Medium and method for producing a bone marrow reconstitution

Abstract

The present invention relates to a culture medium and a culture method for obtaining, in a single step and in the same culture container, cells differentiated into osteoblasts and adipocytes as well as a network of organized endothelial cells from the same pool of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitor cells, and endothelial cells, selected beforehand and amplified simultaneously in the same culture container from a single sample. The invention also relates to a composition obtained by said method, to a bone marrow reconstitution, and to uses thereof.

Description

[0001] TITLE: Medium and process for producing bone marrow reconstitution

[0002] The present invention relates to a culture medium and a culture method for obtaining, in a single step and in the same culture container, cells differentiated into osteoblasts and adipocytes, as well as an organized endothelial cell network, from a single pool of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors, and endothelial cells, previously selected and amplified simultaneously in the same culture container from a single sample. It also relates to a composition obtained by said method, a bone marrow reconstitution, and their uses.

[0003] The present invention also relates to a culture medium and a culture method for culturing and / or differentiating mesenchymal stem cells into osteoblasts and adipocytes, as well as for organizing an endothelial cell network. It also relates to a composition obtained by said method, bone marrow reconstitution, and their uses.

[0004] The ex vivo reproduction of adult human bone marrow is increasingly described in the literature as a way to overcome the costly and time-consuming limitations of animal models, which are hampered by the species barrier. Studies have begun to highlight 3D co-culture models that integrate the osteoblastic and endothelial compartments, generally using cell lines. For example, the endothelial compartment, which plays an active role in hematopoietic stem cell (HSC) proliferation, is often incorporated via HUVEC-type endothelial cell lines. Other models require a mouse step to enable vascularization or functional approaches.Furthermore, these co-cultures are most often performed in several stages (first, osteoblastic differentiation of mesenchymal stem cells, then assembly with a second pool of endothelial cells). Moreover, bone marrow adipose tissue is often overlooked in these studies despite its increasing functional importance described in the literature.

[0005] The inventors have created a novel model that comprehensively incorporates all the cellular and non-hematopoietic microenvironmental parameters of human bone marrow. It includes the medullary adipose tissue, the osteoblastic compartment, and the vascular compartment, also known as the endothelial compartment. The culture medium developed by the inventors allows for the simultaneous collection of these three non-hematopoietic compartments of human bone marrow, specifically from a single sample, in a single step, within the same culture vessel, without the use of cell lines. This makes it possible to generate ex vivo human bone marrow containing the osteoblastic, adipocyte, and endothelial compartments in 2D, and also in 3D, notably through the use of a biomaterial. Furthermore, through self-organization, this ex vivo bone marrow can form a 3D spheroid / organoid-type structure.

[0006] Furthermore, in previous studies, the differentiation of mesenchymal stem cells into adipocytes and osteoblasts required carrying out these two differentiations separately in two different media. The medium developed by the inventors not only allows these two differentiation pathways to occur simultaneously but also promotes the organization of an endothelial network within the same medium and culture well, without subsequent cell assembly, thus greatly simplifying the manipulations required, particularly for bone marrow reconstitution.

[0007] Thus, the invention relates to a culture medium for obtaining, in a single step and within the same culture container, cells differentiated into osteoblasts and adipocytes, as well as an organized endothelial cell network, from a single pool of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells, comprising: a) fetal bovine serum (FBS), and / or platelet lysate (PL), b) 10-100 pM ascorbic acid, c) 10-100 ng / mL of Bone Morphogenetic Protein 7 (BMP7), d) 1-10 pg / mL insulin, e) 1-50 pg / mL apotransferrin, f) 1-100 ng / mL vascular endothelial growth factor (VEGF), and optionally g) 0.01-0.5% (v / v) of intralipids.Thus the invention relates to a culture medium for the culture and / or differentiation into osteoblasts and adipocytes of mesenchymal stem cells, and optionally allowing the organization of an endothelial cell network, comprising: a) fetal bovine serum (FBS), and / or platelet lysate (PL), b) 10 - 100 pM of ascorbic acid, c) 10 - 100 ng / mL of "Bone Morphogenetic Protein 7" (BMP7), d) 1 - 10 pg / mL of insulin, e) 1 - 50 pg / mL of apotransferrin, and f) 1 - 100 ng / mL of vascular endothelial growth factor (VEGF).

[0008] In a second aspect, the invention relates to an in vitro culture method for mesenchymal stem cells comprising the steps of: a) Seeding the mesenchymal stem cells in a culture medium as defined above; and b) culturing said mesenchymal stem cells.

[0009] The invention relates to a method for the in vitro culture of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells, comprising the steps of: a) Seeding the mesenchymal stem cells and / or the mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells in a culture medium as defined above; and b) culturing said mesenchymal stem cells and / or said mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells.

[0010] Preferably this process allows obtaining in a single step, in the same culture container, cells differentiated into osteoblasts and adipocytes, as well as a network of organized endothelial cells, from the same pool of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells.

[0011] The invention relates to a method for producing bone marrow reconstitution comprising the in vitro culture method of mesenchymal stem cells according to the invention. The invention relates to a method for producing bone marrow reconstitution comprising the in vitro culture method of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors, and endothelial cells according to the invention.

