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    • 专利摘要:
    The present invention relates to the use of CD73, CD105, CD44 and / or CD10 to determine the osteogenic potential of differentiated cells in vitro. The invention further relates to a method for determining the osteogenic potential of differentiated cells in vitro comprising measuring the amount of differentiated cells in vitro expressing CD73, CD105, CD10 and / or CD44, and / or measuring the amount. of CD73, CD105 and / or CD44 expressed by differentiated cells in vitro. The invention also relates to a method of selecting a subject for the preparation of in vitro differentiated cells of a chondro-osteoblastic line comprising harvesting MSCs from a biological sample of a subject; obtaining differentiated cells in vitro from MSCs; determining the osteogenic potential of differentiated cells in vitro by a method described herein; and selecting the subject to prepare cells differentiated in vitro from chondro-osteoblast lines if the cells differentiated in vitro have osteogenically useful potential.
    公开号:BE1026600B1
    申请号:E20195630
    申请日:2019-09-25
    公开日:2020-12-07
    发明作者:Sandra Pietri；Troy Delphine De；Sylvain Normand；Laure Hertzog；Caroline Trus
    申请人:Bone Therapeutics Sa；
    IPC主号:
    专利说明:

    METHODS AND USES FOR DETERMINING OSTEOGENIC POTENTIAL
    FIELD OF THE INVENTION The invention relates to methods and uses for determining the osteogenic potential of differentiated cells in vitro. More particularly, the invention relates to methods and uses for determining the osteogenic potential of differentiated cells in vitro, comprising measuring one or more cellular markers. BACKGROUND OF THE INVENTION The transplantation of stem cells capable of undergoing osteogenic differentiation, of cells engaged in osteogenic differentiation or of cells having a capacity for bone formation is a promising route for the treatment of bone-related diseases, in particular when the treatment requires the production of new bone tissue.
    Mesenchymal stem cells (MSCs) have already been used to treat bone disorders - (Gangji et al., 2005 Expert Opinion Biol Ther 5: 437-42). However, although these relatively undifferentiated stem cells can be transplanted, they are not linked to an osteoblastic lineage and their contribution to bone tissue formation is believed to be primarily mediated by paracrine effects. In addition, the amount of CSM that can be obtained from subjects for therapeutic use is often unsatisfactory.
    Several methods of in vitro expansion of MSCs and of obtaining, from MSCs, osteoprogenitors, osteoblasts or cells of osteoblastic phenotype have been developed. Cells obtained by such methods can have variable osteogenic potential in vitro and in vivo. Therefore, the amount of new bone tissue produced in vivo upon transplantation of these cells is not always predictable and, in some cases, not optimal for clinical purposes.
    It is necessary to determine, prior to transplantation of cells cultured in vitro, such as cells derived from MSCs differentiated in vitro, whether the cells have clinically useful osteogenic potential.
    SUMMARY OF THE INVENTION As confirmed by the experimental part, which illustrates some representative embodiments of the invention, the inventors have realized that the osteogenic potential of differentiated cells in vitro, such as cells derived from mesenchymal stem cells (MSCs) , can be evaluated by determining a specific expression profile of the cell surface markers of said cells. More particularly, the inventors have found that by measuring the amount of differentiated cells in vitro which express one or more of CD73, CD105, CD10 or CD44, preferably all CD73, CD105, CD10 and CD44, and by measuring the amount of 'one or more of CD73, CD105 or CD44, preferably all CD73, CD105 and CD44 expressed by the cells, it can be determined whether the cells have osteogenic potential, more specifically the osteogenic potential which makes the cells useful in clinical settings .
    Further, the current inventors have also discovered that by using the method for determining the osteogenic potential of CSM-derived cells as described herein, it is possible to select a particularly suitable subject as a CSM donor to prepare cells derived from CSM of the chondro-osteoblastic line.
    Accordingly, in one aspect, the invention relates to the use of one or more of CD73, CD105 or CD44 to determine the osteogenic potential of differentiated cells in vitro.
    Preferably, the invention provides for the use of CD73, CD105, CD44 and CD10 to determine the osteogenic potential of differentiated cells in vitro.
    In another aspect, the invention relates to a method for determining the osteogenic potential of differentiated cells in vitro comprising measuring the amount of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44, and measuring the amount of differentiated cells in vitro. amount of one or more of CD73, CD105 or CD44 expressed by the differentiated cells in vitro.
    Preferably, the invention relates to a method for determining the osteogenic potential of differentiated cells in vitro comprising measuring the amount of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44, and / or measuring the amount of a or more of CD73, CD105 or - CD44 expressed by differentiated cells in vitro.
    In another aspect, the invention relates to a method of selecting a subject for the in vitro preparation of differentiated cells of the chrondro-osteoblastic lineage, the method comprising: collecting CSM from a biological sample of a subject: obtaining differentiated cells in vitro from CSM; - determination of the osteogenic potential of differentiated cells in vitro by a method as taught here; and selecting the subject for the preparation of in vitro differentiated cells of the chondroosteoblast line if the differentiated cells in vitro have clinically useful osteogenic potential.
    These aspects, as well as other aspects and preferred embodiments of the invention are described in the following sections and in the appended claims.
    The subject matter of the appended claims is expressly incorporated into this specification.
    DESCRIPTION OF THE FIGURES Figure 1 illustrates bone neoformation on a coronal section of a murine bone cranial vault, demonstrated by murine and human fluorochromes binding to calcium, 2 weeks after administration of excipient alone (control condition) , CSM-derived bone-forming A cells (generated with FGF-2 and TGFB1) or CSM-derived bone-forming B cells (generated with FGF-2, TGFB1 and heparin). Figure 2 illustrates the quantification of bone formation (%) performed on coronal sections of a murine cranial vault, 2 weeks after administration of excipient alone (negative control), of bone-forming A cells derived from MSCs (generated with FGF-2 and TGFB1) or bone-forming B cells (generated with FGF-2, TGFB1 and heparin). Figure 3 illustrates the double immunostaining (immunofluorescence) of murine and human type I collagens performed on coronal sections of murine cranial vault 2 weeks after administration of MSC-derived bone-forming B cells (generated with FGF- 2, TGFB1 and heparin). Figure 3a illustrates the dual immunostaining of anti-human and anti-murine type I collagen (fusion) while Figures 3b and 3c show the immunostaining of anti-human and anti-murine type I collagen, respectively.
    Figure 4 illustrates the histological staining of coronal sections of a murine bony cranial vault, 2 weeks after administration of excipient alone, MSCs, MSC-derived bone-forming α cells generated with FGF-2 and TGFB1 (bf A cells), or bone-forming B cells - generated with FGF-2, TGFB1 and heparin (bf B cells), derived from CSM. (A) Calcium-bound fluorochromes were sequentially injected intraperitoneally (alizarin red - calcein green - calcein blue - tetracycline) to highlight new bone formation (arrows) and assess the dynamics of bone formation; (B) immunofluorescence (IF) of human + murine type I collagen; (C) Murine type I collagen IF; (D) IF of human type I collagen.
    A double immunofluorescence of anti-human and anti-murine type I collagen was carried out to allow the detection of the human and murine type I collagen secreted by the bone matrix; (E) ALP + Goldner staining: ALP: detection of osteoblastic activity in black (complete lines and areas), Masson Goldner's trichrome: detection of osteoid (non-mineralized bone tissue) in black dotted lines, mineralized bone in dark gray ; (F) Tartrate-resistant acid phosphatase (TRAP): detection of osteoclastic activity in dark gray / black.
    FIG. 5 represents images illustrating the new bone formation on coronal sections of the murine bone cranial vault 2 weeks after administration of the excipient alone; CSM; MSC-derived bone-forming A cells generated with FGF-2, TGFB1 (b-f A cells); or MSC-derived bone-forming B cells generated with FGF-2, TGFP1 and heparin (b-f B cells). New bone formation is demonstrated by fluorescence (marked by the sequential integration of different fluorochromes: alizarin red - green calcein - blue calcein - yellow tetracycline). The red, green and blue colorations appear in light gray and the thickness of the new bone formation is indicated by double arrows.
    The yellow colorations have been surrounded by dotted lines.
    Figure 6 is a graph illustrating the total area of newly formed bone (mean + SEM, * p <0.05) measured on murine skull vault sections 2 weeks after administration of MSC (dark gray) or cells. B bone formers (light gray). Figure 7 illustrates the safranin orange staining of the cartilage matrix (surrounded by dashed lines) of mineralized nodules performed on sagittal sections of the bone-murine cranial vault one day (D1) after administration of formative B cells. bone and over time (D7, D14, D21) up to 28 days (D28) after administration.
    Figure 8 illustrates the effect of CSM-derived cells in a model of segmental femoral defect of subcritical size. (A) is a graph illustrating the measurement of defect size on x-ray images on day (OD) of surgery or article administration and over time (1, 2, 3, 4, 5 weeks) up to 6 weeks (6W) after administration of the vehicle alone, bone-forming A cells (bf A cells) or bone-forming B cells (bf B cells); mean + SEM, ** p <0.01, *** p <0.001; (B) represents representative radiographic images of segmental femoral defects at OD and 6W after administration of the excipient alone or of bone-forming B cells (b-f B cells); (C) is a graph illustrating the measurement of bone repair volume by microscopic tomography (micro-CT) at 6W after administration of the excipient alone (n = 7) and of bone-forming B cells (n = 8); mean + SEM, p <0.05 Figure 9 illustrates the flow cytometric gating strategy used in Example 5. Figure 10 illustrates the CD73 (top panel) and CD44 (bottom panel) expression levels analyzed. by flow cytometry by λ, B and C cells of CSM. n = 12, 6, 22, 15 (CD73) and n = 22, 8, 22, 18 (CD44) for MSCs and bone-forming cells A, B and C respectively, where n represents the number of individual tests.
    Figure 11 illustrates osteoinduction and osteogenesis evaluated by radiographic analysis.
    A: Osteoinduction (A, left panel) is evaluated by measuring the value of the intensity of the gray which is directly correlated with the opacity of the bone and therefore with its thickness.
    Osteogenesis (A, right panel) is assessed by measuring the area of the nodule which appears to be more refractive by radiographic imaging.
    B: Bone opacity is significantly higher for cryopreserved C bone-forming cells ("BF C cells") than for excipients (n = 20 (excipient) and n = 34 (BF C cells from 5 different lots) C: The surface area of the osteogeny is significantly greater than that of the excipient in which no mineralized nodule was observed (n = 20 (excipient) and n = 34 (BF C cells from 5 different lots). DE: Osteoinduction with (Fig.
    SD) or without (FIG. 8E) osteogenesis (represented by absolute bone formation) is significantly higher for cryopreserved C cells ("B-F C cells") than for excipients.
    Mann Whitney U test: *** p <0.001. F: in addition to osteoinduction activities, cryopreserved C bone-forming cells ("BF C cells") promote high osteogenic activity indicated by the presence of at least one mineralized nodule in 4/5 5 bone marrow donors (or batches) and 65% of the mice (n = 20 (excipient) and n = 34 (BF C cells from 5 separate batches). Figure 12 illustrates the coronal histological section 4 weeks after the administration of bone-forming cells C cryopreserved or excipient.
    Cryopreserved bone-forming C cells exhibit a dual mechanism of action: (1) "osteoinduction": stimulation of host bone formation by paracrine secretion leading to intramembrane ossification and (ii) "osteogenesis" ": promotion of bone formation in a" direct "way (from the origin of the donor, human) by endochondral ossification.
    Figure 13 illustrates the histological analysis of the skull of mice 4 weeks after receiving a single injection of cryopreserved C bone-forming cells.
    C - cryopreserved bone-forming cells exhibit osteoinductive and osteogenic ("fluo") properties. Human bone formation ("human type I collagen") has been demonstrated in mineralized nodules (osteogenesis). The activity of osteoblasts ("ALP" indicated by black arrows in the third panel) and osteoclasts ("TRAP", indicated by black arrows in the fourth panel) was mainly detected in the mineralized nodules showing that the process of Bone remodeling of the nodules was still ongoing 4 weeks after their administration.
    No osteoid ("Goldner's Masson's trichrome stain") was found indicating that the bone formation process is complete.
    Figure 14 illustrates the effect of cryopreservation of bone-forming C cells ("B-F C cells") in a segmental femoral defect model of subcritical size.
    Radiographs represent segmental femoral defects on day 0 through week 10 after administration of the vehicle alone or cryopreserved C-forming cells.
    Figure 15 illustrates the effect of cryopreservation of bone-forming C cells in a sub-critical size femoral segmental defect model (sub-CSD model). The graph represents the percentage of bone repair on the X-ray images on the day of surgery or treatment administration ("WO") and up to 10 weeks ("W10") after vehicle administration. alone, or on cryopreserved C bone-forming cells ("BF C cells"); mean + SEM, *** p <0.001 (ANOV A). Figure 16 illustrates the effect of cryopreservation of bone-forming C cells in a sub-critical size femoral segmental defect model (sub-CSD model). The graph represents the score
    RUS determined from X-ray images on the day of surgery ("WO") and up to 10 weeks ("W 10") after administration of the vehicle alone, or bone-forming cells C cryopreserved (BF C cells); mean + SEM, ** p <0.01, *** p <0.001 (bilateral repeated measures ANOVA). DETAILED DESCRIPTION OF THE INVENTION In the present context, the singular forms "a", "a" and "the / the" include both singular and plural references, unless clearly contraindicated in the context. The terms "comprising", "comprises" and "composed of", in the present context, are synonymous with "comprising", "comprises" or "containing", "contains", and are inclusive or open and do not exclude additional method members, elements or steps not mentioned The terms further cover the terms "consisting of" and "consisting essentially of", which have well-established meanings in patent terminology.
    The recitation of numerical intervals by limits includes all the numbers and fractions included within the respective intervals, as well as the mentioned limits.
    The terms "about" or "approximately" used herein to denote a measurable value such as a parameter, quantity, time duration and the like, are intended to cover variations of the specified value, such as variations of + 10% or less, preferably + 5% or less, more preferably + 1% or less, and still more preferably + 0.1% or less, of the specified value, as far as such variations are suitable for the present invention. It should be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.
    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, are clear in themselves, through other examples, the terms cover in particular a reference to any of said members, or to two or more of said members, such as, for example, three or more, four or more, five or more, six or more, seven or more, or even more, of said members, and to all of said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
    The discussion of the background of the invention is included herein to explain the background of the invention. This should not be taken as an admission that any of the documents referred to were published, known or common knowledge in any country on the priority date of any of the claims.
    Throughout this disclosure, various publications, patents and published patent specifications are referred to by an identifying citation. All documents cited in this specification are incorporated by reference in their entirety. In particular, the teachings or sections of those documents specifically referenced herein are incorporated by reference.
    Unless defined otherwise, all 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 additional guidance, 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, this connotation is intended to apply throughout this specification, i.e. i.e., also in the context of other aspects or embodiments of the invention, unless stated otherwise.
    In the following sections, different aspects or embodiments of the invention are defined in more detail.
    Each aspect or embodiment thus defined may be combined with one or more other aspect (s) or embodiment (s), unless clearly indicated otherwise.
    In particular, any characteristic indicated as being preferred or advantageous may be combined with any other characteristic or with the other characteristics indicated as being preferred or advantageous.
    In the present specification, the expression "an embodiment" means that a particular feature, structure or feature described in the context of the embodiment is included in at least one embodiment of the present invention.
    Therefore, occurrences of the phrase "in one embodiment" at various places in this specification do not necessarily all refer to the same embodiment, but can.
    In addition, the particular elements, structures or features (areas) may be combined in any suitable manner, as will be apparent to those skilled in the art from this disclosure, into one or more embodiments.
    Additionally, although some embodiments described herein include some features, but not others, which are included in other embodiments, the feature combinations of different embodiments are intended to be included in. the scope of the invention and to form different embodiments, as understood by the authors of the invention.
    For example, in the appended claims, any of the claimed embodiments can be used in any combination.
    The present inventors have realized that certain cell surface markers are useful for determining the osteogenic potential of differentiated cells in vitro.
    More particularly, the inventors have observed that the quantity of cells differentiated in vitro expressing one or more of CD73, CD105, CD10 or CD44, preferably all of CD73, CD105, CD10 and CD44, and the quantity of one or more of CD73 , CD105 or CD44, preferably all CD73, CD105 and CD44, expressed by differentiated cells in vitro, can be used to determine the osteogenic potential of differentiated cells in vitro.
    Throughout the description, references to "CD73", "CD105", "CD44" or "CD10" denote the respective peptides, polypeptides, proteins or nucleic acids, as they appear from the context, commonly referred to herein. art under these designations.
    These terms cover peptides, polypeptides, proteins or nucleic acids of any organism in which they are found, and in particular animals, preferably warm-blooded animals, more particularly vertebrates, but more particularly mammals, including humans and non-human mammals, more particularly humans.
    The term "protein" used in this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymer chains of amino acid residues linked by peptide bonds.
    The term can encompass naturally produced, recombinant, semi-synthetically, or synthetically produced proteins.
    The term also encompasses proteins which carry one or more co- or post-expression type modifications of the polypeptide chain (s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, l ubiquitination, signal peptide elimination, N-terminal Met elimination, conversion of pro-enzymes or pre-hormones into active forms, etc.
    The term also includes variants or mutants of proteins which carry variations in amino acid sequence with respect to a corresponding native protein, such as, for example, deletions, additions and / or substitutions of amino acids.
    The term encompasses both whole proteins and parts or fragments of proteins, for example, parts of natural proteins which result from the transformation of these whole proteins.
    The term "polypeptide" used in this specification generally encompasses polymer chains of amino acid residues linked by peptide bonds.
    Thus, since a protein is only composed of a single polypeptide chain, the terms "protein" and "polypeptide" can be used interchangeably herein to refer to such a protein.
    The term is not limited to the minimum length of the polypeptide chain.
    The term can encompass naturally produced, recombinant, hemi-synthetically or synthetically produced polypeptides.
    The term also encompasses polypeptides which carry one or more co- or post-expression type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination. , elimination of the signal peptide, elimination of the N-terminal Met, conversion of pro-enzyme or pre-hormones into active form, etc.
    The term also includes variants or mutants of polypeptides which carry variations in amino acid sequence from a corresponding native polypeptide, such as, for example, deletions, additions and / or substitutions of amino acids.
    The term encompasses both whole polypeptides and parts or fragments of polypeptides, for example the naturally occurring polypeptide parts which result from the processing of these whole polypeptides.
    The term "peptide" used in this specification preferably refers to a polypeptide as used in this specification, consisting essentially of 50 amino acids or less, for example 45 amino acids or less, preferably 40 amino acids or less. , for example 35 amino acids or less, more preferably 30 amino acids or less, for example 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.
    The term “nucleic acid” used in the present description generally denotes a polymer (preferably a linear polymer) of any length, composed essentially of nucleoside units.
    A nucleoside unit generally comprises a heterocyclic base and a sugar group.
    Heterocyclic bases can include, among others, purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are prevalent in natural nucleic acids, other natural bases (eg xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (eg methylated), unnatural or derivative bases.
    Typical modified nucleobases include, but are not limited to, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 - substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
    In particular, 5-methylcytosine substitutions have been shown to increase the stability of the nucleic acid duplex and may be preferred as base substitutions in antisense agents, for example, even more when combined with modifications. 2'-O-methoxyethyl sugar.
    The sugar groups can include, inter alia, the pentose - (pentofuranose) groups such as ribose and / or 2-deoxyribose common in natural nucleic acids, or the arabinose, 2-deoxyarabinose, triose or hexose groups, as well as modified or substituted sugar groups (such as 2'-O-alkylated, for example), 2'-O-methylated or 2'-O-ethylated sugars such as ribose; 2'-O-alkyloxyalkylated sugars, for example, 2'-O-methoxyethylated sugars such as ribose; or 2'-0.4'-C-alkylene linked, for example, 2'-0.4'-C-methylene or 2'-0.4'-C-ethylene linked such as ribose; 2'-fluoro-arabinose, etc). Nucleoside units can be linked to each other by any of a number of known internucleoside linkages, including, among others, the phosphodiester linkages common in naturally occurring nucleic acids, and subsequent modified phosphate or phosphonate linkages, such as phosphorothioate. , alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged phosphorothio phosphate, bridged phosphorothio phosphate; and other siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate linkages such as 3'-N-carbamate, morpholino, borano, thioether, 3'-thioacetal, and internucleoside sulfone.
    Preferably, the internucleoside linkages can be phosphate-based linkages, including modified phosphate-based linkages, such as phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof.
    The term "nucleic acid" also encompasses all other polymers containing nucleobases such as nucleic acid mimetics including, without limitation, peptide nucleic acids (PNAs), phosphate grouped peptide nucleic acids (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (chDNA), tricycloDNA (ctDNA), nucleic acids with backbones with alkyl or amino links (Kurreck 2003 (Eur J Biochem) 270: 1628-1644)). "Alkyl", as used herein, covers in particular the lower hydrocarbon fractions, for example linear or branched C1-C4 hydrocarbons, saturated or unsaturated, such as methyl, l ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, isopropyl.
    Nucleic acids as defined herein can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof.
    A modified nucleoside can include a modified heterocyclic base, a modified sugar moiety, a modified internucleoside linkage, or a combination thereof.
    The term "nucleic acid" further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, including mRNA, mRNA, eDNA, genomic DNA, products of. amplification, oligonucleotides, synthetic DNA (eg, chemically synthesized), RNA, and DNA / RNA hybrids.
    A nucleic acid may be of natural origin, for example present in nature or isolated from nature, may be recombinant, i.e. produced by recombinant DNA technology, and / or may be, partially or wholly, chemically or biochemically synthesized.
    A "nucleic acid" can be double stranded, partially double stranded, or single stranded.
    When it is single-stranded, the nucleic acid can be the sensitive strand or the antisense strand.
    In addition, the nucleic acid can be circular or linear.
    By way of further guidance, CD73 is also known in the art as ecto-5 "- nucleotidase, NT, eN, NT5, NTE, eNT, ESNT and CALJA.
    CD73 is a cell surface enzyme linked to glycosyl-phosphatidylinositol (GPD). For example, the human CD73 gene is annotated under the NCBI Genbank (http://www.ncbi.nlm.nih.gov/) Gene ID 4907 and the human CD73 protein is annotated under Uniprot (www.uniprot. org) under the accession number P21589.1. Human CD73 mRNA and protein sequences are annotated under the following NCBI Genbank accession numbers: CD73 mRNA (NM_002526.3; NM_001204813.1), CD73 protein (NP_002517.1; NP_001191742.1). By way of further guidance, CD105 is also known in the art as endoglin (ENG), END, FLJ41744, HHT1, ORW and ORWI.
