EP4308688A1

EP4308688A1 - Medium and method for producing a bone marrow reconstitution

Info

Publication number: EP4308688A1
Application number: EP22716252.6A
Authority: EP
Inventors: Mélanie Alexandra GADELORGE; Nicolas Lucien ESPAGNOLLE; Frédéric DESCHASEAUX
Original assignee: Ecole Nationale Veterinaire De Toulouse Envt; Institut National de la Sante et de la Recherche Medicale INSERM; Francais du Sang Ets; Universite Toulouse III Paul Sabatier
Current assignee: Ecole Nationale Veterinaire De Toulouse Envt; Institut National de la Sante et de la Recherche Medicale INSERM; Francais du Sang Ets; Universite Toulouse III Paul Sabatier
Priority date: 2021-03-18
Filing date: 2022-03-18
Publication date: 2024-01-24
Also published as: FR3120875A1; US20240158752A1; CA3212094A1; WO2022195104A1; CN117716023A

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

TITLE: Medium and Method for Producing Bone Marrow Reconstitution

The present invention relates to a culture medium and a culture method making it possible to obtain, in a single step and in the same culture container, cells differentiated into osteoblasts and adipocytes as well as a network of endothelial cells organized from the same pool of mesenchymal stem cells and/or of 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 process, a reconstitution of bone marrow and their uses.

The present invention also relates to a culture medium and a culture method allowing the culture and/or the differentiation into osteoblasts and adipocytes of mesenchymal stem cells as well as the organization of a network of endothelial cells. It also relates to a composition obtained by said process, a reconstitution of bone marrow and their uses.

The reproduction of adult human bone marrow ex vivo is increasingly described in the literature in order to overcome animal models which are costly, time consuming and dependent on the species barrier. Studies have begun to highlight 3D models of cocultures grouping the osteoblastic and endothelial compartments, generally from cells derived from lineages. For example, the endothelial compartment, playing an active role in hematopoietic stem cell (HSC) proliferation, is often incorporated through HUVEC-like endothelial cell lines. Other models require a mouse step to allow for vascularization or functional approaches. In addition, these cocultures are usually carried out in several stages (osteoblastic differentiation of mesenchymal stem cells in the first stage, then assembly with a second pool of endothelial cells in a second stage). Furthermore, medullary adipose tissue is often ignored in these studies despite its increasing functional importance described in the literature.

The inventors have created a new model that comprehensively incorporates all the cellular and non-hematopoietic micro-environmental parameters of a marrow human bone. It includes the medullary adipose tissue, the osteoblastic compartment and the vascular compartment, also called the endothelial compartment. The culture medium developed by the inventors makes it possible to obtain these 3 non-hematopoietic compartments of human bone marrow simultaneously and in particular from a single sample, in a single step, in the same culture container, without the use of cell line. This makes it possible to generate an ex vivo human bone marrow comprising the osteoblastic, adipocyte and endothelial compartments in 2D but also in 3D, in particular via the use of a biomaterial. Furthermore, by self-organization this ex vivo bone marrow can form a spheroid/organoid type 3D structure.

Moreover, in previous studies, the differentiation of mesenchymal stem cells into adipocytes and osteoblasts required carrying out the two differentiations separately in two different media. Not only does the medium developed by the inventors make it possible to carry out these two differentiation pathways at the same time, but also to promote the organization of an endothelial network in the same medium, and in the same culture well, without assembling cells at posteriori, thus greatly simplifying the manipulations necessary in particular for carrying out bone marrow reconstitution.

Thus the invention relates to a culture medium making it possible to obtain 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 of a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells, comprising: a) fetal bovine serum (FCS), and/or platelet lysate (LP), b) 10 - 100 mM ascorbic acid, c) 10 - 100 ng/mL 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) intralipids. Thus the invention relates to a culture medium for the culture and/or the differentiation into osteoblasts and into adipocytes of mesenchymal stem cells, and optionally allowing the organization of a network of endothelial cells, comprising: a) fetal calf serum (SVF), and/or platelet lysate (LP), b) 10 - 100 mM 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).