[0012] The invention also relates to a reconstitution of bone marrow obtained by the process according to the invention. The invention further relates to a reconstitution of bone marrow comprising osteoblasts, adipocytes, and vessel-forming endothelial cells. The invention also relates to a reconstitution of bone marrow comprising osteoblasts, adipocytes, and a network of endothelial cells.

[0013] The invention also relates to a composition, preferably injectable, comprising cells obtained by the process of the invention.

[0014] The invention also relates to a bone marrow reconstitution according to the invention or a composition according to the invention for its use in the treatment of diseases, in particular diseases affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis.

[0015] Finally, the invention relates to the use of a bone marrow reconstitution according to the invention, as a model for the study of physiology, pathophysiology, compound testing and / or testing of physical and mechanical conditions.

[0016] The invention also relates to the use of a bone marrow reconstitution according to the invention, in a prosthesis or a medical device.

[0017] Detailed description of the invention

[0018] Cultural environment

[0019] The term "medium for the culture and / or differentiation of mesenchymal stem cells into osteoblasts and adipocytes" refers to a medium suitable for the culture and / or differentiation of mesenchymal stem cells into osteoblasts and adipocytes. Preferably, this medium is suitable for the culture and / or differentiation of mesenchymal stem cells into osteoblasts and adipocytes and for the formation of a vascular network by endothelial progenitors and / or endothelial cells.

[0020] The term "medium for the culture and differentiation of mesenchymal stem cells into osteoblasts and adipocytes" refers to a medium suitable for the simultaneous culture and differentiation of a single pool of mesenchymal stem cells into osteoblasts and adipocytes, in a single step, within the same culture vessel, without cell assembly. Preferably, this medium is a culture and differentiation medium for mesenchymal stem cells that allows for the organization of an endothelial cell network.

[0021] Preferably, this medium allows the simultaneous differentiation of mesenchymal stem cells into osteoblasts and adipocytes, and the organization of an endothelial network, in a single step, within a single culture vessel, without subsequent cell assembly. It can be presented in various forms but is preferably liquid and allows the culture of eukaryotic cells, particularly mammalian cells and especially human cells.

[0022] As intended here, the term "culture" refers to the multiplication of cultured cells.

[0023] The term "differentiation" refers to the acquisition by cells cultured in a culture medium of cellular characteristics that are not present in the cells initially used to seed the cell culture medium. As intended here, "differentiation" specifically refers to the acquisition of characteristics that commit cells, in particular, to the adipocyte or osteoblastic pathway.

[0024] Mesenchymal stem cells, also called mesenchymal stromal cells (MSCs), are stromal cells of mesodermal origin. They are phenotypically characterized by the co-expression of several markers, such as CD73, CD90, CD105, and CD146, and the absence of expression of other markers, particularly CD45 and CD34. They can be derived from bone marrow, adipose tissue, or umbilical cord blood of mammals. Mesenchymal stem cells can be derived from rodents or primates, and are particularly common in murine and human populations.

[0025] In a preferential mode, mesenchymal stem cells are derived from primary cultures. By "primary culture" we mean a cell culture derived directly from the tissue and / or cells of an individual.

[0026] Endothelial progenitors are cells engaged in endothelial differentiation but which are not yet recognizable as endothelial cells under a microscope. They are phenotypically characterized by the expression of a number of markers such as CD133, CD34, CD31, and VEGFR2.

[0027] The term "endothelial cells" refers to cells that are fully differentiated within the endothelial pathway and therefore recognizable as endothelial cells under a microscope. They are phenotypically characterized by the expression of a number of markers such as CD31, VE-Cadherin, von Willebrand factor, and VEGFR2.

[0028] Endothelial progenitors and endothelial cells have the ability to organize themselves into endothelial cell networks, or vascular networks, and thus organize themselves into vessels.

[0029] The endothelial progenitors and endothelial cells of the invention can, for example, be obtained from bone marrow mononuclear cells.

[0030] The term "individual" refers to a subject of an animal species, particularly mammals. According to one embodiment of the invention, the individual is a primate or a rodent, preferably a mouse or a human being.

[0031] Osteoblasts are defined as cells expressing the markers Runx2, DSX, ESP, BSP, DLX5 and / or Osterix (OSX). The osteoblastic phenotype can be assessed by phase-contrast microscopy by evaluating the level of mineralization, by alizarin red staining, by immunohistochemistry by demonstrating alkaline phosphatase (ALP) activity through a chemical reaction with naphthol AS-BIphosphate, and by immunofluorescence with the detection of osteocalcin, osteopontin, ALP and OSX.

[0032] The term "adipocytes" refers to cells expressing the markers LPL, PPARγ, AdipoQ, and / or cells detectable with a fluorescent probe called Bodipy, which labels lipids present in lipid vacuoles. The adipocyte nature of the cells can be verified by phase-contrast microscopy, which shows the presence of lipid vacuoles, and by immunohistochemical staining with Toil Red O, which labels lipids present in lipid vacuoles.

[0033] The culture medium according to the invention is composed of a basic medium supplemented by various compositions and / or compounds.

[0034] Preferably, the base medium is suitable for culturing eukaryotic cells, such as mammalian cells, and in particular human cells. Such culture media are well known to those skilled in the art. Preferably, the base medium is chosen from DMEM, MEM-a, Ham's F-12, RPMI 1640, IMDM, and combinations thereof. Preferably, the base medium is MEM-a.