    CD105 is a type I membrane glycoprotein located on the surface of the cell.
    By way of example, the human CD705 gene is annotated under the Genbank NCBI gene ID 2022 and the human CD105 protein is annotated under Uniprot under the accession number P17813.2. The mRNA and protein sequences of human CD105 are annotated under the following NCBI Genbank accession numbers: CD105 ((NM_001114753.2; NM_0001183; NM_001278138.1), protein CD105 (NP_001108225.1; NP_000109.1; NP_001265067. 1). By means of additional guidance, CD44 is also known in the art as the original cell adhesion molecule (HCAM), Pgp-1 (phagocytic glycoprotein-1), Hermes antigen, receptor lymphocyte origin (LHR), ECM-IIL HUTCH-1, IN, MC56, MDU2, MDU3, MDU3, Pgpl, CDW44, CSPG8, HCELL, Hutchch-I and ECMR-II. CD44 is an involved cell surface glycoprotein in cell-cell interactions, cell adhesion and migration.For example, the human CD44 gene is annotated under the order number NCBI Genbank Gene ID 960 and the human CD44 protein is annotated under Uniprot under the number order P16070.3. The mRNA and protein sequences of human CD44 are annotated under the following NCBI Genbank order numbers s: CD44 mRNA (NM_000610.3, NM_001001389.1, NM_001001390.1, NM_001001391.1, NM_001001392.1, NM_001202555.1, NM_001202556.1, NM_001202557.1), protein CD44 (NP_000601.3, NP_001001389.1, NP 001001390.1, NP_001001391.1, NP 001001391.1, NP_001189484.1, NP_001189484.1, NP_001189484.1, NP_001189485.1, NP_001189486.1).
    By way of further guidance, CD10 is also known in the art as membrane metalloendopeptidase (MME), NEP, SFE, CALLA, CMT2T and SCA43. CD10 is a glycoprotein - transmembrane type II. For example, the human CD / 0 gene is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) Gene ID 4311 and the human CD10 protein is annotated under Uniprot under the number accession P08473.2. Human CD10 mRNA and protein sequences are annotated with the following NCBI Genbank accession numbers: CD10 mRNA (NM_00090202.3, NM_007287.2, NM_007288.3, NM_007289.3, NM_001354642.1, NM_001354643.1, NM_001354644.1), CD10 protein (NP_000893.2, NP_009218.2, NP_009219.2, NP_009220.2, NP_009220.2, NP_001341571.1, NP_001341572.1, NP_001341573.1). The reader is reminded that when the Genbank or Uniprot entries provide the sequence of polypeptides or precursor proteins, the corresponding mature forms (such as, for example, the absence of signal peptides) should be present on the cell surface of cells.
    The terms "CD73", "CD105", "CD44" and "CD10" encompass in particular peptides, polypeptides, proteins or nucleic acids having a native sequence, that is to say whose primary sequence is the same as that peptides, polypeptides, proteins or nucleic acids present or derived in nature. A skilled person understands that native sequences may differ from species to species due to genetic discrepancies between these species. Additionally, native sequences may differ from individual to individual or within different individuals of the same species due to normal genetic diversity (variation) within a given species. In addition, native sequences may differ between individuals of the same species or even within different individuals due to somatic mutations or post-transcriptional or post-translational modifications. These variants or isoforms of peptides, polypeptides, proteins or nucleic acids are provided herein. Therefore, all sequences of peptides, polypeptides, proteins or nucleic acids found in or derived from nature are considered “native.” The terms include peptides, polypeptides, proteins or nucleic acids when they are made. part of a living cell. A first aspect provides for the use of one or more (eg, one, two or all three) of CD73, CD105 or CD44 to determine the osteogenic potential of differentiated cells in vitro. , it is also intended to use one of CD73, CD105 or CD44 to determine the osteogenic potential of differentiated cells in vitro; the use of two of CD73, CD105 or CD44 to determine the osteogenic potential of differentiated cells in vitro; and the use of the three of CD73, CD105 and CD44 to determine the osteogenic potential of differentiated cells in vitro.
    Certain embodiments allow the use of one or more CD73, CD105, CD44, or CD10 to determine the osteogenic potential of differentiated cells in vitro (eg, one, two, three, or all four).
    Certain embodiments allow the use of CD44 to determine the osteogenic potential of differentiated cells in vitro. Some embodiments allow CD44 and either or both of CD73 and CD105, or both, to be used to determine the osteogenic potential of differentiated cells in vitro. Some representations allow the use of CD44 and one or more CD73, CD105 or CD10 to determine the osteogenic potential of differentiated cells in vitro (eg, one, two, or all three).
    Some embodiments allow CD10 to be used to determine the osteogenic potential of differentiated cells in vitro. Some embodiments allow CD10 and one or more CD73, CD105, or CD44 (eg, one, two, or all three) to be used to determine the osteogenic potential of differentiated cells in vitro.
    The term "osteogenic potential" as used herein refers to the ability of cells to (trans) differentiate into cells secreting bone matrix or the ability of cells to secrete bone matrix (i.e. that is to say without the need for a (trans) differentiation step), in vivo, and possibly in vitro. The term encompasses the ability of cells to form bone tissue through intramembrane ossification or endochondral ossification. The ability of cells to form bone tissue through intramembrane ossification generally represents the ability of cells to form bone tissue without the need for a calcified cartilage matrix as a carrier. The ability of cells to form bone tissue by endochondral ossification typically represents the ability of cells to form bone tissue by first forming a calcified cartilage matrix and then using said calcified cartilage matrix as a carrier for bone tissue formation. The term does not cover the osteoinductive potential of cells, which represents the ability of cells to attract other cells secreting bone matrix and / or to induce (trans) differentiation of other cells by secreting bone matrix. Those skilled in the art will understand that while the current objective is to determine the osteogenic potential of differentiated cells in vitro, the cells can, but need not necessarily, in addition to the osteogenic potential, also have an osteoinductive potential.
    In particular, the osteogenic potential is the potential of cells to form a bone matrix by endochondral ossification.
    The term "endochondral ossification" used throughout the application refers to a process of bone tissue formation in which the first chondrocytes form an extracellular cartilaginous matrix, which is then used by the osteoblasts as a carrier for depositing the bone matrix. .
    During endochondral ossification, some chondrocytes can transform into osteoblasts.
    In preferred embodiments, all CD73, CD105 and CD44 are used to determine the osteogenic potential of differentiated cells in vitro.
    In particular, CD10 is used in addition to one or more of CD73, CD105 and CD44, preferably in addition to all CD73, CD105 and CD44, to determine the osteogenic potential of differentiated cells in vitro.
    Therefore, the use of CD73, CD105, CD44 and CD10 is also expected to determine the osteogenic potential of differentiated cells in vitro.
    Another aspect provides a method for determining the osteogenic potential of differentiated cells in vitro comprising (a1) measuring the amount of differentiated cells in vitro expressing one or more (eg, one, two, three or four) of CD73, CD105 , CD10 or CD44, and / or (a2) measuring the amount of one or more (eg, one, two or all three) of CD73, CD105 or CD44 expressed by differentiated cells in vitro.
    Therefore, a method for determining the osteogenic potential of differentiated cells in vitro is also provided herein, comprising (a1) measuring the amount of differentiated cells in vitro expressing one of CD73, CD105, CD10 or CD44, preferably measuring the amount of differentiated cells in vitro expressing two of CD73, CD105, CD10 or CD44, preferably by measuring the amount of differentiated cells in vitro expressing three of CD73, CD105, CD10 or CD44, preferably by measuring the amount of cells differentiated in vitro expressing all four of CD73, CD105, CD10 and CD44; and / or (a2) measure the quantity of one of the CD73, CD105 or CD44 expressed by the differentiated cells in vitro, preferably measure the quantity of two of the CD73, CD105 or CD44 expressed by the differentiated cells in vitro, more preferably measure the amount of the three of CD73, CD105 or CD44 expressed by the differentiated cells in vitro.
    Therefore, a method for determining the osteogenic potential of differentiated cells in vitro is also provided herein, comprising (a1) measuring the amount of differentiated cells in vitro expressing the four CD73, CD105, CD10 and CD44, and / or (a2 ) the measurement of the quantity of the three CD73, CD105 or CD44 expressed by the differentiated cells in vitro. In particular, the method taught here does not involve the measurement of other markers in addition to CD73, CD105, CD10 and CD44.
    In some cases, the methods taught herein include measuring the amount of differentiated cells in vitro expressing one or more (eg, one, two, three or four) of CD73, CD105, CD10 or CD44.
    In some cases, the methods taught here consist of measuring the amount of differentiated cells in vitro expressing CD44. In some cases, the methods taught herein include measuring the amount of differentiated cells in vitro expressing CD44 and either CD73 and CD105, or both. In some cases, the methods taught herein include measuring the amount of differentiated cells in vitro expressing CD44 and one or more cells (eg, one, two or all three) of CD73, CD105 or CD10.
    In some cases, the methods taught here consist of measuring the amount of differentiated cells in vitro expressing CD10. In some cases, the methods taught herein include measuring the amount of differentiated cells in vitro expressing CD10 and one or more cells (eg, one, two or all three) of CD73, CD105 or CD44.
    In some cases, the methods taught herein include measuring the amount of one or more CD73, CD105 or CD44 expressed by the differentiated cells in vitro (eg, one, two or three). In some cases, the methods taught here consist in measuring the amount of CD73, CD105 and CD44 expressed by the differentiated cells in vitro.
    In some cases, the methods taught here consist of measuring the amount of CD73 expressed by the differentiated cells in vitro. In some cases, the methods taught herein include measuring the amount of CD73 and CD105 and CD44 expressed by the differentiated cells in vitro, or any of them. In some cases, the methods taught herein include measuring the amount of CD73 and one or more CD105, CD44 or CD10 expressed by cells - differentiated in vitro (eg, one, two or all three).
    In some cases, the methods taught here consist of measuring the amount of CD105 expressed by the differentiated cells in vitro. In some cases, the methods taught herein include measuring the amount of CD105 and CD73 and CD44 expressed by the differentiated cells in vitro, or any of them. In some cases, the methods taught herein include measuring the amount of CD105 and one or more CD73, CD44 or CD10 expressed by the differentiated cells in vitro (eg, one, two, or all three).
    In some cases, the methods taught here consist of measuring the amount of CD44 expressed by the differentiated cells in vitro. In some cases, the methods taught herein include measuring the amount of CD44 and CD73 and CD105 expressed by the differentiated cells in vitro, or any of them. In some cases, the methods taught 1ci include measuring the amount of CD44 and one or more CD73, CD105 or CD10 expressed by the differentiated cells in vitro (eg, one, two or all three). In some cases, the methods taught here include measuring the amount of one or more CD73, CD105, CD44, or CD10 on the cell surface or expressed by the differentiated cells in vitro (eg, one, two, three or four) . In some cases, the methods taught here include measuring the amount of CD73, CD105, CD44 and CD10 on the cell surface or expressed by differentiated cells in vitro.
    In some cases, the methods taught here consist of measuring the amount of CD10 expressed by the differentiated cells in vitro.
    In some cases, the methods taught herein include measuring the amount of CD10 and one or more CD73, CD105 or CD44 (eg, one, two or all three) expressed by the differentiated cells in vitro.
    In some instances, the methods taught herein include (a1) measuring the amount of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44, and / or (a2) measuring the amount of one or more cells (by example, one, two or all three) of CD73, CD105 or CD44 expressed by differentiated cells in vitro.
    In some embodiments, the methods taught herein include (a1) measuring the amount of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44, and / or (a2) measuring the amount of one or more cells. (eg, one, two, three or four) of CD73, CD105, CD44 or CD10 expressed by differentiated cells in vitro.
    The terms "expression" or "expression" used in this specification generally cover the generation of any transcription or translation product, such as RNA, peptides, polypeptides and proteins, by a cell, as well as the presentation of peptides, polypeptides or proteins on the cell surface.
    By means of additional guidelines, when a cell is said to be positive for or expresses or comprises the expression of a given gene, peptide, polypeptide or protein, such as CD73, CD105, CD105, CD10 or CD44, a competent person would conclude the presence or proof of a distinct signal for that gene, peptide, polypeptide or protein by performing a measurement that can detect or quantify the gene, peptide, polypeptide or protein in or on the cell.
    The presence or proof of the distinct signal for the gene, peptide, polypeptide or protein would be suitably concluded on the basis of a comparison of the result of the measurement obtained for the cell with a result of the same measurement made for one. negative control (eg, a cell known to not express the marker) and / or a positive control (eg, a cell known to express the marker). A molecule or analyte such as a peptide, polypeptide, protein or nucleic acid, or a group of two or more molecules or analytes such as two or more peptides, polypeptides, proteins or nucleic acids, is "measured" in a sample when the presence or absence and / or the amount of said molecule or analyte or said group of molecules or analytes is detected or determined in the sample, preferably substantially to the exclusion of other molecules or analytes. The terms "amount", "amount" and "level" are synonymous and generally well understood in the art. The terms used herein can refer in particular to an absolute quantification of a number of cells, of a peptide, of a polypeptide, protein or nucleic acid in a sample, or relative quantification of a number of cells, peptide, polypeptide, protein or nucleic acid in a sample, for example , by rapp ort to another value such as the one shown here. The amount of a peptide, polypeptide or protein can also be represented by the activity of a peptide, polypeptide or protein.
    … The activity of a peptide, polypeptide or protein in a sample can also be expressed in absolute terms, eg. ex. in enzymatic units by volume or in relative terms.
    An absolute quantity of a peptide, of a polypeptide, of a protein or of a nucleic acid in a sample can be advantageously expressed in weight or in molar quantity, or more generally in concentration, for example in weight per volume or in moles by volume.
    A relative amount of a peptide, polypeptide, protein or nucleic acid in a sample can be advantageously expressed as an increase or decrease or an increase in fold by relative to said other value, such as relative to a reference value as described elsewhere. To perform a relative comparison between a first and a second parameter (for example, first and second magnitudes), it is not necessary to first determine the absolute values of said first and second parameters. For example, a measurement method can produce quantifiable readings (such as, for example, signal strengths) for said first and second parameters, wherein said readings are a function of the value of said parameters, and wherein said readings can be directly compared to produce a relative value for the first parameter versus the second parameter, without the real need - to convert the readings to the absolute value of the respective parameters beforehand.
    A relative quantity of cells can be expressed as a percentage (fraction) of the total number of cells analyzed, more particularly of the total number of cells for which the expression of one or more of the CD73, CD105, CD10 or CD44 cells is determined. Therefore, measuring the amount of differentiated cells in vitro expressing one or more cells (eg, one, two, three, or four) of CD73, CD105, CD105, CD10 or CD44, as taught herein, may typically involve (i) determine the expression (that is to say the presence) of CD73, CD105, CD10 and / or CD44 by the differentiated cells in vitro, (ii) count the number of differentiated cells in vitro which express CD73, CD105, CD10 and / or CD44 in step (1); and (iii) calculating the fraction of in vitro differentiated cells which express CD73, CD105, CD10 and / or CD44 in step (1) relative to the total number of in vitro differentiated cells examined in step (i). The amount of differentiated cells in vitro which express one or more of CD73, CD105, CD105, CD10 or CD44 can be measured by any means known in the art. For example, by flow cytometry.
    Accordingly, in particular embodiments, the amount of in vitro differentiated cells expressing one or more (eg, one, two, three or four) of CD73, CD105, CD10 or CD44 is the fraction of the differentiated cells determined in vitro. to express one or more of CD73, CD105, CD10 or CD44 relative to all the differentiated cells in vitro analyzed.
    The determination of the presence and / or the measurement of the quantity of one or more CD73, CD105, CD10 or CD44 by differentiated cells in vitro can be carried out by any existing, available or conventional detection and / or quantification method. used to measure the presence or absence (for example, the reading being present or absent, or the detectable amount or the absolute or relative amount of a peptide, polypeptide, protein or nucleic acid in or on a cell or cell population) . For example, these methods may include biochemical analysis methods, immunoassay methods, mass spectrometric analysis methods, chromatography methods, or combinations of these methods.
    In particular, the measurement of the presence and / or the quantity of one or more of CD73, CD105, CD10 or CD44 comprises the measurement of peptides, polypeptides or proteins CD73, CD105, CD10 or CD44 or of CD73 mRNA, CD105, CD10 or CD44 or both.
    In preferred embodiments, measuring the presence and / or amount of one or more of CD73, CD105, CD10 or CD44 comprises measuring the peptides, polypeptides or proteins CD73, CD105, CD10 or CD44.
    Since each of the peptides, polypeptides or proteins CD73, CD105, CD10 and CD44 is usually expressed on the surface of the cell, the skilled person will understand that if reference is made to one or more of CD73, CD105, CD10 or CD44 to the cell surface, the peptide, polypeptide or protein is in the form is intended for one or more peptides, polypeptides or - proteins CD73, CD105, CD10 or CD44, or that reference is made to one or more peptides, polypeptides or proteins CD73, CD105, CD10 or CD44 on the cell surface.
    In particular, measuring the presence and / or quantity of one or more CD73, CD105, CD10 or CD44 comprises measuring CD73, CD105, CD10 or CD44 peptides, polypeptides or proteins on the cell surface.
    More particularly, the presence and / or the quantity of one or more peptides, polypeptides or proteins CD73, CD105, CD10 or CD44 in an undenatured form on the cell surface of living cells is measured.
    More particularly, measuring the presence and / or amount of one or more peptides, polypeptides or proteins CD73, CD105, CD10 or CD44 comprises the use of a technique which uses one or more agents capable of binding specifically respectively to CD73, CD105, CD10 or CD44, preferably where the one or more agents are, each independently, one or more antibodies, fragments of antibodies, carriers of antibody-like proteins or aptamers.
    These methods may include affinity-based assay methods, in which the ability of an assay to detect and / or quantify a peptide, polypeptide, protein, or nucleic acid is conferred by specific binding between an agent. detectable and / or quantifiable binder and i) the peptide, polypeptide, protein or nucleic acid. The binder can be an immunological (antibody) or non-immunological binder. Examples of antibodies capable of binding to human CD73 include, but are not limited to, antibodies available from the following suppliers ("#" stands for catalog number): BD Biosciences (mouse monoclonal antibody conjugated to allophycocyanin (APC), No. 560847; Fluorescein-conjugated mouse monoclonal antibody (FITC), No. 561254; R-phycoerythrin (PE) conjugated mouse monoclonal antibody No. 55027), Abcam (mouse monoclonal antibody, No. ° ab54217; rabbit monoclonal, # ab79423; mouse monoclonal # ab34199), R&D systems (biotinylated polyclonal goat antibody, # BAF1182; mouse monoclonal, # MAB1182; PE conjugate polyclonal goat antibody, # FAB8160P). Examples of antibodies capable of binding to human CD105 include, but are not limited to, antibodies available from the following suppliers ("#" stands for catalog number): BD Biosciences (APC Conjugated Mouse Monoclonal Antibody, # 562408 ; PE-labeled mouse monoclonal antibody, # 560839), Abcam (mouse monoclonal, # ab156756; mouse monoclonal, # ab2529; (Alexa Fluor® 488 conjugate monoclonal, # FAB10971G; Alexa Fluor® 647 conjugate monoclonal) , # FAB10971R; goat polyclonal, # AF1097) Examples of antibodies capable of binding to human CD44 include, but are not limited to, antibodies available from the following vendors ("#" stands for catalog number): BD Biosciences (mouse monoclonal antibody - PE conjugate, # 550989; mouse monoclonal antibody FITC conjugate, # 555478), Abcam (polyclonal rabbit, # ab157107, mouse monoclonal PE conjugate, # ab46793; mouse monoclonal PE conjugate, # ab58754), R systems & D (rat monoclonal conjugate Alexa Fluor®, # FAB6127 S; mouse monoclonal PE conjugate, # FAB3660P). Examples of antibodies capable of binding to human CD10 include, but are not limited to, antibodies available from the following suppliers ("#" stands for catalog number): BD Biosciences (PE-conjugated mouse monoclonal antibody, # 555375) , Abcam (rabbit monoclonal, # ab79423; rabbit polyclonal, # ab82073; mouse monoclonal PE conjugate, # ab210380), R&D systems (mouse monoclonal Alexa Fluor®, # FAB1182N; biotinylated goat, # BAF1182). Affinity-based assay methods, such as immunoassay methods, include but are not limited to immunohistochemistry, immunocytochemistry, flow cytometry, mass cytometry, fluorescence activated cell sorting (FACS), fluorescence microscopy, fluorescence cell sorting using microfluidic systems,
    techniques based on (immuno) affinity adsorption such as affinity chromatography, magnetic activated cell sorting or bead cell sorting using microfluidic systems, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and techniques based on ELISPOT, radioimmunoassay (RIA), Western blot, etc.
    Other techniques for detecting and / or quantifying peptides, polypeptides or proteins can be used, optionally in conjunction with one of the analytical methods described above. These methods include, but are not limited to, mass spectrometry (MS) techniques, chemical extraction separation, isoelectric focusing (IEF) including capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP) , capillary electrochromatography (CEC), etc, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), chromatography Micellar electrokinetics (MEKC), free flow electrophoresis (FFE), etc.
    The presence or absence and / or the amount of a nucleic acid, for example at the level of nhRNA, pre-mRNA, mRNA or cDNA, can be detected using monitoring tools. Standard quantitative measurement known in the art. Non-limiting examples include hybridization-based analysis, microarray expression analysis, digital gene expression (DGE), RNA in situ hybridization (FISH), Northern blot analysis and others ; PCR, RT-PCR, RT-qPCR, end point PCR, digital PCR or others; detection of supported oligonucleotides, pyrosequencing, - polonymic cycle sequencing by synthesis, simultaneous bidirectional sequencing, monomolecular sequencing, monomolecular real-time sequencing, true monomolecular sequencing, sequencing of nanopores assisted by hybridization, sequence by synthesis or the like.
    In other examples, it is possible to use any combination of methods such as those described in this document.
    In particular, CD73, CD105, CD105, CD10 or CD44 denote peptides, polypeptides or proteins and the expression of one or more of CD73, CD105, CD10 or CD44 denotes the expression of the cell surface of one or several of CD73, CD105, CD10 or CD44, respectively. The expression of the cell surface of a peptide, polypeptide or protein is preferably determined by flow cytometry.