In a second aspect, the invention relates to a process for the in vitro culture of mesenchymal stem cells comprising the steps consisting in: a) seeding the mesenchymal stem cells in a culture medium as defined above; and b) culturing said mesenchymal stem cells.

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 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.

Preferably, this method makes it possible to obtain 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.

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

The invention also relates to a reconstitution of bone marrow obtained by the method according to the invention. The invention also 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.

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

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

Finally, the invention relates to the use of a bone marrow reconstitution according to the invention, as a model for the study of physiology, physiopathology, the test of compounds and/or the test of physical and mechanical conditions.

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

Detailed description of the invention

Culture centre

By “medium for the culture and/or the differentiation into osteoblasts and into adipocytes of mesenchymal stem cells”, is meant a medium suitable for the culture and/or the differentiation into osteoblasts and into adipocytes of mesenchymal stem cells. Preferably, this medium is a medium for the culture and/or the differentiation into osteoblasts and into adipocytes of mesenchymal stem cells and for the formation of a vascular network by endothelial progenitors and/or endothelial cells.

The term "medium for the culture and differentiation into osteoblasts and into adipocytes of mesenchymal stem cells" means a medium suitable for the culture and simultaneous differentiation into osteoblasts and into adipocytes of the same pool of cells mesenchymal strains, in a single step, in the same culture container, without cell assembly. Preferably, this medium is a medium for the culture and differentiation into osteoblasts and into adipocytes of mesenchymal stem cells allowing the organization of a network of endothelial cells.

Preferably, this medium allows the simultaneous differentiation of mesenchymal stem cells into osteoblasts and into adipocytes. 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, in a single culture container, without assembly of cells a posteriori. It can be in different forms but is preferably liquid and allows the culture of eukaryotic cells, in particular of mammalian cells and more particularly of human cells.

As intended herein, the term "culture" refers to the multiplication of cultured cells.

The term "differentiation" relates to the acquisition by cells cultured in a culture medium of cellular characteristics which are not present in the cells initially used to inoculate the cell culture medium. As provided here, “differentiation” designates in particular the acquisition of characteristics involving in particular here the cells in the adipocyte or osteoblastic pathway.

By “mesenchymal stem cells” also called “mesenchymal stromal cells”, or MSCs, is meant stromal cells of mesodermal origin. They are characterized phenotypically by the co-expression of a certain number of markers such as for example CD73, CD90, CD105, CD146, and the absence of expression of other markers, in particular CD45 and CD34. They can be derived from bone marrow, adipose tissue, or umbilical cord blood from mammals. Mesenchymal stem cells can be derived from rodents or primates, and in particular are of murine or human origin.

In a preferential mode, the mesenchymal stem cells are derived from primary cultures. By "primary culture" is meant a culture of cells derived directly from the tissue and/or cells of an individual.

By “endothelial progenitors” is meant cells engaged in endothelial differentiation but which are not yet recognizable as cells. endothelial cells under the microscope. They are characterized phenotypically by the expression of a certain number of markers such as for example CD133, CD34, CD31, VEGFR2.

By "endothelial cells" is meant cells completely differentiated in the endothelial pathway and therefore recognizable as endothelial cells under the microscope. They are characterized phenotypically by the expression of a certain number of markers such as for example CD31, VE-Cadherin, von Willebrand factor, VEGFR2.

Endothelial progenitors and endothelial cells have the ability to organize themselves into a network of endothelial cells, or even a vascular network, and therefore to organize themselves into vessels.

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

By “individual” is meant a subject of an animal species, in particular of mammals. According to one mode of the invention, the individual is a primate or a rodent, preferably a mouse or a human being.