[0035] In one embodiment, the base medium according to the invention is supplemented with fetal bovine serum (FBS), preferably 0.5 to 5% (v / v) of FBS, and more particularly 2% (v / v) of FBS. This medium may further comprise intralipids, preferably 0.01% to 1% (v / v) of intralipids, preferably 0.01 to 0.5% (v / v) of intralipids, and more particularly 0.04% (v / v) of intralipids.

[0036] In a second embodiment, the base medium according to the invention is supplemented with platelet lysate (PL), preferably 0.5 to 5% (v / v) of PL, and more particularly 1% (v / v) of PL.

[0037] In a third embodiment, the base medium according to the invention is supplemented with fetal bovine serum (FBS) and platelet lysate (PL). This medium may further comprise intralipids, preferably from 0.01% to 1% (v / v) intralipids, preferably from 0.01% to 0.5% (v / v) intralipids, and more particularly 0.04% (v / v) intralipids.

[0038] Fetal bovine serum and platelet lysate are preferably sterile before being used in the culture medium.

[0039] The base medium according to the invention is also supplemented with 10 to 100 pM of ascorbic acid, 10 to 100 ng / mL of "Bone Morphogenetic Protein 7" (BMP7), 1 to 10 pg / mL of insulin, 1 to 50 pg / mL of apotransferrin, and 1 to 100 ng / mL of vascular endothelial growth factor (VEGF).

[0040] Preferably, the medium according to the invention is supplemented with 30 to 70 pM ascorbic acid, more preferably with 45 to 55 pM ascorbic acid.

[0041] Preferably, the medium according to the invention is supplemented with 30 to 70 ng / mL of BMP7, more preferably with 45 to 55 ng / mL of BMP7. Preferably, BMP7 according to the invention is a recombinant human protein.

[0042] Preferably, the medium according to the invention is supplemented with 3 to 7 pg / mL of insulin, more preferably with 4.5 to 5.5 pg / mL of insulin. Preferably, the insulin according to the invention is recombinant human insulin.

[0043] Preferably, the medium according to the invention is supplemented with 8 to 12 pg / mL of apotransferrin, more preferably with 9 to 11 pg / mL of apotransferrin. Preferably, the apotransferrin according to the invention is recombinant human apotransferrin.

[0044] Preferably, the medium according to the invention is supplemented with 1 to 20 ng / mL of vascular endothelial growth factor (VEGF), more preferably with 5 to 15 ng / mL of VEGF. Preferably, the VEGF according to the invention is recombinant human VEGF.

[0045] The proteins used in the medium are preferably of recombinant origin and used in purified form.

[0046] The culture medium according to the invention can be sterilized or filtered before use. The culture medium according to the invention can be used in various culture processes.

[0047] in vitro culture method

[0048] The invention relates to an in vitro culture method for mesenchymal stem cells comprising the steps of: a) seeding the mesenchymal stem cells in a culture medium as defined above; and b) culturing said mesenchymal stem cells.

[0049] The invention also relates to a method for in vitro culture of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells, comprising the steps of: a) seeding the mesenchymal stem cells and / or the mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells in a culture medium as defined above; and b) culturing said mesenchymal stem cells and / or said mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells.

[0050] Preferably, these methods allow for obtaining, in a single step and within the same culture container, cells differentiated into osteoblasts and adipocytes, as well as an organized endothelial cell network, from a single pool of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors, and endothelial cells. In these methods according to the invention, endothelial progenitors and endothelial cells can also be cultured in the culture medium, simultaneously with said mesenchymal stem cells.

[0051] In one embodiment, the culture takes place in an adherent or non-adherent monolayer, or in suspension, or in the presence of a biomaterial (in solid or gel form). In another embodiment of the methods according to the invention, the cells are cultured in suspension.

[0052] Culture can be performed continuously or discontinuously, in batch, fed-batch or perfused bioreactor or even within a microfluidic chip.

[0053] In the in vitro culture methods according to the invention, part of the mesenchymal stem cells is differentiated into osteoblasts and another part of the mesenchymal stem cells is differentiated into adipocytes, preferably simultaneously, in a single step, in the same medium and the same culture container, without a posteriori cell assembly, and preferably from a single sample.

[0054] Preferably, in the in vitro culture methods according to the invention, the endothelial progenitors and endothelial cells organize themselves into vessels in the culture medium, at the same time as said mesenchymal stem cells differentiate.

[0055] Preferably, all the cells in the process according to the invention come from the same animal species, in particular a mammal. According to one embodiment of the invention, the cells are rodent or primate cells, preferably human cells.

[0056] Endothelial progenitors and / or endothelial cells can be cultured in the culture medium, at the same time as said mesenchymal stem cells.

[0057] Endothelial progenitors and / or endothelial cells present in the same initial cell pool as the mesenchymal stem cells and from the same donor, can be cultured in the culture medium, at the same time as said mesenchymal stem cells.

[0058] In a preferred mode, the cells in the culture processes according to the invention are primary cells. In a preferred mode, the mesenchymal stem cells are derived from primary cultures.

[0059] In a preferential mode, endothelial progenitors and endothelial cells are derived from primary cultures.