    In particular, the method as taught herein comprises (a1) measuring the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro; and / or (a2) measuring the amount of one or more of CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro. Therefore, also provided herein is the method as taught herein comprising (a1) measuring the fraction of differentiated cells in vitro expressing one of CD73, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro, preferably measuring the fraction of differentiated cells in vitro expressing two of CD73, CD105, CD10 or CD44 at the cell surface of differentiated cells in vitro, more preferably measuring the fraction of differentiated cells in vitro expressing three of CD73, CD105 , CD10 or CD44 at the cell surface of differentiated cells in vitro, more preferably the measurement of the fraction of differentiated cells in vitro expressing all of the CD73, CD105, CD10 and CD44 at the cell surface of differentiated cells in vitro; and / or (a2) measuring the amount of one of CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro, preferably measuring the amount of two of CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro , more particularly to measure the total amount of CD73, CD105 and CD44 on the cell surface of differentiated cells in vitro.
    In certain embodiments, the methods as taught herein may include: (a1) measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44 at the cell surface of differentiated cells in vitro; (a2) measuring the amount of one or more CD73, CD105 or CD44 (eg, one, two or all three) on the cell surface of differentiated cells in vitro; (b1) comparing the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured in (a1) with a cutoff value representative of cells with known osteogenic potential; - (b2) the comparison of the quantity of one or more CD73, CD105 or CD44 (for example, one, two or all three) measured in (a2) with one or more respective threshold values representative of cells having an osteogenic potential known; (c1) determining the deviation of the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured in (al) from said cutoff value; - (c2) determining the deviation of the amount of any one (for example, one, two or all three) of CD73, CD105 or CD44 as measured in (a2) from said threshold value; and (d) using said spread to determine a particular osteogenic potential of differentiated cells in vitro.
    In certain embodiments, the methods as taught herein may include: (a1) measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44 on the cell surface of differentiated cells in vitro; (a2) measuring the amount of one or more CD73, CD105, CD44 or CD10 (eg, one, two, three or four) on the cell surface of differentiated cells in vitro;
    (b1) comparing the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured in (a1) with a cutoff value representative of cells with known osteogenic potential; (b2) comparing the amount of one or more CD73, CD105, CD44 or CD10 (eg, one, two, three or four) measured in (a2) with one or more respective cutoff values representative of cells having a known osteogenic potential; (c1) determining the deviation of the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured in (a1) from said cutoff value; (c2) determining the deviation of the amount of any one (eg, one, two, three or all four) of CD73, CD105, CD44 or CD10 as measured in (a2) from said value threshold; and (d) using said spread to determine a particular osteogenic potential of differentiated cells in vitro.
    In particular, measuring the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro; and / or the measurement of the amount of one or more CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro is performed using a technique selected from the group consisting of flow cytometry, mass cytometry, fluorescence activated cell sorting, fluorescence microscopy, affinity separation, magnetic cell separation, microfluidic separation, and combinations thereof.
    Flow cytometry covers the methods by which individual cells in a cell population are analyzed for their optical properties (for example, light absorbance, light scattering, and fluorescence properties) when they are pass in a narrow stream in single file through a laser beam.
    Flow cytometry methods include fluorescence activated cell sorting (FACS) methods by which a population of cells with particular optical properties is separated from other cells.
    Elemental continuous flow cytometry, or mass cytometry, based on mass spectrometry, allows cells to be analyzed by replacing fluorochrome-labeled binding reagents with mass-labeled binding reagents, i.e. marked with an element or isotope of defined mass.
    In these methods, the labeled particles are introduced into a mass cytometer, where they are atomized and ionized individually.
    The individual particles are then subjected to elemental analysis which identifies and measures the abundance of the mass marks used.
    The identities and quantities of isotopic elements associated with each particle are then stored and analyzed.
    Due to the resolution of elemental analysis and the number of elemental isotopes that can be used, it is possible to simultaneously measure up to 100 or more parameters on a single particle.
    Fluorescence microscopy broadly encompasses the methods by which individual cells in a cell population are analyzed under a microscope for their fluorescence properties.
    Fluorescence microscopy methods can be manual or, preferably, semi-automatic or automated.
    Affinity separation, also known as affinity chromatography, generally encompasses techniques involving specific interactions of cells present in a mobile phase, such as an appropriate liquid phase (eg, a cell population in aqueous suspension) with a stationary phase, such as an appropriate solid phase, and hence adsorption of cells into a stationary phase, followed by separation of the stationary phase from the remainder of the mobile phase and recovery (e.g. elution) of adsorbed cells from the stationary phase.
    Affinity separation can be columnar, or alternatively, can involve batch processing, in which the stationary phase is collected / separated from the liquid phases by appropriate techniques, such as centrifugation or the application of a magnetic field (e.g. example, when the stationary phase comprises a magnetic substrate, such as magnetic particles or beads). Accordingly, the separation of magnetic cells is also contemplated here.
    Microfluidic systems enable precise and high throughput detection, quantification and / or sorting of cells, exploiting a variety of physical principles.
    Cell sorting on microchips offers many advantages by reducing the size of the equipment required, eliminating potentially biologically hazardous aerosols, and simplifying the complex protocols typically associated with cell sorting.
    The term "microfluidic system", as used in this specification, generally refers to systems having one or more microchannels of fluid.
    Microchannels refer to fluid channels having cross-sectional dimensions the largest of which are generally less than 1mm, preferably less than 500 µm, more preferably less than 400 µm, more preferably less than 300 µm, more preferably less than 200. µm, for example, 100 µm or less.
    These microfluidic systems can be used to manipulate fluids and / or objects such as droplets, bubbles, capsules, particles, cells, etc.
    Microfluidic systems can allow, for example, unlabeled cell sorting (reviewed in Shields et al., Lab Chip. 2015, vol. 15: 1230-1249) or unlabeled cell sorting (for example, using a or more fluorescent binding agents based on conjugate labels (eg, one or more fluorophore binding agents, such as one or more antibodies conjugated with beads, such as antibody (s) conjugated to beads). In particular, measurement of the fraction of cells differentiated in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro; and measurement of the amount of one or more of CD73, CD105 or CD44 on the Cell surface of differentiated cells in vitro is performed using flow cytometry.
    Cells differentiated in vitro can be labeled with fluorochrome-conjugated antibodies (eg, PE, PE, PE-Cy 7, PE-Cy5, APC, APC-Cy7, Alexa Fluor 647 °, Alexa Fluor 700®, FITC, Pacific Blue, Alexa Fluor 488 ° for one or more of CD73, CD105, CD10 or CD44 and then excited by a laser at a certain wavelength (fluorochrome excitation wavelength) to emit the light at different wavelengths (fluorochrome emission wavelength). For example, differentiated cells in vitro can be labeled with an allophycocyanin conjugated antibody (APC) against CD105 (BD Biosciences ”, Cat No: 562408), an antibody conjugated to an APC against CD73 (BD Biosciences ”, Cat No: 560847), an antibody conjugated to phycoerythrin (PE) against CD10 (BD Biosciences”, Cat No: 555375) and / or PE conjugated antibody against CD44 (BD Biosciences ”, Cat No: 550989). The skilled person will understand that the laser wavelength and the excitation wavelength of the fluorochrome used to label the antibody must be compatible. For example, the excitation wavelength for FITC is 488nm and the emission wavelength is 500-560nm; the excitation wavelength for PE is 488-561nm and the emission length is 560-595nm. The presence or amount of more than one of CD73, CD105, CD105, CD10 or CD44 can be measured simultaneously using antibodies which are each conjugated to a different fluorochrome which emits different light at a different wavelength.
    Most cells do not naturally emit fluorescent light. Therefore, if a fluorochrome conjugated antibody is bound to one or more of CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro, a fluorescent light signal will be picked up when this cell passes through the laser beam of the cytometer. in flux. For each of the fluorochrome conjugated antibodies used in the flow cytometric analysis, a positivity threshold can be set. For example, a positivity threshold can be set at 1% of the positivity of the control isotype antibody.
    The amount of one or more CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro can be represented by the average or average intensity of the fluorescent signal emitted by the differentiated cells in vitro. The mean or median intensity of the fluorescent signal is determined from the fluorescent signal detected for each cell of the entire cell population analyzed (i.e., including cells that did not emit a signal. fluorescent light). More specifically, the amount of one or more CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro can be represented by the normalized median fluorescence intensity (nMFI). Normalized MFI is usually determined by dividing the MFI of the entire analyzed cell population labeled with one or more fluorochrome-conjugated antibodies by the MFI of the negative control (for example, cells labeled with one or more fluorochrome-conjugated isotype antibodies , such as immunoglobulin G (IgG), conjugated to FITC, APC and PE).
    The "normalized median fluorescence intensity" or "nMFI" as used herein refers to the ratio of the nMFI of the entire analyzed cell population labeled with one or more fluorochrome conjugated antibody (MFI marker_channel ) and MFI of the cell population labeled with one or more fluorochrome-conjugated anti-isotype antibodies (MFI isotype_channel), such as the control of immunoglobulin G (IgG) conjugated to a fluorochrome such as FITC, APC or PE. are proportional to the amount of markers present on the cell surface of a population of interest. The (n) MFI is generally related to the wavelength at which the fluorescent signal emission is measured. For example, the lengths excitation wavelengths can be 488nm for FITC, 488nm for PE and 633nm for APC. For example, the emission wavelengths can be 530nm for FITC, 580nm for PE and 660nm for APC.
    The flow cytometer can count the total number of labeled in vitro differentiated cells that pass through the laser as well as the number of labeled in vitro differentiated cells that emit light at a certain wavelength. This information can be used to determine the amount, preferably the fraction, of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44, also known as percentage of fluorescent positive cells (PPFC).
    Those skilled in the art will understand that before measuring the presence or quantity of one or more CD73, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro, the population of cells of interest can be distinguished from other cells or debris according to their forward and lateral diffusion properties, for example by gating strategies.
    Analysis of flow cytometry data can be performed on a fixed number of events - detected (for example, a number of cells passing through the laser of the flow cytometer). For example, analysis of flow cytometry data can be performed on 10,000 events from the population of carrier cells.
    Flow cytometry can be performed using any cytometer known in the art. For example, using FACS Cantoll (BD Biosciences®).
    Analysis of the flow cytometry data can be performed using any flow cytometry software known in the art. For example, FACS Diva software ”8.0 (BD Biosciences”). In particular, the method as taught herein comprises (b1) comparing the amount of differentiated cells in vitro expressing one or more (eg, one, two, three or four) of CD73, CD105, CD105 or CD44 such as measured as described elsewhere in this document with a baseline or cutoff value representing cells of known osteogenic potential and (b2) comparing the amount of one or more (e.g., one, two or all three) of CD73, CD105 or CD44 expressed by the differentiated cells in vitro as measured as described elsewhere in this document with a reference value or a cut-off value representing cells of known osteogenic potential.
    Therefore, the method as taught is also provided herein, comprising (b1) comparing the amount of differentiated cells in vitro expressing all of CD73, CD105, CD10 and CD44 as measured as described elsewhere in the present document with a reference value or a cut-off value representing cells with known osteogenic potential and (b2) the comparison of the amount of all differentiated cells in vitro CD73, CD105 and CD44 expressed by the cells as measured as described in this document with a reference or cut-off value representing cells with known osteogenic potential.
    In more particular embodiments, the method as taught herein comprises (b1) comparing the fraction of differentiated cells in vitro expressing one or more (eg, one, two, three or four) of CD73, CD105, CD105 or CD44 as measured as described elsewhere herein with a cutoff value representing cells of known osteogenic potential and / or (b2) comparing the amount of one or more (eg, one, two or all) of CD73, CD105 or CD44 expressed by differentiated cells in vitro as measured as described herein with a cutoff value representing cells with known osteogenic potential.
    Therefore, the method as taught herein includes (b1) comparing the fraction of differentiated cells in vitro expressing all CD73, CD105, CD10 and CD44 as measured as described elsewhere in this document with a cutoff value representing the cells with known osteogenic potential; and / or (b2) comparing the total amount of CD73, CD105 and CD44 expressed by differentiated cells in vitro as measured as described elsewhere in this document with a cutoff value representing cells of known osteogenic potential.
    In more particular embodiments, the method as taught herein comprises (b1) comparing the fraction of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44 as measured as described elsewhere in this document with a cutoff value representing cells of known osteogenic potential; and / or (b2) comparing the amount of one or more (eg, one, two or three) CD73, CD105 or CD44 expressed by the differentiated cells in vitro as measured as described elsewhere in this document with a cutoff value representing cells with known osteogenic potential.
    In some instances, the method taught herein includes (b1) comparing the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured as described elsewhere in this document with a cutoff value representing cells having known osteogenic potential; and / or (b2) comparing the amount of one or more (eg, one, two, three or all four) of CD73, CD105, CD44 or CD10 expressed by the differentiated cells in vitro as measured as described elsewhere, with a cut-off value representing cells of known osteogenic potential.
    Cells having known osteogenic potential may be cells having some degree of osteogenic potential (eg, no osteogenic potential, low osteogenic potential, high osteogenic potential, or a desired degree of osteogenic potential).
    The degree of osteogenic potential can be expressed by the amount of bone matrix formed and / or by the rate of bone matrix formation in vitro or in vivo. The amount of bone matrix and / or the rate of bone matrix formation can be determined by any method known in the art, such as bone histomorphometry, histology (eg, collagen I, Masson Goldner trichrome) and immunofluorescence (eg, collagen I, tetracycline, bone staining). For example, the thickness of newly mineralized bone, the surface area of newly formed bone, or the presence of at least one mineralized nodule can be assessed in vivo after administration of the cells to mice by subcutaneous injection.
    For example, the degree of osteogenic potential of cells can be determined by measuring the osteogenic activity of these cells. The osteogenic activity of differentiated human cells in vitro can be measured in vivo, for example by determining the presence of at least one mineralized nodule (for example, of human origin or of mixed human-murine origin) after administration of the cells to mice by subcutaneous injection into the bony cranial vault. The osteogenic activity of human - differentiated cells in vitro can be measured in vivo, for example by evaluating the thickness of newly mineralized nodules (eg, of human origin or mixed human-murine) after administration of the cells to mice by subcutaneous injection into the cranial vault, or by assessing the degree of bone repair in a mouse model of subcritical segmental femoral defect (sub-CSD).
    For example, a quantity of human cells, such as 2.5 x 10 ° cells formulated in 100 µl of excipient, can be administered to nude mice by a single subcutaneous administration to the bone of the cranial vault. To mark new bone formation over time, calcium-binding fluorochromes such as alizarin red (red), calcein (green), calcein (blue), and tetracycline (yellow) can be administered sequentially to mice by intraperitoneal injection 3 days before and 4, 8 and 12 days after cellular administration of the cells, respectively. Mice can be euthanized 2 weeks after cell administration and the cranial vault of each mouse can be removed to assess bone forming properties by histomorphometry (eg, quantification of bone formation). The initial and final thicknesses of the cranial vault can be used to calculate the percentage of neosseous formation after administration of the cells. In addition, bone forming properties can also be assessed by immunofluorescence (eg, murine or human origin of bone formation). Osteoblastic activity can be assessed on sections of the cranial vault using the ALP enzyme activity detection method. Osteoclastic activity can be assessed on sections of the cranial vault using TRAP enzyme activity detection methods. The state of mineralization of newly formed bone can be assessed using Masson Goldner's trichrome stain on ALP-stained sections of the cranial vault, for example using commercially available kits (e.g. example, Bio-Optica®). Cartilage formation can be assessed using an orange safranin stain on the sagittal paraffin sections of the cranial vault.
    In another example, in vitro differentiated human cells, such as 1.25 x 10 ° cells formulated in a 50 µl excipient, can be administered locally to mice at the site of the bone defect by percutaneous injection one day after being subjected. with a segmental femoral defect of subcritical size. Bone repair can be quantified by radiography. The size of the bone defect can be quantified by measuring the distance between the two edges of the bone defect.
    In particular, cells with known osteogenic potential may be cells known to form bone matrix without the need for calcified cartilage matrix as a template (eg, cells known to form bone by intramembranous ossification) or cells. known to form a bone matrix by first forming a calcified cartilage matrix and then using said calcified cartilage matrix as a template for forming bone (eg, cells known to form bone by endochondrial ossification). The type of bone formation (eg, endochondral ossification or intramembrane ossification) can be determined by any method known in the art, such as bone histomorphometry, histology (eg, collagen I, Masson Goldner trichrome, safranin-orange, SOX9, ID-type collagen and immunofluorescence (eg, collagen I, tetracycline bone staining, ID-type collagen. For example, the presence of at least one mineralized nodule after administration of cells to mice by subcutaneous injection as described elsewhere in this document may indicate that the bone matrix has formed by endochondral ossification.
    In particular, cells with known osteogenic potential are cells known to form bone by endochondral ossification. For example, cells with known osteogenic potential can be human cells which form at least one mineralized nodule of human origin in vivo after administration of the cells to mice by subcutaneous injection, the subcutaneous injection preferably being performed on the bone of the cranial vault.
    In particular, cells with known osteogenic potential are cells (eg, human cells) which show an increase in bone formation in vivo (eg, of human origin) of at least about 20% (about 1, 2 times or more), or at least about 30%
    (about 1.3 times or more), or at least about 40% (about 1.4 times or more), or at least about 50% (about 1.5 times or more), or at least at least about 60% (about 1.6 times or more), or at least about 70% (about 1.7 times or more), or at least about 80% (about 1.8 times or more), or d '' at least about 90% (about 1.9 times or more), or at least about 100% (about 2 times or more) after administration of the cells, for example, to mice by subcutaneous injection, compared to to the bone formation observed upon administration of control cells (eg cells without osteogenic potential or cells with low osteogenic potential) or a vehicle, eg to mice by subcutaneous injection, preferably in which the subcutaneous injection is performed on the bone of the cranial vault.
    For example, undifferentiated MSCs obtained from the same donor as cells with known osteogenic potential can be used as control cells.
    Among the non-limiting examples of cells known to have a low osteogenic potential, mention may be made of cells derived in vitro from differentiated MSCs (hereinafter referred to as "cell product A" or "bone-forming cells A") obtained as follows: Bone marrow white blood cells are seeded at a density of 50,000 cells / cm ”in the culture medium, and incubated at 37 ° C in a humid incubator containing 5% CO. 4 days after cell seeding, the nonadherent cells are removed and the medium is renewed with a conventional culture medium supplemented with 5% Octaserum (autologous serum 50:50 and OctaPlasLG® (Octapharma)), FGF-b (CellGenix), TGFP-1 (Humanzyme 7 days and 11 days after inoculation, half of the culture medium is removed and replaced with fresh medium.
    The cells are grown during the primary culture for 14 days.
    On day 14, cells are removed by detachment, for example, with Trypzean (Lonza), and by vortexing and pipetting (passage 1: PI). Intermediate cells are cryopreserved in freezing medium comprising culture medium. , 10% Octaserum (autologous serum 50:50 and OctaPlasLG® (Octapharma), 10% DMSO) and stored in liquid nitrogen.
    For secondary culture, cells are thawed and reseeded at a density of 1144 cells / cm2. Cells are grown during secondary culture for 14 days.
    On day 28, cells are removed by detachment, eg, with Trypzean (Lonza), and by vortexing and pipetting up and down (passage 2: P2). To obtain the final cell product, the cells are resuspended, for example, in OctaPlasLG®, to a final concentration of 25 x 10 ° cells / ml.
    This cellular product is referred to herein as “cellular product A” or “bone-forming cells A.” Among the non-limiting examples of cells known to have high osteogenic potential, there may be mentioned cells derived in vitro from differentiated MSCs. (hereinafter referred to as "cell product B" or "bone-forming cells B") obtained as follows: white blood cells from bone marrow are inoculated at a density of 50,000 cells / cm 'in a conventional culture medium comprising 5% OctaPlasLG® (Octapharma), 0.1 IU / ml heparin (LEO Pharma), FGF-b (CellGenix) and
    TGFP-1 (Humanzyme), and incubated at 37 ° C in a humidified incubator containing 5% CO. 4 days after seeding, the non-adherent cells are removed and the medium is renewed with culture medium. 7 days and 11 days after seeding, half of the culture medium is removed and replaced with fresh medium to renew the growth factors.
    The cells are grown during the primary culture for 14 days.
    On day 14, cells are removed by detachment, eg, with Trypzean (Lonza), and by vortexing and pipetting (passage 1: PI). Intermediate cells are cryopreserved (eg, in CryoStor® CS10 (BioLife). Solutions)) and stored in liquid nitrogen.
    Then the intermediate cells are thawed and reseeded for secondary culture at a density of 286 cells / cm2. Cells are grown during secondary culture for 14 days.
    On the 28th day, the cells are removed by detachment, for example with Trypzean (Lonza), and by vortexing and pipetting up and down (passage 2: P2). To obtain the final cell product, the cells are resuspended, for example, in OctaPlasLG®, to a final concentration of 25 x 10 ° cells / ml.
    This cellular product is referred to herein as "cell product B" or "bone forming cells B". In particular, cells having known osteogenic potential are cells having clinically useful osteogenic potential.
    In some embodiments, the threshold value of (b1) and / or said respective threshold values of (b2) may be threshold values representative of cells having clinically useful osteogenic potential.
    The term "clinically useful", when used in connection with the osteogenic potential of cells, refers to a degree of osteogenic potential of cells which enables cells to form, upon transplanting cells into a subject, a bone matrix into one. amount and / or by a mechanism (eg, endochondral ossification or intramembrane ossification) which is of therapeutic significance to a subject, such as one which provides the subject with relevant clinical benefit, such as to a subject with musculoskeletal disease or disorder related to bones.
    In some cases, cells differentiated in vitro may have clinically useful osteogenic potential if at least 50% of animals (eg, in mice) form mineralized nodules (eg, of human origin or mixed human-murine) after administration of the cells to animals (eg, mice) by subcutaneous injection into the cranial vault.
    In some manifestations, cells differentiated in vitro may have clinically useful osteogenic potential if at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 or at least 95% of animals (eg, mice) form mineralized nodules (eg, human or mixed human / mouse origin) after administration of the cells to animals (eg, mice) by subcutaneous injection into the cranial vault.
    Non-limiting examples of musculoskeletal diseases can include local or systemic disorders, such as any type of osteoporosis or osteopenia, eg, primary, postmenopausal, senile, corticosteroid, bisphosphonates, and radiotherapy; any secondary osteonecrosis, mono or multisite; any fracture, for example, non-union, mal-union, delayed union fractures or compression, maxillofacial fractures; conditions requiring bone fusion (eg, fusions and reconstruction of the spine); congenital bone defect; bone reconstruction, for example, after traumatic injury or cancer surgery, and craniofacial bone reconstruction; traumatic arthritis, focal defect in cartilage and / or joints, focal degenerative arthritis; osteoarthritis, degenerative arthritis, gonarthrosis and coxarthrosis; osteogenesis imperfecta; osteolytic 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 McCune-Albright syndrome; rheumatoid arthritis ; spondyloarthropathies, including ankylosing spondylitis, psoriatic arthritis, enteropathic arthropathy and undifferentiated spondylitis and reactive arthritis; systemic lupus erythematosus and related syndromes; scleroderma and related disorders; Sjôgren syndrome; systemic vasculitis, including giant cell arteritis (Horton's disease), Takayasu's arteritis, polymyalgic rheumatic arthritis, ANCA-associated vasculitis (Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome), Behcet's syndrome and other polyarteritis and related diseases (such as polyarteritis nodosa, Cogan syndrome and Buerger's disease); arthritis accompanying other systemic inflammatory diseases, including amyloidosis and sarcoidosis; crystal arthropathies, including gout, calcium pyrophosphate dihydrate disease, disorders or syndromes associated with joint deposition of calcium phosphate or calcium oxalate crystals; chondrocalcinosis and neuropathic arthropathy; Felty's syndrome and Reiter's syndrome; Lyme disease and rheumatosis.
