further relates to compositions comprising cells derived from MSC or the population of cells derived from MSC presently described and further comprising one or more other components. For example, components can be included which can maintain or increase the viability of cells. By way of example and without limitation, such components can comprise salts to ensure substantially isotonic conditions, pH stabilizers such as one or more buffer system (s) (for example, to ensure a substantially pH neutral (such as a phosphate or carbonate buffer system), carrier proteins such as, for example, albumin, media comprising base media and / or media supplements, serum or plasma, nutrients , carbohydrate sources, preservatives, stabilizers, antioxidants or other materials known to those of skill in the art. The invention further relates to methods of producing said compositions by mixing cells derived from MSC or population of cells derived from respective MSC with said one or more additional components as described above. The compositions can be, for example, liquid or can be semi-solid or solid (for example, can be frozen compositions or can exist in gel form or can exist on a solid support or scaffold, etc.). Cryopreservatives such as, inter alia, dimethyl sulfoxide (DMSO) are known in the art.
    The terms "composition", "formulation", or "preparation" can be used interchangeably at present.
    In particular embodiments, the composition is a pharmaceutical composition 18/5662 comprising cells derived from MSC or the population of cells derived from MSC as presently defined, and optionally one or more pharmaceutically acceptable excipients.
    The term “pharmaceutically acceptable”, in the present context, is in accordance with the related art and means: compatible with the other components of a pharmaceutical composition and not deleterious for the recipient thereof.
    In the present context, “vehicle” or “excipient” includes any and all of the solvents, diluents, buffers (for example, neutral saline buffer or phosphate buffered saline), solubilizers, colloids, dispersion media, vehicles, fillers, chelating agents (for example, EDTA or glutathione), amino acids (for example, glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colors, flavors, flavorings, thickeners, agents for obtaining an effect delay, coatings, antifungal agents, preservatives, stabilizers, antioxidants, tone control agents, agents delaying absorption, and the like. The use of such media and agents for active pharmaceutical substances is known in the art. Such materials must be non-toxic and must not interfere with cell activity.
    The precise nature of the vehicle, excipient or other material will depend on the route of administration. For example, the composition may be in the form of an aqueous solution acceptable parenterally, which is pyrogen-free and has a suitable pH, isotonicity and stability. For general principles in medicinal formulation, the reader can consult Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn and W. Sheridan ed., Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister and P. Law, Churchill Livingstone, 2000.
    Liquid pharmaceutical compositions can generally comprise a liquid vehicle such as water or a pharmaceutically acceptable aqueous solution. For example, physiological saline, tissue or cell culture medium, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
    The composition may include one or more cell protection molecules, cell regeneration molecules, growth factors, antiapoptotic factors, or factors that regulate gene expression in cells. Such substances can make cells independent of their environment.
    Such pharmaceutical compositions may further contain components ensuring the viability of the cells contained therein. For example, the compositions may comprise a suitable buffer system (for example, a phosphate or carbonate buffer system) to obtain a desirable pH, most often a
    L,. . . . ... BE2Q18 / 5662 pH close to neutral, and can include enough salt to ensure iso-osmotic conditions for cells to avoid osmotic stress. For example, a suitable solution for this purpose may be a phosphate buffered saline (PBS), a solution of sodium chloride, a solution for injection of Ringer or a solution for injection of Ringer-lactate, as is known in the art . In addition, the composition can include a carrier protein, e.g. albumin (e.g., bovine or human albumin), which can increase cell viability.
    Other suitable pharmaceutically acceptable vehicles or additives are known to those skilled in the art and, for example, can be chosen from proteins such as collagen or gelatin, carbohydrates such as starch, polysaccharides, sugars ( dextrose, glucose and sucrose), cellulose derivatives such as sodium or calcium carboxymethylcellulose, hydroxypropylcellulose or hydroxypropylmethylcellulose, pregelatinized starches, agar pectin, carrageenan, clays, hydrophilic gums (acacia gum, guar gum, gum arabic and xanthan gum), alginic acid, alginates, hyaluronic acid, polyglycolic and polylactic acid, dextran, pectins, synthetic polymers such as a water-soluble acrylic polymer or polyvinylpyrrolidone , proteoglycans, calcium phosphate and the like.
    If desired, a cell preparation can be administered on a support, scaffold, matrix or material to enhance tissue regeneration. For example, the material can be a granular ceramic or a biopolymer such as gelatin, collagen or fibrinogen. Porous matrices can be synthesized according to standard techniques (e.g. Mikos et al., Biomaterials 14: 323, 1993; Mikos et al., Polymer 35: 1068, 1994; Cook et al., J. Biomed. Mater. Res. 35: 513, 1997). Such a support, scaffolding, matrix or material can be biodegradable or non-biodegradable. Therefore, cells can be transferred and / or cultured on a suitable substrate, such as a porous or non-porous substrate, to produce implants. For example, cells which have proliferated or which are differentiated in culture dishes can be transferred onto three-dimensional solid supports in order to cause them to multiply and / or continue the differentiation process by incubation of the solid support in a liquid nutritive medium. of the invention, if necessary. The cells can be transferred to a three-dimensional solid support, for example, by impregnating said support with a liquid suspension containing said cells. The impregnated supports thus obtained can be implanted in a subject. Such impregnated supports can also be re-cultivated by immersing them in a liquid culture medium, before being finally implanted. The solid three-dimensional support is not necessarily biocompatible to be able to be implanted in a human. It can be biodegradable or non-biodegradable.
    The cells or cell populations can be administered in a way that their 18/5662 peBme to survive, grow, spread and / or differentiate into desired cell types such as, for example, hepatocytes. The cells or cell populations can be grafted or can migrate to and be grafted to the desired organ such as, for example, the liver. Grafting of cells or cell populations to other locations, tissues or organs such as the liver, spleen, pancreas, renal capsule, peritoneum or omentum may be considered.
    In one embodiment, the preparation of pharmaceutical cells as defined above can be administered in the form of a liquid composition. In embodiments, the cells or the pharmaceutical composition comprising these can be administered systemically, topically, into an organ, at a site of organ dysfunction or injury, or at a site of tissue damage.
    Preferably, the pharmaceutical compositions can comprise a therapeutically effective amount of the desired cells. The term "therapeutically effective amount" means an amount which can induce a biological or therapeutic response in a tissue, system, animal or human being, which is sought by a researcher, a veterinarian, a doctor or another clinician and, in particular, may relieve one or more of the local or systemic symptoms or characteristics of a disease or condition to be treated. Therapeutically effective amounts may be determined by a qualified physician, taking due account of the nature of the cells desired, the condition and severity of the disease and the age, size and condition of the subject.
    The invention further relates to methods of producing said pharmaceutical compositions by mixing the cells of the invention with one or more additional components as described above, as well as with one or more pharmaceutical excipients as described above.
    The invention further relates to an arrangement or kit of components comprising a surgical instrument or device for the administration of cells derived from MSCs or the population of cells derived from MSCs as presently described or the pharmaceutical compositions as presently defined. a subject, such as, for example, systemically, for example, by injection, and further comprising cells derived from MSC or the population of cells derived from MSC as presently described or pharmaceutical compositions as presently defined.
    In one embodiment, the pharmaceutical composition as defined above can be administered in the form of a liquid or viscous composition.
    In related aspects, the invention relates to cells derived from MSCs or populations of cells derived from MSCs defined above or the pharmaceutical composition comprising
    BE2018 / 5662 said cells derived from MSC or population of cells derived from MSC for use as a medicament. In related aspects, the invention relates to cells derived from CSM or populations of cells derived from CSM defined above or the pharmaceutical composition comprising said cells derived from CSM or population of cells derived from CSM for use in the treatment of a subject needing a CSM-derived cell transplant. In related aspects, the invention relates to a method of treating a subject in need of a MSC-derived cell transplant comprising admitting to said subject a therapeutically effective amount of cells derived from defined MSCs or cell populations derived from CSM defined above or from the pharmaceutical composition comprising said cells derived from CSM or population of cells derived from CSM to the subject. In related aspects, the invention relates to the use of cells derived from CSM or population of cells derived from CSM defined above or composition comprising said cells derived from CSM or a population of cells derived from CSM for the manufacture of a drug for the treatment of a subject in need of a MSC-derived cell transplant.
    In this context, the terms "treat" or "treatment" mean both therapeutic treatment and prophylactic or preventive measures, the objective being to prevent or slow (decrease) an undesirable physiological change or disorder. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, decrease in extent of disease, stabilized (i.e., no worsening) condition, delay or slowing the progression of the disease and the onset of complications, the improvement or palliation of the pathological condition. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment.
    The term "subject in need of a MSC-derived cell transplant" in the present context includes subjects, such as mammalian or human subjects, who may benefit from the treatment of a given condition, preferably a condition or disease as described above. Such subjects will typically include, without limitation, those who have been diagnosed with the condition, those who are likely to have or develop the condition in question and / or those in whom the disease is to be prevented.
    The term "transplant" or "cell transplant" has its usual meaning and, in particular, denotes the administration of cells to a subject. The term "cell transplant" can be used interchangeably with "cell therapy". A cell transplant can be performed by any technique known in the art. By way of example, and without limitation, cells can be transplanted by infusion into a subject. Typically, a cell infusion may be performed parenterally, for example, intravascularly, subcutaneously, intradermally or intramuscularly, preferably by
    BE2018 / 5662 intravascular. Cells can be administered, for example, and without limitation, systemically, topically, or at the site of an injury. It may be clear that, depending on the specific application, the targeted tissues, an adjustment of the therapeutic objective or of the type of cells can be carried out accordingly, taking into account the routes of administration, as well as the formulations, concentrations, etc. .
    The homogeneous and small cell size of the cells derived from MSC as presently described leads to an acute toxicity reduced or eliminated after intravenous administration of said cells to a subject. Consequently, cells derived from MSCs as described herein are particularly suitable for intravascular or percutaneous administration.
    Therefore, in particular embodiments, the cells derived from MSC or population of cells derived from MSC defined above or the pharmaceutical composition can be administered to said subject in need of a transplant of cells derived from MSC percutaneously or intravascular.
    In addition, the inventors have discovered that cells derived from MSC of osteochondroblastic line or osteoblastic line would be obtained by the methods as presently described which have more powerful osteoforming properties.
    Consequently, in a particular embodiment, said affection or disease is a musculoskeletal disease.
    The term "musculoskeletal disease" in this context means any type of bone disease, muscle disease, joint disease or chondrodystrophy, the treatment of which may benefit from the administration of the present pharmaceutical formulation to a subject having the disease. In particular, such a disease can be characterized, for example, by reduced bone and / or cartilage formation or excessive resorption of bone and / or cartilage, reduced number, viability or function of osteoblasts or osteocytes present in the bone and / or chondroblasts or chondrocytes present in the cartilage, a decrease in bone mass and / or cartilaginous mass in a subject, thinning of the bones, decrease in resistance or elasticity of bones, etc.
    Nonlimiting examples of musculoskeletal diseases can include local or systemic disorders, such as any type of osteoporosis or osteopenia, for example primary, post-menopausal, senile, corticosteroid-induced, biphosphonate-induced and radiotherapy; any type of secondary, mono- or multisite osteonecrosis, any type of fracture, for example, unconsolidated, badly consolidated, delayed consolidation or compression fractures, maxillofacial fractures; conditions requiring bone fusion (for example, vertebral fusion and reconstruction); congenital bone defect; bone reconstruction, for example, after a traumatic injury or
    BE2018 / 5662 cancer surgery and cranio-facial bone reconstruction; traumatic arthritis, focal cartilage and / or joint damage, degenerative focal arthritis; osteoarthritis, degenerative arthritis, gonarthrosis and coxarthrosis; imperfect osteogenesis; osteolytic bone cancer; Paget's disease; endocrinological disorders; hypophosphatemia; hypocalcemia; renal osteodystrophy; osteomalacia; adynamic bone disease, hyperparathyroidism, primary hyperparathyroidism, secondary hyperparathyroidism; periodontal disease; Gorham-Stout disease and McCuneAlbright syndrome; rheumatoid arthritis; spondyloarthropathies, including ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis and undifferentiated spondylitis and reactive arthritis; systemic lupus erythematosus and related disorders; scleroderma and associated disorders; Sjogren's syndrome; systemic, including giant cell arteritis (Horton's disease), Takayasu, polymyalgia rheumatica, vasculitis associated with ANCA Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome), Behçet syndrome and others polyarteritis and associated disorders (such as polyarteritis nodosa, Cogan syndrome and Buerger's disease); arthritis accompanying other systemic inflammatory diseases, including amyloidosis and sarcoidosis; crystalline arthritis, including gout, calcium pyrophosphate dihydrate crystal disease, disorders or syndromes associated with joint deposition of calcium phosphate or calcium oxalate crystals; chondrocalcinosis and neuropathic arthropathy; Felty syndrome and Reiter syndrome; Lyme disease and rheumatic fever.
    arteritis vasculitis (such as
    In a particular embodiment, said condition or disease is a bone disorder.
    Consequently, the term “bone disorder”, in the present context, designates any type of bone disease, the treatment of which can benefit from the transplantation of cells having osteoforming properties, for example, osteochondroprogéniteurs, osteoprogéniteurs, preosteoblasts, osteoblasts or cells having an osteoblast phenotype to a subject having the disorder. In particular, such disorders can be characterized, for example, by reduced bone formation or excessive bone resorption, by reduced number, viability or function of osteoblasts or osteocytes present in bone, reduced bone mass in a subject, thinning of the bones, alteration of the elasticity of the bones, etc.