[0060] Preferably, mesenchymal stem cells, endothelial progenitors and endothelial cells are derived from primary cultures, preferably from the same individual.

[0061] In one embodiment according to the invention, the mesenchymal stem cells, endothelial progenitors, and endothelial cells are derived from a single sample taken from an individual. Preferably, the mesenchymal stem cells, endothelial progenitors, and endothelial cells are derived from the same primary culture in the same well, from a single sample.

[0062] In one embodiment according to the invention, mesenchymal stem cells, endothelial progenitors and endothelial cells are derived from a single sample taken from an individual.

[0063] In one embodiment according to the invention, the cells are all taken from the same sample on a single subject.

[0064] In one embodiment according to the invention, the cells are taken from a healthy individual, that is to say, not suffering from a disease, in particular a disease affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis.

[0065] In another embodiment of the invention, the cells used are taken from an individual suffering from a disease, in particular a disease affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis, such as bone marrow aplasia, myelodysplastic syndrome, primary immunodeficiency, or a hematological disorder.

[0066] In one embodiment of the process, step a) is preceded by step a0) in which mesenchymal stem cells (MSCs), endothelial progenitors, and endothelial cells are selected and amplified together in the same medium and, preferably, in the same culture container. This medium is preferably EGM2 (Endothelial Growth Medium 2). This step lasts between 3 and 30 days, preferably between 5 and 25 days, and more particularly between 10 and 20 days.

[0067] The temperature of the culture process is selected to allow cell growth. Typically, a cell culture temperature is between 30°C and 38°C. The oxygen concentration is also selected to allow cell growth. Typically, the oxygen concentration is between 10% and 30%, preferably between 15% and 25%. Similarly, the carbon dioxide concentration is between 2% and 8%.

[0068] In one embodiment according to the invention, the cells are cultured in 2 dimensions or in 3 dimensions. The inventors have notably developed two distinct 3D culture approaches. In the first, the different cell types form spheroids or organoids by self-organization; in the second, the cells are deposited on a 3D support.

[0069] Thus, in one embodiment of the method according to the invention, the cells are seeded in step a) onto a three-dimensional support, preferably in the form of spheroids or a three-dimensional support. The support can be any type of support suitable for culturing the cells in question. In particular, the support can be a gel, such as a hydrogel, or a solid. It can be formed from a silicone polymer (such as polydimethylsiloxane PDMS), a resin (such as DS 3000), and / or a calcium biomaterial. In a particular embodiment, the support is integrated into a microfluidic chip. Preferably, the support is a calcium biomaterial, preferably based on tricalcium phosphate and / or hydroxyapatite, and more particularly, the calcium biomaterial is formed from p-TCP.

[0070] In one embodiment of the invention, the differentiation of mesenchymal stem cells into osteoblasts and adipocytes, as well as the organization of a vascular network, are carried out simultaneously on the biomaterial or within the organoid, preferably in a single step, in a single culture container, from a single sample, without subsequent cell assembly.

[0071] Regarding culture on a 3D support, inoculation, culture, and differentiation can be carried out in a perfused bioreactor. This method ensures good cell homogeneity on the biomaterial and allows for maintenance in culture for at least 3 weeks thanks to the oxygen and nutrients supplied by the perfusion. This is particularly the case in microfluidic chip cultures, which are perfused microbioreactors.

[0072] The inventors observed that in the medium according to the invention, mesenchymal stem cells differentiated into osteoblasts and adipocytes, and endothelial progenitors and endothelial cells formed vessels, thus reconstituting the microenvironment of bone marrow in vitro. The inventors observed that in the medium according to the invention, mesenchymal stem cells differentiated into osteoblasts and adipocytes, and that a network of endothelial cells organized itself, thus reconstituting the microenvironment of bone marrow in vitro.

[0073] Method for producing bone marrow reconstitution

[0074] Thus the invention relates to a method for producing a reconstituted bone marrow comprising the in vitro culture method of mesenchymal stem cells described above.

[0075] The invention relates to a method for producing a bone marrow reconstitution comprising the in vitro culture method of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells described above.

[0076] In one embodiment, these processes are preceded by a cell expansion step, which may last from 1 to 2 weeks.

[0077] Preferably, the cells are cultured in the culture medium according to the invention for 4 to 20 days, more preferably for 7 to 15 days.

[0078] The temperature of the culture processes is selected to allow cell growth. Typically, a temperature for cell culture is between 30°C and 38°C.

[0079] The oxygen concentration is selected to allow cell culture. Typically, the oxygen concentration is between 10 and 30%, preferably between 15 and 25%. Similarly, the carbon dioxide concentration is between 2 and 8%.

[0080] In a preferred embodiment of the invention, the method for producing a bone marrow reconstitution includes an in vitro culture method for mesenchymal stem cells comprising the steps of: a) seeding the mesenchymal stem cells in a culture medium according to the invention with endothelial progenitors and endothelial cells; and b) culturing said cells.

[0081] In this embodiment, the cells are cultured in step b) for 4 to 20 days, more preferably for 7 to 15 days. In a preferred embodiment of the invention, the method for producing a bone marrow reconstitution includes an in vitro culture method for mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells comprising the steps of: a) seeding the mesenchymal stem cells and / or the mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells in a culture medium according to the invention; and b) culturing said cells.