    By way of example, but not limited to, bone disorders which may benefit from a transplant of cells with clinically useful osteogenic potential may include local or systemic disorders, such as any type of osteoporosis or osteopenia. , for example, primary, postmenopausal, senile, corticosteroid, any secondary, single or multi-site osteonecrosis, any fracture, for example, non-union, malunion, fractures or delayed compression, conditions that require bone fusion (for example, fusions and reconstruction of the spine), maxillofacial fractures, bone reconstruction, e.g. after traumatic injury or cancer surgery, craniofacial bone reconstruction, osteogenesis imperfecta, osteolytic bone cancer, Paget's disease, endocrinological disorders, hypophosphatemia , hypocalcemia, renal osteodystrophy, osteomalacia, adynamic disease, rheumatoid arthritis, primary hyperparathyroidism, seco hyperparathyroidism ndaire, periodontal disease, Gorham-Stout disease and McCune-Albright syndrome.
    Among the non-limiting examples of cells having known clinically useful osteogenic potential include "cell product B" or "bone-forming cells B" obtained as described elsewhere in this document. Other non-limiting examples of cells having potential Known clinically useful osteogenic are "cell product C" or "bone-forming cells C" obtained as described elsewhere in this document.
    In particular, cells with known clinically useful osteogenic potential are "B cell product" or "B bone-forming cells" cells obtained as described elsewhere. In particular, cells with known clinically useful osteogenic potential are cells " cell product C "or" bone forming cells C "obtained as described elsewhere, including" cell product C - cryo "or" bone forming cells C cryo (conserved) ".
    In particular, the reference value for the amount of differentiated cells in vitro expressing one or more of the CD73, CD105, CD10 or CD44 cells can be determined by determining the amount of differentiated cells in vitro expressing one or more of the CD73, CD105 cells, CD10 or CD44, respectively, in reference cells (eg, cells whose osteogenic potential is clinically useful), thereby producing a reference value. Likewise, the reference value for the amount of one or more CD73, CD105 or CD44 expressed by the differentiated cells in vitro can be determined by determining the amount of one or more CD73, CD105 or CD44, respectively, in reference cells (eg, cells known to have clinically useful osteogenic potential), thereby producing a reference value.
    One or more reference values obtained from one or more types of reference cells can be used to determine a threshold value or a threshold value generally known in the art to ensure a certain degree of osteogenic potential of the cells, of preference for clinically useful osteogenic potential.
    In particular, the reference values or cut-off values indicated in (b1) comparing the quantity (or fraction) of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44, preferably all CD73, CD105, CD10 and CD44, as measured as described elsewhere with a baseline or cutoff value representing cells of known osteogenic potential; and / or (b2) compare the amount of one or more CD73, CD105 or CD44, preferably all CD73, CD105 and CD44, expressed by the differentiated cells in vitro as measured as described elsewhere, to a value of reference or a cut-off value representing cells having a known osteogenic potential, are reference values or cut-off values representing cells having a useful osteogenic potential.
    By extensive research, the current inventors have discovered that at least 90% of a cell population of differentiated cells in vitro having osteogenic potential, and in particular of a cell population of differentiated cells in vitro having high osteogenic potential and forming a bone matrix in vivo by endochondral ossification, expresses one or more of CD73, CD105, CD10 and CD44, preferably CD73, CD105 and CD44. In addition, these in vitro differentiated cells also express an increased amount of CD73 and / or CD44 and a reduced amount of CD105 compared to the amounts of CD73, CD44 and CD105, respectively, in MSCs.
    Accordingly, in particular embodiments, the method as taught herein comprises (cl) determining a deviation or no deviation in the amount (or fraction) of the differentiated cells in vitro expressing one or more of the CD73. , CD105, CD10 or CD44, preferably all CD73, CD105, CD10 and CD44, measured as described elsewhere herein from the baseline or cutoff value representing cells with known osteogenic potential, and / or (c2) find a cart or no deviation of the amount of any of CD73, CD105 or CD44, preferably all CD73, CD105 and CD44, expressed by differentiated cells in vitro as measured as described elsewhere herein from the baseline value or the cutoff value representing cells with known osteogenic potential, and (d) assigning the deviation or no deviation found in (cl) and / or (c2) to a particular determination of the osteogenic potential of the differentiated cells s in vitro.
    Therefore, the method as taught herein comprising (cl) determining a deviation or no deviation of the amount (or fraction) of the differentiated cells in vitro expressing all CD73, CD105, CD10 and CD44 measured as described. elsewhere from the baseline or cutoff value representing cells with known osteogenic potential, and / or (c2) determining a deviation or no deviation in the amount of all CD73, CD105 and CD44 expressed by the cells - differentiated in vitro measured as described elsewhere in this document from the reference value or the cut-off value representing cells with known osteogenic potential, and (d) assign it to the deviation or no deviation found in (cl) and / or (c2) for the particular determination of the osteogenic potential of differentiated cells in vitro.
    In some cases, the methods taught here may include (cl) finding a deviation or no deviation in the amount (or fraction) of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44, as measured as described elsewhere herein. from the baseline or cutoff value representing cells with known osteogenic potential, and / or (c2) find a deviation or no deviation in the amount of either (e.g., one, two, or all three) of CD73, CD105 or CD44; preferably all of CD73, CD105 and CD44; expressed by differentiated cells in vitro as measured as described elsewhere from the baseline or cutoff representing cells with known osteogenic potential, and (d) assign the deviation or no deviation found in (cl) and / or (c2) to a particular determination of the osteogenic potential of the differentiated cells in vitro.
    In some cases, the methods taught herein may include (c) finding deviation or no deviation in the amount (or fraction) of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44, as measured as described elsewhere in this document from the reference value or limit value representing cells with known osteogenic potential, and / or (c2) find a deviation or no deviation in the amount of either (for example, a , two, three or all four) of CD73, CD105, CD44 or CD10; preferably all of CD73, CD105, CD44 and CD10; expressed by the differentiated cells in vitro as measured as described elsewhere from the baseline or cut-off representing cells with known osteogenic potential, and (d) assign the deviation or no deviation found in (c1) and / or (c2) to a particular determination of the osteogenic potential of the differentiated cells in vitro.
    A "deviation" of a first value from a second value can generally cover any direction (for example, increase: first value> second value; or decrease: first value <second value) and any degree modification.
    For example, a deviation may include a decrease from a first value of at least about 10% (about 0.9 times or less), at least 20% (about 0.8 times or less), at least at least 30% (about 0.7 times or less), at least 40% (about 0.6 times or less), at least 50% (about 0.5 times or less), or at least about 60 % (about 0.4 times or less), or at least about 70% (about 0.3 times or less), or at least about 80% (about 0.2 times or less), or at least about 90% (about 0.1 times or less), compared to a second value with which a comparison is made.
    For example, a deviation may include an increase from a first value of at least about 10% (about 1.1 times or more), at least 20% (about 1.2 times or more), at least at least 30% (about 1.3 times or more), at least 40% (about 1.4 times or more), at least about 50% (about 1.5 times or more), at least 60% (about 1.6 times or more), at least 70% (about 1.7 times or more), or at least about 80% (about 1.8 times or more), or at least about 90% (about 1.9 times or more), or at least about 100% (about 2 times or more), or at least about 150% (about 2.5 times or more), or at least about 200% (about 3 times or more), or at least about 500% (about © times or more), or at least about 700% (about 8 times or more), or the like, with respect to one second value with which a comparison is made.
    Preferably, a deviation can refer to a statistically significant alteration observed.
    For example, a deviation may relate to an observed alteration that is outside the margins of error of the reference values in given reference cells (expressed, for example, by standard deviation or standard error, or by a predetermined multiple of these, for example, £ 1xSD or + 2xSD or
    + 3xSD, or + 1xSE or + 2xSE or + 3xSE). Deviation can also refer to a value that is outside of a reference range defined by values in given reference cells (for example, outside of a range that includes 240%, 50%, 2260% , 270%, 275% or 280% or 285% or 290% or 295% or even 2100% of the values in given reference cells).
    In another embodiment, a deviation can be concluded if an observed alteration is beyond a given threshold or threshold. For example, a deviation can be concluded if an observed alteration is less than, equal to or greater than a given threshold or threshold.
    Particularly the embodiments, the method as taught herein comprises, consists essentially of, or consists of: (a1) measuring the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro; (a2) measuring the amount of one or more CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro; (b1) comparing the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44 as measured in (a1) with a cutoff value representing cells with known osteogenic potential; (b2) comparing the amount of one or more CD73, CD105 or CD44 measured in (a2) with one or more respective cut-off values representing cells of known osteogenic potential; (c1) finding a deviation or no deviation of the fraction of in vitro differentiated cells expressing one or more of CD73, CD105, CD10 or CD44 as measured in (a1) from said cutoff value; (c2) finding a deviation or no deviation of the amount of any one of CD73, CD105 or CD44 measured in (a2) from said threshold value; and (Qd) assigning said deviation or no deviation to a particular determination of the osteogenic potential of differentiated cells in vitro.
    Therefore, the method as taught herein comprises, consists essentially of, or consists of, or consists of: (a1) measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44 on the cell surface of differentiated cells in vitro; (a2) measuring the amount of CD73, CD105 and CD44 on the cell surface of differentiated cells in vitro;
    (b1) comparing the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured in (a1) with a cutoff value representing cells having known osteogenic potential; (b2) comparing the amount of CD73, CD105 and CD44 measured in (a2) with one or more respective cut-off values representing cells of known osteogenic potential; (c1) finding a deviation or no deviation of the fraction of in vitro differentiated cells expressing CD73, CD105, CD105, CD10 and CD44 as measured in (a1) from said cutoff value; (c2) finding a deviation or no deviation of the amount of CD73, CD105 and CD44 measured in (a2) from said threshold value; and (d) assigning said deviation or no deviation to a particular determination of the osteogenic potential of differentiated cells in vitro.
    In particular embodiments, in which the reference cells are cells which are known to lack clinically useful osteogenic potential, - the same quantity or identical or reduced fraction of the cells differentiated in vitro expressing one or more CD73, CD105, CD105, CD10 or CD44, as measured as described elsewhere, compared to cutoff values known to have no clinically useful osteogenic potential as described elsewhere indicates that cells differentiated in vitro have no useful osteogenic potential; or an increased amount or fraction of cells differentiated in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44, as measured as described elsewhere, over the cutoff value representing cells known to have no clinically useful osteogenic potential as described elsewhere indicates that cells differentiated in vitro have useful osteogenic potential, and / or - the same or reduced amount of CD73, CD44 and / or CD10 expressed by differentiated cells in vitro, as measured as described elsewhere in this document, with respect to the respective threshold values of representation of cells known to have no clinically useful osteogenic potential, as described elsewhere in this document, and the same or increased amount of CD105 expressed by cells differentiated in vitro, as measured as described elsewhere, against the respective cutoff values representing an osteogenic potential known to have no clinically useful osteogenic potential as described in another document, indicate that the cells differentiated in vitro have no useful osteogenic potential; or - an increased amount of any of CD73, CD44 and / or CD10 expressed by differentiated cells in vitro as measured as described elsewhere in this document over respective cutoff values representing cells known to have no clinically useful osteogenic potential as described elsewhere in this document, and / or a reduced amount of CD105 expressed by differentiated cells in vitro as measured as described elsewhere in the document relative to the respective cutoff values of representative cells known to not possess No clinically useful osteogenic potential as described elsewhere in the document indicates that cells differentiated in vitro exhibit useful osteogenic potential, preferably in which the expression of one or more of CD73, CD105, CD10 or CD44 represents the expression of CD73, CD105, CD10 or CD44, respectively, on the cell surface.
    In particular embodiments, in which the reference cells are cells known to have a desired osteogenic potential, preferably a clinically useful osteogenic potential, a decrease in the amount or fraction of cells differentiated in vitro expressing one or more more of CD73, CD105, CD105, CD10 or CD44, as measured as described elsewhere, relative to the cut-off value representing cells known to have a desired osteogenic potential as described elsewhere indicates that cells differentiated in vitro do not have the potential desired osteogenic, or - the same amount or the same or greater amount or fraction of the differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44, as measured as described elsewhere, compared to cut-off cells known to having a desired osteogenic potential as described elsewhere, indicates that the differentiated cells These in vitro have the desired osteogenic potential, preferably clinically useful osteogenic potential; and / or - a decreased amount of any of the CD73, CD44 and / or CD10 expressed by the differentiated cells in vitro as measured as described elsewhere in this document relative to the respective cut-off values representing cells known to have a desired osteogenic potential as described elsewhere, and / or an increased amount of CD105 expressed by differentiated cells in vitro as measured as described elsewhere, relative to the respective cutoff values of cells known to have desired osteogenic potential, as described elsewhere, indicates that cells differentiated in vitro do not possess the desired osteogenic potential or - the same or increased amount of CD73, CD44 and / or CD10 expressed by the differentiated cells in vitro, as measured as described elsewhere in this document, with respect to the respective threshold values of representation of cells known to have osteogenic potential as desired, as described elsewhere in this document, and the same or a reduced amount of CD105 expressed by differentiated cells in vitro, as measured as described elsewhere, relative to the respective cutoff values representing cells recognized to have a Desired osteogenic potential, as described elsewhere, indicates that cells differentiated in vitro have useful osteogenic potential, preferably clinically osteogenic potential, preferably in which the expression of one or more of CD73, CD105, CD10 or CD44 represents expression of CD73, CD105, CD10 or CD44, respectively, at the cell surface. In particular embodiments, in which the reference cells are cells which are known to lack clinically useful osteogenic potential, - the same or a reduced amount of CD10 expressed by the differentiated cells in vitro, as measured as described elsewhere in this document, compared to the respective representational cutoff values of cells known to have no clinically useful osteogenic potential, as described elsewhere, indicates that cells differentiated in vitro have no useful osteogenic potential ; or - an increased amount of CD10 expressed by differentiated cells in vitro, as measured as described elsewhere in this document, relative to the respective cutoff values representing cells known to have no clinically useful osteogenic potential , as described elsewhere, indicates that cells differentiated in vitro have useful osteogenic potential, preferably in which expression of CD10 represents expression of CD10 on the cell surface.
    In particular embodiments, in which the reference cells are cells known to have a desired osteogenic potential, preferably a clinically useful osteogenic potential, - a decrease in the amount of CD10 expressed by the differentiated cells in vitro, such as measured as described elsewhere in this document, against respective cutoff values representing cells known to have a desired osteogenic potential, as described elsewhere, indicates that cells differentiated in vitro do not have the desired osteogenic potential, or the same amount or increased amount of CD10 expressed by the differentiated cells in vitro, as measured as described elsewhere in this document, compared to the respective threshold values of representation of cells known to have a desired osteogenic potential, as described elsewhere, indicates that cells differentiated in vitro have the potential desired osteogenic, preferably clinically useful osteogenic potential,
    preferably wherein the expression of CD10 represents the expression of CD10 on the cell surface.
    Some embodiments relate to methods as taught herein, in which: - an identical or increased fraction of the differentiated cells in vitro, as measured in (a1) relative to the cutoff value of (b1), indicates that the cells differentiated in vitro have clinically useful osteogenic potential; and -Ja the same or an increased amount of CD73, CD44 and / or CD10 measured in (a2) compared to the respective cutoff values of (b2), and the same amount or a reduced amount of CD105 measured in (a2) compared to at the respective cut-off value of (b2) indicates that the cells differentiated in vitro have clinically useful osteogenic potential.
    In some embodiments, said cut-off value of (b1) is 90% of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the cell surface; and wherein said cut-off value of (b2) is a normalized median fluorescence intensity (CD73 nMFD of 500, CD44 nMFI of 100, CD105 nMFI of 150 and / or CD10 nMFI of 40. In certain embodiments, said cutoff value of (b1) is 90% of in vitro differentiated cells expressing CD73, CD105, CD10 and CD44 on the cell surface; and wherein said cutoff value of (b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 100, an nMFI for CD105 of 150 and an nMFI for CD10 of 50. In some embodiments, said cut-off value of (b1) is 90% of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44 on the cell surface; and wherein said cutoff value of (b2) is a normalized nMFI for CD73 of 500, an nMFI for CD44 of 150, an nMFI for CD105 of 150 and / or an nMFI for CD10 of 40. In some embodiments, said cut-off value of (b1) is 90% of differentiated cells in vitro expressing CD 73, CD105, CD10 and CD44 on the cell surface; and wherein said cut-off value of (b2) is a normalized nMFI for CD73 of 500, an nMFI for CD44 of 150, an nMFI for CD105 of 150 and / or an nMFI for CD10 of 50. In some cases, the amount of CD73 , CD105 and CD44 expressed by differentiated cells in vitro is measured.
    In some embodiments, said cutoff value of (b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 100 and an nMFI for CD105 of 150. In some cases, the amount of CD73, CD105, CD44 and CD10 expressed by the differentiated cells in vitro is measured.
    In some embodiments, said threshold value of (b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 100, an nMFI for CD105 of 150, and an nMFI for CD10 of 40. In some embodiments, said threshold value of ( b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 150, an nMFI for CD105 of 150, and an nMFI for CD10 of 40. In some embodiments, said threshold value of (b2) is an nMFI for CD73 of 500. , an nMFI for CD44 of 100,
    an nMFI for CD105 of 150, and an nMFI for CD10 of 40. In some embodiments, said threshold value of (b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 150, an nMFI for CD105 of 150 and an nMFI for CD10 of 50.
    In particular, the cutoff value of (b1) comparing the fraction of in vitro differentiated cells expressing one or more of CD73, CD105, CD10 or CD44 with a cutoff value representing cells with known osteogenic potential, preferably clinically useful osteogenic potential known, is 90%, 91%, 92%, 93%, 94% and 95%; 96%; 97%, 98% or 99%, preferably 90%, of cells differentiated in vitro expressing one or more of CD73, CD105, CD10 or CD44, preferably where the expression of one or more of CD73, CD105, CD10 or CD44 represents the expression of CD73, CD105, CD10 or CD44, respectively on the cell surface. Therefore, the cut-off value of (bl) comparing the fraction of in vitro differentiated cells expressing all of CD73, CD105, CD10 and CD44 with a cut-off value representing cells with known osteogenic potential, preferably known clinical useful osteogenic potential , is 90%, 91%, 92%, 93%, 94%, 95%; 96%; 97%, 98% or 99%, preferably 90%, of in vitro differentiated cells expressing all CD73, CD105, CD10 and CD44, preferably in which the expression of all CD73, CD105, CD10 and CD44 represents the expression of CD73, CD105, CD10 and CD44, respectively at the cell surface. In a particular embodiment, if approximately 90%, 91%, 92%, 93%, 94%, 94 Jo, 95%, 96%, 96%, 97%, 98% or 99%, preferably 90% or plus cells differentiated in vitro express one or more of CD73, CD105, CD10 or CD44, preferably all of CD73, CD105, CD10 and CD44, these cells have the desired osteogenic potential, preferably useful osteogenic potential. Therefore, if about 90% or more of the cells differentiated in vitro express all of CD73, CD105, CD105, CD10 and CD44, the cells differentiated in vitro have the desired osteogenic potential, preferably clinically useful osteogenic potential.
    In particular, the cutoff value of (b2) comparing the amount of one or more CD73, CD105, CD44 and / or CD10 expressed by cells differentiated in vitro at the cell surface with a cutoff value representing cells with osteogenic potential known, preferably a known clinical useful osteogenic potential, is an nMFIcp73 of 500, 550, 600, 650, 700, 700, 750, 800, 850 or 900, preferably an nMFlep73 of 500, an nMFIcp44 of 100, 110, 120 , 130, 140, 150, 200, 250, 300 or 350, preferably an nMFICD44 of 100, an nMFlepios of 180, 170, 160, 150, 150, 140, 130, 120, 110 or 100, preferably an nMFlcp105 of 150 and / or an nMFlepio of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60, preferably an nMFlcpio of 50; preferably in which nMFlep73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, nMFlcpa4 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE, the nMFIcp10s is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC, and / or the nMFIcp is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE. Therefore, the cut-off value of (b2) of (b2) comparing the amount of CD73, CD105 and CD44 expressed by cells differentiated in vitro at the cell surface with a cut-off value representing cells with known osteogenic potential, preferably a known clinical useful osteogenic potential is an nMFlcp73 of 500, 550, 600, 650, 700, 750, 800, 850 or 900, preferably an nMFlecp73 of 500, an nMFlIcp44 of 100, 110, 120, 130, 140, 150, 200, 250, 300 or 350, preferably an nMFlep4 of 100 and an nMFlcpios of 180, 170, 160, 150, 140, 130, 120, 110 or 100, preferably an nMFlep105 of 150, preferably where the nMFlçp73 is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC, the nMFlcpa is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE, and nMFlep1os is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC. In some representations, the cutoff value of (b2) comparing the amount of one or more CD73, CD105, CD44 and CD10 expressed by cells differentiated in vitro at the cell surface with a cutoff value representing cells with known osteogenic potential , preferably a known clinical useful osteogenic potential, is an nMFlcp735 of 500, 550, 600, 650, 700, 700, 750, 800, 850 or 900, preferably an nMFIcp; 3 of 500, an nMFlcpa4 of 100, 110, 120, 130, 140, 150, 200, 250, 300 or 350, preferably an nMFlIcp44 of 100, an nMFlop1os of 180, 170, 160, 150, 150, 140, 130, 120, 110 or 100, preferably an nMFICD105 of 150 and an nMFlcpio of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60, preferably an nMFlcpio of 50; preferably in which the nMFlcp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, the nMFIcp44 is measured with an excitation wavelength of 488 nm and emission length of 580 nm for PE, nMFlep105 is measured with excitation length of 633 nm and emission length of 660 nm for APC and nMFlop1o is measured with excitation length of 488 nm and an emission length of 580 nm for PE. In particular, the cut-off value of (b2) comparing the amount of CD10 expressed by cells differentiated in vitro at the cell surface with a cut-off value representing cells with known osteogenic potential is an nMFIcpi0 of 10, 15, 20, 25 or 30, preferably an nMFlop10 of
    20. In particular, the cut-off value of (b2) comparing the amount of CD10 expressed by cells differentiated in vitro at the cell surface with a cut-off value representing cells with clinically useful osteogenic potential is an nMFIcp10 of 40, 45, 50 , 55 or 60, preferably an nMFIcpio of 40; more preferably an nMFlepio of 50; even more preferably an nMFIcp10 of 60. Preferably, the nNMFlop10 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE.