    By way of example, but not limited to, bone disorders which can benefit from a graft of cells derived from MSCs having bone formation properties (for example, cells of osteoblastic line) obtained by the method according to the present invention can include local or systemic disorders, such as, any type of osteoporosis or osteopenia, for example, primary, postmenopausal, senile, corticosteroid-induced, any type of secondary, mono- or multisite osteonecrosis, any type of fracture , for example Bdes 18/5662 unconsolidated, poorly consolidated, delayed consolidation or compression fractures, conditions requiring bone fusion (for example, vertebral fusions and reconstructions), maxillofacial fractures, bone reconstruction , after traumatic injury or cancer surgery, cranio-facial bone reconstruction, osteogenesis imperfecta, cancer osteolytic bones, Paget's disease, endocrinological disorders, hypophosphatemia, hypocalcemia, renal osteodystrophy, osteomalacia, adynamic bone disease, rheumatoid arthritis, hyperparathyroidism, primary hyperparathyroidism, secondary hyperparathyroidism periodontal disease, Gorham-Stout disease and McCune-Albright syndrome.
    The cells derived from MSCs, the population of cells derived from MSCs and the pharmaceutical compositions described herein can be used alone or in combination with any of the therapies or active compounds known for the respective disorders. Administration can be simultaneous or sequential in any order, as described elsewhere.
    If the cells are derived from a heterologous source (i.e., non-autologous, non-homologous or non-allogenic), concomitant immunosuppression therapy can typically be administered, for example, using immunosuppressive agents, such as than cyclosporine or tacrolimus (FK506).
    The amount of cells to be administered will vary depending on the subject being treated. In a preferred embodiment, the quantity of cells to be administered is between 10 2 and 10 10 or between 10 2 and 10 9 , or between 10 3 and 10 10 or between 10 3 and 10 9 , or between 10 4 and 10 10 or between 10 4 and 10 9 , for example between 10 4 and 10 8 , or between 10 5 and 10 7 , for example, about 1x10 5 , about 5x10 5 , about 1x10 6 , about 5x10 6 , about 1x10 7 , about 5x10 7 , approximately 1x10 8 , approximately 5x10 8 , approximately 1x10 9 , approximately 2x10 9 , approximately 3x10 9 , approximately 4x10 9 , approximately 5x10 9 , approximately 6x10 9 , approximately 7x10 9 , approximately 8x10 9 , approximately 9x10 9 or approximately 1x10 10 cells can be administered to a human subject. In additional embodiments, between 10 6 and 10 8 cells per kg of body weight, for example, about 1x10 7 to 9x10 7 cells per kg of body weight, for example, about 1x10 7 , about 2x10 7 , about 3x10 7 , approximately 4x10 7 , approximately 5x10 7 , approximately 6x10 7 , approximately 7x10 7 , approximately 8x10 7 , approximately 9x10 7 or approximately 1x10 8 cells per kg of body weight may be administered to a human subject. For example, such a number of cells or such a number of cells per kg of body weight can denote, in particular, the total number of cells to be administered to a subject, said administration being able to be appropriately distributed in one or more doses ( for example, divided into 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses or more) administered over one or more days (for example, over 1, 2, 3, 4 or 5 days or more) . However, the precise determination of a therapeutically effective dose may be based on individual factors for each patient, including their height, age, size, 4.-1- 414 I. II '· 4 4 .BE2018 / 5662 tissue damage, and the time since lesions have occurred, and can be readily determined by those skilled in the art from this description and knowledge in the art.
    Suitably, in a composition for administration, the cells may be present at a concentration of between about 10 4 / ml and about 10 9 / ml, preferably between about 10 5 / ml and about 10 8 / ml, even more preferably between about 1x10 6 / ml and about 1x10 8 / ml, even more preferably between about 1x10 7 / ml and about 1x10 8 / ml, such as, for example, about 7.5x10 7 / ml. The reduced cell size of the MSC-derived cells as described herein provides an adjustable and / or high cell concentration. Consequently, if the composition is a liquid composition, the volume of the composition comprising cells derived from MSCs obtained by the method as presently described for administration to the subject needing a transplant of cells derived from MSCs is less than the volume of the composition comprising cells derived from MSC obtained by other methods.
    Therefore, the aspects and embodiments of the present invention cover, and this specification describes, the subject matter as described in any one and all of the following statements:
    Declaration 1. Method for obtaining cells derived from mesenchymal stem cells from mesenchymal stem cells (MSC) comprising contacting MSC in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    Declaration 2. Procedure according to declaration 1, comprising the steps of:
    (a) culture of MSCs recovered from a biological sample of a subject in a medium comprising FGF-2, TGF-β and heparin or a derivative or analog thereof thereof at a concentration of at least 0.01 IU / ml;
    (b) elimination of the non-adherent material and further culture of the adherent cells in the medium comprising FGF-2, TGF-β and heparin or a derivative or analog thereof at a concentration of at least 0 , 01 IU / ml, so as to obtain the cells derived from MSC.
    Declaration 3. Method according to declaration 1 or 2, in which TGF-β is chosen from the group consisting of TGF-β! TGF ^ 2, TGF ^ 3, and mixtures thereof; preferably where TGF-β is TGF-β!
    Statement 4. A method for obtaining cells derived from CSM having improved transplant properties from CSM, the method comprising a size reduction step, wherein said size reduction step is characterized by contacting
    CSM or cells derived from CSM in vitro or ex vivo with heparin or an E or 18/5662 derivative analog thereof at a concentration of at least 0.01 IU / ml.
    Declaration 5. Method according to any one of declarations 1 to 4, in which the concentration of heparin or of a derivative or analog thereof is at least 0.05 IU / ml, preferably approximately 0.1 IU / ml.
    Declaration 6. Method according to any one of declarations 1 to 5, in which the heparin or the heparin derivative or the analog is chosen from the group consisting of an unfractionated heparin (HNF); a low molecular weight heparin (LMWH), such as enoxaparin, dalteparin, nadroparin, tinzaparin, certoparin, reviparin, ardeparin, parnaparin, bemiparin, or mixtures thereof ; a heparinoid, such as heparan sulfate, dermatan sulfate, chondroitin sulfate, acharan sulfate, keratan sulfate, or mixtures thereof, such as danaparoid; a heparin salt; a heparinoid salt; a heparin fragment; a heparinoid fragment; and mixtures thereof.
    Declaration 7. Method according to any one of declarations 1 to 6, in which the average diameter of the cells derived from CSM in suspension is less than 25 μm, for example less than 24 μm, such as between 20 μm and 25 μm.
    Declaration 8. Method according to any one of declarations 1 to 7, in which the diameter of each cell derived from CSM in suspension is less than 35 μm.
    Declaration 9. Method according to any one of declarations 1 to 8, in which at least 60% of the cells derived from MSC in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm) and in which at most 5 % of cells derived from CSM in suspension have a diameter greater than 35 μm.
    Declaration 10. Method according to any one of declarations 1 to 9, in which the cells derived from MSC are of osteochondroblastic line.
    Declaration 11. Method according to any one of declarations 1 to 10, in which the cells derived from MSC are of osteoblastic or chondroblastic line, preferably of osteoblastic line.
    Declaration 12. Method according to any one of declarations 1 to 11, in which the MSCs are further brought into contact with, for example when the medium additionally comprises one or more elements among the plasma, serum or a substitute of these.
    Declaration 13. Method for obtaining cells derived from CSM from CSM comprising bringing CSM into contact in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or analog thereof , in which the average diameter of the cells derived from CSM in suspension is less than 25 μm, for example less than 24 μm or, for example, between 20 μm and 25 μm.
    Declaration 14. Method according to declaration 13, in which the diameter of each cell derived from suspended CSM is less than 35 μm.
    Declaration 15. Method for obtaining cells derived from CSM from CSM comprising contacting CSM in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or analog thereof , in which at least 60% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm) and in which at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 .mu.m.
    Declaration 16. Method according to any one of declarations 13 to 15, in which the MSCs are brought into contact with heparin or a derivative or analog thereof at a concentration of at least 0.01 IU / ml .
    Statement 17. A method according to any of Claims 13 to 16, wherein TGF-ß is selected from the group consisting of TGF-ß1, TGF-ß2, TGF-ß3, and mixtures thereof; preferably in which TGF-ß is TGF-ß1.
    Declaration 18. Population of cells derived from MSCs obtainable by in vitro or ex vivo expansion of MSCs, in which the average diameter of cells derived from MSCs in suspension is less than 25 μm, for example less than 24 μm, such as between 20 μm and 25 μm.
    Declaration 19. Population of cells derived from MSC according to Declaration 18, in which the diameter of each cell derived from MSC in suspension is less than 35 μm.
    Declaration 20. Population of cells derived from MSCs obtainable by in vitro or ex vivo expansion of MSCs, in which at least 60% of the cells derived from MSCs in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm ) and in which at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm.
    Declaration 21. Population of cells derived from MSC according to any one of declarations 18 to 20, in which the cells derived from MSC can be obtained by a method comprising bringing CSM into contact in vitro or ex vivo with FGF-2, TGF-ß and heparin or a derivative or the like thereof.
    Declaration 22. Population of cells derived from MSCs according to Declaration 21, in which MSCs are contacted with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    Declaration 23. Population of cells derived from MSC according to any one of declarations 18 to 22, in which the cells derived from MSC are of osteochondroblastic line.
    Statement 24. Population of MSC-derived cells according to any one of claims 18 to 23, wherein the cells derived from MSC are of osteoblastic or chondroblastic line, preferably of osteoblastic line.
    Statement 25. A population of MSC-derived cells according to any one of claims 18 to 24, wherein the MSCs are further contacted with one or more of plasma, serum or a substitute thereof.
    Declaration 26. Population of cells derived from MSC according to any one of declarations 18 to 25, in which TGF-β is chosen from the group consisting of TGF-β! TGFβ2, TGF ^ 3, and mixtures thereof; preferably in which TGF-β is TGF-β!
    Statement 27. Population of cells derived from MSC of osteochondroblastic line according to any of claims 23 to 27, wherein substantially all of the cells derived from MSC of osteochondroblastic line are positive for CD90, CD105, CD73, CD63 and CD166; substantially all cells derived from MSC of the osteochondroblastic line are negative for CD45, CD14 and CD19; at least 70% of the cells derived from MSC of osteochondroblastic line are positive for alkaline phosphatase (ALP); and less than 10% of the cells derived from MSC of osteochondroblastic line are positive for HLA-DR.
    Declaration 28. Pharmaceutical composition comprising the population of cells derived from MSC as defined in any one of declarations 18 to 27.
    Declaration 29. Population of cells derived from MSC according to any of declarations 18 to 27 or pharmaceutical composition according to declaration 28 for use as a medicament.
    Declaration 30. Population of cells derived from MSC for use according to Declaration 29, the population of cells derived from MSC being present at a concentration of between approximately 1 × 10 7 / ml and approximately 1 × 10 8 / ml, preferably 7, 5 x 10 7 cells / ml.
    Declaration 31. Population of cells derived from MSC according to any one of declarations 18 to 27 or pharmaceutical composition according to declaration 28 for use in the treatment of a subject in need of a transplant of cells derived from MSC.
    Declaration 32. Population of cells derived from MSC for use according to any one of declarations 29 to 31, the population of cells derived from MSC or the pharmaceutical composition being suitable for percutaneous or intravascular administration.
    Although the invention has been described with reference to specific embodiments thereof, it is obvious that many alternatives, modifications and variations will appear to those skilled in the art on reading the description given above. Consequently, it is §E2018 / 5662 years the spirit and the broad scope of the appended claims.
    The above aspects and embodiments are further supported by the following nonlimiting examples.
    EXAMPLES
    Example 1: Process for Obtaining Osteoforming Cells Derived from Small MSCs
    1. Experimental procedures
    1.1 Collection of human bone marrow and cultures of human bone marrow MSC at 60 ml of human bone marrow aspirations (BM) are obtained from the iliac crest of 8 healthy voluntary donors. After collection, the white bone marrow globules are counted, seeded at a density of 50,000 cells / cm 2 in a conventional culture medium containing 1% of penicillin-streptomycin and incubated at 37 ° C. in a humidified atmosphere containing 5% CO 2 . After 24 hours, the non-adherent cells are removed by rinsing with saline phosphate buffer (PBS) (Lonza BioWhittaker®) and fresh medium is added. The culture medium is replaced every 2 to 3 days. Adherent cell colonies are grown until 80% cell confluence is reached. The cells are then detached with trypsin-EDTA (TrypZean® EDTA, Lonza BioWhittaker®). The trypsin activity is neutralized with Dulbecco's phosphate buffered saline (DPBS). The cells are counted and subcultured for additional culture. The culture medium is replaced every 2 to 3 days until 80% of cell confluence is reached. The mesenchymal stem cells (MSCs) are detached as described above.