[0082] In this embodiment, the cells are cultured in step b) for 4 to 20 days, more preferably for 7 to 15 days.

[0083] The inventors have demonstrated at the protein level the presence of the three medullary compartments in their bone marrow reconstitution.

[0084] Bone marrow regeneration

[0085] The invention relates to a bone marrow reconstitution that can be obtained by the production processes according to the invention.

[0086] The invention also relates to a reconstitution of bone marrow comprising osteoblasts, adipocytes and vessel-forming endothelial cells.

[0087] The invention also relates to a bone marrow reconstitution comprising osteoblasts, adipocytes, and a network of endothelial cells. Preferably, the cells of the bone marrow reconstitution are in direct contact. In particular, the bone marrow reconstitution according to the invention includes vessels.

[0088] Bone marrow reconstitution can therefore be two-dimensional or three-dimensional and may include the support or biomaterial used in its formation. Preferably, the reconstitution is carried out in a pharmaceutically acceptable medium.

[0089] The bone marrow reconstitution of the invention is typically adapted for implantation in the body of an individual.

[0090] Composition. The invention also relates to a composition comprising the cells obtained by the culture process according to the invention. The composition according to the invention is preferably liquid, and more preferably injectable. In this composition, the cells may be in dispersed suspension or in the form of spheroids. In the latter case, the spheroids have an average diameter of less than 500 µm.

[0091] The term "spheroids" refers to a grouping of cells linked together in three dimensions. Preferably, a spheroid comprises 500,000 to 750,000 cells, or alternatively, 1,000 to 500,000 cells. The spheroids of the composition according to the invention have an average diameter of between 50 µm and 750 µm, preferably between 100 µm and 500 µm.

[0092] In one embodiment the composition is a pharmaceutical composition which includes at least one cell obtained by the culture process according to the invention in a pharmaceutically acceptable medium.

[0093] "Pharmaceutically acceptable" here refers to compositions and molecular entities that do not produce adverse, allergic, or otherwise undesirable reactions when administered to a subject. A pharmaceutically acceptable excipient or vehicle is thus an encapsulating material, a diluent, a carrier, or any other non-toxic liquid, semi-solid, or solid formulation aid.

[0094] The compositions of the invention are typically prepared to suit the method of administration. The acceptable pharmaceutical excipients are typically determined partly by the composition being administered, as well as by the particular technique used to administer the composition.

[0095] The compositions of the invention are preferably liquid and adapted to the route of administration.

[0096] The pharmaceutical composition according to the invention may also include at least one other active compound, for example, a calcium biomaterial. Indeed, calcium biomaterials are known to be osteoinductive. They are already used, in particular, for filling bone defects.

[0097] Use of bone marrow reconstitution or composition

[0098] The invention relates to the reconstitution of bone marrow according to the invention or the composition according to the invention for use in the treatment of diseases. Preferably, the diseases are diseases affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis.

[0099] Indeed, bone marrow reconstitution or the composition according to the invention could be used to promote hematopoiesis in various pathological situations, and in particular to allow ectopic hematopoiesis, which would allow hematopoietic "normalization" in these patients.

[0100] By "treatment" or "to treat" is meant herein to achieve, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, improving a clinical symptom or indicator associated with the disease, delaying, inhibiting or preventing the progression of the disease, or partially or totally delaying, inhibiting or preventing the occurrence of a relapse of the disease.

[0101] A method for treating a disease affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis is thus proposed, in which a therapeutically effective amount of a bone marrow reconstitution according to the invention or of the composition according to the invention, is administered to a subject suffering from a disease affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis.

[0102] The term "subject" here refers to a mammal, preferably a primate and most preferably a human. Preferably, the subject treated within the framework of the invention suffers from a disease affecting the integrity of the bone marrow, and / or a disorder of hematopoiesis.

[0103] A disease affecting the integrity of the bone marrow and / or a disorder of hematopoiesis is defined as a disease in which the bone marrow is damaged and / or a disease related to an excess, deficiency, or dysregulation of hematopoiesis in an individual. Examples of such diseases include aplastic anemia, myelodysplastic syndrome, primary immunodeficiency, and hematological malignancies such as leukemia, lymphoma, or myeloma. A therapeutically effective amount is defined as a quantity of compound or reconstitution sufficient to destroy, modify, control, or eliminate the disease. A therapeutically effective amount also refers to a quantity that delays or minimizes the progression of the disease. It also refers to the quantity that provides a therapeutic benefit in the treatment or management of the disease.Finally, the term "therapeutically effective amount" means an amount of the composition or reconstitution, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease, including an improvement in the symptoms associated with the disease. The therapeutically effective amount naturally depends on the product administered, the route of administration, the therapeutic indication, the patient's age, and their condition.

[0104] Determining the route of administration and the appropriate dosage for each individual is within the capabilities of a person skilled in the art.

[0105] The dosage depends on the individual case and, as is well known to those skilled in the art, must be tailored to individual circumstances to achieve an effective therapeutic amount and optimum effect. The therapeutically effective dose level is specific to each patient and will depend particularly on a variety of factors, including the disorder being treated and its severity, the patient's age, body weight, general health, sex, and diet, the time of administration, the route of administration, the duration of treatment, any medications used in combination, and similar factors well known in the medical field.