    The recitations "nMFI for CD73" or "nMFlcp3" used herein refer to the ratio of the MFI of the entire analyzed cell population labeled with an APC conjugated antibody against CD73 (eg, BD Biosciences ”, Cat No: 560847 ) and that of the cell population labeled with control IgG conjugated with APC (eg, BD Biosciences ”, Cat No: 555751). Preferably, nMFICD73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC.
    The recitations "nMFI for CD44" or "nMFlcpaa" used herein refer to the ratio of the MFI of the entire analyzed cell population labeled with an antibody conjugated PE against CD44 (eg, BD Biosciences ”, Cat No: 550989 ) and that of the cell population labeled with IgG conjugated with PE (eg, BD Biosciences®, Cat No .: 556650). Preferably, nMFIcp44 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE.
    The recitation "nMFI for CD105" or "nMFlep105" as used herein refers to the ratio of the MFI of the entire analyzed cell population labeled with APC conjugate antibodies against CD105 (eg, BD Biosciences ”, Cat N No .: 562408) and MFI of the cell population labeled with IgG control conjugated with APC (e.g. BD Biosciences®, Cat No: 555751). Preferably, nMFlcp10s is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC.
    The recitations "nMFI for CD10" or "nMFlcpio" used herein refer to the ratio of the MFI of - the entire analyzed cell population labeled with an antibody conjugated PE against CD10 (eg, BD Biosciences ”, Cat No: 555375) and that of the cell population labeled with IgG conjugated with PE (eg, BD Biosciences®, Cat No: 556650). Preferably, the nMFIcp10 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE.
    In particular, an nMFlcp75 of 500, 550, 600, 650, 700, 750, 800, 850 or 900, preferably 500, or more, expressed by differentiated cells in vitro as measured as described elsewhere indicates that differentiated cells in vitro. vitro have the desired osteogenic potential, preferably clinically useful osteogenic potential.
    In particular, an nMFIcp4 of 100, 110, 120, 130, 140, 150, 200, 250, 300 or 350, preferably 100, or more, expressed by differentiated cells in vitro as measured as described elsewhere, indicates that the cells differentiated in vitro have the desired osteogenic potential, preferably clinically useful osteogenic potential.
    In particular, an nMFlcpi0s of 180, 170, 160, 150, 140, 130, 120, 110 or 100, preferably 150, or less, expressed by differentiated cells in vitro, as measured as described elsewhere, indicates that the cells differentiated in vitro have the desired osteogenic potential, preferably clinically useful osteogenic potential.
    In particular, an nMFlcp1o of at least 10, at least 15, at least 20, at least 25, at least 30, at least 30, at least 40, at least 45, at least 50, at least 50, at least 55 or at least 60, preferably at least 50, expressed by the differentiated cells in vitro as measured as described elsewhere indicates that these cells have the desired osteogenic potential, preferably useful osteogenic potential.
    In preferred embodiments, an nMFlIcp73 of 500 or more, an nMFIcp44 of 100 or more, and an nMFlcpios of 150 or less expressed by the differentiated cells in vitro, as measured as described elsewhere, indicate that the differentiated cells in vitro. vitro have the desired osteogenic potential, preferably a clinically useful osteogenic potential, preferably in which nMFlcp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, nMFlep44 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE, and / or nMFlcpi05 is measured with an excitation length of 633 nm and an emission length of 660 nm for APC.
    In particular, the cutoff value of (b2) comparing the amount of one or more CD73, CD105, CD44 and / or CD10 expressed by cells differentiated in vitro at the cell surface with a cutoff value representing cells with osteogenic potential known, preferably a known clinical useful osteogenic potential, is an nMFlcp75 of 500, an nMFlcpa4 of 100, an nMFlop105 of 150 and / or an nMFlop10 of 40, preferably in which the nMFIcps3 is measured with a wavelength of excitation of 633 nm and an emission wavelength of 660 nm for APC, nMFlop4 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE, nMFI; 9s is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC, and / or nMFlop10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In particular, the cut-off value of (b1) comparing the fraction of in vitro differentiated cells expressing one or more of CD73, CD105, CD10 or CD44 at the cell surface with a cut-off value representing cells with known osteogenic potential, preferably one known clinical useful osteogenic potential is 90% of cells differentiated in vitro expressing one or more of CD73, CD105, CD10 or CD44; and / or the cutoff value of (b2) comparing the amount of CD73, CD105, CD44 and / or CD10 expressed by cells differentiated in vitro on the cell surface with a cutoff value representing cells with known osteogenic potential, preferably a known clinical useful osteogenic potential, is an nMFIçp73 of 500, an nMFlepy of 100, an nMFlcpios of 150 and / or an nMFlep, o of 40, preferably in which the nMFlçp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for the APC, the nMFlçp44 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE, the nMFI,; os is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC, and / or nMFlcp10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In particular, the differentiated cells in vitro are obtained or derived from pluripotent stem (PS) cells, such as mammalian PS and human PS, preferably human PS.
    The term "differentiated cells in vitro", as used in the specification, refers to all cells which have been cultured in vitro under conditions suitable for allowing cells to switch from one cell type to another. Differentiation of cells may involve culturing MSCs under conditions capable of inducing differentiation of cells to the desired cell type, more typically culturing cells in a medium comprising one or more agents (eg, growth factors). capable of inducing cell differentiation to the desired cell type. The term "stem cell" generally denotes a cell that is unspecialized or - relatively less specialized and proficient in proliferation, capable of self-renewal, that is to say of proliferating without differentiation, and of which the offspring can give. birth to at least one relatively more specialized cell type. The term encompasses stem cells capable of substantially unlimited self-renewal, i.e. in which the progeny of a stem cell or at least part of it - here substantially retains the non-specialized or - relatively less specialized phenotype, the differentiation potential and the proliferative capacity of the parent stem cell, as well as the stem cells which exhibit limited self-renewal, i.e. in in which the ability of the products or part of the progeny to continue multiplication and / or to differentiate is markedly reduced compared to the parent cell. r example, a stem cell can give rise to descendants capable of differentiating along one or more lines to produce more and more specialized cells, these descendants and / or more and more specialized cells being able to be them even stem cells as defined herein, or even to produce terminally differentiated cells, i.e. fully specialized cells, which may be postmitotic. Included in the definition of mPS cells are embryonic stem cells of various types, exemplified without limitation by murine embryonic stem cells, as described by Evans & Kaufman 1981 (Nature 292: 154-6) and Martin 1981 (PNAS 78: 7634 -8); rat pluripotent stem cells, for example, described by lannaccone et al. 1994 (Dev Biol 163: 288-292); hamster embryonic stem cells, for example, as described by Doetschman et al. 1988 (Dev Biol 127: 224-227); rabbit embryonic stem cells, for example, as described by Graves et al. 1993 (Mol Reprod Dev 36: 424-433); porcine pluripotent stem cells, as described by Notarianni et al. 1991 (J Reprod Fertil Suppl 43: 255-60) and Wheeler
    1994 (Reprod Fertil Dev 6: 563-8); ovine embryonic stem cells, for example, as described by Notarianni et al. 1991 (supra); bovine embryonic stem cells, for example, as described by Roach et al. 2006 (Methods Enzymol 418: 21-37); human embryonic stem cells (hES), as described by Thomson et al. 1998 (Science 282: 1145-1147); human embryonic germ cells (hEG), as described by Shamblott et al. 1998 (PNAS 95: 13726); embryonic stem cells from other primates such as Rhesus stem cells, as described by Thomson et al. 1995 (PNAS 92: 7844-7848) or the marmoset stem cells, for example described by Thomson et al. 1996 (Biol Reprod 55: 254-259). Other types of mPS cells are also included in the term, as are cells of mammalian origin capable of producing offspring comprising derivatives of all three germ layers, whether derived from embryonic, fetal, or other tissues. sources.
    The mPS cells are preferably not from a malignant source.
    A cell or cell line is from a "non-malignant source" if it was established from primary tissue which is not cancerous or modified by a known oncogene.
    It may be desirable for mPS to maintain a normal karyotype throughout prolonged culture under appropriate conditions.
    It may also be desirable, but not always necessary, for mPS to retain a substantially undefined self-renewal potential under appropriate in vitro conditions.
    In this document, the term "pluripotent" refers to the ability of a cell to give rise to cell types originating from the three germ layers of an organism, namely - mesoderm, endoderm and l. 'ectoderm, and potentially capable of giving rise to any cell type in an organism, although not able to grow throughout the organism.
    More particularly, the differentiated cells in vitro are obtained or derived from mesenchymal stem cells (MSC), embryonic stem cells (ESC) or induced pluripotent stem cells (iPS). More particularly, the uses or methods taught here - differentiated cells in vitro are obtained or derived from CSM.
    The term "mesenchymal stem cell" or "MSC" used herein denotes an adult stem cell derived from the mesoderm which is capable of generating cells of mesenchymal lineages, generally of two or more mesenchymal lineages, more generally three or more mesenchymal lineages, by example, chondro-osteoblastic (bone and cartilage), osteoblastic (bone), - chondroblastic (cartilage), myocytic (muscle), tenocytic (tendon), fibroblastic (connective tissue), adipocyte (fat) and stromogen (medullary stroma). CMS can be isolated from a biological sample, preferably a biological sample from a human subject, e.g. bone marrow, trabecular bone, blood, umbilical cord, placenta, fetal yolk sac, skin (dermis), in particular fetal and adolescent skin, periosteum, dental pulp, tendon and adipose tissue.
    The terms "biological sample" or "sample", as used herein, mean a sample obtained from a biological source, for example from an organism, such as an animal or human subject, a cell culture. , a tissue sample, etc. A biological sample from an animal or human subject refers to a sample taken from an animal or human subject and comprising cells thereof. The biological sample from an animal or human subject may include one or more tissue types and may include cells of one or more tissue types. Methods of obtaining biological samples from an animal or human subject are well known in the art, e.g. example, tissue biopsy or blood sampling Human MSC, its isolation, in vitro expansion and differentiation has been described in, for example, US Pat. No.
    5,486,359; US Pat. No. 5,811,094; US Pat. No. 5,736,396; US Pat. No. 5,837,539; or US Pat. No. 5,827,740. Any CSM described in the art and isolated by any method described in the art may be suitable in the present method. In particular, MSCs can be defined as exhibiting the capacity for trilineage mesenchymal differentiation in vitro into osteoblasts, adipocytes and chondroblasts (Dominici et al., 2006, vol. 8, 315).
    - The term "embryonic stem cells" or "ESC", as used herein, refers to pluripotent stem cells which are derived from an embryo, for example from the inner cell mass of the blastocyst, and which are capable, under suitable conditions, to produce progeny of different cell types which are derivatives of the three germ layers, Le., endoderm, mesoderm and ectoderm, according to a standard test accepted by the art, such as the ability to form a teratoma in SCID mice, or the ability to form identifiable cells of the three germ layers in tissue culture. The term "hES cells" covers pluripotent stem cells which are derived from a human embryo at the blastocyst stage, or before substantial differentiation cells in three germ layers. ES cells, especially hES cells, are usually derived from the inner cell mass of blastocysts or blastocysts in third. The derivation of hES cell lines from the morula stage has been documented and the ES cells thus obtained can also be used in the invention (Strelchenko et al. 2004. Reproductive BioMedicine Online 9: 623-629). The term "induced pluripotent stem cells" or "iPS cells" as used herein refers to pluripotent stem cells generated from adult cells by reprogramming. IPS cells can renew themselves and give rise to cell types from all three germ layers of an organism, i.e. mesoderm, endoderm and ectoderm, and potentially from all or part of an organism, but cannot grow in the whole organism. Examples of iPS cells are those taught inter alia by Yamanaka et al. 2006 (Cell 126: 663-676) and Yamanaka et al. 2007 (Cell 131: 861-872).
    The term "CSM", "ESC" or "iPS" also encompasses progeny of CSM, ESC or iPS, respectively, for example, progeny obtained by in vitro or ex vivo proliferation
    (propagation / expansion) of CSM, ESC or 1iPS, respectively, obtained from a biological sample from an animal or human subject.
    The term "adult stem cell", as used herein, means a stem cell present in an organism or obtained from an organism (eg isolated from an organism) in the fetal stage or preferably after birth. (e.g., in particular, but not limited to a human organism, at least one month after birth, e.g., at least 2 months, at least 3 months, e.g. at least 4 months, at least 5 months, e.g. at least 6 months after birth, such as for example 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), as per example after reaching majority.
    For example, adult stem cells can be obtained from human subjects who would otherwise be described under the conventional terms "infant", "child", "young", "adolescent" or "adult". Unless otherwise indicated, the terms "subject", "donor" or "patient" are used interchangeably and denote animals, preferably vertebrates, preferably mammals, and more particularly human patients and non-human mammals.
    Preferred patients are human subjects.
    Animal subjects include prenatal forms of animals, such as fetuses.
    In particular, cells differentiated in vitro are human cells embodying the uses or methods taught herein.
    Current methods and protocols may preferably deviate from populations of pluripotent stem cells (eg, mPS or hPS cell populations) which are "undifferentiated", ie in which a substantial proportion (eg. , at least about 60%, preferably at least about 70%, preferably at least about 70%, still more preferably at least 80%, still more preferably at least about 90% and up to 100%) of the cells of the population of stem cells exhibit characteristics (eg, morphological characteristics and / or markers) of undifferentiated mPS cells, clearly differentiating them from cells undergoing differentiation.
    Undifferentiated mPS cells are generally easily recognized by those skilled in the art and can appear in two dimensions from a microscopic view with high nuclear / cytoplasmic ratios and prominent nucleoli, like compact colonies with sharp edges.
    It is understood that colonies of undifferentiated cells within the population can often be surrounded by neighboring cells which are more differentiated.
    Nevertheless, undifferentiated colonies persist when the population is cultured or transmitted under suitable conditions known per se and the undifferentiated individual cells constitute a substantial proportion of the cell population.
    Undifferentiated mPS cells can express specific embryonic stage 3 and 4 antigens (SSEA) and markers detectable using antibodies called Tra-1-60 and Tra-1-81 (Thomson et al. 1998, supra) . Undifferentiated mPS cells can also typically express Nanog, Oct-4 and TERT.
    Undifferentiated mPS cells can also include expression of alkaline phosphatase (AP) (eg, determined by an appropriate AP activity assay). In particular, the cells differentiated in vitro are of the chondro-osteoblastic line (bone and cartilage), of the osteoblastic line (bone), such as for example osteochondrogens and / or osteoprogenitors and / or preosteoblasts and / or osteoblasts and / or osteocytes, etc. . ; chondroblastic line (cartilage), such as, for example, osteochondrogens and / or chondrogens and / or chondrogens and / or prechondroblasts and / or chondroblasts and / or chondroblasts and / or chondrocytes; adipogenic line (fat); myogenic (muscle); tenogenic (tenocytes); fibroblastic (connective tissue), such as, for example, fibroblasts, fibrocytes or synovium (synovial fluid). The term "chondro-osteoblastic line", as used herein with reference to cells differentiated in vitro, can refer to cells which have the ability to differentiate into cells of the osteoblastic line, such as osteochondrogens, osteoprogenitors. and / or preosteoblasts and / or osteoblasts and / or osteoblasts and / or osteocytes, etc., or in cells of the chondroblast lineage, such as osteochondrogens, chondrogens and / or prechondroblasts and / or chondroblasts and / or chondroblasts and / or chondrocytes.
    The skilled person will understand that cells will differentiate either into cells of the osteoblast lineage (eg, pre-osteoblasts or osteoblasts) or into cells of the chondroblast lineage (eg, pre- or chondroblasts) depending on the conditions to which they are exposed. , such as physical, chemical or - biological factors, such as growth factors.
    In particular, the cells differentiated in vitro are cells of the osteoblastic lineage (eg, osteoprogenitors, pre-osteoblasts, osteoblasts or -ostéocytes) or chondroblasts (chondrogenerators and / or pre-chondroblasts or chondroblasts). The differentiation of MSCs into cells of the chondro-osteoblastic, osteoblastic or chondroblastic line may involve the culture of MSCs under conditions capable of inducing the differentiation of MSCs towards cells of the chondro-osteoblast, osteoblastic or chondroblastic line, more generally the culture of MSCs in a medium comprising one or more agents (eg, growth factors) capable of inducing differentiation of MSCs towards cells of the chondro-osteoblast, osteoblastic or chondroblastic line.
    Protocols for differentiating MSCs into cells of the chondro-osteoblast, osteoblastic or chondroblast lineage include the method of differentiating MSCs into "bone B cells" as described elsewhere herein and the method of differentiating MSCs into "cell product C" or " bone cells C "which is as follows: white blood cells from bone marrow are seeded at a density of 50,000 cells / cm in a conventional culture medium comprising 5% OctaPlasLG® (Octapharma) 0.1 IU / ml heparin ( LEO Pharma), FGF-b (CellGenix) and TGFB-1 (Humanzyme)) and incubated at 37 ° C in a humidified incubator containing 5% CO, 4 days after seeding, non-adherent cells are removed and the medium is renewed with a culture medium. 7 days and 11 days after seeding, half of the culture medium is removed and replaced with fresh medium to renew the growth factors.
    The cells are grown during the primary culture for 14 days.
    On day 14, cells are removed by detachment, for example, with Trypzean (Lonza), and by vortexing and pipetting (passage 1: PI). Intermediate cells are cryopreserved (eg in CryoStor® CS10) and stored in liquid nitrogen.
    Then the intermediate cells are thawed and re-seeded for secondary culture at a density of 572 cells / cm2. Cells are grown during secondary culture for 10 days.
    On day 24, cells are removed by detachment, for example, with Trypzean (Lonza), and by vortexing and pipetting up and down (passage 2: P2). Intermediate cells are cryopreserved (eg in CryoStor® CS10) and stored in liquid nitrogen.
    Subsequently, the intermediate cells are thawed and re-seeded for tertiary culture at a density of 572 cells / em 2. The cells are cultured in tertiary culture for 10 days.
    On day 34, cells are removed by detachment, for example, with Trypzean (Lonza), and by vortexing and pipetting (passage 3: P3). To obtain the final cell product, the cells are resuspended, for example, in OctaPlasLG®, at a final concentration of 25 x 10 ° cells / ml.
    This cell product will hereinafter be referred to as “cell product C - fresh.” At the end of the tertiary culture, the cells are also cryopreserved for long-term storage.
    To do this, the cells are resuspended in a cryopreservation medium to achieve the desired concentration (25 x 10 ° cells / ml). The cell suspension is then transferred into cryotubes which are stored in liquid nitrogen.
    This cellular product will be referred to hereinafter as “C-cryo cellular product”. The cryopreservation medium can be: CryoStor® CS10 (BioLife Solutions), or 50% (v / v) CryoStor® CS10 (BioLife Solutions Inc) and 50% (v / v) of human serum albumin (Octapharma), or 95% (v / v) of CryoStor® CS10 (BioLife Solutions Inc) and 5% (v / v) of human serum albumin (Octapharma), or 80% (v / v) of Hypothermosol® (BioLife Solutions Inc ) 10% (v / v) of DMSO, and 10% (v / v) of human albumin (Octapharma) Other protocols for differentiating MSCs into cells of the osteoblastic or chondroblastic lineage include, among others, WO 2009 / 087213; WO 2007/093431; and REGER, R.L. et al.
    Differentiation and Characterization of Human MSCs'. In: Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology), edited by D.J.
    Prockop et al.
    Humana Press, 2008, vol. 449, p. 93-107; VERMURI, M.C. et al (Eds.). Tests and applications of mesenchymal stem cells (methods in molecular biology). Humana Press, 2011, vol. 698, in particular pages 201 to 352). The term "growth factor" as used herein means a biologically active substance which influences the proliferation, growth, differentiation, survival and / or migration of various cell types and which can affect the changes. developmental, morphological and functional of an organism, alone or modulated by other substances. A growth factor can generally act by binding, as a ligand, to a receptor (for example, a surface or intracellular receptor) present in cells responsive to growth factor. A growth factor can be a protein entity comprising one or more polypeptide chains. For example and without limitation, the term "growth factor" includes members of the fibroblast growth factor family (FGF), from the family of bone morphogenetic proteins (BMP), from the family of platelet-derived growth factors (PDGF), from the family of fac Transformative growth factors beta (TGFP), of the family of nerve growth factors (NGF) and of the family of epidermal growth factors (EGF), family of insulin-like growth factors (IGF), family of factors Growth Differentiation (GDF), Hepatocyte Growth Factor (HGF) Family, Hematopoietic Growth Factor (HeGF) Family, Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF), Angiopoietin, Factor Family endothelial vascular growth (VEGF), glucocorticoid family and the like. The skilled person will understand that the growth factor or combination of growth factors can be any growth factor or combination of growth factors known to be capable of inducing differentiation of MSCs to a desired cell type. The skilled person will understand that in vitro methods for inducing the differentiation of MSCs to a desired cell type (e.g., to cells of osteochondroblastic lineage, - osteoblastic or chondroblastic) can produce a substantially pure cell population (i.e. i.e. composed mainly) of the desired cell type. Without limitation, the cell population thus derived may contain at least 90% (by number) of the desired cell type, such as, for example, 91%, 292%, 293%, 294%, 95%,> 95%, 296 %, 296%, 97%, 98%,> 99%, or 100% of the desired cell type.
    Those skilled in the art will understand that, as the method taught here relates to the evaluation of the osteogenic potential of differentiated cells in vitro, the differentiated cells in vitro as provided herein are generally cultured under conditions capable of inducing the differentiation of pluripotent cells. , such as CSM, in cells of the chondro-osteoblast, osteoblastic or chondroblastic lineage. Likewise, cells differentiated in vitro as provided herein are generally not grown under conditions capable of inducing differentiation from pluripotent cells, such as CSM, to cells of the myocyte lineage ( muscle), tenocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic stroma (marrow).