    1.2 Osteoforming cells derived from MSC and cell culture and plasma preparation
    As described above, 20 to 60 ml of heparinized bone marrow (BM) is obtained from the iliac crest of 8 healthy voluntary donors. After collection, the bone marrow is seeded in culture flasks at a fixed white blood cell density (50,000 cells / cm 2 ) and cultured either
    - to obtain osteoforming B cells derived from CSM derived from CSM: a conventional culture medium supplemented with 5% v / v of plasma treated with solvent / detergent (S / D) (Octaplas®, Octapharma AG, human origin), 0 , 1 IU / ml heparin (Heparin LEO, LEO Pharma SA, Belgium, lot A17605), basic fibroblast growth factor (FGF-2) and transforming growth factor beta (TGF-βΙ);
    - to obtain osteoforming cells derived from CSM Z: a conventional 18/5662 cuBure medium supplemented with 5% v / v of plasma treated with solvent / detergent (S / D) (Octaplas®, Octapharma AG, human origin) and basic fibroblast growth factor (FGF-2); or
    - to obtain osteoforming cells A derived from MSC: a conventional culture medium supplemented with 5% v / v of plasma treated with solvent / detergent (S / D) (Octaplas®, Octapharma AG, human origin), growth factor basic fibroblasts (FGF-2) and beta transforming growth factor (TGF-ß1).
    The cells are cultured in a humidified atmosphere at 37 ° C containing 5% CO 2 . The MSCs are left to settle before an initial change of environment. The medium is replaced every 3 or 4 days. At the end of the primary culture, the cells are detached, using a trypsin / EDTA solution for 1 to 5 min at 37 ° C., counted and subcultured for secondary culture in culture flasks in the same medium. At the end of the secondary culture, the osteoforming cells derived from MSCs are collected and washed with PBS.
    1.3 Characterization of cells in vitro
    1.3.1 Counting and viability of cells
    The density and viability of the cells are determined by means of a trypan blue exclusion assay. After collection, the cells are diluted 1: 2 with trypan blue (0.4%, Lonza BioWhittaker®) and the cell viability is analyzed using a Bürker chamber (Sigma-Aldrich®) and an inverted microscope (AE31, Motic®). Cell viability is also analyzed by flow cytometry using amino-actinomycin D (7-AAD, BD Biosciences®) and BD FACSCanto II ™ and BD FACSDiva ™ (Becton Dickinson®) software. After collection, 50,000 cells are incubated in the dark for 10 min at room temperature in PBS-bovine serum albumin (BSA) 1% (Lonza BioWhittaker®) with 2.5 μl of 7-AAD.
    1.3.2 Expression of markers
    1.3.2.1 Analysis by flow cytometry
    The osteoforming cells derived from MSC obtained after secondary culture, as described in section 1.1 above, are collected and cell surface markers are analyzed by flow cytometry (BD FACSCanto II ™ software and BD FACSDiva ™; Becton Dickinson, States -United). The cells are incubated with the following conjugated monoclonal antibodies: anti-CD73, anti-CD90, anti-CD105 and anti-CD166 (which are mesenchymal markers, and should be highly expressed by MSCs or cells derived from MSCs), CD3, anti-CD34 and anti-CD45 (which are hematopoietic markers and should be substantially absent from MSCs or cells derived from MSCs), anti-CD44, antiCD 51/61, anti-CD49a-e, anti-CD29 (which are adhesion markers), anti-CD40, anti-CD86 and anti-HLA-DR (which are markers of immunogenicity), and anti-alkaline phosphatase (ALP) 18/5662 for 15 min at room temperature, and then washed with phosphate buffered saline (PBS) before centrifugation and resuspended in 0.3 ml of PBS.
    For the characterization of cell surface markers CD105, CD73, CD10 and CD44, 50,000 cells at a concentration of 1x10 6 cells / ml in 1% PBS - BSA are incubated for 10 min in the dark with 5 μl of antibodies. After this incubation time, the cells are washed once with PBS. The different antibodies used for extracellular staining are as follows: antibodies against CD105 conjugated to allophycocyanin (APC) (BD Biosciences®, ref. N ° 562408), CD73 (BD Biosciences®, ref. N ° 560847), antibodies against CD10 conjugated to phycoerythrin (PE) (BD Biosciences®, ref. N ° 555375), CD44 (BD Biosciences®, ref. N ° 550989). Non-specific staining is determined by incubation of cells with an immunoglobulin G (IgG) control conjugated to FITC, APC and PE (all from BD Biosciences®, ref. No. 556649; 555751; 556650 respectively). Before analysis, a sort of individuals and population of interest is carried out. The flow cytometry analysis is performed on 10,000 events of the sorted population using FACS CantolI (BD Biosciences ® ) and FACS Diva ® 8.0 (BD Biosciences ® ) software. The adjustment parameters used for the analysis are applied automatically with beads (BD CompBeads Plus ® , ref. N ° 560497). For each conjugate, the positivity threshold is fixed at 1% of positivity of the isotype control antibody and the positivity of each marker is determined. The median fluorescence intensity (MFI) of the total analyzed population is also determined and divided by the MFI of the control antibody of the corresponding isotype to obtain the normalized MFI (nMFI).
    Table 1: presentation of the suppliers and of the catalog numbers of the antibodies used in the examples
    Antibody Provider Catalog number Anti-ALP BD Biosciences 561433 Anti-CD166 BD Biosciences 560903 Anti-CD3 BD Biosciences 555340 Anti-CD34 BD Biosciences 555824 Anti-CD40 BD Biosciences 555588 Anti-CD44 BD Biosciences 550989 Anti-CD45 BD Biosciences 555485
    BE2018 / 5662
    Anti-CD49a BD Biosciences 559596 Anti-CD49b BD Biosciences 555669 Anti-CD49c BD Biosciences 556025 Anti-CD49d BD Biosciences 555503 Anti-CD49e BD Biosciences 555617 Anti-CD51 / 61 BD Biosciences 550037 Anti-CD73 BD Biosciences 561254 Anti-CD29 BD Biosciences 556048 Anti-CD86 BD Biosciences 555660 Anti-CD90 R&D System FAB7335P Anti-HLA-DR BD Biosciences 555558 Anti-CD105 BD Biosciences 562408 Anti-CD10 BD Biosciences 555375 Anti-HLA-DR-DPDQ BD Biosciences 555558 HLA-ABC BD Biosciences 555552
    1.3.2.2 ALP staining
    The cells are spread at the end of the manufacturing process at 60,000 cells / cm 2 in their respective culture medium and placed in a humidified incubator (37 ° C - 5% CO 2 ). ALP staining is performed after 24 h on adherent cells. The cells are fixed with citrate-buffered acetone and incubated with an ALP staining solution composed of 4% v / v of naphthol alkaline phosphate AS-MX (Sigma; ref. 855) and 96% v / v RR solid blue salt solution (Sigma; ref. FBS25) for 30 min in the dark.
    1.3.2.3 Measurement of ALP enzyme activity
    The ALP enzyme activity is measured by a biochemical assay based on the hydrolysis of p-nitrophenyl phosphate (pNPP). After being dephosphorylated by ALP, the pNPP becomes yellow and can be detected by a spectrophotometer at 410 nm. The ALP enzymatic activity of the cells is determined relative to a standard curve on the basis of the alkaline phosphatase activity of purified calf intestine. ALP activity is expressed in units of ALP / mg of protein. A unit of ALP hydrolyzes 1 pmol of pNPP in 1 min at 37 ° C.
    rpqOI8 / 5662
    1.3.3 Quantitative chain reaction by reverse transcription (RT-qPCR)
    After collection, the cells are stored at -80 ° C. in the form of dry pellets (500,000 cells) until extraction of RNA. Total RNA is extracted using the RNeasy® Mini kit (Qiagen®) according to the manufacturer's instructions. The RNA concentration is measured using DropSense® 16 (Trinean®). Reverse transcriptions are made from 1 µg of total RNA extracts, using the PrimeScript® RT reagent kit (Takara®) according to the manufacturer's instructions. The qPCRs are performed using Premix Ex Taq® (Takara®) from 2 µl of cDNA according to the manufacturer's instructions. The expression levels of the following genes of interest have been quantified: The expression levels of the following genes of interest have been quantified: RUNX2 (sense: GGTTCCAGCAGGTAGCTGAG (SEQ ID NO: 1), antisense: AGACACCAAACTCCACAGCC (SEQ ID NO : 2)), SOX9 (S: TAAAGGCAACTCGTACCCAA (SEQ ID NO: 3), A: ATTCTCCATCATCCTCCACG (SEQ ID NO: 4), BMP2 (S: GGAACGGACATTCGGTCCTT (SEQ ID NO: 5), A:
    CACCATGGTCGACCTTTAGGA (SEQ ID NO: 6)), ALPL (S:
    ACCATTCCCACGTCTTCACATTTG (SEQ ID NO: 7), A: AGACATTCTCTCGTTCACCGCC (SEQ ID NO: 8)), MMP13 (S: TGGAATTAAGGAGCATGGCGA (SEQ ID NO: 9), A: AACTCATGCGCAGCAACAAG (SEQ ID NO: 10)), CHIL
    TGGGTCTCAAAGATTTTCCAAGA (SEQ ID NO: 11), A: GCTGTTTGTCTCTCCGTCCA (SEQ ID NO: 12)), DCN (S: AAAATGCCCAAAACTCTTCAGG (SEQ ID NO: 13), A:
    GCCCCATTTTCAATTCCTGAG (SEQ ID NO: 14)), OCN (S: AAGGTGCAGCCTTTGTGT (SEQ ID NO: 15), A: GCTCCCAGCCATTGATACAG (SEQ ID NO: 16)), SPON1 (S:
    CCTGCGGAACTGCCAAGTA (SEQ ID NO: 17), A: CACGGGTGAGCCCAATTCT (SEQ ID NO: 18)), POSTN (S: TTTGGGCACCAAAAAGAAAT (SEQ ID NO: 19), A:
    TTCTCATATAACCAGGGCAACA (SEQ ID NO: 20)). The qPCRs are performed in duplicate using a LightCycler® 480 (Roche®). Normalization is carried out using the geometric mean obtained from three household genes: RPL13A (S:
    CATAGGAAGCTGGGAGCAAG (SEQ ID NO: 21), A: GCCCTCCAATCAGTCTTCTG (SEQ ID NO: 22)), TBP (S: AACAACAGCCTGCCACCTTA (SEQ ID NO: 23), A:
    GCCATAAGGCATCATTGGAC (SEQ ID NO: 24)), HPRT (S: CCCTGGCGTCGTGATTAGT (SEQ ID NO: 25), A: GTGATGGCCTCCCATCTCCTT (SEQ ID NO: 26)). A comparison between the different products of cells derived from MSCs from the same donors is carried out by calculation of the gene expression (change factor) using the 2-AACt method for each gene of interest (Schmittgen and Livak, 2008, 3 ( 6), 1101-8; Nature Protocols, 3 (6), 11011108).
    Statistical analysis is carried out using JMP® software (13.1.0). The RT-qPCR data expressed as a change factor are subjected to a logarithmic transform and Student's tests (with α = 0.05) are carried out to assess the statistical significance B 5êS 18/5662 differences observed between the types of cells.
    The statistical significance is represented graphically as a function of the p (p) value obtained: * for p <0.05, ** for p <0.01, and *** for p <0.001.
    1.3.4 Multiplex dosing
    After collection, the cells are spread at a density of 50,000 cells / cm 2 . After 48 hours of incubation at 37 ° C in a humidified atmosphere containing 5% CO 2 , the cell culture supernatants are collected, centrifuged (5 min at 1500 rpm at room temperature) and stored at -80 ° C . The supernatants are analyzed by Luminex® assay using Human Magnetic Luminex® assays (R&D System®). The multiplex premix is prepared to measure (R&D System®). The following secreted factors are studied: BMP-2, COL1A1, MMP13, OPN, OPG, SPARC, RANKL, CHI3L1. The assay is carried out in accordance with the manufacturer's instructions and the analyzes are carried out using MAGPIX® (R&D System®) and Bio-Plex Manager 5.0TM (Bio-Rad®) software.
    1.4 Cell size measurement
    The osteoforming cells derived from MSC obtained after secondary culture as described in section 1.1 above are collected and suspended in PBS with 0.4% trypan blue at a cell density of 12.5 × 10 6 cells per ml . 10 μl of the cell suspension are placed on a graduated slide (Motic ® ), then protected by a coverslip to be placed under an inverted microscope at 40X magnification (AE31; Motic). The images acquired with a camera (Moticam) placed on the microscope are analyzed by the Motic Image Plus 2.02 software in order to measure the diameters of the cells. At least one hundred cells are measured to consider the analysis statistically significant.
    The size of the osteoforming cells derived from MSCs obtained at different times from the ex vivo culture is also analyzed by flow cytometry (BD FACS Canto II ™ and BD FACS Diva ™ software; Becton Dickinson, United States). Briefly, on day 21, 23, 26 and 28 after starting the ex vivo cell culture as described in section 1.1, the cells are collected, suspended in phosphate buffered saline (PBS) at a cell density 1.10 6 cells per ml and analyzed with the flow cytometer for front diffusion measurement (FSC) (expressed in relative fluorescence unit). The front scattering measures the light scattered in the direction of the laser path and, therefore, gives a relative size for the cells passing through the flow chamber.
    2. Results BE2018 / 5662
    2.1 Expression profile of cellular markers
    Analysis by flow cytometry shows that the cell identities based on the expression profiles of cell surface markers of osteoforming cells A (generated with FGF 2 and TGF-β 1) and of osteoforming cells B (generated with FGF- 2, TGF ^ 1 and heparin; embodiment of the present invention) are comparable.