[0106] Preferably, the bone marrow reconstitution or the composition according to the invention is administered subcutaneously or intrafemorally.

[0107] In another aspect, the invention relates to the use of a bone marrow reconstitution as defined above in a biomedical application, whereby the biomedical application is preferably selected from among prostheses and medical devices. The invention therefore relates to the use of a bone marrow reconstitution as defined above in a prosthesis or a medical device.

[0108] In another aspect, the invention relates to the use of a bone marrow reconstitution as defined above, as a model for the study of physiology, pathophysiology, testing of compounds, and / or physical and / or mechanical conditions.

[0109] By "studying physiology" we mean the study of the mechanisms involved in cell interactions, the functioning and development of bone marrow throughout life.

[0110] Indeed, the mechanisms involved in cell interactions within human bone marrow remain poorly understood, both in physiology and pathology. Understanding these mechanisms is limited due to the lack of in vitro tools for studying human bone marrow in a comprehensive context that integrates its microenvironmental aspects. Specifically, the use of primary cell cultures in the reconstitution according to the invention allows for a closer approximation of in vivo conditions. The bone marrow reconstitution of the invention can be used to study the role of its constituent cellular and humoral components.

[0111] Another aspect of the invention relates to the use of a bone marrow reconstitution according to the invention to study the cellular and / or molecular mechanisms involved in the differentiation of mesenchymal stem cells.

[0112] Since the reconstitution can be formed from a sample of a donor, it can be used to study variations due to age, sex, etc. It can also serve as a bone marrow model for a particular stage of life, such as an aged bone marrow model, a young bone marrow model, a fetal bone marrow model, etc.

[0113] By "studying the pathophysiology" we mean studying the impact of diseases on the characteristics of the bone marrow, such as cell morphology, cell growth, the formation or disappearance of vessels, the expression of certain proteins, etc. In this embodiment, the reconstitution then includes at least one cell type model of the disease studied and / or a cell type from an individual suffering from the disease studied.

[0114] Thus, the invention also relates to the use of a bone marrow reconstitution according to the invention as a model of pathological bone marrow. In a particular embodiment, one or more of the cell types of the reconstitution are model cell types of pathologies. By "model cell types of pathologies," we mean cell types derived from animal models reproducing pathologies that appear spontaneously or are induced by genetic engineering methods (such as transgenesis) or with pharmacological tools in order to reproduce the characteristics of cells from individuals affected by these particular pathologies.

[0115] According to a particular embodiment, one or more of the cell types of the reconstitution are derived from an individual suffering from the disease under study.

[0116] Preferably the pathologies studied according to the invention are pathologies having or being suspected of having an influence on the bone marrow such as: bone marrow aplasia, myelodysplastic syndrome, primary immunodeficiency, hematological diseases such as leukemia, lymphoma or myeloma.

[0117] The reconstitution according to the invention can also be used to study pathologies developing at a particular time in an individual's life. "Testing molecules" refers to studying the impact of these molecules on the bone marrow. In this embodiment, at least one molecule to be tested is applied to the reconstitution according to the invention, and after a period of exposure or incubation, the reconstitution is analyzed to determine the changes caused by said at least one tested molecule. These changes may, in particular, relate to cell morphology, cell growth and death, the formation or disappearance of blood vessels, the expression of certain proteins, cell differentiation, etc.

[0118] By "molecules" we mean molecules with preventive, therapeutic or diagnostic aims targeting the cellular and molecular components of the bone marrow

[0119] Another aspect of the invention relates to the use of a bone marrow reconstitution according to the invention to study the efficacy and / or toxicity of a drug candidate.

[0120] In one embodiment, the invention relates to the use of a bone marrow reconstitution according to the invention to study the efficacy and / or toxicity of a drug candidate for a specific individual. Indeed, a bone marrow reconstitution prepared from an individual's sample can serve as a model to study the efficacy and / or toxicity of a drug candidate for that particular individual. This type of analysis can be performed, in particular, within the framework of personalized medicine, to define the most appropriate treatment for an individual.

[0121] By "testing physical and / or mechanical conditions," we mean studying the impact of these conditions on bone marrow reconstitution. In this embodiment, at least one physical or mechanical condition is applied to the reconstitution according to the invention. After a period of exposure or incubation, the reconstitution is analyzed to determine the changes caused by said at least one physical or mechanical condition tested. These changes may, in particular, relate to cell morphology, growth and cell death, the formation or disappearance of blood vessels, the expression of certain proteins, cell differentiation, etc. The result of adding the physical or mechanical condition can be studied by comparing it to a reconstitution to which the condition has not been applied.

[0122] By "physical condition", we mean in particular the use of waves such as magnetic, electromagnetic or ultrasound waves.

[0123] The term "mechanical condition" refers in particular to pressure, contraction, stretching, gravity, weightlessness, and shear. The invention relates to the use of a bone marrow reconstitution according to the invention as a model for the long-term study of hematopoiesis and / or mesenchymal stem cell differentiation.

[0124] By "long term" we mean more than 3 days, more than 10 days, more than 15 days, more than 20 days and preferably up to 21 days.

[0125] The term "study of hematopoiesis" refers to the study of the proliferation and differentiation of blood cells.