    Another aspect relates to a method for determining the osteogenic potential of differentiated cells in vitro comprising, consisting essentially of or consisting of:
    - measurement of the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro; - measuring the quantity of one or more CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro; and - determine that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express one or more of CD73, CD105, CD10 or CD44, and if the cells differentiated in vitro have one or more of a nMFI for CD73 of at least 500, nMFI for CD44 of at least 100 or nMFI for CD105 of 150 or less, preferably in which nMFlcp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, nMFlopa is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE, and / or the nMFlep105s is measured with an excitation length of 633 nm and an emission length of 660 nm for APC.
    Preferably, another aspect relates to a method for determining the osteogenic potential of differentiated cells in vitro comprising, consisting essentially of or consisting of: - measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the cell surface of differentiated cells in vitro; - measuring the quantity of one or more CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro; and - determine that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express CD73, CD105, CD10 and CD44, and if the cells differentiated in vitro have one or more of an nMFI for CD73 d '' at least 500, an nMFI for CD44 of at least 100 or an nMFI for CD105 of not more than 150, preferably in which the nMFlcp73 is measured with an excitation wavelength of 633 nm and a length emission wavelength of 660 nm for APC, nMFlep44 is measured with an excitation wavelength of 488 nm and emission length of 580 nm for PE and / or nMFlcp10s is measured with a excitation length of 633 nm and emission length of 660 nm for APC.
    In some embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential if at least 90% of the differentiated cells in vitro express CD73, CD105, CD105 and CD44, and whether the differentiated cells in vitro have one or more of an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 150 or an nMFI for CD105 of at most 150, preferably in which the nMFlcp73 is measured with a wavelength of excitation of 633 nm and emission wavelength of 660 nm for APC, nMFlepy is measured with excitation wavelength of 488 nm and emission length of 580 nm for PE and / or nMFlcp10s is measured with an excitation length of 633 nm and an emission length of 660 nm for APC. In some cases, the methods for determining the osteogenic potential of differentiated cells in vitro may comprise, consist essentially of or consist of: - measurement of the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the surface cellular differentiated cells in vitro; - measuring the quantity of one or more CD73, CD105, CD44 or CD10 on the cell surface of differentiated cells in vitro; and - determine that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express CD73, CD105, CD10 and CD44, and if the cells differentiated in vitro have one or more of an nMFI for CD73 d '' at least 500, an nMFI for CD44 of at least 100, an nMFI for CD105 of at most 150 or an nMFI for CD10 of at least 40, for example, at least 50, at least 55 or at least 60, of preference in which nMFICD73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, nMFlcpa4 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE, the nMFIcpi0s is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC and / or the nMFlop10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In some embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential if at least 90% of the differentiated cells in vitro express CD73, CD105, CD10 and CD44, and whether the differentiated cells in vitro have one or more nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100, an nMFI for CD105 of at most 150 or an nMFI for CD10 of at least 50, preferably in which the nMFlep73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for the APC, the nMFIcpa4 is measured with an excitation wavelength of 488 nm and a wavelength emission of 580 nm for PE, the nMFlcpios is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC and / or the nMFlIep10 is measured with a wavelength of d excitation of 488 nm and an emission length of 580 nm for PE.
    In some cases, the methods taught herein may include determining that cells differentiated in vitro have osteogenic potential if at least 90% of cells differentiated in vitro express CD73, CD105, CD10 and CD44, and if cells differentiated in vitro have or more nMFI for CD73 of at least 500, one nMFI for CD44 of at least 150, one nMFI for CD105 of 150 or one nMFI for CD10 of 40, for example, at least 50, at least 55 or at least 60, preferably in which nMFlcp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, nMFlcp4 is measured with an excitation wavelength of 488 nm and emission length of 580 nm for PE, nMFlcp10s is measured with excitation wavelength of 633 nm and emission wavelength of 660 nm for APC and / or nMFIep10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In some embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential if at least 90% of the differentiated cells in vitro express CD73, CD105, CD10 and CD44, and whether the differentiated cells in vitro have one or more of nMFI for CD73 of at least 500, nMFI for CD44 of at least 150, nMFI for CD105 of 150 or more or nMFI for CD10 of at least 50, preferably in which nMFlcp73 is measured With an excitation wavelength of 633 nm and an emission wavelength of 660 nm for the APC, the nMFIcpa4 is measured with an excitation wavelength of 488 nm and a length of emission wave of 580 nm for PE, the nMFlcpios is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC and / or the nMFlop10 is measured with a wavelength excitation of 488 nm and an emission length of 580 nm for PE.
    In one aspect, the invention relates to a method for determining the osteogenic potential of differentiated cells in vitro, the method comprising, consisting essentially of or consisting of: - measuring the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro; - measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - determining that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express one or more of CD73, CD105, CD10 or CD44, and if the cells differentiated in vitro have an nMFI for CD10 of at least 40, for example at least 50, at least 55 or at least 60, nMFICD10 is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE.
    In certain preferred embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential if at least 90% of the differentiated cells in vitro express one or more of CD73, CD105, CD10 or CD44, and whether cells differentiated in vitro have an nMFI for CD10 of at least 50, preferably nMFlep10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In one aspect, the invention provides a method for determining the osteogenic potential of differentiated cells in vitro, the method comprising, consisting essentially of or consisting of:
    - measurement of the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the cell surface of differentiated cells in vitro; - measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - determining that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express CD73, CD105, CD10 and CD44, and if the cells differentiated in vitro have an nMFI for CD10 of at least 40, for example, at least 50, at least 55 or at least 60, nMFlep10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In certain preferred embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential if at least 90% of the differentiated cells in vitro express CD73, CD105, CD10 and CD44, and whether the differentiated cells in vitro vitro have an nMFI for CD10 of at least 50, preferably nMFlep10 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    Furthermore, the invention relates to a method for determining the osteogenic potential of differentiated cells in vitro, the method comprising, consisting essentially of or consisting in: measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - the determination of the osteogenic potential of differentiated cells in vitro which exhibit an nMFI for CD10 of at least 40, the nMFlcp is preferably measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    In some preferred embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential if the differentiated cells in vitro have an nMFI for CD10 of at least 50, preferably nMFlop10 is measured with a excitation wavelength of 488 nm and emission length of 580 nm for PE.
    Preferably, another aspect relates to a method for determining the osteogenic potential of differentiated cells in vitro comprising, consisting essentially of or consisting of: - measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the cell surface of differentiated cells in vitro; - measuring the amount of CD73, CD105 and CD44 on the cell surface of differentiated cells in vitro; and - determining that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express CD73, CD105, CD10 and CD44, and if the cells differentiated in vitro have an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100 and an nMFI for CD105 of at least 150, preferably in which the nMFlcp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of
    660 nm for the APC, nMFlep4, is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE, and / or nMFlcp10s is measured with an excitation length of 633 nm and an emission length of 660 nm for APC.
    In some embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential, in particular clinically useful osteogenic potential, if at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the cells differentiated in vitro express one or more CD73, CD105, CD10 or CD44 and if they have been modified in vitro, the differentiated cells: - one or more of an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100 or an nMFI for CD105 of at most 150 ; - an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100 and an nMFI for CD105 of at most 150; - one or more of an nMFlcp73 of at least 550, at least 600, at least 650, at least 700, at least 750, at least 750, at least 800, at least 850 or at least 900; an nMFlIcp4s of at least 110, at least 120, at least 130, at least 140, at least 140, at least 150, at least 200, at least 250, at least 300 or at least 350; or an nMFlep105 of 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; - an nMFlçcp73 of at least 550, at least 600, at least 650, at least 650, at least 700, at least 750, at least 800, at least 850 or at least 900; an nMFlep4 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; and an nMFlep105 of 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; one or more of an nMFI for CD73 of at least 700, an nMFI for CD44 of at least 200 or an nMFI for CD105 of at most 150; and / or - an nMFI for CD73 of at least 700, an nMFI for CD44 of at least 200 and an nMFI for CD105 of at most 150, preferably in which the nMFlIcp73 is measured with a wavelength of excitation of 633 nm and an emission wavelength of 660 nm for the APC, the nMFIcp44 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE, and / or nMFlepios is measured with an excitation length of 633 nm and an emission length of 660 nm for APC.
    In some embodiments, the methods taught herein may include determining that the differentiated cells in vitro have osteogenic potential, in particular clinically useful osteogenic potential, if at least 90%, such as at least 91%, at least 92%, at least 93%, at least
    94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, of cells differentiated in vitro expresses CD73, CD105, CD10 and CD44, and whether the differentiated cells in vitro have: one or more of an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100 or an nMFI for CD105 of at most 150; - an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100 and an nMFI for CD105 of at most 150; one or more of an nMFlcp73 of at least 550, at least 600, at least 650, at least 700, at least 700, at least 750, at least 800, at least 850 or at least 900; an nMFlcpa4 of at least 110, at least 120, at least 130, at least 140, at least 140, at least 150, at least 200, at least 250, at least 300 or at least 350; or an nMFlop105s of 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: - an nMFlçp73 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 nMFIcp44 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; and an nMFlepios of at most 180, at most 170, at most 160, at most 150, at most 140, at most 140, at most 130, at most 120, at most 110 or at most 100; one or more of an nMFI for CD73 of at least 700, an nMFI for CD44 of at least 200 or an nMFI for CD105 of at most 150; and / or - an nMFI for CD73 of at least 700, an nMFI for CD44 of at least 200 and an nMFI for CD105 of at most 150, preferably in which the nMFlcp73 is measured with a wavelength of excitation of 633 nm and an emission wavelength of 660 nm for the APC, the nMFlçp44 is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE, and / or nMFlepios is measured with an excitation length of 633 nm and an emission length of 660 nm for APC.
    The current inventors have discovered that the method of determining the osteogenic potential of differentiated cells in vitro as taught herein can be used to screen for donors of MSCs which comprise MSCs which can be differentiated in vitro into cells having clinically useful osteogenic potential. .
    Accordingly, another aspect relates to a method of selecting a subject to prepare in vitro differentiated cells of chrondro-osteogenic lineage, the method comprising: collecting MSCs from a biological sample of a subject;
    obtaining differentiated cells in vitro from MSCs; -determining the osteogenic potential of differentiated cells in vitro by a method as taught here; and selecting the subject for the preparation of in vitro differentiated cells of the chondroosteoblastic lineage if the differentiated cells in vitro have clinically useful osteogenic potential.
    In particular, the recovery of CSM from a biological sample of a subject can be performed as described elsewhere in this document.
    In particular, obtaining differentiated cells in vitro from CSM can be carried out as described elsewhere in this document.
    In particular embodiments, the subject is a human subject.
    Those skilled in the art will understand that the definitions and particular embodiments described in this document apply to all of the methods and uses described in this document.
    The present application also provides aspects and embodiments as set forth in the following statements: Statement 1: Use of one or more of CD73, CD105 or CD44 to determine the osteogenic potential of differentiated cells in vitro.
    Statement 2: Method for determining the osteogenic potential of differentiated cells in vitro, - comprising measuring the amount of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44, and measuring the amount of one or several of CD73, CD105 or CD44 expressed by differentiated cells in vitro.
    Statement 3: The method according to statement 2, the method comprising: (a1) measuring the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44 on the cell surface of differentiated cells in vitro ; (a2) measuring the amount of one or more of CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro; (b1) comparing the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD105, CD10 or CD44 as measured in (a1) with a cutoff value representing cells of known osteogenic potential; (b2) comparing the amount of one or more of the CD73, CD105 or CD44 measured in (a2) with one or more respective cut-off values representative of cells of known osteogenic potential;
    (c1) the identification or not of a deviation of the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44 as measured in (a1) from said limit value; (c2) whether or not there is a deviation of the amount of any of the CD73, CD105 or CD44 measured in (a2) from said threshold value; and (d) assigning said deviation to a particular determination of the osteogenic potential of differentiated cells in vitro.
    Statement 4: Method according to Statement 3, wherein said cutoff value of (b1) and said respective cutoff values of (b2) are cutoff values representing cells having clinically useful osteogenic potential.
    Statement 5: Method according to statement 4, in which: - a decrease in the fraction of differentiated cells in vitro measured in (al) compared to the limit value of (b1) indicates that the cells differentiated in vitro have no clinically useful osteogenic potential, or - an identical or increased fraction of cells differentiated in vitro, as measured in (a1) relative to the cut-off value of (b1), indicates that cells differentiated in vitro have useful osteogenic potential on the clinical plan; and - a decrease in the amount of any one of CD73 and CD44 measured in (a2) compared to the respective threshold values of (b2), and / or an increase in the amount of CD105 measured in (a2) compared to the respective cut-off value of (b2) indicates that cells differentiated in vitro do not have any clinically useful osteogenic potential, or - the same or increased amount of CD73 and CD44 measured in (a2) compared to the values respective thresholds of (b2), and the same or a reduced amount of CD105 measured in (a2) relative to the respective threshold value of (b2) indicates that the cells differentiated in vitro have useful osteogenic potential.
    Statement 6: A method according to any of statements 3 to 5, wherein said cutoff value of (b1) is 90% of differentiated cells in vitro expressing one or more of CD73, CD105, CD10 or CD44 on the cell surface; and wherein said cut-off value of (b2) is a normalized median fluorescence intensity (nMFI) for CD73 of 500, an nMFI for CD44 of 100 and / or an nMFI for CD105 of 150, preferably where nMFlcp73 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, nMFlep44 is measured with an excitation wavelength of 488 nm and a wavelength emission of 580 nm for PE, and / or nMFIcp10 is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC.
    Statement 7: Method according to any one of statements 1 to 6, in which the quantity of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44, and the quantity of CD73, CD105 and CD44 expressed by the differentiated cells in vitro are measured. vitro.
    Statement 8: A method according to statement 7, wherein said cutoff value of (b1) is 90% of in vitro differentiated cells expressing CD73, CD105, CD10 and CD44 on the cell surface; and wherein said threshold value of (b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 100 and an nMFI for CD105 of 150, preferably where the nMFIcps3 is measured with an excitation wavelength of 633 nm and an emission wavelength of 660 nm for APC, the nMFlcpu is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE, and the nMFlcpi05 is measured with an excitation wavelength of 633 nm and an emission length of 660 nm for APC.
    Statement 9: Method according to any one of statements 1 to 8, in which the cells differentiated in vitro are obtained from mesenchymal stem cells (MSCs). Statement 10: A method according to any one of statements 1 to 9, wherein the cells - differentiated in vitro are human cells.
    Statement 11: A method of selecting a subject for the in vitro preparation of differentiated cells of the chrondro-osteoblast lineage, the method comprising: collecting MSCs from a biological sample of a subject; obtaining differentiated cells in vitro from CSM; - determination of the osteogenic potential of the differentiated cells in vitro by a method as defined in any one of statements 1 to 10; and -selecting a subject for the preparation of in vitro differentiated cells of the chondroosteoblast lineage if the differentiated cells in vitro have clinically useful osteogenic potential.
    Statement 12: Method according to statement 11, in which the subject is a human subject. Statement 13: Use of CD73, CD105, CD44 and CD10 to determine the osteogenic potential of differentiated cells in vitro.
    Statement 14: A method for determining the osteogenic potential of differentiated cells in vitro, comprising measuring the amount of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44, and measuring the amount of one or more of CD73, CD105 or CD44 expressed by differentiated cells in vitro.
    Statement 15: The method according to statement 14, the method comprising:
    (a1) measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD10 and CD44 on the cell surface of differentiated cells in vitro; (a2) measuring the amount of one or more of CD73, CD105 or CD44 on the cell surface of differentiated cells in vitro; (b1) measuring the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 as measured in (a1) with a cutoff value representing cells having known osteogenic potential; (b2) comparing the amount of one or more CD73, CD105 or CD44 measured in (a2) with one or more respective cut-off values representing cells of known osteogenic potential; (c1) determining the deviation of the fraction of in vitro differentiated cells expressing CD73, CD105, CD105, CD10 and CD44 as measured in (al) from said cutoff value; (c2) determining the deviation of the amount of any one of CD73, CD105 or CD44 measured in (a2) from said threshold value; and (d) assigning said deviation to a particular determination of the osteogenic potential of differentiated cells in vitro. Statement 16: Method according to statement 15, wherein said cutoff value of (b1) and said respective cutoff values of (b2) are cutoff values representing cells having clinically useful osteogenic potential.
    Statement 17: Method according to any one of statements 14 to 16, in which the amount of one or more CD73, CD105, CD44 or CD10 on the cell surface of differentiated cells in vitro is measured.
    Statement 18: Method according to statement 16 or 17, in which: - an identical or increased fraction of the differentiated cells in vitro, as measured in (al) compared to the cut-off value of (b1), indicates that the differentiated cells in vitro have clinically useful osteogenic potential; and - the same or an increased amount of CD73, CD44 and / or CD10 measured in (a2) compared to the respective threshold values of (b2), and the same amount or a reduced amount of CD105 measured in (a2) compared to at the respective cut-off value of (b2) indicates that the cells differentiated in vitro have clinically useful osteogenic potential.
    Statement 19: Method according to statements 15 to 18, wherein said cut-off value of (b1) is 90% of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the cell surface; and wherein said cut-off value of (b2) is a median normalized fluorescence intensity (nMFI) for CD73 of 500, an nMFI for CD44 of 100, an nMFI for CD105 of 150 and an nMFI for CD10 of 40.
    Statement 20: Method according to statements 15 to 18, wherein said cut-off value of (b1) is 90% of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 on the cell surface; and wherein said cut-off value of (b2) is a median normalized fluorescence intensity (nMFI) for CD73 of 500, an nMFI for CD44 of 150, an nMFI for CD105 of 150 and an nMFI for CD10 of 40.
    Statement 21: Method according to any one of statements 14 to 20, in which the amount of CD73, CD105 and CD44 expressed by the differentiated cells in vitro is measured.
    Statement 22: Method according to statement 21, wherein said cutoff value of (b2) is an nMFI for CD73 of 500, an nMFI for CD44 of 100 and an nMFI for CD105 of 150.
    Statement 22: Use according to statement 13 or the method according to any of statements 14 to 22, wherein the cells differentiated in vitro are obtained from mesenchymal stem cells (MSCs).
    Statement 24: Use according to statement 13 or the method according to any one of statements 14 to 23, wherein the cells differentiated in vitro are human cells. Statement 25: A method of selecting a subject for the in vitro preparation of differentiated cells of the chrondro-osteoblastic lineage, the method comprising: harvesting CSM from a biological sample of a subject; obtaining differentiated cells in vitro from CSM; the determination of the osteogenic potential of the differentiated cells in vitro by a method as defined in any one of statements 14 to 24; and - selecting the subject for the preparation of differentiated cells in vitro of the chondroosteoblastic line if the cells differentiated in vitro have a clinically useful osteogenic potential.
    Statement 26: Method according to statement 25, in which the subject is a human subject. Statement 27: Method for determining the osteogenic potential of differentiated cells in vitro, the method comprising, consisting essentially of, or consisting of: - measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - determining the osteogenic potential of differentiated cells in vitro which exhibit an nMFI for CD10 of at least 40, the nMFIcpio is preferably measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description. . Therefore, it is intended to cover all such alternatives, modifications and variations as follows within the spirit and general scope of the appended claims. The aspects and embodiments of the invention disclosed herein are supported by the following non-limiting examples.
    EXAMPLES Example 1: Method for Obtaining Mesenchymal Stem Cells (MSC) and Cells Derived from MSC Mesenchymal Stem Cells Undifferentiated MSCs were prepared by taking samples of human bone marrow (BM) from the iliac crest of healthy voluntary donors. health. After harvesting, white blood cells in the bone marrow were counted, seeded at a density of 50,000 cells / cm "in conventional culture medium and incubated at 37 ° C in a humidified incubator containing 5% CO. After 24h, the culture medium was removed and fresh cell culture medium was added. The culture medium was replaced every 2-3 days. When more than half of the colonies reached 80% confluence or when some colonies reached 100% confluence, cells were harvested (passage 1: PI). During this first passage, the cells were directly cryopreserved in CryoStor® CS10 (BioLife Solutions Inc.). To complete the culture process, the MSCs were thawed, seeded at 572 cells / cm for the second crop and cultivated. Cells were removed when more than half of the colonies reached 80% confluence or when some colonies reached 100% confluence to obtain MSCs in passage 2 (P2). This cellular product is referred to herein as "CSM".
    - Cell product A (also referred to herein as bone-forming cells A) Conventional culture medium comprising 5% Octaserum (autologous serum 50:50 and OctaPlasLG® (Octapharma)), FGF-b (CellGenix), TGFB- 1 (Humanzyme).
    Freezing medium: 80% conventional culture medium, 10% Octaserum [autologous serum 50:50 and OctaPlasLG® (Octapharma)] and 10% DMSO.
    MSC-derived in vitro differentiated cells referred to herein as "cell product A" were prepared by removing human BM samples from the iliac crest of healthy volunteer donors. After harvesting, white blood cells in the bone marrow were counted, seeded at a density of 50,000 cells / em ”in the culture medium and incubated at 37 ° C in a humidified incubator containing 5% CO 3. 4 days after seeding the cells, the non-adherent cells were removed and the medium was renewed with culture medium. 7 days and 11 days after seeding, half of the culture medium was removed and replaced with fresh medium.
    Cells were grown during primary culture for 14 days.
    On day 14 cells were removed by detachment with Trypzean (Lonza) and by swirling and pipetting up and down (passage 1: PI). Intermediate cells were cryopreserved in CryoStor® CS10 (BioLife Solutions Inc.) or freezing medium and stored in liquid nitrogen.
    For secondary culture, cells were thawed and re-seeded at a density of 1144 cells / cm2. The cells were grown for 14 days during the secondary culture.
    On day 28, cells were removed by detaching with Trypzean (Lonza) and by vortexing and pipetting up and down (passage 2: P2). To obtain the final cell product, the cells were resuspended in OctaPlasLG® at a final concentration of 25x10 ° cells / ml.
    This cellular product is referred to herein as “cellular product A”. Cell product B (also referred to herein as bone forming B cells) Conventional culture medium comprising 5% OctaPlasLG® (Octapharma), 0.1 IU / ml heparin (LEO Pharma), FGF-b (CellGenix ), TGFB-1 (Humanzyme). MSC-derived cells by in vitro differentiation were obtained from the aspiration of iliac crest bone marrow from healthy volunteer human donors.
    After harvesting, white blood cells in the bone marrow were counted, seeded at a density of 50,000 cells / em ”in the culture medium and incubated at 37 ° C in a humidified incubator containing 5% CO 3. Four days after seeding the cells, non-adherent cells were removed and the medium was renewed with culture medium.
    Seven days and eleven days after seeding, half of the culture medium was removed and replaced with fresh medium to renew the growth factors.
    Cells were grown during primary culture for 14 days.
    On day 14 cells were removed by detachment with Trypzean (Lonza) and by swirling and pipetting up and down (passage 1: PI). Intermediate cells were cryopreserved in CryoStor® CS10 (BioLife Solutions Inc.) and stored in liquid nitrogen.