By “osteoblasts” is meant cells expressing the Runx2, DSX, ESP, BSP, DLX5 and/or Osterix (OSX) markers. The osteoblastic phenotype can be evaluated under a phase-contrast microscope by assessing the level of mineralization, by staining with alizarin red, by immunohistochemistry by demonstrating alkaline phosphatase (ALP) activity thanks to a chemical reaction with naphthol AS - BIphosphate, by immunofluorescence with demonstration of osteocalcin, osteopontin, PAL and OSX.

By “adipocytes” is meant cells expressing the LPL, PPARy, AdipoQ markers and/or cells detectable with a Bodipy fluorescent probe marking the lipids present in the lipid vacuoles. The adipocyte nature of the cells can be verified by phase contrast microscopic examination showing the presence of lipid vacuoles, and by immunohistochemical staining with oil red O marking the lipids present in the lipid vacuoles.

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

Preferably, the basal medium makes it possible to cultivate 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-α, Ham's F-12, RPMI 1640, IMDM and combinations thereof. Preferably the basal medium is MEM-α.

In one embodiment, the basic medium according to the invention is supplemented with fetal calf serum (SVF), preferably from 0.5 to 5% (v/v) of S VF, and more particularly 2% ( v/v) of SVF. This medium may further comprise intralipids, preferably from 0.01% to 1% (v/v) of intralipids, preferably from 0.01 to 0.5% (v/v) of intralipids, and more particularly 0.04% (v/v) intralipids.

In a second embodiment, the basic medium according to the invention is supplemented with platelet lysate (LP), preferably from 0.5 to 5% (v/v) of LP, and more particularly 1% (v/ v) LP.

In a third embodiment, the basic medium according to the invention is supplemented with fetal calf serum (FCS) and platelet lysate (LP). This medium may further comprise intralipids, preferably from 0.01% to 1% (v/v) of intralipids, preferably from 0.01 to 0.5% (v/v) of intralipids, and more particularly 0.04% (v/v) intralipids.

The fetal bovine serum and the platelet lysate are preferably sterile before being used in the culture medium.

The basal medium according to the invention is also supplemented with 10 to 100 mM 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 apotransferrin, and 1 to 100 ng/mL vascular endothelial growth factor (VEGF).

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

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.

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 a recombinant human insulin.

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. Of Preferably, the apotransferrin according to the invention is a recombinant human apotransferrin.

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 a recombinant human VEGF.

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

The culture medium according to the invention can be sterilized or filtered before being used. The culture medium according to the invention can be used in various culture methods.

In vitro culture process

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

The invention also relates to a process 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 consisting in: 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.

Preferably, these methods make it possible to obtain in a single step, in the same culture container, cells differentiated into osteoblasts and into adipocytes, as well as a network of organized endothelial cells, from the same pool of stem cells 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, at the same time as said mesenchymal stem cells.

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 form or in gel form). In another embodiment of the methods according to the invention, the cells are cultured in suspension.

The culture can be carried out continuously or discontinuously, in batch, in fed-batch or in a perfused bioreactor or even within a microfluidic chip.

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 assembly of cells a posteriori, and preferably from a single sample.

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

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

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

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

In a preferential mode, the cells of the culture methods according to the invention are primary cells. In a preferential mode, the mesenchymal stem cells are derived from primary cultures.

In a preferential mode, the endothelial progenitors and the endothelial cells are derived from primary cultures.

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

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

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

In one embodiment according to the invention, the cells all come from the same sample taken from a single subject.

In one embodiment according to the invention, the cells come 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.

In another embodiment according to the invention, the cells used come from an individual suffering from a disease, in particular a disease affecting the integrity of the bone marrow and/or linked to a hematopoiesis disorder, such as aplastic anemia, myelodysplastic syndrome, primary immune deficiency, or blood disease.

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

The temperature of the culture process is selected to allow the culture of the cells. Typically a temperature for culturing the cells is between 30°C and 38°C. The oxygen concentration is selected to allow cell culture. Typically the oxygen concentration is between 10 and 30%, preferentially from 15 to 25%. Similarly, the carbon dioxide concentration is between 2 and 8%.