[0126] For the purposes of this application, the term "comprising" should be interpreted as covering all the specifically mentioned features, as well as any additional unspecified features. Furthermore, the use of the term "comprising" also describes an embodiment in which no features other than those specifically mentioned are present (e.g., "consisting of").

[0127] The present invention will be illustrated in more detail by the figures and examples below.

[0128] FIGURES

[0129] Figure 1 shows the relative expression of genes involved in the osteoblastic, adipocyte, and vascular (endothelial cell) lineages present in bone marrow reconstitutions after culture in 2D models. After selection and amplification in EGM2 medium, the cells are cultured without passage, in the same culture vessel, for 14 days in 2D in the medium according to the invention (with SVF and intralipids). Gene expression is quantified by Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR) and calculated after normalization with a reference gene using the 2D method. ACT , n=5.

[0130] Figure 2 shows the relative expression of genes involved in the osteoblastic, adipocyte, and vascular (endothelial cell) lineages present in bone marrow reconstitutions after culture in 2D models. After selection and amplification in EGM2 medium, the cells are cultured without passage, in the same culture vessel, for 14 days in 2D in either a medium with FBS and intralipids, a medium with LP without intralipids, or in their amplification medium (EGM2 medium), corresponding to the CTRL condition. Gene expression is quantified by Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR) and calculated after normalization with a reference gene using the 2D method. ACTn=3. Figure 3 shows the relative expression of genes corresponding to prohematopoietic factors present in bone marrow reconstitutions after culture in 2D models. After selection and amplification in EGM2 medium, cells are cultured without passage, in the same culture vessel, for 14 days in 2D in either a medium with FBS and intralipids, a medium with LP without intralipids, or in their amplification medium (EGM2 medium), corresponding to the CTRL condition. Gene expression is quantified by Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR) and calculated after normalization with a reference gene using the 2-method. ACT , n=3.

[0131] Figure 4 shows the number of CD34 CD38- cells, the most immature hematopoietic cells, obtained after co-culture for 14 days of hematopoietic stem cells (HSCs) with bone marrow reconstitutions obtained according to the process described in the invention.

[0132] Example:

[0133] Example 1:

[0134] Mesenchymal stem cells (MSCs), endothelial progenitors and endothelial cells from the same bone marrow sample are selected and amplified using the adhesion properties of these cell types and the use of Endothelial Growth Medium 2 (Promocell) for culture.

[0135] After 14 days of culture, these cells are detached and used to generate different bone marrow models, in 2 and 3 dimensions. For this purpose, the cells are cultured in a medium that allows the differentiation, within the same culture, of some MSCs into osteoblasts (bone compartment) and others into adipocytes (adipose compartment), while also allowing the organization of a vascular network. The medium used here consists of a MEMa base supplemented with 2% fetal bovine serum, 50 µM ascorbic acid, 0.04% (v / v) intralipids, 50 ng / mL Bone Morphogenetic Protein 7, 5 pg / mL insulin, 10 pg / mL apotransferrin, and 10 ng / mL VEGF.

[0136] For the 3D models, two technologies are used: spheroid generation based on cellular self-organization and the use of a cell / biomaterial complex infused in a bioreactor where the biomaterial is PTCP. The inventors demonstrated the presence of the different compartments through gene analysis (RT-qPCR). Figure 1 presents these results for the 2D model.

[0137] The inventors also demonstrated the presence of the different compartments through fluorescence analyses (immunofluorescence and fluorescent probes), targeting specific markers for each compartment (Osterix for osteoblasts, CD31 for endothelial cells, Bodipy for adipocytes). These different markers were detected in the 2D model, the spheroid model (3D model), and the pTCP biomaterial model in a perfused bioreactor (3D model).

[0138] Example 2:

[0139] Bone marrow reconstitutions were performed as in example 1 but different compositions of the differentiation medium were tested.

[0140] Cells recovered from primary bone marrow culture in EGM2 are seeded at 20,000 cells / cm³ 2 The cells are cultured in EGM2 medium and left in this medium for 4 to 6 days at 37°C and 5% CO2. The EGM2 medium is then replaced with one of the two differentiation media (SVF or LP) described below. The differentiation medium is renewed twice a week. After 14 days in the differentiation medium, the culture is stopped for gene analysis.

[0141] The so-called SVF medium is composed of a MEMa medium base supplemented with 2% (v / v) fetal bovine serum, 50 pM ascorbic acid, 0.04% (v / v) intralipids, 50ng / mL Bone Morphogenetic Protein, 7.5 pg / mL insulin, 10 pg / mL apotransferrin and 10 ng / mL VEGF.

[0142] The so-called LP medium is composed of a MEMa medium base supplemented with 1% (v / v) platelet lysate (LP), 50 pM ascorbic acid, 50 ng / mL Bone Morphogenetic Protein, 7.5 pg / mL insulin, 10 pg / mL apotransferrin and 10 ng / mL VEGF.

[0143] The simultaneous generation of the 3 medullary compartments was assessed in a 2D culture by gene analysis (see figure 2).