    Then, the intermediate cells were thawed and re-seeded for secondary culture at a density of 286 cells / cm2. The cells were grown for 14 days during the secondary culture.
    On day 28, cells were removed by detachment with Trypzean (Lonza) and by vortexing and pipetting up and down (passage 2: P2). To obtain the final cell product, the cells were resuspended in OctaPlasLG® at a final concentration of 25x10 ° cells / ml.
    This cellular product is referred to herein as "cellular product B".
    Cell product C (i.e. cell product C fresh products and cell products C cryo; also referred to herein as bone-forming C cells) Conventional culture medium comprising 5% OctaPlasLG® (Octapharma), 0.1 IU / ml heparin (LEO Pharma), FGF-b (CellGenix) and TGFB-1 (Humanzyme). 20 to 60 ml of human bone marrow (BM) were taken from the iliac crest of a healthy voluntary donor.
    After harvesting, white blood cells in the bone marrow were counted, seeded at a density of 50,000 cells / em ”in the culture medium and incubated at 37 ° C in a humidified incubator containing 5% CO 3. 4 days after seeding the cells, the non-adherent cells were removed and the medium was renewed with culture medium. 7 days and 11 days after seeding, half of the culture medium was removed and replaced with fresh medium to renew the growth factors.
    Cells were grown during primary culture for 14 days.
    On day 14, cells were detached with Trypzean (Lonza) and by swirling and pipetting up and down and then collected (passage 1: PI). Intermediate cells were cryopreserved (in CryoStor® CS10) and stored in liquid nitrogen.
    Each stock of cells came from a donor, with no pooling between donors.
    Then the intermediate cells were thawed and re-seeded for secondary culture at a density of 572 cells / cm2. The cells were grown for 10 days during the secondary culture.
    On day 24 cells were removed by detachment with Trypzean (Lonza) and by swirling and pipetting up and down (passage 2: P2). Intermediate cells were cryopreserved (in CryoStor® CS10) and stored in liquid nitrogen.
    Subsequently, the intermediate cells were thawed and re-seeded for tertiary culture at a density of 572 cells / cm 2. Cells were grown during tertiary culture for 10 days.
    On day 34 cells were removed by detachment with Trypzean (Lonza) and by swirling and pipetting up and down (passage 3: P3). To obtain the final cell product, the cells were resuspended in OctaPlasLG® at a final concentration of 25x10 ° cells / ml.
    This cell product will hereinafter be referred to as “cell product C - fresh.” At the end of the tertiary culture, the cells were also cryopreserved for long term storage.
    The cells were resuspended in cryopreservation medium to achieve the desired concentration (25x10 ° cells / ml). The cell suspension was then transferred to cryotubes which were stored in liquid nitrogen.
    This cellular product will be referred to herein as “C-cryo cell product” or “cryo (preserved) C bone-forming cells”, also abbreviated as “BF C cells.” The cryopreservation medium was: -CryoStor® CS10 ( BioLife Solutions Inc.), or
    -50% (v / v) of CryoStor® CS10 (BioLife Solutions Inc.) and 50% (v / v) of human serum albumin (Octapharma), or -CryoStor® CS10 (BioLife Solutions Inc.) and 5% ( v / v) human serum albumin (Octapharma), or -80% (v / v) of Hypothermosol® (BioLife Solutions Inc.), 10% (v / v) of DMSO and 10% (v / v) human serum albumin (Octapharma). Example 2 In Vivo Bone Formation Properties of MSC-Derived Chondro-osteoblast Cells Materials and Methods CSM cell culture, bone-forming A, B and C cells were prepared as described in Example 1. Mice 9-10 week old female NMRI-Nude (nu / nu) mice were purchased from Janvier SAS (Le Genest-St-Isle, France) and housed under standard conditions with food and water in libidum. 196 mice were used in total for the present study.
    Cranial vault bone formation model in mice 12 week old female NMRI-Nude (nu / nu) mice (n = 137) were anesthetized with isoflurane (IsoFlo®) and received a single sub-administration. skin of MSCs, bone-forming A cells (cultured with FGF-2 and TGFB1) or bone-forming B cells (2.5 x 10 ° - cells in 100 µl per mouse or vehicle (100 µl) on the bone of the cranial vault.
    To mark new bone formation over time, calcium binding fluorochromes were sequentially administered to mice.
    Alizarin red (red), calceins (green and blue) and tetracycline (yellow) (all from Sigma-Aldrich®) were injected intraperitoneally 3 days before and 4, 8 and 12 days after administration cellular, respectively.
    Body weight, general clinical signs and clinical signs of laboratory animals were monitored at the site of administration for two weeks after administration.
    The mice were euthanized 2 weeks after cell administration by cervical dislocation and the cranial vault of each mouse was removed to assess the bone forming properties of the bone cells by radiographic imaging, histomorphometry (quantification of bone formation) and - immunofluorescence. .
    Sample incorporation and histological sectioning For histomorphometry, ALP, TRAP (tartrate-resistant acid phosphatase), Masson's Trichrome Goldner stains and immunofluorescence, the cranial vaults were fixed and dehydrated by successive incubations in baths of 70%, 80% and 90% ethanol for 12 hours each, at 4 ° C and with gentle stirring, in hydroxyethylmethacrylate plastic resin (HistoResin, Leica ”). Four 8 µm thick coronal sections were made using a microtome (Leica®, RM2255). For saffranin orange staining and immunoperoxidase, the bony cranial vaults were fixed in 3.7% formaldehyde for 24 hours, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) pH 7.4 for three days and incorporated in paraffin.
    Seven 7 µm thick coronal and sagittal paraffin sections were cut using a microtome (Leica®, RM2255). Immunofluorescence staining - Evaluation of human and murine collagen I by immunofluorescence was performed on plastic coronal histological sections of the cranial vault 4 µm thick.
    Briefly, after a permeabilization step using 1X PBS / 0.3% Triton solution for 30 minutes at room temperature (RT), the histological sections were incubated for 1 hour at RT in the blocking solution. (ie PBS / BSA / horse serum / Triton “) to non-specific binding sites.
    The histological slides were then incubated overnight at 4 ° C with primary anti-human and anti-mouse mouse collagen I antibodies (Abcam; # ab138492 and Abcam; # ab21286 respectively). After 3 rinsing steps in PBS for 5 min at RT, blocking was performed with the blocking solution for 1 hour at RT.
    The secondary antibodies diluted in the blocking solution were then added for 2 hours at RT in the dark.
    The secondary antibodies Alexa Fluor ”488 donkey anti-rabbit IgG H&L (ThermoFisher, # A21206) and Alexa Fluor” Cy3® Goat anti-mouse IgG H&L (Abcam; # ab97035) were used to visualize murine collagen I in green and the human collagen I in red.
    The slides were then rinsed 3 times in 1X PBS for 5 minutes at RT and incubated with NucBlue® solution for 1 minute at RT to stain the nucleus.
    Finally, the slides were briefly rinsed once in PBS and then mounted in the GlycerGel® reagent. As a negative control for immunofluorescence, the omission of the primary antibody was performed on the adjacent histological slide.
    Histological staining The osteoblastic and osteoclastic activities were evaluated on sections of the cranial vault using the ALP and TRAP methods, respectively, for the detection of enzyme activity.
    For ALP staining, 4 µm thick coronal sections of cranial vaults were incubated for 1 hour with a solution of rapid blue RR salt (Sigma-Aldrich®) and Naphthol AS-MX alkaline phosphate (Sigma-Aldrich® ). TRAP staining was performed on 8 µm thick calvary coronal sections using the TRAP (Acid Phosphatase, Leukocyte) kit, (Sigma-Aldrich®) according to the manufacturer's instructions.
    To assess the state of mineralization of the newly formed bone, - Masson Goldner Trichrome staining was performed on the sections of the cranial vaults stained with ALP using a kit (Bio-Optica®) according to the manufacturer's instructions.
    To demonstrate the formation of cartilage, an orange safranin stain was performed on sagittal paraffin sections of the cranial vault with a thickness of 7 µm.
    Briefly, after dewaxing, the histological sections were incubated successively in Weigert's hematoxylin (Klinipath®) for 10 min, 0.1% rapid green (Klinipath®) for 5 min, 1% acetic acid (VWR Chemicals) for 15 dry and 0.1% safranin-orange (Fluka® ref: 84120) for 5 min.
    After dehydration the slides were mounted with glass coverslips using Pertex® (HistoLab®). Digital images were taken with an optical microscope (Leica®) and Leica ”LAS EZ software.
    Immunoperoxidase After deparaffinization, 7 µm thick coronal or sagittal paraffin paraffin sections were successively incubated with 2.5% hyaluronidase (Sigma-Aldrich®) for 30 minutes at 37 ° C, in HO, 3% (Sigma-Aldrich®) for 30 minutes at room temperature, in PBS 0.3% Triton X-100 (Sigma-Aldrich®) for 30 minutes at room temperature and in blocking solution (i.e. PBS / BSA / horse serum / Triton) for 1 hour at room temperature.
    Sections were incubated overnight at 4 ° C with mouse anti-human type I collagen primary antibodies (Abcam, ab90395), rabbit anti-murine type I collagen primary antibodies (Abcam, ab21286) or rabbit anti-Ku80 primary antibodies (Abcam, ab80592). Staining was visualized using a Vectastain kit (Vector Laboratories, PK6200) and 3.3 'diaminobenzidine (Vector Laboratories), according to the manufacturer's instructions.
    Sections were counterstained with Mayer's hematoxylin (Klinipath®). The slides were mounted with Pertex® glass coverslips. Quantification of bone formation by radiographic analysis (bone-forming C cells) During euthanasia, ex vivo radiographic imaging of the cranial vault of each mouse placed side by side was performed using the Faxitron® device. MX-20. Digital images were taken - at 1.5X magnification in manual mode with voltage set to 35kV, exposure time 4.8 seconds, brightness / contrast of 8300/6000. The radiographic images generated are grayscale images with gray intensity values ranging from 0 (black region) to 255 (white region) and are directly proportional to the radio-opacity and therefore to the opacity or l bone thickness.
    The intensity value of the gray level of the osteoinductive part of the formation - bone (mineralized nodules rejected from selection) on the parietal bones (manual selection) was analyzed using the histogram of the AdobePhotoshop® software. X-ray imaging and AdobePhotoshop® software were also used to quantify the surface area of mineralized nodules (manual selection).
    Histomorphometric Analyzes of Bone Cranial Vaults: Quantification of Bone Formation Quantification of bone formation (ie absolute bone formation) was performed on tissues embedded in plastic.
    The absolute thickness of the newly formed bone (from basal mineralization front fluorescent labeled with alizarin red to fluorescent bone neoformation labeled with calcein and tetracycline) with and without mineralized nodules was measured (in µm) on section. coronal 4 µm thick with ZEN® software (Zeiss). For each animal, 4 measurements of absolute thicknesses were carried out on 5 independent levels, with a distance of 200 μm between each level.
    First, the average thickness (with or without nodules) + standard deviation (i.e., the average of the 4 measurements per level over the 5 levels) was calculated for each animal.
    Quantification of the surface of newly formed bone on histological images (ImageJ ”software) For the surface analysis of osteoinductive and osteogenic nodules, digital images of 6 independent levels taken every 2 levels after the coronal suture were taken at From histological sections in plastic resin (4 µm) of the cranial vault, using a combination of multiple fluorescent filters and brightfield fluorescent microscope (Zeiss Axioscope A1, Zeiss, Germany). At each level measured, the selection of osteoinduced bone neoformation was defined manually in stitches images using the Image) ”software. The mineralized and total surfaces of this selection were measured (in mm ”). The same procedure was carried out for the mineralized and total surfaces of the osteogenic nodules.
    For osteoinduction and osteogenic nodules, the average of the total surface area and the average of the mineralized surface were then calculated per experimental animal and per group.
    The total surface area of new bone formation was finally calculated by adding the osteoinduced bone surfaces to the surfaces of the osteoinductive nodules.
    Statistical analyzes The results were expressed as mean + standard deviation (SD or SD). Statistical analyzes were performed using JMP® software (SAS Institute Inc.) or GaphPad Prism ”. Differences between groups were considered statistically significant when p <0.05. Results Bone-forming cells λ (generated with FGF-2 and TGFf1) and bone-forming cells B (generated with FGF-2, TGFB1 and heparin) both showed bone formation. significantly higher than the controls (vehicle) 2 weeks after administration (Figures 1-2, Table 1). More particularly, Figure 3 shows that the B bone-forming cells exhibit osteoinductive (homogeneous bone formation of murine origin on the cranial vault) and osteogenic (mineralized nodules of human and murine origin). Table 1: Quantification of bone formation (%) in sections of the murine cranial vault. The murine cranial vaults were treated with an excipient (negative control), bone-forming cells A or bone-forming cells B.% bone formation Nb. of lots Nb. of animals Mean + SD Abbreviations: SD: standard deviation The osteoinductive properties (that is, the amount of newly formed murine bone after implantation) were equivalent for bone-forming cells A and B (Figures 1-2). It is very interesting to note that the B bone-forming cells of the present invention exhibited potent osteogenic and osteoinductive properties, as evidenced by the large amount of newly formed human and murine bones after implantation (human Coll IF staining and murine, figure 3).
    The presence of nodules was observed in 7/8 donors and 80% of mice of bone-forming cells B and in 4/11 donors and 20% of mice of bone-forming cells A. No nodules were - observed after administration of CSM or excipient. In addition to osteoinduction activities, bone-forming B cells promote high osteogenic activity evidenced by the presence of large mineralized nodules observed in 80% of treated mice, while bone-forming cells A show low activity. osteogenic, ie, small nodules in only% of the treated mice (Table 2).
    Table 2: Quantification of the presence of mineralized nodules on the murine cranial vault two weeks after administration to the cranial vault of the excipient, CSM, bone-forming A cells or bone-forming B cells.
    Frequency of Donor Batch Animal Osteogenesis
    Formative cells 4/10 (40%) 4/11 (36%) 9/45 (20%) bone To formative cells 7/8 (88%) 7/8 (88%) 37/46 (80%) bone B Abbreviations: MSC: mesenchymal stem cells; NA: not applicable.
    Histologic staining of coronal sections of murine cranial vault two weeks after administration (vehicle only, CSM, bone-forming A cells (generated with FGF-2 and TGFB1; bf A cells) or bone-forming B cells ( generated with FGF-2, TGFB1 and heparin; bf B cells)) revealed that all the treated conditions (MSC, bf A cells and bf B cells) have high osteoinductive potential with moderate remodeling activity (ALP and TRAP staining) in bones formed by osteoinduction.
    Interestingly, the mineralized nodules seen in mice treated with bone-forming B cells consisted of both murine (host) and human - (donor) bone tissue (as evidenced by collagen-type staining. I human and murine), which demonstrates that the nodules were formed by these two processes: osteogenesis (bone formation from the donor) and osteoinduction (bone formation from the host). In addition to strong osteoblastic and osteoclastic activity (ALP + TRAP staining), the nodules exhibited osteoid tissue (non-mineralized tissue) suggesting that bone formation was still progressing two weeks after administration, while the osteoinduction process observed in all conditions was finished (Figure 4).
    Figure 4 shows that human bone formation (i.e. osteogenesis) (observed with anti-human type I collagen staining) and high osteoblastic and osteoclastic activities (observed with ALP + Goldner staining) and TRAP respectively) were detected - mainly in mouse nodules administered with bone-forming B cells, showing that the process of bone formation was continuous and not completed after 2 weeks, unlike osteoinduction of A-forming cells bone and CSM which seemed to be over. All the conditions treated (MSC, b-f A cells, b-f B cells) exhibited a high osteoinduction potential with moderate remodeling activity (ALP and TRAP staining) in bone formation - osteoinduced (Figure 4).
    Bone neoformation was evaluated by fluorescence two weeks after treatment with excipient only, CSM, b-f λ cells or b-f B cells (Figure 5). To this end, mice were administered fluorescent dyes that bind to bone calcium (i.e., red alizarin, green and blue calceins, yellow tetracycline) at specific times to mark newly formed bone. The last fluorochrome administered was tetracycline, which was administered 12 days after the cells were administered.
    As shown in Figure 5, the nodules of mice given bone-forming B cells were predominantly stained with tetracycline fluorochrome (yellow spots have been circled in dotted lines in Figure 5) confirming a more advanced stage of formation. compared to the osteoinduction observed in the osteoinduced formation (alizarin red (red), calcein (green) and calcein blue (blue): these spots appear in light gray and the double arrows indicate the thickness of bone formation). The new bone formation of the treated mice was evaluated by quantifying the surface area of the new bone on histological images (ImageJ® software). The total area of the newly formed bone was determined by adding the osteo-induced areas and the areas of bone nodule for each level analyzed and each mouse. The results show that the bone-forming B cells (n = 7 mice, shown in light gray in Figure 6) significantly increased bone neoformation 2 weeks after administration of the cells by approximately 2 times more than CSM cells. (n = 6 mice, shown in dark gray in Figure 6; Table 3). This difference is due to the strong osteogenic property of bone-forming B cells and the absence of such a property for MSCs. Table 3: Total bone neoformation is measured on coronal sections including osteoinduction and osteogenic formation. Type of | Number Osteoinduction Osteogenesis (nodules) | Total (animal cell osteoinduction + osteogeny) (from Surface Surface Surface Surface Surface Surface even mineralized | total mineralized | total mineralized | total donor) (mm ) | (mn ) |) am) am) | mm) (mean | (mean | (mean (mean (mean (mean + SD) + SD) | + SD) + SD) | + sDm) + SD) CSM 0.42 + 0.09 | 0.57 + 0.42 + 0.09 | 0.57 +
    0.17 0.17 b-f 7 0.43 + 0.16 | 0.59 + 0.22 + 0.19 | 0.57 + 0.65 + 0.30 | 1.16 + cells 0.25 0.53 0.71
    B Abbreviations: MSC: mesenchymal stem cells; SD: standard deviation In addition, evaluation of new bone formation over time by histological staining revealed that the nodules observed on the top of the skull of mice given bone-forming B cells ossified by a mechanism of endochondral ossification. In Figure 7, the safranin-orange stain shows the proteoglycan matrix (specific for cartilage) (area surrounded by dotted lines), nuclei, bone tissue and cytoplasm.
    Unlike bone formation by osteoinduction through intramembrane ossification, bone nodules were produced by endochondral ossification, with cartilage matrix occurring between 1 week and 3 weeks after administration (Figure 7). Immunohistochemical stains of human collagen type I, murine collagen type I and human nucleus (Ku80) performed 4 weeks after administration of bone-forming B cells confirmed the presence of human bone in the nodules.
    In addition, Ku80 staining revealed that bone-forming B cells were grafted into the bone matrix (nodules) and developed into osteocytes after in vivo administration.
    Mice administered the C cryo cell product showed greater bone formation than controls 4 weeks after administration (Figure 11 A-C). Bone opacity was significantly higher for C cryo bone-forming cells than for the excipient (Figure 11B). The area of the osteogeny was significantly greater than that of the excipient in which no mineralized nodule was observed (Figure 11C). Histomorphometric measurements of osteoinduction with or without osteogenesis (represented by absolute bone formation) were significantly higher for cryo C bone-forming cells than for excipients (Figure 11D and 11E). In addition, in addition to osteoinduction activities, cryo-osseous C bone-forming cells favored strong osteogenic activity as evidenced by the presence of mineralized nodules.
    This osteogenic activity was observed in 4/5 bone marrow donors (or production of batches) and 65% of the mice (FIG. 11F). A donor / lot was considered osteogenic (positive) when at least one mineralized nodule was observed in one mouse per group.
    No nodule was observed after administration of the vehicle.
    More particularly, the C cryo cell products exhibited osteoinductive (homogeneous bone formation of murine origin on the cranial vault) and osteogenic (mineralized nodules of human and murine origin) (Figure 12). Intramembrane ossification of the host was induced along the surface of the cranial vault (Figures 12 and 13). More particularly, the osteoinductive cryo C cells exhibit osteoinductive and osteogenic properties (FIG. 13, “fluo”). Double immunostaining - mouse / human type I collagen (Figure 13, “Human type I collagen”) revealed the presence of host and donor bone (osteogeny). Osteoblast activities (Figure 13, “ALP + ") and osteoclasts (Figure 13," TRAP ") were especially detected in mineralized nodules showing that the process of bone remodeling of the nodules was still ongoing 4 weeks after their administration.
    This observation depended on the size of the nodule: the larger the nodule, the more ALP and TRAP activities are still present 4 weeks after its administration.
    A weak osteoid
    (Figure 13, "Goldner's Masson trichrome stain") was shown indicating that the bone formation process is complete.
    Therefore, cryopreserved C bone-forming cells increase new bone formation.
    This demonstrates the utility of cell products and cell composition as described in the specification and examples for the treatment of bone defects of flat bones as well as long bones.
    Example 3: In vivo mouse subcritical size segmental femoral defect (sub-CSD) repaired by cryopreserved bone-forming cells A, bone-forming cells B and C-bone-forming cells C.
    Experimental procedures Cell culture Bone-forming cells A, bone-forming cells B, and cryopreserved bone-forming cells C were prepared as described in Example 1. Sub-CSD segmental femoral model The surgery was performed. been 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 = 73) were anesthetized by 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), placed in a prone position on a hot plate.
    After the application of a 6-hole titanium micro-locking plate (RISystem AG ”) fixed with 4 or 5 screws on the anterior aspect of the left femur, a 2 mm long mid-shaft femoral osteotomy was performed. with a Gigli saw and a device (RISystem AG ”). As a preventive drug, antibiotics (Baytril®, 10 mg / kg body weight) were administered the day before the operation (in drinking water) and analgesics (hydrochloride - buprenorphine, Temgesic ”, Schering-Plow, 0.1 mg / kg body weight) the day before and every 12 hours for at least 3 days after the procedure.
    MSC-derived cells (2.25 x 10 ° cells in a volume of 30 µl per mouse) or the vehicle (control group) were administered on the day of the procedure (immediately after closing the wound with sutures. surgical), locally at the site of the bone defect, by percutaneous injection with a 50 µl Hamilton syringe.
    Mice were euthanized 6 or 10 weeks after administration of cells or excipients by cervical dislocation.
    The left femur of each mouse was dissected, removed, and maintained at 0.9% NaCl at room temperature until radiographic imaging.