    The populations of osteoforming cells A and B both express the mesenchymal markers CD73, CD90, CD105, CD63, CD166 and do not express the hematopoietic markers CD45, CD34 and CD3 (less than 5% of the cell population expresses these markers) (Tables 2 and 3). The osteoforming cells B (i) continue to express low levels of MHC class II cell surface receptor such as HLA-DR and (ii) strongly express ALP. The low immunogenicity represented by the low expression of HLA-DR advantageously allows a cell transplant, for example, to allogeneic subjects (Table 5). In addition, the osteoforming cells A and the osteoforming cells B strongly express the adhesion markers CD49e, CD44 and the enzyme ALP on their surface compared to undifferentiated MSCs (Tables 3 and 4). The high expression of this last marker (ALP) indicates the orientation towards the osteoblastic line of osteoforming cells. In addition, the high expression of ALP highlights the orientation of the osteoforming B cells towards the osteoblastic line (compared to undifferentiated MSCs). Table 6 further shows that the expression of ALP is higher for cells cultured in the presence of heparin (osteoforming cells B) than for cells cultured in the absence of heparin (osteoforming cells A).
    Table 2: expression profile of markers of MSC populations and osteoforming cells derived from MSC.
    % Marker expression (mean ± SD) CSM Z osteoforming cells generated with FGF-2 Osteoformor A cells generated with FGF-2 and TGF-ß1 Osteoformor B cells generated with FGF-2, TGF-ß1 and heparin CD44-FITC 98 ± 2 (N = 3) 100 ± 1 (N = 16) 100 ± 1 (N = 11) 100 ± 1 (N = 16) CD51 / 61 19 ± 18 (N = 10) 50 ± 17 (N = 8) 13 ± 12 (N = 8) 32 ± 31 (N = 8) CD34-FITC 3 ± 2 (N = 3) 1 ± 1 (N = 3) ND ND CD34-APC 2 ± 1 (N = 6) 3 ± 1 (N = 5) ND ND CD49-FITC 8 ± 8 (N = 10) 44 ± 14 (N = 7) 25 ± 13 (N = 7) 42 ± 18 (N = 6)
    CD45-FITC 2 ± 1 (N = 6) 2 ± 1 (N = 11) 1 ± 1 (N = 11) -2 ± 1 (N = 6) CD166-PE 97 ± 3 (N = 10) 98 ± 2 (N = 8) 97 ± 3 (N = 9) 96 ± 6 (N = 8) CD73-PE 99 ± 1 (N = 6) 100 ± 1 (N = 12) 100 ± 1 (N = 11) 100 ± 1 (N = 8) CD29-APC 100 ± 1 (N = 8) 100 ± 1 (N = 7) 100 ± 1 (N = 10) / 100 ± 1 (N = 8) ALP-PE 20 ± 7 (N = 13) 70 ± 19 (N = 17) 69 ± 18 (N = 16) 91 ± 8 (N = 10) Intra-PE ALP 19 ± 13 (N = 11) 63 ± 22 (N = 10) 59 ± 22 (N = 10) 80 ± 13 (N = 8) HLA-DR N / A 63 ± 20 (N = 10) 6 ± 6 (N = 22) 3 ± 2 (N = 8)
    Abbreviations: ALP: alkaline phosphatase; APC: allophycocyanin; FGF-2: fibroblast growth factor 2; FITC: fluorescein isothiocyanate; HLA-DR: human leukocyte antigen - DR isotype; MSC: mesenchymal stem cells; NA: not available; ND: not determined; PE: phycoerythrin; AND: standard deviation; TGF-βΙ: growth factor 5 transforming beta 1
    Table 3: Expression profile of cell surface markers of MSC populations and osteoforming cells derived from MSC
    Marker expression (in%) Statistics CSM Osteoforming cells s A B osteoforming cells CD73-APC Average 100.0 100.0 100.0 AND 0.0 0.0 0.0 NOT 6 11 22 CD90-PE Average 100.0 99.9 99.9 AND 0.1 0.2 0.2 NOT 8 12 22 CD105-APC Average 100.0 99.8 100.0 AND 0.0 0.5 0.1 NOT 8 12 20 CD45-APC Average 0.4 0.3 1.0 AND 0.2 0.2 2.9 NOT 8 12 19 CD34-APC Average 0.6 1.0 1.6
    Marker expression (in%) Statistics CSM Osteoforming cells s A -B osteoforming cells AND 0.4 0.6 1.8 NOT 8 12 22 CD3-PE Average 0.2 0.1 0.2 AND 0.1 0.1 0.1 NOT 6 10 17 HLA-DR-PE Average 0.7 1.0 1.8 AND 1.2 0.6 2.0 NOT 8 12 22 HLA-DR / DP / DQFITC Average 1.0 1.6 1.6 AND 0.4 1.1 1.1 NOT 8 12 22 ALP-PE Average 40.7 88.7 94.8 AND ND 5.6 6.6 NOT 1 5 10 CD49e-PE Average 92.7 99.6 99.8 AND 20.5 1.1 0.5 NOT 8 12 19 CD44-PE Average 99.9 99.7 100.0 AND 0.2 0.5 0.0 NOT 8 12 22 CD10 Average 19.6 99.6 98.8 Standard deviation 14 0.4 1.5 NOT 10 12 25
    Abbreviations: ALP: alkaline phosphatase; APC: allophycocyanin; FITC: fluorescein isothiocyanate; HLA-DR: human leukocyte antigen - DR isotype; HLA-DR / DP / DQ:
    human leukocyte antigen - DR / DP / DQ isotypes; CSM: cells
    BE2018 / 5662 mesenchymal strains; ND: not determined; PE: phycoerythrin; AND: standard deviation
    Table 4: ALP expression rate of a population of MSC and osteoforming cells derived from MSC evaluated by different methods
    Statistics CSM Osteoforming cells s A B osteoforming cells Population positive for ALP-PE (%) Average 40.7 88.7 94.8 AND ND 5.6 6.6 NOT 1 5 10 ALPPE cell surface expression rate (nMFI) Average 2.4 19.8 56.1 AND ND 10.8 27.4 NOT 1 5 10 ALP enzyme activity (mU / mg total protein) Average 176.3 671.9 874.7 AND 252.9 305.8 772.9 NOT 3 9 26
    Abbreviations: ALP: alkaline phosphatase; MSC: mesenchymal stem cells; ND: not determined; nMFI: normalized median fluorescence intensity; PE: phycoerythrin; AND: standard deviation
    Table 5: comparisons of the expression of the immunogenicity surface marker
    HLA-DR (FACS) on osteoforming cells derived from MSC using different culture conditions
    Cell population
    HLA-DR (%) mean ± SD
    Z osteoforming cells generated with FGF-2
    Osteoformor A cells generated with FGF-2 and TGFβ1 ± 20 (N = 10) ± 6 (N = 22)
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    Osteoformor B cells generated with FGF-2, TGF-ß1 and heparin 3 ± 2 (N = 8) Osteoformor A cells generated with FGF-2 and TGFß1 1.0 ± 0.6 (N = 12) Osteoformor B cells generated with FGF-2, TGF-ß1 and heparin 1.8 ± 2.0 (N = 22)
    Abbreviations: FGF-2: fibroblast growth factor 2; HLA-DR: human leukocyte antigen - linked to antigen D; MSC: mesenchymal stem cells; AND: standard deviation; TGF-βΙ: growth factor transforming beta 1
    Table 6: ALP expression rate of MSC populations and of osteoforming cells generated under different culture conditions
    Cell population % of ALP + cells (flow cytometry) % of intra + ALP cells (flow cytometry) Enz. ALP (mUI / mg total protein) ALP staining CSM (witness) 20 ± 7(N = 13) 19 ± 13(N = 11) 108 ± 86(N = 2) N / A cells 70 ± 19 63 ± 22 877 ± 680 1.8 ± 0.4 osteoformers Zgenerated with FGF-2 (N = 17) (N = 10) (N = 6) (N = 23) cells 69 ± 18 59 ± 22 495 ± 466 1.2 ± 0.4 osteoformers Agenerated with FGF-2 and TGF-ß1 (N = 16) (N = 10) (N = 17) (N = 13) cells 91 ± 8 80 ± 13 1.016 ± 685 2.0 ± 0.0 osteoformers Bgenerated with FGF-2, TGF-ß1 and heparin (N = 10) (N = 8) (N = 14) (N = 22)
    Abbreviations: ALP: alkaline phosphatase; FGF-2: fibroblast growth factor ^ 0 ; 18/5662
    MSC: mesenchymal stem cells; NA: not available; TGF-βΙ: growth factor transforming beta 1
    The expression profile of cell surface markers is not only characterized by the presence of markers on the cell surface (percentage of population positivity), but also by analysis of the quantity of markers expressed on the cell surface (median of normalized fluorescence for the population) of different markers. These analyzes highlight differences between the different osteoforming cells derived from MSC.
    Osteoforming B cells grown in the presence of heparin express a higher level of ALP than MSCs and osteoforming A cells cultured in the absence of heparin (Table 7, ALP-PE nMFI results), which confirms the orientation towards the osteoblastic line of osteoforming cells.
    The expression of the mesenchymal markers CD73 and CD105 on the cell surface are also dependent on the cell types. The osteoforming cells generated in the presence of heparin (osteoforming cells B) express a higher CD73 and CD105 level than the osteoforming cells A (Table 7).
    Table 7: additional results of expression of cell surface marker of MSC populations and of osteoforming cells derived from MSC
    Marker expression (in nMFI) Statistics CSM Osteoforming cells s A B osteoforming cells ALP-PE Average 2.4 19.8 56.1 AND - 10.8 27.4 NOT 1 5 10 CD73-APC Average 234.8 130.7 646.3 AND 84.3 80.1 138.8 NOT 6 11 22 CD105-APC Average 207.7 26.6 59.1 AND 67.6 15.2 13.1 NOT 8 12 20 CD44-PE Average 139.8 62.0 156.6 AND 57.5 19.1 40.7
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    NOT 8 12 22 CD49e-PE Average 81.0 22.5 33.5 AND 51.4 9.9 11.0 NOT 8 12 19 HLA-ABC-FITC Average 26.1 21.6 80.2 AND - 5.2 17.4 NOT 1 4 8 CD10-PE Average 0.8 36.2 32.2 AND 1.1 16.4 16.8 NOT 8 12 22
    Abbreviations: ALP: alkaline phosphatase; APC: allophycocyanin; FGF-2: fibroblast growth factor 2; FITC: fluorescein isothiocyanate; HLA-ABC: ABC human leukocyte antigen; MSC: mesenchymal stem cells; NA: not available; ND: not determined; PE: _phycoerythrin; AND: standard deviation; TGF-P1: transforming growth factor beta 1
    2.2 RT-qPCR and multiplex dosing
    The analysis shows that the RUNX2, SOX9, ZNF521, ALPL, BMP2, OPG, POSTN, CHI3L1, MMP13, CADM1, CX43, CD10, WISP1 genes coding for osteochondroblastic markers, and the DCN, SPON1 genes coding for proteins of bone or cartilage matrix are significantly overexpressed in osteoforming cells A and B compared to MSCs (Table 8). Likewise, the gene expression of DKK1 coding for an osteochondrogenesis inhibitor is significantly downregulated in osteoforming cells A and B compared to MSCs (Table 8).
    The expression of the KI67 and PCNA genes coding for proliferation markers is significantly downregulated in both osteoforming cells A and B compared to MSCs, and the gene expression of markers associated with apoptosis BCL2 and BAX is equivalent in all cell types (Table 8).
    Compared to osteoforming cells A, osteoforming cells B (statistical significance represented graphically according to the value p (p) obtained: * for p <0.05, ** for p <0.01 and *** for p <0.001) :
    - express higher levels of the PPARG gene (***) (coding for a protein involved in adipogenesis);
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    - - express higher levels of the CD73 (***), BMP2 (***) genes (coding for osteochondroblastic proteins);
    - - express higher levels of the COL1A1 (***), BGN (***), SPARC (***), ALPL (*), BCL2 (***) genes (coding for osteochondroblastic proteins).
    Regarding genes that are overexpressed in osteoforming B cells compared to osteoforming cells A, PPARG, MMP13, BMP2 are also significantly overexpressed in osteoforming B cells compared to MSCs, while CD73 has the same expression rate in osteoforming B cells and in MSCs.
    Regarding genes that are downregulated in osteoforming cells
    B compared to osteoforming cells A (that is to say, COL1A1, BGN, SPARC, BCL2), all have the same level of expression in osteoforming cells B as in MSCs with the exception of ALPL, which is still overexpressed in osteoforming B cells compared to MSCs.