[0144] The results show no significant difference between the two differentiation media tested. Each medium allows the in vitro differentiation of some MSCs into osteoblasts and others into adipocytes, while maintaining the CD31-positive cells corresponding to the endothelial compartment (Figure 2). Furthermore, the inventors observed a trend: the SVF-based medium appears to differentiate more MSCs into adipocytes, and the LP-based medium more MSCs into osteoblasts. These results suggest adaptability / flexibility of the process. Indeed, several studies show that MSC differentiation is modified by physical and mechanical constraints, which must be taken into account for 3D models where these constraints differ from standard 2D models (variable rigidity of biomaterials, variable contraction of spheroids / organoids, etc.).The production process could therefore make it possible to adapt to these constraints by adapting the differentiation environment.

[0145] On the other hand, the older the bone marrow, the greater the proportion of adipose tissue and the more its functionality decreases, along with a decline in vascularization. Conversely, the younger the bone marrow, the greater the bone density and the stronger the vascularization. Thus, modulating the composition of the culture medium could allow for the study of aging at the level of the hematopoietic niche.

[0146] Furthermore, the results show an increase in the expression of key pro-hematopoietic factors in bone marrow reconstitutions generated in vitro using the method according to the invention, compared to the CTRL condition (Figure 3). These factors are secreted or expressed in vivo by the bone marrow microenvironment at the level of hematopoietic niches and are necessary for the establishment of hematopoiesis in the bone marrow, indicating the functionality of the bone marrow reconstitutions according to the invention.

[0147] Example 3:

[0148] To enhance the functional study of in vitro bone marrow reconstitutions, the inventors tested the addition of hematopoietic stem cells to these bone marrow reconstitutions, in order to evaluate in vitro hematopoiesis (figure 4).

[0149] Bone marrow reconstitution formation: After selection and amplification in EGM2 medium, mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors, and endothelial cells are cultured without transition, in the same culture vessel, for 14 days in 2D in either a medium with FBS and intralipids, a medium with LP without intralipids, or in their amplification medium (EGM2 medium), corresponding to the "EGM2" condition. In parallel, after pre-selection and pre-amplification in MEMa medium with FBS, mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors, and endothelial cells are cultured under the same conditions as before (same cell density, culture time, etc.) for 14 days in 2D in MEMa medium with FBS.This medium being a reference medium used in most publications for the culture of mesenchymal stem cells, it corresponds to the condition “MES”.

[0150] Co-culture with hematopoietic stem cells (HSCs): HSCs are sorted from placental blood units based on the CD34-positive marker. They are then co-cultured with previously generated 2D bone marrow reconstitutions for 14 days in IMDM medium + 10% SF + 1 pM hydrocortisone. At the end of the co-culture, all cells are collected, counted, and then analyzed by flow cytometry (n=3). Initial results show better maintenance and proliferation of the most immature hematopoietic cells (CD34+CD38+) with the bone marrow reconstitutions according to the invention than under control conditions. Indeed, in many models, most of these cells differentiate rapidly and are poorly maintained.

Claims

DEMANDS 1) A culture medium that allows for the single-step production, in the same culture vessel, of cells differentiated into osteoblasts and adipocytes, as well as an organized endothelial cell network, from a single pool of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells, comprising: a) fetal bovine serum (FBS) and / or platelet lysate (PL), b) 10–100 pM ascorbic acid, c) 10–100 ng / mL Bone Morphogenetic Protein 7 (BMP7), d) 1–10 pg / mL insulin, e) 1–50 pg / mL apotransferrin, and f) 1–100 ng / mL vascular endothelial growth factor (VEGF), and optionally g) 0.01-0.5% (v / v) of intralipids. 2) Method for in vitro culture of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells comprising the steps of: a) seeding the mesenchymal stem cells and / or the mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells in a culture medium as defined in claim 1; and b) culturing said mesenchymal stem cells and / or the mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells. 3) A culture method according to claim 2, wherein part of the mesenchymal stem cells is differentiated into osteoblasts and another part of the mesenchymal stem cells is differentiated into adipocytes simultaneously, in a single step, in the same culture medium and container, without a posteriori cell assembly. 4) A culture method according to any one of claims 2 to 3, wherein endothelial progenitors and endothelial cells organize into vessels in the culture medium, at the same time as said mesenchymal stem cells differentiate. 5) A culture method according to any one of claims 2 to 4, wherein the cells are primary cells. 6) A culture method according to any one of claims 2 to 5, wherein the cells are all taken from the same sample on a single subject. 7) A culture method according to any one of claims 2 to 6, wherein in step a) the cells are seeded in 3 dimensions in the form of spheroids or on a 3-dimensional support. 8) A method for producing a bone marrow reconstitution comprising the in vitro culture method of mesenchymal stem cells and / or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells according to any one of claims 2 to 7, wherein the cells are cultured in step b) for 4 to 20 days. 9) Reconstitution of bone marrow obtained by the process of claim 8. 10) Reconstitution of bone marrow including osteoblasts, adipocytes and vessel-forming endothelial cells. 11) Composition comprising cells obtained by the process of any one of claims 2 to 6. 12) Bone marrow reconstitution according to any one of claims 9 to 10 or composition according to claim 11 for its use in the treatment of diseases, in particular diseases affecting the integrity of the bone marrow and / or related to a disorder of hematopoiesis. 13) Use of a bone marrow reconstitution as defined in claim 9 or claim 10, as a model for the study of physiology, pathophysiology, compound testing, and / or testing of physical and mechanical conditions. 14) Use of a bone marrow reconstitution as defined in claim 9 or claim 10, in a prosthesis or medical device.

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