In one embodiment according to the invention, the cells are cultured in 2 dimensions or in 3 dimensions. The inventors have in particular developed two distinct 3-dimensional (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.

Thus, in one embodiment of the method according to the invention, the cells are seeded in step a) on a 3-dimensional support, preferably in the form of spheroids or on a 3-dimensional support. Thus the support can be any support allowing the culture of the cells considered. 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 b-TCP.

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 assembly of cells a posteriori.

Concerning the culture on a 3-dimensional support, the seeding as well as the culture and the differentiations can be carried out in a perfused bioreactor. This embodiment ensures good cellular homogeneity on the biomaterial and allows maintenance in culture for at least 3 weeks thanks to the supply of oxygen and nutrients provided by the perfusion. This is particularly the case in cultures on a microfluidic chip which are perfused microbioreactors.

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

Method for producing a reconstitution of bone marrow Thus the invention relates to a method for producing a reconstitution of bone marrow comprising the method of in vitro culture of mesenchymal stem cells described above.

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

In one embodiment, these methods are preceded by a cell expansion step, possibly lasting 1 to 2 weeks.

Preferably, the cells are cultured in the culture medium according to the invention for 4 to 20 days, more preferentially from 7 to 15 days.

The temperature of the culture processes is selected in order to allow the culture of the cells. Typically a temperature for culturing the cells is between 30°C and 38°C.

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

In a preferred mode of the invention, the process for producing a bone marrow reconstitution comprises a process for the in vitro culture of mesenchymal stem cells comprising the steps consisting in: 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.

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 mode of the invention, the process for producing a bone marrow reconstitution comprises a process 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 consisting in: a) inoculating 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.

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

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

Bone marrow reconstruction

The invention relates to a reconstitution of bone marrow capable of being obtained by the production methods according to the invention.

The invention also 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. Preferably the cells of the bone marrow reconstitution are in direct contact. In particular, the reconstitution of bone marrow according to the invention comprises vessels.

The reconstitution of bone marrow can therefore be in two or in three dimensions and possibly include the support or the biomaterial having served for its formation. Preferably the reconstitution is included in a pharmaceutically acceptable medium.

The bone marrow reconstitution of the invention is typically suitable for implantation into an individual's body.

Composition The invention also relates to a composition comprising the cells obtained by the culture method according to the invention. The composition according to the invention is preferably liquid, and more preferably injectable. In this composition the cells can 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.

By “spheroids”, we mean a group of cells linked together in the three dimensions of space. Preferably, a spheroid comprises from 500 to 750,000 cells, or else from 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.

In one embodiment, the composition is a pharmaceutical composition which comprises at least one cell obtained by the culture method according to the invention in a pharmaceutically acceptable medium.

By "pharmaceutically acceptable" herein is meant compositions and molecular entities that do not produce side, allergic, or otherwise undesired 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 auxiliary.

The compositions of the invention are typically prepared to suit the mode of administration. Acceptable pharmaceutical excipients are typically determined in part by the composition being administered, as well as the particular technique used to administer the composition.

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

The pharmaceutical composition according to the invention can also comprise 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 fillings of loss of bone substance.

Use of bone marrow reconstitution or composition

The subject of the invention is the reconstitution of bone marrow according to the invention or the composition according to the invention for its use for the treatment of diseases. Preferably, the diseases are diseases affecting the integrity of the bone marrow and/or linked to a disorder of hematopoiesis.

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

By “treatment” or “treating”, we mean here achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, improving a clinical symptom or an indicator associated with the disease, delaying, inhibit or prevent the progression of the disease, or partially or totally delay, inhibit or prevent the occurrence of a relapse of the disease.

A method for treating a disease affecting the integrity of the bone marrow and/or linked to a haematopoiesis disorder is thus proposed, in which a therapeutically effective quantity of a reconstitution of bone marrow 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 linked to a haematopoiesis disorder.

By “subject” is meant here a mammal, preferably a primate and 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 from a hematopoiesis disorder.