    Table 8: gene expression profile of populations of MSCs and osteoforming cells (expressed as a change factor compared to the mean values of MSCs - the statistical significance is graphically represented as a function of the value p (p) obtained: * for p <0.05; ** for p <0.01; *** for p <0.001; NS: not statistically significant)
    Gene expression (factor ofchange from average values ofCSM) Statistics CSM Osteoforming cells A B osteoforming cells Mesenchymal markers CD73 Average 1.00 0.36 ** 1.12 (NS) AND N / A 0.01 0.24 NOT 1 3 6 CD105 Average 1.00 0.55 (NS) 0.48 * AND N / A 0.10 0.12 NOT 1 3 6 Master genes of differentiation RUNX2 Average 1.04 147 *** 1.64 *** AND 0.27 0.27 0.43 NOT 7 9 29 SOX9 Average 1.16 2.95 *** 2.03 **
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    Gene expression (factor ofchange from average values ofCSM) Statistics CSM Osteoforming cells A LJI-2-UB osteoforming cells AND 0.73 1.42 0.87 NOT 7 9 29 PPARG Average 1.17 6.93 *** 3.05 *** AND 0.75 2.16 1.83 NOT 7 9 29 ZNF521 Average 1.45 49.85 *** 63.88 *** AND 1.18 29,10 23.11 NOT 7 9 29 DKK1 Average 1.00 0.02 *** 0.01 *** AND N / A 0.01 0.01 NOT 1 3 6 Markers associated with theextracellular matrix Spon1 Average 1.09 576.25 *** 539.25 *** AND 0.45 397.77 339.77 NOT 6 9 29 COL1A1 Average 1.03 2.09 *** 0.88 (NS) AND 0.27 0.41 0.41 NOT 7 9 29 BGN Average 1.00 2.18 *** 1.26 (NS) AND 0.05 0.37 0.36 NOT 7 9 29 DCN Average 1.08 9.80 *** 7.31 *** AND 0.42 3.30 3.22 NOT 6 9 29 SPARC Average 1.01 2 21 *** 0.91 (NS) AND 0.15 0.59 0.33 NOT 7 9 29 IBSP Average 1.11 8.24 (NS) 14.34 ** AND 0.46 11.29 15.38 NOT 7 9 28
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    Gene expression (factor ofchange from average values ofCSM) Statistics CSM Osteoforming cells A B osteoforming cellsOCN Average 1.02 1.29 (NS) 1.50 ** AND 0.22 0.33 0.58 NOT 7 9 29 Osteochondrogenic markers ALPL Average 1.49 13.83 *** 8.91 *** AND 1.61 5.98 6.73 NOT 7 9 29 MMP13 Average 1.32 216.27 *** 2739.98 *** AND 1.26 254.24 2886.30 NOT 7 9 29 Cx43 Average 1.06 3.20 *** 2 74 *** AND 0.38 0.98 0.96 NOT 7 9 29 OPN Average 1.70 6.32 (NS) 4.47 (NS) AND 1.71 6.60 11.23 NOT 7 9 29 OPG Average 1.12 2.79 * 1.48 (NS) AND 0.53 2.43 0.78 NOT 7 9 29 BMP2 Average 1.04 10.76 *** 32.69 *** AND 0.29 6.19 25.88 NOT 7 9 29 Postn Average 1.14 5.70 *** 3.70 *** AND 0.71 1.42 1.81 NOT 7 9 29 WISP1 Average 1.11 3.16 *** 2.13 ** AND 0.53 1.21 0.92 NOT 7 9 29 CADM1 Average 1.70 43.28 *** 19.55 *** AND 1.87 27.53 22.12
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    Gene expression (factor ofchange from average values ofCSM) Statistics CSM Osteoforming cells A LJI-2-UB osteoforming cells NOT 7 9 29 CHI3L1 Average 1.84 430.19 *** 775.23 *** AND 2.21 309.05 462.38 NOT 7 9 29 CD10 Average 1.00 62.65 *** 57.96 *** AND N / A 14.43 30.91 NOT 1 3 6 Markersproliferation KI67 Average 1.27 0.13 *** 0.14 *** AND 1.13 0.26 0.13 NOT 7 9 29 PCNA Average 1.09 0.63 ** 0.62 *** AND 0.50 0.06 0.23 NOT 7 9 29 Markers associated withapoptosis BCL2 Average 1.08 3.43 *** 0.97 (NS) AND 0.46 0.97 0.46 NOT 7 8 29 BAX Average 1.01 1.65 * 1.92 ** AND 0.15 0.18 2.43 NOT 7 9 29
    The analysis of cell secretion shows that the osteoforming B cells secrete higher amounts of the proteins CHI3L1 and MMP13 involved in osteochondrogenesis than my osteoforming cells A and MSCs (Table 9) and secrete an amount of DKK1 involved in inhibition. osteogenesis lower than my osteoforming A cells and MSCs. No significant difference was observed between the cell types for the quantity of COL1A1 secreted (Table 9).
    Table 9: Secretion profile of populations of MSCs and of osteoforming cells derived from MSCs (expressed as a factor of change compared to the mean values of MSCs - the statistical significance is graphically represented in terms of dB3 $ a 18/5662 p value (p) obtained: * for p <0.05; ** for p <0.01; *** for p <0.001; NS: not statistically significant)
    Protein secretion (pg / ml) STATISTICSes CSM Osteoforming cells A B osteoforming cells COL1A1 Average 57347 79822 (NS) 79216 (NS) AND 32288 48969 41636 NOT 5 6 11 CHI3L1 Average 64533 123800 (NS) 303436 ** AND 32242 70909 232874 NOT 5 6 11 DKK1 Average 3679 4979 (NS) 1609 *** AND 1287 1913 850 NOT 5 6 11 MMP13 Average 180 294 693 *** AND 54 80 (NS) 545 NOT 5 6 11
    2.3 Cell size
    The cell size measurements obtained using (i) the Motic Image Plus 2.0 / 3.0 software and (ii) the FSC analysis by flow cytometry confirm that the osteoforming cells derived from CSM generated with FGF-2, TGF-ß1 and heparin (osteoforming B cells) are smaller and more homogeneous than osteoforming cells derived from MSCs generated with 10 FGF-2 and TGF-β! without heparin (e.g. osteoforming cells A) (Tables 10 and
    11, Figures 1 and 11).
    It is very interesting to note that the vast majority of osteoforming B cells (> 70%) do not exceed 25 μm in diameter and <5% of these exceed 35 μm in diameter (Tables 8 and 9). On the other hand, the population of osteoforming cells obtained without the addition of heparin (osteoforming cells A) comprises only 20% of cells which do not exceed 25 μm in diameter and 41% of cells with a diameter greater than 35 μm (FIG. 1 and table 10). As detailed further in Example 3, such large diameter cells could prove to be deleterious for the subject's transplant.
    Table 10: Distribution of cell sizes of osteoforming cells A and B
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    % of cells with a diameter <25 μm > 35 μm Osteoformor A cells generated with FGF-2 and TGF-ß-1 20% 41% Osteoformor B cells generated with FGF-2, TGF-ß-1 and heparin 70% 5%
    Abbreviations: FGF-2: fibroblast growth factor 2; TGF-βΙ: growth factor transforming beta 1
    Table 11: CSM cell size distribution and osteoforming cells derived from CSM
    % of cells with a diameter <25 μm > 35 μm CSM 89.9% 1.3% Osteoforming cells A 35.4% 26.9% B osteoforming cells 73.7% 3.3%
    Abbreviations: CSM: mesenchymal stem cells
    Table 12: diameter of CSM and osteoforming cells derived from CSM
    Cell diameter (μm) Average ± AND (N) Min - max CSM (witness) 22.4 ± 4.9 (N = 101) 13.6 -38.0 Osteoformor A cells generated with FGF-2 and TGF-ß1 34.1 ± 9.9 (N = 699) 15.9 -67 Osteoformor B cells generated with FGF-2, TGF-ß1 and heparin 23.3 ± 6.8 (N = 1170) 12.1 -74.5 Report (osteoforming cells B / osteoforming cells A) 0.68
    Abbreviations: FGF-2: fibroblast growth factor 2; MSC: mesenchymal stem cells; AND: standard deviation; TGF ^ 1: transforming growth factor beta 1
    Table 13: diameter of CSM and osteoforming cells derived from CSM
    Cell diameter (μm) Average ± AND Min - max NOT CSM 19.2 ± 4.8 9.8 -41.8 450 Osteoforming cells A 30.2 ± 9.9 11.4 -67 1205
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    ------- BEZtTl B osteoforming cells 22.4 ± 6.4 7.9 -74.5 1744
    Abbreviations: CSM: mesenchymal stem cells; AND: standard deviation
    FSC flow cytometry assays are used to assess the relative average cell size of osteoforming B cells generated with FGF-2, TGF-ß1 and heparin at different times during in vitro culture and, therefore, at different confluences cells, namely 45%, 70%, 90% and 100% confluence. Table 14 shows that the cell size of osteoforming B cells is stable and ultimately increases with the confluence of cell cultures. In other words, Table 14 shows that the average size of osteoforming B cells from a more confluent cell flask is larger than that of a less confluent culture flask. It should be noted that FSC flow cytometry assays compare the average cell size of different samples without, however, providing information on the absolute values of the average cell size.
    Table 14: FSC value by flow cytometry of osteoforming B cells collected at different times and confluences during an in vitro culture
    Total cell culture time Confluence Relative mean fluorescence unit D21 45% 69.307 D23 70% 65.228 D26 90% 77.349 D28 100% 91.124
    Example 2: specificity of the method for preparing cells derived from MSC of example 1
    1. Experimental procedures
    1.1 Cell culture and plasma preparation
    The cell culture and the plasma preparation are carried out as described in Example 1.
    For the tests relating to the comparison between heparin and the analogues thereof, the conventional culture medium is supplemented with (i) 5% v / v of plasma S / D; (ii) basic FGF-2; (iii) TGF-ß1; and (iv) 0.1 IU / ml of unfractionated heparin (UFH), dalteparin, heparan sulphate or danaparoid.
    For tests relating to the comparison between heparin and other anticoagulants, the conventional culture medium is supplemented with (i) 5% v / v of plasma S / D; (ii) basic FGF-2;
    (iii) TGF-β; and (iv) 1 IU / ml of heparin (Heparin LEO, LEO Pharma SA, Belgium, lot A17B05) 018/5662 100 IU / ml of heparin, 2 mg / ml of ethylenediaminetetraacetic acid (EDTA) or 0.1 mg / ml Actilyse®.
    For the tests relating to the comparison between the S / D plasma and the serum, the conventional culture medium is supplemented with (i) 5% v / v of S / D plasma or 5% of serum; (ii) basic FGF-2; (iii) TGF-β; and (iv) 0.1 IU / ml heparin.
    For the tests relating to the comparison between the presence or absence of S / D plasma and serum, the conventional culture medium is supplemented with (i) 5% v / v of S / D plasma, 5% v / v serum or 0% S / D plasma and 0% serum; (ii) basic FGF-2; (iii) TGF-β; and (iv) 0.1 IU / ml heparin.
    2. Results
    2.1 Heparin vs. analogues thereof
    Figure 2 and Table 15 show that the heparin present in the culture medium can be replaced by derivatives thereof (heparinoid compounds), namely by dalteparin, heparan sulfate or a mixture of glycosaminoglycans such than the danaparoid comprising heparan sulfate. The 4 heparin derivatives have the same effects on cell viability and the expression profile of markers as heparin (Table 15).
    Table 15: osteoforming cells derived from MSCs generated using different heparinoids: HNF, dalteparin, danaparoid and heparan sulfate; used at 0.1 IU / ml.
    Viability CSM hematopoietic immuno osteo CD73 CD90 CD3 CD34 CD45 HLA-DR CD40 CD86 ALP97 ± 2 99 ± 1 98 ± 1 0 ± 1 1 ± 1 4 ± 3 2 ± 1 0 ± 1 2 ± 2 88 ± 16 heparin (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 3) (N = 4) (N = 4) (N = 4)98 ± 2 98 ± 1 98 ± 1 0 ± 1 0 ± 1 4 ± 4 3 ± 3 0 ± 1 1 ± 2 94 ± 3 dalteparin (N = 3) (N = 3) (N = 3) (N = 3) (N = 3) (N = 3) (N = 3) (N = 3) (N = 3) (N = 3)97 ± 2 99 ± 1 98 ± 1 0 ± 1 2 ± 3 5 ± 4 4 ± 2 0 ± 1 3 ± 2 89 ± 15 Danaparoïd (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 3) (N = 4) (N = 4) (N = 4) Sulfate 97 ± 2 99 ± 1 99 ± 1 2 ± 3 1 ± 1 4 ± 1 4 ± 2 1 ± 2 4 ± 4 92 ± 11 heparan (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 3) (N = 4) (N = 4) (N = 4)
    membership
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    CD29 CD44 CD49a CD49b CD49d CD49e CD51 /CD61 CD54 LJI 2 tCD166 heparin 99 ± 1(N = 4) 98 ± 1(N = 4) 25 ± 6(N = 4) 53 ± 7(N = 4) 79 ± 3(N = 4) 97 ± 4(N = 4) 50 ± 23(N = 4) 70 ± 19(N = 4) 97 ± 3(N = 4) dalteparin 98 ± 1(N = 3) 98 ± 1(N = 3) 29 ±14(N = 3) 61 ± 3(N = 3) 78 ±15(N = 3) 98 ± 1(N = 3) 26 ± 19(N = 3) 65 ± 11(N = 3) 96 ± 1(N = 3) Danaparoïd 99 ± 1(N = 4) 99 ± 1(N = 4) 32 ± 9(N = 4) 69 ±21(N = 4) 88 ± 6(N = 4) 98 ± 2(N = 4) 39 ± 23(N = 4) 74 ± 10(N = 4) 97 ± 3(N = 4) Heparan sulfate 99 ± 1(N = 4) 98 ± 1(N = 4) 32 ± 8(N = 4) 75 ±15(N = 4) 88 ± 3(N = 4) 98 ± 2(N = 4) 41 ± 18(N = 4) 79 ± 10(N = 4) 98 ± 2(N = 4)
    2.2 Heparin vs. other anticoagulants
    Figure 3 shows that heparin and its derivatives (at a concentration of 1 IU / ml or
    100 IU / ml) cannot be replaced by other anticoagulants such as EDTA 5 2 mg / ml (E8008, Sigma-Aldrich, lot RNBBB7793), Actilyse® 0.1 mg / ml (Boehringer Ingelheim, lot
    001 408).