    Quantification of bone repair by radiological analyzes In vivo radiographic imaging of the left femur of each mouse was performed using the Faxitron® MX-20 device just after the operation to check the fixation of the plate, the size of the segmental femoral defect and to obtain a referral every two weeks. Digital images were taken in mediolateral and anteroposterior view at 5X magnification in manual mode with voltage set to 35 kV, exposure time of 4.8 seconds, brightness of 4,300 and contrast of 7,100 An ex vivo radiography was performed on left femurs taken during euthanasia, 6 weeks after cell administration. For α bone-forming cells and B bone-forming cells, the size of the defect was quantified for each mouse over time by measuring the distance (µm) between the two edges of the bone defect at three locations ( right, middle and left of the defect) on mediolateral and anteroposterior radiographic images (6 measurements in total), using the ImageJ® software. The average of the six measurements was calculated for each mouse at each point in time.
    For C bone-forming cells, the size of the defect was quantified for each mouse over time by measuring the distance (µm) between the two edges of the bone defect at two locations (both cortices) on mediolateral radiographic images. and anteroposterior (4 measurements in total), using the Image software) ”. The average of the four measurements was calculated for each mouse at each point in time.
    The RUS (radiographic union score) adapted for the sub-CSD model is a semi-quantitative measure based on the presence or absence of new bone formation, bypass surgery and a fracture line (radiographic images anteroposterior and mediolateral). The score corresponds to the sum of 4 scores determined on 2 cortical defect sites on the two views (total of 4 scores ranging from 1 to 4 each). The score therefore varies from 4 (no sign of healing) to 16 (complete fusion).
    The fusion score is a binary score that assesses the rate of fusion between the edges of the femoral defect.
    The radiological criteria used to define the fusion are the visualization of the defect bypass in at least 3 cortices (Cekig E et al, Acta Orthop Traumatol Turc. 2014, 48 (5), 533-40). The score is 0 (no merge) or 1 (merge). For this parameter, only the last point in time has been analyzed (W10, here).
    Tomography (micro-CT) analyzes After sampling for euthanasia, the left femurs were fixed with 3.7% formaldehyde and transferred to the Center for Microscopy and Molecular Imaging (CMMI, ULB, Gosselies, Belgium) for analyzes micro-CT. Samples were scanned using a microPET / CT nanoScan® PET / CT multimodal camera (Mediso) and NuclineTM v2.01 software (Mediso). Scans were performed using semicircular scan, maximum zoom, 35 kVp tube voltage, 720 projections per gantry rotation, 300 ms exposure time per projection, pixel binning of the detector. 1 to 1. The scan lengths in the X and Y dimensions have been adapted for each acquisition. The total duration of the micro-CT scan was 3 minutes 42 seconds. Each micro-CT scan was post-reconstructed with a 40 µm side cubic voxel using a Shepp-Logan filter and 8 regular sample multisampling mode. The dimensions of the X and Y images were adapted to each reconstruction. The size of the Z images corresponded to the scan length defined for acquisition. A qualitative assessment of bone repair was performed on the micro-CT images after reorienting the bone with the Z axis (scanner axis) and cropping the image from one screw proximal to another in the femoral bone on the Z axis, and as narrow as possible in the transverse (XY) plane. Then, a 3D projection rendering at Maximum Intensity (MIP) was produced. To quantitatively assess bone repair, a virtual cylinder 2 mm in diameter and 2 mm in axial length was placed in the defect space on the micro-CT scanners and the mean bone volume was assessed in this cylinder by thresholding. voxels with radiological intensity greater than or equal to 1500 HU.
    Results B bone-forming cells and cryo bone-forming C cells improved repair of the subcritical segmental femoral defect in mice.
    In the subcritical size segmental femoral defect (sub-CSD) model in NMRI-Nude mice, B bone-forming cells (n = 12 mice, 2 lots) improved fracture repair, as shown a significant reduction in the size of the bone defect relative to the excipient (n = 11 mice) and to bone-forming cells λ (n = 4 mice) from 2 to 6 weeks after administration (FIG. 8A).
    X-rays of segmental femoral defects at OD and 6W after administration of the vehicle, λ cells (not shown) or B cells showed a reduction in the size of the bone defect in mice receiving B cells according to one embodiment of the invention compared to mice - receiving the excipient (Figure 8B) or λ cells (not shown).
    The bone repair volumes of the segmental femoral defect were quantified by tomography (micro-CT) at 6W after administration of the excipient and B bone-forming cells. The results confirmed that the B-bone-forming cells induced a better bone repair than the excipients (Figure 8C).
    Cryopreservation of bone-forming cells C significantly improved and accelerated the percentage of bone fracture repair in the sub-CSD segmental femoral model (Figures 14 and 15) compared to the vehicle from 2 to 10 weeks after administration ( n = 38 mice; 19 treated and 19 controls with vehicle, 2 batches; p <0.001). In addition, the RUS score was significantly higher for the cryopreserved group of bone-forming cells C than for the group of excipients (Figure 16). Finally, the rate of fusion was also improved with fusion in 9/19 (47%) mice 10 weeks after administration of cryo C bone-forming cells, compared to no fusion after administration of excipient.
    Example 4: In vitro cellular characterization of cells derived from CSM exhibiting osteogenic potential in Examples 2 and 3 Materials and methods Cell culture CSM, cell product λ and cell product B, fresh cell product C and cell products C cryo are obtained as described. in Example 1. Analysis by Flow Cytometric Characterization of the cell surface markers was carried out by flow cytometry. 50,000 cells at a concentration of 1x10 ° cells / ml in PBS - 1% BSA were incubated for 10 min in the dark with antibody Sul.
    After this incubation period, the cells were washed once with PBS.
    The different antibodies used for extracellular staining are: allophycocyanin (APC) conjugated antibodies against CD105 (BD Biosciences ”, Cat No: 562408), CD73 (BD Biosciences®, Cat No: 560847), Phycoerythrin ( PE) - conjugated antibodies against CD10 (BD Biosciences®, Cat No: 555375), CD44 (BD Biosciences®, Cat No: 550989). Nonspecific staining was determined by incubation of cells with FITC, APC and PE conjugated Immunoglobulin G (IgG) control (all BD Biosciences®, Cat No .: 556649; 555751; 556650 respectively). Prior to analysis, evaluation of the singlets and the population of interest was performed, as depicted in Figure 9. Flow cytometry analysis was performed on 10,000 events from the closed population to 1. Using FACSCanto "II (BD Biosciences®) and FACSDiva" 8.0 software (BD Biosciences®). The adjustment parameters used for the analysis were performed automatically with beads (BD CompBeads Plus®, Cat No. 560497) For each conjugate, the positivity threshold was set at 1% of the positivity of the control isotype antibody and the positivity of each marker was determined.
    The fluorescence median (MFI) of the entire analyzed population was also determined and divided by the MFI of the corresponding isotype control antibody to obtain the normalized IMF (nMFD.
    Results The flow cytometric analysis revealed that the general cell identity based on the expression profiles of the cell surface markers of cell product A, cell product B (generated with comparative methods according to the prior art) and C cell products with or without final cryopreservation (generated with a method illustrating the invention) were comparable.
    All expressed the mesenchymal markers CD73, CD90 and CD105 and did not express the hematopoietic markers CD45, CD34 and CD3 (less than 5% of the cell population expressed these markers) (Table 4). Cell product λ, cell product B and cell products C (with or without final cryopreservation) express low levels of MHC class II cell surface receptor, such as HLA-DR. The low immunogenicity represented by a low expression of HLA-DR allows advantageous cell grafting, for example on allogeneic subjects (Table 4). In addition, cell product A, cell product B and cell products C (with or without final cryopreservation) strongly express the ALP enzyme on their surface compared to undifferentiated MSCs (Tables 4 and 5). The high expression of ALP underlines the commitment of cell product A, cell product B and cell products C (with or without final cryopreservation) to the osteoblastic line. Cell product A, cell product B and cell products C (with or without final cryopreservation) also strongly expressed the CD10 cell marker compared to undifferentiated MSCs (Table 4). Table 4: Expression profile of cell surface markers of comparative cell products (i.e. CSM, cell product A, cell product B) and cell products illustrating the invention (i.e. cellular products C) Cellular product C | Product | cryopreserve Product Expression | Product vop Statisti- cellu- marker (in CSM | cellu- | cellu- CS10 ques C 2, CS10 HTS%) milk A | milk B diluted fresh CS10 5% HS | 10% DMSO
    HSA A 10% HSA 1: 1 Avg | 100. e 0 100.0 | 100.0 [100.0 | 100.0 | 100.0 | 100.0 | 100.0 CD73-APC Standard deviation 0.1 So re Average | 100. e 0 99.7 99.5 99.8 CD90-PE Standard deviation 0.1 10.2 0.2 0.2 0.7 0.2 0.4 Se re Mean | 100. CD105-APC e 0 99.8 100.0 100.0 | 100.0 | 100.0 | 100.0 Heat | oo jos jor jos joo [oo | oo that
    Cell product C | Product | cryopreserved Product Expression | Product yop y Statisti- cellu- marker (in CSM | cellu- | cellu- CS10 ques C ‚, | CS10 | HTS%) milk A | B diluted fresh CS10 5% HS | 10% DMSO
    HSA A 10% HSA 1: 1 ve | | | | | | | AT
    NC CE EC EN EE Average 1.3 1.0 1.0 ND | ND ND CD45-FITC Standard deviation 0.7 0.3 0.3 0.1 Nop qe qe [oP BB Average 0.4 10.3 1.0 CD45-APC Standard deviation 0.2 10.2 2.9 ND ND ND ND ND ND Ns [> | ” be bp jp pe Average 1.0 1.6 21 1.6 2.8 3.3 1.5 CD34-APC Standard deviation 0.4 1.8 1.6 2.2 22 1.0 hee eer B Average 0.2 10.1 0.2 0.1 0.1 CD3-PE Standard deviation 0.1 0.1 0.1 0.1 0.1 0.1 0.1 hee ee EE Medium- HLA-DR-PE ne 0.7 1.0 1.8 1.6 1.4 1.4 1.4 pan [zee [eo fre [ree nen
    Cellular product C P it 2 4 Product expression | Product roduct | cryopreserved Statisti- cellu- marker (in CSM | cellu- | cellu- CS10 ques mil C ‚, | CS10 | HTS%) wafer B diluted fresh CS10 5% HS | 10% DMSO
    HSA A 10% HSA 1: 1 type we | L LL LL LL
    NC CC EC CO EN EN Medium- HLA- ne 10 | 1.6 1.6 20 1.5 1.8 1.8 1.6 DR / DP / DP / DQ | Deviation -FITC type 04 | 11 1.1 1.6 0.7 0.2 hohe ke klerk kf Average 40.7 | 88.7 94.8 96.2 96.2 | 97.7 98.7 98.4 ALP-PE Standard deviation 5.6 4.4 3.6 20 1.7 Average 92.7 99.8 100.0 99.8 CD49e-PE Standard deviation 20.5 | 1.1 0.5 0.4 0.1 0.2 0.4 0.2 hk ep kle Ef Average 99.7 100.0 | 100.0 | 100.0 | 100.0 100.0 CD44-PE Standard deviation 0.2 0.5 0.1 0.1 0.2 0.1 hk ER kle bk Average- CD10-PE ne 19.6 98.8 99.3 99.5 | 99.3 99.1 98.9 en eas eee ee
    Cell product C | Product | cryopreserved Product Expression | Product vop Statisti- cellu- marker (in CSM | cellu- | cellu- CS10 ques C 2, CS10 HTS%) milk A | laireB diluted fresh CS10 5% HS | 10% DMSO
    HSA A 10% HSA 1: 1 we | T LL | 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; MSC: mesenchymal stem cells; ND: not determined; PE: phycoerythrin; SD: standard deviation Table 5: ALP expression levels of comparative cell products (i.e. CSM, cell product λ, cell product B) and cell products illustrating the invention (i.e. cellular products C) Cryopreservation of ‚| Cellular product C Product Product | Product Statisti- cellu- HTS CSM | cellu- CS10 ques laire C…, | CS10 | 10% D laire A lar B diluted fresh CS10 5% HS | MSO
    HSA A 10% H 1: 1
    SA Average 40.7 | 88.7 94.8 96.2 96.2 198.7 | 97.7 | 98.4 ALP population - positive PE deviation (%) type 5.6 44 3.6 20 1.7 | Medium- Level ne 2.4 19.8 56.1 60.3 380 157.7 142.7 141.2 expression of the surface | Std Dev cell ALP- 10.8 27.4 38.9 242 | 43.1 | 372 131.3 PE (nMF])
    Medium enzymatic activity ne 176.3 | 671.9 874.7 895.5 801.5 IND 719.9 | 633.6 Abbreviations: ALP: alkaline phosphatase; ND: not determined; PE: phycoerythrin; The expression profile of the cell surface markers was characterized not only by the presence of markers at the cell surface (percentage of positivity of the population) but also by the analysis of the quantity of markers expressed at the cell surface (median normalized fluorescence of the population) of different markers. These analyzes revealed some differences between the different cells derived from CMS.
    Cell product B and cell products C (with or without final cryopreservation) cultured in the presence of heparin express a higher level of ALP than MSCs and cell product A - cultured in the absence of heparin (ALP results -PE nMFD) enhancing their commitment to the osteoblastic lineage of bone-forming cells.
    The expression of the mesenchymal markers CD73 and CD105 on the surface of cells also depended on cell types. Cell products generated in the presence of heparin (cell product B and cell products C with or without final cryopreservation) expressed higher levels of CD73 and CD105 than cell product A. In addition, cell products C appeared to possess more CD73 and CD105 on their surface than cell product B, especially when cell product C did not undergo final cryopreservation (Table 6). The differentiated cell products A, B and C express a higher amount of CD10 cell marker than the undifferentiated MSCs. In addition, C cell products have more CD10 on their surface than A and B cell products (Table 6).
    In view of the foregoing, it appears that the cell product B and the cell products C (with or without final cryopreservation), the products B and C of which showed an osteoinductive potential and a high osteogenic potential in Examples 2 and 3, can be distinguished from the cellular product A, the product of which showed an osteoinductive potential and a low osteogenic potential in - examples 2 and 3, depending on the quantity expressed of one or more of CD73, CD105, CD10 or CD44 and / or the amount expressed on the cell of one or more of the CD73, CD105 or CD44 More particularly, on the basis of Tables 4 and 6, it appears that at least 90% of the cells of the cell product B and of the cell products C express CD73, CD105, CD10 and CD44 on the cell surface (Table 4) and that cells of cell product B and cell products C have an nMFlcp73 of at least 500, an nMFlcpa44 of at least 100 (or 150) and an nMFlep105s of 150 (ta bleau 6).
    On the other hand, the cells of the cell product A have an nMFlcp73 less than 500 and an nMFlop44 less than 100 (or less than 150); and MSCs have an nMFlcp73 less than 500 and an nMFlep105 greater than 150 (Table 6). Accordingly, the amount of differentiated cells in vitro expressing CD73, CD105, CD105, CD10 and CD44 in combination with the amount of CD73, CD105 and CD44 expressed by differentiated cells in vitro is appropriate and sufficient to distinguish cells from the B cell product. and C cell products of CSM cells or λ cell product cells.
    Similarly, the amount of differentiated cells in vitro expressing CD73, CD105, CD10, and CD44 in combination with the amount of CD73, CD105 and CD44 expressed by differentiated cells in vitro is appropriate and sufficient to determine whether the cells differentiated in vitro have an osteogenic potential, and in particular to determine whether they have a high osteogenic potential.
    Table 6: Other Cell Surface Marker Expression Results of Comparative Cell Products (ie CSM, Cell Product A, Cell Product B) and Cell Product C Cryopreservation of Product Expression | Product Product | cell product C in Statisti- cellu- CSM | cellu- cellu-. CS10 marker | cs10 HTS (nMFI) Diluted C Whey C | CS10 ss [so gs | 10% DMSO © 10% HSA 1: 1 Medium 2.4 19.8 56.1 60.3 38.0 157.7 42.7 41.2 ALP-PE Standard deviation 10.8 27.4 38.9 24.2 | 43.1 37.2 31.3 Medium 2348 1130.7 | 646.3 | 996.9 703.9 17778 17192 784.2 CD73- APC | Standard deviation 84.3 80.1 138.8 | 181.1 150.0 | 73.3 47.3 Ns Ju | æ is Jo be hb (5 | Average 207.7 126.6 59.1 99.7 70.2 1746 72.6 67.0 D105- We © Standard deviation 67.6 | 15.2 13.1 27.0 13.1 12.3 4.2 3.9 N Is Je Jo [is Jo | 2 hb B | Medium- CD44-PE [ne 139.8 162.0 156.6 | 362.8 378.4 185.5 1 | 227.5 261.3 2504 [2053 147.7
    Cryopreservation of Product Expression | Product Product | cell product C in Statisti- cellu- CSM | cellu- cellu-. CS10 marker | ques laire A | area B laire C diluted Cs10 HTS (fresh nMFD | CS10 [es | scmsa 10% DMSO 1: 1 ° 10% HSA twe | | | | LL LL 1 |
    N le In [2 Js Jo »Lb Lb |
    Way-
    ne 81.0 122.5 33.5 44.3 39.6 135.7 35.2 36.87 CD49e-PE | Standard deviation 51.4 11.0 7.4 3.3 3.5 10.0
    N the [2 Jo Js qe | Lb Lb |
    Way-
    ne 26.1 121.6 80.2 115.7 104.7 | 71.5 79.5 70.1 HLA-
    difference
    Nh | # I bs qe | Lb Lb |
    Way-
    ne 36.2 32.2 64.0 59.3 163.8 64.1 57.9 CD10-PE | Standard deviation 1.1 16.4 16.8 38.5 30.5 | 45.5 35.4 32.9
    N I I | m Js Job | |; | Abbreviations: ALP: alkaline phosphatase; APC: allophycocyanin; FITC: fluorescein isothiocyanate; HLA-ABC: Human Leukocyte ABC Antigen; HLA-DR: Human leukocyte antigen - DR isotype; MSC: mesenchymal stem cells; NA: not available; ND: not determined; PE: phycoerythrin; SD: standard deviation
    Additionally, nMFI flow cytometric analysis revealed that CD73 and CD44 protein expressions were higher in C bone-forming cells than in other cell types (Figure 10), including D-forming cells. 'os B.
    权利要求:
    Claims (12)
    [1]
    AMENDED CLAIMS BE2019 / 5630 l. A method for determining the osteogenic potential of differentiated cells in vitro, the method comprising: - measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - Ja determination of the osteogenic potential of differentiated cells in vitro which exhibit an nMFI for CD10 of at least 40, the nMFlcou is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    [2]
    2. The method of claim 1, wherein the method comprising determining the osteogenic potential of differentiated cells in vitro which exhibit an nMFI for CDIO of at least 50.
    [3]
    3, The method according to claim 1 or 2, in laguello the method comprising: - measuring the fraction of differentiated cells in vitro expressing one or more of CD73, CD105, CDIO or CD44 on the cell surface of differentiated cells in vitro; - measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - determining that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express one or more of the CD73, CD105, CD19 or CD44, and if the cells differentiated in vitro have an nMFI for CD10 of ' at least 40, nMFloni is measured with an excitation wavelength of 488 nm and an emission wavelength of 580 nm for PE.
    [4]
    4 The method according to any one of claims 1 to 3, wherein the method comprising: - reading the fraction of differentiated cells in vitro expressing CD73, CD105, CD105, CDIG and CD44 on the cell surface of differentiated cells in vitro; - measuring the amount of CD10 on the cell surface of differentiated cells in vitro; and - determine that the cells differentiated in vitro have an osteogenic potential if at least 90% of the cells differentiated in vitro express CD73, CD195, CDI10 and CD44, and if the collules differentiated in vitro have an nMFI for CD10 of at least 40, nMFlopi is measured with an excitation wavelength of 488 nm and an emission length of 580 nm for PE.
    [5]
    5, The method according to any one of claims! to 4, wherein the method further comprising measuring the amount of one or more of CD73, CD105 or CD44 expressed by the differentiated cells in vitro.
    [6]
    6. The method according to claim 5, wherein the method comprising determining that the differentiated cells in vitro have osteogenic potential if the differentiated cells in vitro have an nMFI for CD10 of at least 40, or if the differentiated cells in vitro have. one or more of an nMFI for CD73 of at least 500, of an nMFI for CD44 of at least 100 or of an nMFI for CD105 of 150 or less, in the acile the nMF1 (p73 is measured with a length of excitation wave of 633 nm and emission wavelength of 660 nm for APC, nMFlop4s is measured with an excitation wavelength of 488 nm and emission length of 580 nm for PE, ct / or nMFlomios is measured with an excitation length of 633 nm and an emission length of 660 nm for APC.
    [7]
    7. The method of any one of claims 1 to 6, wherein the method further comprising measuring the amount of CD73, CD105 and CD44 expressed by the differentiated cells in vitro.
    [8]
    8. The method according to claim 7, wherein the method comprising determining that the differentiated cells in vitro have osteogenic potential if the differentiated cells in vitro have an nMFI for CDIO of at least 40, and whether the differentiated cells in vitro have. an nMFI for CD73 of at least 500, an nMFI for CD44 of at least 100 and an nMFI for CD105 of 150 or less, in which the nMFlcp73 is measured with an excitation wavelength of 633 nm and a length of d emission wave of 660 nm for APC, nMFlcoa is measured with an excitation wavelength of 488 nm and emission length of 580 nm for PE, and nMFlopios is measured with length d excitation of 633 nm and emission length of 660 nm for APC.
    [9]
    9. The method according to any one of claims 1 to 8, wherein the cells differentiated in vitro are obtained from mesenchymal stem cells (MSCs).
    [10]
    10. The method according to any one of claims | to 9, wherein the cells differentiated in vitro are human cells.
    [11]
    11. A method of selecting a subject for the in vitro preparation of differentiated chrondro-osteoblastic hgnea cells the method comprising: “collecting CSM from a biological sample of a subject: obtaining differentiated cells in vitro apart from CSM; da determination of the osteogenic potential of differentiated cells in vitro by a method as defined in any one of claims 1 to 10; and selecting the subject for the preparation of in vitro differentiated cells of the chondro-osteoblastic lineage if the cells differentiated in vitro have a similarly useful osteogenic potential.
    [12]
    12. The method of claim 11, wherein the subject is a human subject.
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    同族专利:
    公开号 | 公开日
    SG11202102874UA|2021-04-29|
    AU2019349633A1|2021-05-27|
    JP2022502034A|2022-01-11|
    KR20210076018A|2021-06-23|
    EP3856932A1|2021-08-04|
    WO2020064793A1|2020-04-02|
    US20210404961A1|2021-12-30|
    CN113260713A|2021-08-13|
    BE1026600A1|2020-04-03|
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    法律状态:
    2021-02-03| FG| Patent granted|Effective date: 20201207 |
    优先权:
    申请号 | 申请日 | 专利标题
    EP18196721|2018-09-25|
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