    2.3 Plasma vs. serum
    FIG. 4 shows that the S / D plasma can be replaced by serum to generate osteoforming cells derived from MSCs according to the invention. Consequently, it appears that heparin or analogues thereof are essential elements in the process of preparing osteoforming B cells.
    Example 3: In Vitro Mineralization Capacity of Cells Derived from CSM B Obtained by the Process of Example 1
    1. Experimental procedures
    The mineralization assay evaluates the capacity of cells to generate a mineralized matrix in vitro by culturing them in osteogenic medium for several weeks. The mineralized matrix is then stained using alizarin S red (ARS) staining.
    Briefly, the osteoforming B cells derived from MSC obtained after culture co8me 18/5662 described in section 1.1 of test 1 above are collected and spread in a basic αMEM medium (Lonza) supplemented with penicillin-streptomycin (Lonza) and 5% serum per well at 60,000 cells / cm 2 in a 48-well plate until they reach confluence (1 to 2 days). Then, the medium is replaced by an osteogenic medium comprising basic α-MEM medium (Lonza) supplemented with penicillin-streptomycin (Lonza), 5% serum, dexamethasone 10 -8 M (Sigma), 50 μg / ml of acid. ascorbic (Sigma) and 5 mM βglycerophosphate (Sigma). The osteogenic medium is replaced every 3 ± 2 days by freshly prepared osteogenic medium.
    ARS staining is performed on day 21 and on day 28 after osteogenic induction, as follows: the cells are washed with phosphate-buffered saline, incubated with 4% formaldehyde at room temperature for 15 minutes, washed with phosphate-buffered salt and then washed with demineralized water. The cells are then exposed for 10 minutes at room temperature to an ARS solution (20 g / l) pH 4.2. The cells are washed with demineralized water until the washing solution is clear, and observed macroscopically and microscopically. The wells are placed under an inverted microscope (AE31; Motic). The images taken with a camera (Moticam) placed on the microscope are analyzed in order to evaluate the red-orange coloration of the calcium deposited in the wells.
    2. Results
    The macroscopic and microscopic observations show a positive ARS staining, with an increase in the ARS staining from day 21 to day 28 and, consequently, an increase in the calcium / phosphate deposit over time (FIG. 5). These results show that osteoforming B cells generated with FGF-2, TGF-ß1 and heparin are able to synthesize a bone matrix and to mineralize it by deposition of calcium and phosphate. More particularly, these results show that the osteoforming B cells have high osteogenic properties.
    Example 4 Capability of In Vitro Chondrogenesis of Cells Derived from CSM B Obtained by the Method of Example 1
    1. Experimental procedures
    Under specific culture conditions, cells derived from MSCs obtained by the method of Example 1 can undergo differentiation into chondrocytes. These conditions include the three-dimensional conformation of cells into aggregates where high cell density and intercellular interactions contribute to the mechanism of chondrogenesis. Briefly, the osteoforming cells B derived from MSC obtained after secondary culture as described in section 1.1 of Example 1 above are collected and resuspended in chondrogenic differentiation medium and placed in B of 18/5662 96-well plates (non-adherent conical bottom) at a density of 2.5 x 10 5 cells / well. The chondrogenic culture medium consists of Eagle medium modified by Dulbecco (DMEM), rich in glucose (4.5 g / l) (DMEM-HG, Lonza) supplemented with 10% human insulin, human transferrin, and sodium selenite (ITS) (ITS + 1, Sigma-Aldrich), 1% sodium pyruvate (Lonza), dexamethasone 10 -7 M (Sigma), ascorbic acid 1 pM (Sigma) and 10 ng / ml of TGF-ß1 (HumanZyme). The negative control consists of chondrogenic medium without soluble differentiating factors, dexamethasone, ascorbic acid and TGF-ß1. Multiwell plates are then centrifuged at 200 xg for 5 min to form cell aggregates and are then placed at 37 ° C in a humidified atmosphere of 95% air and 5% CO 2 for 3 weeks. The chondrogenic culture medium is replaced every 2 or 3 days. Macroscopic observation 24 hours after the formation of aggregates shows that cell aggregates freely float in the culture media.
    Three weeks after the induction of chondrogenesis, the cell aggregates are collected and treated for histological analysis: the collected cellular aggregates are fixed in 3.7% formaldehyde and included in paraffin wax. Paraffin blocks are cut into 5 μm sections. The sections are stained with (i) hematoxylin and eosin (H&E), (ii) toluidine blue and orange saffron to stain the proteoglycans and (iii) sirius red to stain the collagen fibers . The process consists of standard dewaxing, staining, dehydration and mounting on a glass slide. The colored sections are observed qualitatively under the microscope (cellularity, location and appearance of cells, appearance of the extracellular matrix and content of collagen and proteoglycans).
    2. Results
    Microscopic observations and measurements of the diameter of the aggregates indicate that the aggregates of cells cultivated in chondrogenic medium have a larger size than that of the aggregates of cells cultivated in a control medium (data not shown). These size increases in chondrogenic medium could be associated with (1) the production and accumulation of extracellular matrix by cells and / or (2) cell proliferation in the aggregate.
    Qualitative observation of sections of aggregates stained with hematoxylin and eosin highlights differences in cell morphology between the control medium and the chondrogenic medium (H&E staining, FIG. 6). In fact, in the control medium, “micronuclei” are observed and it is difficult to observe the cell cytoplasm. While in chondrogenic medium, two types of cells can be observed: cells with a flattened nucleus (at the periphery of the aggregates) and, as one moves away from the periphery, cells with a rounded nucleus. The chondrogenic differentiation is confirmed by the coloring of the proteoglycans and of the collagen of the cartilaginous extracellular matrix. In comparison, the control aggregates 18/5662 show no positive staining for all the stains tested (staining with toluidine blue, orange safranine and sirius red, FIG. 6).
    These results show that the osteoforming B cells generated with FGF-2, TGF-βΙ and heparin are capable of producing an abundant extracellular matrix mainly composed of molecules specific to cartilage such as collagen and proteoglycans, when they are cultured in a chondrogenic medium.
    Example 5: In Vivo Safety of Osteoforming Cells A and B Obtained by the Method of Example 1
    1. Experimental procedures
    1.1 Cell culture and plasma preparation
    The cell culture and the plasma preparation are carried out as described in Example 1.
    Prior to administration, osteoforming cells A and B are assessed for viability, cell size, identity (including expression of surface antigens by flow cytometry analysis, ALP enzyme activity and assay by ALP staining) and sterility.
    1.2 Toxicity study in mice
    Twelve week old male and female NMRI-nude mice are given one of the following products by intravenous injection: (i) osteoforming A cells (5 million cells suspended in 200 µl of excipient), (ii) osteoforming A cells (5 million cells suspended in 200 μl of excipient) with heparin (Heparin LEO 100 IU / ml, LEO Pharma SA, lot A17605; 4 units) or (iii) osteoforming B cells (5 million cells in suspension in 200 μl of excipient). The control consists of 200 μl of excipient (alone). The administration of the control and of the experimental products is carried out in the form of a slow bolus, by intravenous injection into the lateral vein of the tail. The duration of the injection is at least 60 seconds. The quantity of cells and the volume administered per animal are constant.
    The animals are observed for a period of up to 6 months, and several parameters are monitored and / or evaluated during the follow-up, among others: mortality and morbidity, body weight of the animals, clinical observations / physical examination, haematological and chemical analysis of the blood and organ collection for histopathological analyzes after euthanasia of animals.
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    1.3 Histopathological analysis
    The lungs of the mice are collected for histopathological analysis. The lungs of the collected mice are treated for histopathological analysis: the samples are dehydrated and included in paraffin wax. Sections are cut 3 to 5 μm thick (cross sections) and stained for hematoxylin and eosin. The slides are submitted to a qualified pathologist to determine the cause (s) of acute mortality.
    2. Results
    2.1 Acute toxicity
    A toxicity study is being conducted to assess the possible adverse effects of intravenous injection of osteoforming A cells (generated with FGF-2 and TGF-βΙ) and small osteoforming B cells (generated with FGF-2, TGF-βΙ and heparin) in mice.
    As described in Table 16, acute mortality (10-35%) is observed after intravenous administration of osteoforming A cells (A-1 and A-2), and the addition of heparin anticoagulant (4 units) in the suspension of cells does not decrease this mortality (A-3 to A-5 with heparin). On the other hand, no acute toxicity is observed after intravenous administration of control (excipient) and of small osteoforming B cells (B-1 to B-4).
    Table 16: Cell size profile of osteoforming cells A and B and acute toxicity observed after intravenous injection in mice
    Batch of osteoforming cells Average (μm) Max (μm) Min (μm) Number ofcell> 30 μm acute toxicity A-1 ND ND ND ND 10% (4/40) A-2 ND ND ND ND 35% (14/40) A-3 + heparin 21.7 34.9 14.1 1 cell / 20 0% (0/20) A-4 + heparin 38.2 57.6 18.6 17 cells / 20 21% (4/19) A-5 + heparin 26.8 53.6 53.6 5 cells / 28 75% (3/4 *) B-1 17.5 29.5 11.6 Any 0% (0/19) B-2 21.5 30.8 16.9 1 cell / 20 0% (0/20) B-3 14.3 21.2 8.7 Any 0% (0/18) B-4 17.7 25.0 13.3 Any 0% (0/17)
    * The injections were stopped after the death of 3 animals (out of the 15 planned).
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    ND: not determined
    2.2 Histopathological examination
    After euthanasia, an autopsy is performed on all animals. The mice having received an injection of small osteoforming B cells have a generally normal pulmonary architecture, without observation of cellular embolization of the alveolar or bronchiolar capillaries (FIG. 7A). Mice injected with osteoforming A cells have pulmonary lesions characterized by moderate to severe disseminated embolization of the alveolar and bronchiolar capillaries by a high number of medium-sized cells (interpreted as infected cells) frequently accompanied by inflammation acute interstitial (Figure 7B): 30% to 50% of the alveolar capillaries and small to medium-sized bronchiolar arterioles are randomly dilated by groups of cells that block all of the vascular lumen. Larger groups of occluded capillaries compress the bronchioles in their vicinity and are surrounded by alveolar collapse. Intra-alveolar and intrabronchiolar microhemorrhages are rarely seen near groups of cells.
    The main characteristic of all the samples observed is a moderate to severe disseminated embolization of the alveolar and bronchiolar capillaries by a high number of injected cells (osteoforming cells A). The number of these cells, as well as the number of occluded alveolar capillaries suggests that the gas exchanges in the alveoli could be strongly disturbed, leading to collapse of the respiratory system. It is very likely that this process is responsible for the death of the animals examined. The vascular congestion and microhemorrhages observed are interpreted as agonal changes.
    No microthrombus formation has been observed in the heart, liver, kidneys or spleen.
    Example 6 Properties of In Vivo Bone Formation of Osteoforming Cells A and B Obtained by the Process of Example 1
    The skull bone formation model consists of a single subcutaneous administration of 2.5 x 10 6 osteoforming cells, formulated in 100 μl of excipient (or 100 μl of excipient alone as a negative control) on the cranial vault. 12-week-old female NMRI-nude mice. At specific times, fluorescent dyes that fix calcium (i.e., alizarin red, calcein green, calcein blue and tetracycline) are administered to mark new bone formation. Alizarin red is administered before the administration of osteoforming cells, while calcein green, calcein blue and tetracycline are administered after the administration of osteoforming cells. Laboratory animals are monitored for body weight, general clinical signs and clinical signs at the site of administration for 2 weeks after administration.
    After 2 weeks, the laboratory animals are euthanized to evaluate the E properties of 18/5662 bone formation of the osteoforming cells by radiography, histomorphometry (quantification of bone formation) and immunofluorescence.
    1. Experimental procedures
    1.1 Cell culture and plasma preparation
    The cell culture and the plasma preparation are carried out as described in Example 1.
    1.2 Mouse
    Female NMRI-nude mice (nu / nu) from 9 to 10 weeks are purchased from Janvier S.A.S. (Le Genest-St-Isle, France) and housed under standard conditions with unlimited food and water. A total of 196 mice are used for this study.
    1.3 Model of skull bone formation in mice
    Twelve-week-old female NMRI-nude (nu / nu) mice twelve weeks old (n = 137) are anesthetized with isoflurane (IsoFlo®) and receive a single subcutaneous administration of MSC, osteoforming A cells (generated with FGF -2 and TGF-ß1) or osteoforming B cells (generated with FGF-2, TGF-ß1 and heparin) (2.5 x 10 6 cells in 100 μl per mouse) or an excipient (100 μl) on 1 'bones of the cranial vault. To mark new bone formation over time, calcium binding fluorochromes are administered sequentially to mice. Alizarin red (red), calceins (green and blue) and tetracycline (yellow) (all of Sigma-Aldrich®) are injected intraperitoneally 3 days before and 4, 8 and 12 days after administration of cells, respectively. Laboratory animals are monitored for body weight, general clinical signs and clinical signs at the site of administration for 2 weeks after administration. The mice can be euthanized 2 weeks after the administration of cells by cervical dislocation and the cranial vault of each mouse is collected to evaluate the properties of bone formation by histomorphometry (quantification of bone formation) and immunofluorescence.
    1.4 Sample inclusion and histological sectioning
    For histomorphometry, ALP, TRAP (tartrate resistant acid phosphatase), Masson-Goldner trichrome stains and immunofluorescence, the cranial vaults are fixed and dehydrated with successive incubations in a 70% ethanol bath, 80% and 90%, for 12 hours at 4 ° C with gentle stirring, and included in plastic resin of hydroxyethyl methacrylate (HEMA) (HistoResin, Leica®). Coronal sections of 4 μm and 8 μm thickness are cut using a microtome (Leica®, RM2255). For staining with orange saffron and immunoperoxidase, the cranial vaults are fixed in 3.7% formaldehyde for 24 hours, decalcified in thick acid.
    BE2018 / 5662 10% ethylenediaminetetraacetic acid (EDTA) pH 7.4 for three days and included in paraffin. Coronal and sagittal paraffin sections of 7 μm cut using a microtome (Leica®, RM2255).
    1.5 Immunofluorescence staining
    The evaluation of human and murine collagen I by immunofluorescence is carried out on coronal plastic histological sections 4 μm thick in the cranial vault. Briefly, after a permeabilization step using a PBS 1X / 0.3% Triton solution for 30 min at room temperature (RT), the histological sections are incubated for 1 hour at RT in the blocking solution (i.e. , PBS / BSA / horse serum / Triton TM ) to saturate non-specific binding sites. The histological slides are then incubated overnight at 4 ° C. with primary antibodies from human anti-collagen I and rabbit mice (Abcam; no. Ab138492 and Abcam; no. Ab21286, respectively). After 3 rinsing steps in PBS for 5 min at RT, blocking is carried out with the blocking solution for 1 hour at RT. The secondary antibodies diluted in the blocking solution are then added for 2 hours at RT in the absence of light. Donkey anti-rabbit IgG secondary antibodies Alexa Fluor® 488 H&L (ThermoFisher, no. A21206) and goat anti-mouse IgG goat Alexa Fluor® Cy3® H&L (Abcam; no. Ab97035) are used to visualize collagen I murine in green and I human collagen in red, respectively. The slides are then rinsed 3 times in 1 × PBS for 5 minutes at RT and incubated with a NucBlue® solution for 1 minute at RT to stain the nucleus. Finally, the slides are briefly rinsed once in PBS and then mounted in the GlycerGel ® reagent. As a negative immunofluorescence control, the omission of the primary antibody is carried out on an adjacent histological slide.
    1.6 Histological staining
    The osteoblastic and osteoclastic activities are evaluated on cranial vaults, respectively, using methods of detection of ALP and TRAP enzymatic activity, respectively. For ALP staining, coronal plastic sections of the cranial vault 4 μm thick are incubated for 1 hour with a solution of solid blue salt RR (Sigma-Aldrich®) and alkaline phosphate of naphthol AS-MX ( Sigma-Aldrich®). TRAP staining is carried out on coronal plastic sections of the cranial vault 8 μm thick using the Acid Phosphatase, Leukocyte (TRAP) kit (Sigma-Aldrich®) in accordance with the manufacturer's instructions. In order to assess the state of mineralization of the newly formed bone, Masson-Goldner trichrome staining is performed on the cranial vault sections stained with ALP using a kit (Bio-Optica®) in accordance with the instructions. from the manufacturer. In order to highlight the formation of cartilage, an orange saffron staining is carried out on sagittal paraffin sections of the cranial vault 7 μm thick. Briefly, after dewaxing, the histological sections are successively incubated in Weigert hematoxylin (Klinipath®) for 10 minutes, 0.1% solid green (Klinipath®) for 5 min, 1% acetic acid (VWR Chemicals ) for 15 seconds and 0.1% orange saffron. (Fluka® ref. 84120) for 5 min. After dehydration, the slides are mounted with glass slides using Pertex® (HistoLab®). Digital images are taken with an optical microscope (Leica®) and Leica® LAS EZ software.
    1.7 Immunoperoxidase
    After dewaxing, coronal or sagittal paraffin sections of the cranial vault 7 μm thick are successively incubated with 2.5% hyaluronidase (SigmaAldrich®) for 30 min at 37 ° C in 3% H2O2 (Sigma -Aldrich®) for 30 min at room temperature, in PBS containing 0.3% Triton X-100 (Sigma-Aldrich®) for 30 min at room temperature and in a blocking solution (i.e. PBS / BSA / horse serum / Triton) for 1 hour at room temperature. The sections are incubated overnight at 4 ° C. with primary antibodies of human anti-collagen type I mice (Abcam, ab90395), primary rabbit antibodies against murine anti-collagen I (Abcam, ab21286) or primary antibodies of anti-Ku80 rabbit (Abcam, ab80592). The staining is visualized using a Vectastain kit (Vector Laboratories, PK6200) and 3,3'-diaminobenzidine (Vector Laboratories), in accordance with the manufacturer's instructions. The sections are counterstained with Mayer's hematoxylin (Klinipath®). The slides are mounted with glass slides using Pertex ® .
    1.8 Histomorphometric analyzes of cranial vaults: quantification of bone formation
    Quantification of bone formation (i.e., percentage of bone formation) is performed on tissue included in plastic. The measurements of the initial thickness (front of basal mineralization marked by fluorescence with alizarin red) and final (bone neoformation marked by fluorescence with calcein and tetracycline) of the cranial vaults are measured (in μm) on a coronal section of 4 μm thick using ZEN® image analysis software (Zeiss). The initial and final thicknesses of the cranial vault are then used to calculate the percentage of new bone formation in each laboratory animal after administration. For each animal, 4 initial and final thickness measurements are made on 5 independent levels, with a distance of 200 μm between each level. Initially, the average of the initial and final thicknesses ± ET (that is to say the average of the 4 measurements per level on the 5 levels) is calculated for each animal. Next, the percentage of bone formation for each mouse is calculated as the ratio of the average final thickness to the average initial thickness.
    1.9 Quantification of the area of newly formed bone on histological images 18/5662 (ImageJ® software)
    For the analysis of the surface of osteoinduction and osteogenic nodules, digital images of 6 independent levels, taken every 2 levels after the coronal suture are taken on histological sections in plastic resin (4 μm) of the cranial vault, using a combination of several fluorescence and bright field filters from the fluorescence microscope (Zeiss Axioscope A1, Zeiss, Germany). At each level measured, the selection of osteoinduced bone neoformation is manually defined in images assembled in bright fields using ImageJ® software. The mineralized and total surfaces of this selection are measured (in mm 2 ). The same procedure is carried out for the mineralized and total surfaces of the osteogenic nodules.
    For osteoinduction and osteogeny nodules, the average of the total surface and the average of the mineralized surface are then calculated by laboratory animal and by group. The total area of new bone formation is finally calculated as the sum of the area of osteoinduction and osteogenic nodules.
    1.10 Statistical analyzes
    The results are expressed as the mean ± standard error on the mean (SEM). Statistical analyzes are performed using JMP® software (SAS Institute Inc.). For in vitro data (flow cytometry, RT-qPCR and multiplex), paired t tests are performed on the values transformed into log10 and for in vivo data, Mann-Whitney U tests are used. Unless otherwise noted, differences between groups are considered to be statistically significant when p <0.05.
    2. Results
    Osteoforming cells A (generated with FGF-2 and TGF-βΙ) and osteoforming cells B (generated with FGF-2, TGF-βΙ and heparin) have a significantly higher bone formation than the controls (excipient) 2 weeks after administration (Figures 8-9, Table 17). More particularly, FIG. 10 shows that the osteoforming B cells have osteoinductive properties (homogeneous bone formation of murine origin on the cranial vault) and osteogenic properties (mineralized nodules of human and murine origin).
    Table 17: Quantification of bone formation (%) on murine cranial vaults. The cranial vaults are treated with an excipient (negative control), osteoforming cells A or osteoforming cells B.
    Number of lots Number of animals % of training
    8/5662
    LJI 2 UboneAverage ± AND excipient - 59 107 ± 2 Osteoforming cells A 10 39 165 ± 19 B osteoforming cells 7 30 158 ± 23
    Abbreviations: AND: standard deviation
    The osteoinductive properties (i.e., the amount of newly formed murine bone post implantation) are equivalent for osteoforming cells A and B (Figures 8 to 9).
    It is very interesting to note that the osteoforming B cells of the present invention exhibit powerful osteogenic and osteoinductive properties, as indicated by the high quantity of newly formed human and murine bone after implantation (coloration of human and murine Coll IF, figure 10).
    The presence of nodules is observed in 7/8 donors and 80% of the mice for osteoforming cells B and 4/11 donors and 20% of the mice for osteoforming cells A.
    No nodule is observed after administration of CSM or excipient. In addition to the osteoinduction activities, the osteoforming B cells therefore promote a high osteogenic activity evidenced by the presence of large mineralized nodules observed in 80% of the treated mice, while the osteoforming A cells exhibit a low osteogenic activity, c ie small nodules in only 20% of the treated mice (Table 18).
    Table 18: Quantification of the presence of mineralized nodules on murine cranial vaults two weeks after administration onto the cranial arch of excipient, MSC, osteoforming cells A or osteoforming cells B
    Occurrence of osteogeny Giver Lot Animal excipient SO SO 0/32 (0%) CSM 0/2 0/2 0/14 (0%) Osteoforming cells A 4/10 (40%) 4/11 (36%) 9/45 (20%) B osteoforming cells 7/8 (88%) 7/8 (88%) 37/46 (80%)
    Abbreviations: CSM: mesenchymal stem cells; N / A: not applicable
    BE2018 / 5662 Histological staining of murine cranial vault coronal sections two weeks after administration (excipient only, MSC, osteoforming cells A (generated with FGF-2 and TGF-P1; cells of A) or osteoforming cells B (generated with FGF- 2, TGF-P1 and heparin; cells of B)) indicates that all of the conditions treated (MSC, cells of A and of B) have a high potential for osteoinduction with moderate remodeling activity (ALP staining and TRAP) in the bone formed by osteo-induction.
    It is interesting to note that the mineralized nodules observed in mice treated with osteoforming B cells consist of murine (host) and human (donor) bone tissue (indicated by a coloration of human and murine type I collagen), which demonstrates that nodules are formed by bone formation processes: osteogeny (donor bone formation) and osteoinduction process (host bone formation). In addition to high osteoblastic and osteoclastic activities (ALP + TRAP staining), the nodules have osteoid tissue (non-mineralized tissue), which suggests that bone formation continued to progress two weeks after administration, while the process of The osteoinduction observed in all conditions was already completed (Figure 12).
    Figure 12 shows that human bone formation (i.e., osteogenesis) (observed with human anti-collagen type I staining) and high activities of osteoblasts and osteoclasts (observed with ALP + Goldner staining and TRAP staining, respectively) are mainly detected in the nodules of mice having received osteoforming B cells, which demonstrates that the process of bone formation in the nodules was in progress and was not finished after 2 weeks , unlike the osteoinduction process of MSC and osteoforming A cells, which seemed to be finished. All the conditions treated (MSC, o-f A cells, o-f B cells) indicate a high potential for osteoinduction with moderate remodeling activity (ALP and TRAP staining) in osteoinduced bone formation (FIG. 12).
    Bone neoformation is evaluated by fluorescence two weeks after treatment with an excipient alone, MSCs, o-f A or o-f B cells (FIG. 13). For this purpose, at specific times, fluorescent dyes binding to calcium (namely alizarin red, green and blue calcein, tetracycline yellow) are administered to mark new bone formation. The last fluorochrome administered is tetracycline, 12 days after administration of the cells.
    As indicated in FIG. 13, the nodules of the mice having received osteoforming B cells are mainly stained with fluorochrome tetracycline (the yellow coloration is dotted with a dotted line in FIG. 13), which confirms a stage of later formation compared to osteoinduction observed in osteoinduced bone formation (alizarin red (red), calcein (green and blue): these colors appear in light gray and a double arrow indicates the thickness of bone formation).
    BE2018 / 5662 The bone neoformation of the treated mice is evaluated by a quantification of the area of newly formed bone on histological images (ImageJ® software). The total area of newly formed bone is determined by adding the osteoinduced surface areas and bone nodules for each level analyzed and each mouse.
    The results show that the osteoforming B cells (n = 7 mice, represented in FIG. 14 in light gray) significantly increase bone neoformation 2 weeks after the administration of the cells, by approximately 2 times compared to MSCs (n = 6 mice, shown in Figure 14 in dark gray; table 19). This difference was due to the property of elevated osteogeny presented by the osteoforming B cells and to the absence of this property in MSCs.
    Table 19: measurement of total bone neoformation including osteoinduction and osteogenic formation on coronal sections
    Cell type (from the same donor) Number of anim Osteoinduction Osteogeny (nodules) Total (osteo-induction+ osteogeny) Mineralized area e (mm 2 ) (mean ± SD) Total area(mm2) (average± AND) Mineralized area e (mm2) (mean ± SD) Total area(mm2) (mean ± SD) Mineralized area e (mm2) (mean ± SD) Total area(mm2) (mean ± SD) CSM 6 0.42 ±0.09 0.57 ±0.17 0 0 0.42 ±0.09 0.57 ±0.17 cell s o-f B 7 0.43 ±0.16 0.59 ±0.25 0.22 ±0.19 0.57 ±0.53 0.65 ±0.30 1.16 ±0.71
    Abbreviations: CSM: mesenchymal stem cells; AND: standard deviation
    In addition, the evaluation of bone neoformation over time using histological staining indicates that the nodules observed at the top of the cranial vault of mice having received osteoforming B cells undergo ossification by means of a mechanism. endochondral ossification. In FIG. 15, the coloring with orange safranine indicates a matrix of proteoglycans (specific to cartilage) (area surrounded by a dotted line); the kernels ; bone tissue; and the cytoplasm. Unlike osteoinduced bone, which is produced by intramembrane ossification, bone nodules are produced by endochondral ossification, the cartilaginous matrix appearing between 1 and 3 weeks after administration (Figure 15).
    Immunohistochemical stains targeting human type I collagen, murine type I collagen, and the human nucleus (i.e., Ku80) performed 4 weeks after administration of osteoforming B cells confirm the presence of human bone in ^ es 18/5662 nodules. In addition, the staining of Ku80 indicates that osteoforming B cells are grafted into the bone matrix (nodules) and become osteocytes after administration in vivo.
    Example 7: Subcritical Size Defect (Sub-CSD) in Femoral Segmentation of Mice Repaired in Vivo Repaired by Osteoforming Cells Obtained by the Process of Example 1
    1. Experimental procedures
    1.1 Subcritical size defect model (sub-CSD) of femoral segmentation
    The surgical intervention is performed under aseptic conditions according to the literature (Manassero et al., 2013, Tissue Engineering, Part C Methods, 19 (4): 271-80; Manassero et al., 2016, Journal of Visualized Experiments ; (116): 52940). Briefly, 13-week-old female NMRI-nude (nu / nu) mice (n = 27) are anesthetized with an intraperitoneal injection of a mixture of dexmedetomidine hydrochloride (Dexdomitor®, Orion Pharma, 1 mg / kg body weight ) and ketamine (Nimatek®, Euronet, 150 mg / kg body weight) and are placed in the prone position on a hot plate. After applying a 6-hole titanium micro-locking plate (RISystem AG®) to the anterior surface of the left femur, a 2 mm long diaphyseal femoral osteotomy is performed using a Gigli saw and a caliper (RISystem AG®). As a preventive treatment, antibiotics (Baytril ® , 10 mg / kg of body weight) are administered the day before the surgery (in drinking water) and analgesics (buprenorphine hydrochloride, Temgesic ® , Schering-Plow, 0, 1 mg / kg body weight) are administered the day before surgery and every 12 hours for at least 3 days after surgery. Cells derived from MSC (2.25 x 10 6 cells in a volume of 30 µl per mouse) or the excipient (control group) are administered on the day of surgery (just after the wound is closed with surgical sutures) , locally at the bone defect site, by percutaneous injection using a 50 pl Hamilton syringe. The mice are euthanized 6 weeks after the administration of cells or of excipient by cervical dislocation. The left femur of each mouse is dissected, collected and stored in 0.9% NaCl at room temperature until radiography.
    1.2 Quantification of bone repair by radiography
    In vivo radiographic imaging of the left femur of each mouse is carried out using the Faxitron® MX-20 device just after the operation in order to control the fixation of the plate, the size of the segmental femoral defect and to obtain a reference, every two weeks up to 6 weeks after the administration of cells or derivatives of MSC or of excipient. Digital images are taken in medio-lateral and anteroposterior views at 4x magnification, in manual mode, with a voltage set to 35 kV, an exposure time
    BE2018 / 5662 of 4.8 seconds, a brightness of 4,300 and a contrast of 7,100. Ex vivo radiography imaging is performed on the left femurs taken during euthanasia, 6 weeks after the administration of the cells. Digital images are taken in medio-lateral and antero-posterior views at 5x magnification, in manual mode, with a voltage set to 26 kV, an exposure time of 15 seconds, a brightness of 4,850 and a contrast of 6 850. The defect size is quantified over time for each mouse by measuring the distance (μm) between the two edges of the bony defect at three locations (right, center and left of the defect) on medio-lateral and antero radiographic images. - later, using ImageJ® software. The average of the six measurements is calculated for each mouse at each time.
    1.3 Analysis by microtomodensitometry (micro-CT)
    After collection at euthanasia, the left femurs are fixed with 3.7% formaldehyde and transferred to the Center for Microscopy and Molecular Imaging (CMMI, ULB, Gosselies, Belgium) for micro-CT analyzes. The samples are examined using a multimodal nanoScan® PET / CT camera (Mediso) and Nucline ™ v2.01 software (Mediso). The scans are acquired using a semicircular scan, maximum zoom, a tube voltage of 35 kVp, 720 projections per rotation of the gantry, an exposure time of 300 ms per projection and a partitioning of the detector pixels 1: 1. The scanning lengths in the X and Y dimensions are adapted for each acquisition. The total duration of the micro-CT is 3 min 42 s. Each micro-CT scan is post-reconstructed with a cubic voxel of 40 μm side by means of a Shepp-Logan filter and a multi-sampling mode of 8 regular samples. The dimensions of the X and Y images are adapted for each reconstruction. The size of the Z images corresponds to the scanning length defined for the acquisition. A qualitative evaluation of bone repair is performed on micro-CT images after reorientation of the bone with the Z axis (axis of the scanner) and cropping of the image from one proximal screw to the other proximal screw in the femoral bone on the Z axis, and as narrow as possible in the transverse plane (XY). Then, a maximum intensity projection (MIP) 3D rendering is generated. In order to quantitatively assess bone repair, a virtual cylinder 2 mm in diameter and 2 mm in axial length is placed in the defect space on micro-CT scans and the average bone volume is evaluated in this cylinder by thresholding of voxels with a radiological intensity greater than 1500 HU.
    2. Results
    2.1 B osteoforming cells improve the repair of subcritical defect in femoral segmentation in mice
    In the subcritical size segmentation defect (CSD) model in NMRInude mice, osteoforming B cells (n = 12 mice, 2 lots) make it possible to improve fracture repair as indicated by a significant reduction in defect size bone 3 by 18/5662 compared to the excipient (n = 11 mice) and to the osteoforming cells A (n = 4 mice) from 2 to 6 weeks after the administration (FIG. 16A).
    Radiographic images of segmental femoral defects on D0 and 6S after the administration of the excipient, osteoforming cells A (not shown) or osteoforming cells B produce a reduction in the size of the bone defect in mice receiving osteoforming cells B according to one embodiment of the invention compared to the mice having received the excipient (FIG. 16B) or osteoforming cells A (not shown).
    The bone repair volumes of femoral segmentation defect are quantified by microtomodensitometry analyzes (micro-CT) at 6S after the administration of the excipient and of osteoforming B cells. The results confirm that the osteoforming B cells induce bone repair greater than that of the excipient (Figure 16C).
    权利要求:
    Claims (15)
    [1]
    1. Method for obtaining cells derived from mesenchymal stem cells (CSM) from CSM, said cells derived from CSM being cells of mesenchymal line obtained by differentiation of CSM in vitro or ex vivo, comprising contacting CSM in vitro or ex vivo with fibroblast growth factor-2 (FGF-2), transforming growth factor beta (TGF-β) and heparin or a derivative or analog thereof at a concentration of at least minus 0.01 IU / ml.
    [2]
    2. Method according to claim 1, comprising the steps of:
    (a) culture of MSCs recovered from a biological sample of a subject in a medium comprising FGF-2, TGF-β and heparin or a derivative or analog thereof thereof at a concentration of at least 0.01 IU / ml;
    (b) elimination of the non-adherent material and further culture of the adherent cells in the medium comprising FGF-2, TGF-β and heparin or a derivative or analog thereof at a concentration of at least 0 , 01 IU / ml, so as to obtain the cells derived from MSC.
    [3]
    3. Method according to claim 1 or 2, in which TGF-β is chosen from the group consisting of TGF-β! TGF ^ 2, TGF ^ 3, and mixtures thereof; preferably where TGF-β is TGF-β!
    [4]
    4. A method for obtaining cells derived from CSM having improved transplantation properties from CSM, the method comprising a size reduction step, in which said size reduction step is characterized by bringing CSM into contact or cells derived from MSC in vitro or ex vivo with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml.
    [5]
    5. Method according to any one of claims 1 to 4, in which:
    - the concentration of heparin or of a derivative or analog thereof is at least
    0.05 IU / ml, preferably about 0.1 IU / ml; and or
    - heparin or the heparin derivative or the analog thereof is chosen from the group consisting of an unfractionated heparin (HNF); a low molecular weight heparin (LMWH), such as enoxaparin, dalteparin, nadroparin, tinzaparin, certoparin, reviparin, ardeparin, parnaparin, bemiparin, or mixtures thereof ; a heparinoid, such as heparan sulfate, dermatan sulfate, chondroitin sulfate, acharan sulfate, keratan sulfate, or mixtures thereof, such as danaparoid; a heparin salt; a heparinoid salt; a heparin fragment; a heparinoid fragment; and mixtures thereof.
    BE2018 / 5662
    [6]
    6. Method according to any one of claims 1 to 5, in which at least 60% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm) and in which at most 5% cells derived from CSM in suspension have a diameter greater than 35 μm.
    [7]
    7. Method according to any one of claims 1 to 6, in which the cells derived from MSC are of osteochondroblastic line, preferably osteoblastic.
    [8]
    8. Method according to any one of claims 1 to 7, in which the CSMs or cells derived from CSMs are further brought into contact with (for example when the medium also comprises one or more of) plasma, serum or a substitute for these.
    [9]
    9. A method for obtaining cells derived from CSM from CSM comprising bringing CSM into contact in vitro or ex vivo with FGF-2, TGF-β and heparin or a derivative or analog thereof, in which at least 60% of the cells derived from CSM in suspension have a diameter equal to or less than 25 μm (D 60 <25 μm) and in which at most 5% of the cells derived from CSM in suspension have a diameter greater than 35 μm .
    [10]
    10. The method according to claim 9, in which:
    - MSCs are brought into contact with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml; and or
    - TGF-β is chosen from the group consisting of TGF-ß1, TGF-ß2, TGF-ß3, and mixtures of these; preferably in which TGF-ß is TGF-ß1.
    [11]
    11. Population of cells derived from MSC, which are cells of mesenchymal line obtained by differentiation of MSC in vitro or ex vivo, in which at least 60% of the cells derived from MSC in suspension have a diameter equal to or less than 25 μm ( D 60 <25 μm) and in which at most 5% of the cells derived from MSC in suspension have a diameter greater than 35 μm.
    [12]
    12. Population of MSC-derived cells according to claim 11, in which:
    - cells derived from CSM can be obtained by a method comprising bringing CSM into contact in vitro or ex vivo with FGF-2, TGF-ß and heparin or a derivative or analog thereof, preferably in which TGF-ß is selected from the group consisting of TGF-ß1, TGF-ß2, TGF-ß3, and mixtures thereof, more preferably in which the TGF-ß is TGF-ß1, preferably in which the MSCs are contacted with heparin or a derivative or the like thereof at a concentration of at least 0.01 IU / ml, and optionally the MSCs are further contacted with one or more of plasma, serum or a substitute thereof; and or
    BE2018 / 5662
    the cells derived from CSM are of osteochondroblastic or osteoblastic line, preferably in which substantially all the cells derived from CSM of osteochondroblastic or osteoblastic line are positive for CD73, CD63 and CD166; substantially all cells derived from MSC of osteochondroblastic or osteoblastic line are negative for CD45; at least 70% of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for alkaline phosphatase (ALP); and less than 10% of the cells derived from MSC of osteochondroblastic or osteoblastic line are positive for HLA-DR.
    [13]
    13. A pharmaceutical composition comprising the population of cells derived from MSC as defined in claim 11 or 12.
    [14]
    14. MSC-derived cell population according to claim 11 or 12 or a pharmaceutical composition according to claim 13 for use as a medicament, preferably for use in the treatment of a subject in need of a transplant of cells derived from MSC .
    [15]
    15. Population of cells derived from MSC for use according to claim 14, in which:
    the population of cells derived from MSC is present at a concentration of between approximately 1 × 10 7 cells / ml and approximately 1 × 10 8 cells / ml, preferably 7.5 × 10 7 cells / ml; and or
    - the population of cells derived from MSC or the pharmaceutical composition is suitable for percutaneous or intravascular administration.
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    EP3697894A1|2020-08-26|
    IL273987D0|2020-05-31|
    CA3079439A1|2019-04-25|
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    WO2019076591A1|2019-04-25|
    IL273987A|2021-06-30|
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    法律状态:
    2019-11-25| FG| Patent granted|Effective date: 20191018 |
    优先权:
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    EP17197605.3|2017-10-20|
    EP17197605|2017-10-20|
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