By "a disease affecting the integrity of the bone marrow and / or a disorder of hematopoiesis", we mean diseases where the bone marrow is damaged and / or diseases related to an excess, a deficiency or a disorder of hematopoiesis in an individual. These diseases include, for example, bone marrow aplasia, myelodysplastic syndrome, primary immune deficiency, and hematology, such as leukaemia, lymphoma or myeloma. By "therapeutically effective amount" is meant herein an amount of composition or reconstitution sufficient to destroy, modify, control or eliminate the disease. A "therapeutically effective amount" also refers to an amount capable of delaying or minimizing the extent of the disease. It also refers to the amount providing therapeutic benefit in the treatment or management of disease. Finally, the term "therapeutically effective amount" means an amount of the composition or reconstitution, alone, or in combination with other therapies, which provides therapeutic benefit in the treatment or management of disease, including an improvement in the symptoms associated with the disease. The therapeutically effective amount naturally depends on the product administered, the mode of administration, the therapeutic indication, the age of the patient and his condition.

The determination of the route of administration and of the appropriate dosage according to the subject is within the abilities of those skilled in the art.

The dosage depends on the individual case and, as is well known to those skilled in the art, must be adapted to the individual circumstances to obtain an effective therapeutic amount and an optimum effect. The therapeutically effective dose level is specific for any patient, and will in particular depend on a variety of factors, including the disorder being treated and the severity of the disorder, age, body weight, general health, gender and the patient's diet, time of administration, route of administration, duration of treatment, drugs used in combination, and the like factors well known in the medical field.

Preferably, the bone marrow reconstitution or the composition according to the invention are administered subcutaneously or intrafemorally.

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

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

By “studying physiology” we mean the study of the mechanisms involved in cell interactions, functioning and development of the bone marrow during life.

Indeed, the mechanisms involved in cellular interactions within human bone marrow remain poorly understood, whether in physiology or pathology. The understanding of these mechanisms is indeed limited due to the absence of an in vitro tool allowing the study of human bone marrow in a global context integrating in particular its microenvironmental aspects. In particular, the use of primary cell cultures in the reconstitution according to the invention makes it possible to approach even more closely the in vivo conditions. The reconstitution of bone marrow of the invention can be used to study the role of the cellular and humoral elements constituting it.

Also, another aspect of the invention relates to the use of a reconstitution of bone marrow according to the invention to study the cellular and/or molecular mechanisms involved in the differentiation of mesenchymal stem cells.

As the reconstruction can be formed from a sample from 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 stage. particular life model, such as aged bone marrow model, young bone marrow model, fetal bone marrow model, etc.

By “studying physiopathology” 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 comprises at least one model cell type of the disease under study and/or a cell type originating from an individual suffering from the disease under study.

Thus the invention also relates to the use of a bone marrow reconstitution according to the invention as a model of pathological bone marrow. According to 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 from animal models reproducing pathologies appearing spontaneously or induced by genetic engineering methods (such as for example transgenesis) or with pharmacological tools in order to reproduce the characteristics of cells individuals affected by these particular pathologies.

According to a particular embodiment, one or more of the cell types of the reconstitution come from an individual suffering from the disease under study.

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 immune deficiency, hemopathies such as leukaemia, lymphoma or myeloma.

The reconstitution according to the invention can also be used to study pathologies developing at a particular time in the life of the individual. By “testing molecules” we mean 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 an exposure or incubation time, the reconstitution is analyzed in order to determine the changes which have been caused by said at least one molecule tested. These changes may in particular concern cell morphology, cell growth and death, the formation or disappearance of vessels, the expression of certain proteins, cell differentiation, etc. ...

By "molecules" we mean molecules for preventive, therapeutic or diagnostic purposes targeting the cellular and molecular actors of the bone marrow.

Also, another aspect of the invention relates to the use of bone marrow reconstitution according to the invention to study the efficacy and/or toxicity of a drug candidate.

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 particular individual. Indeed, a reconstitution of bone marrow made from an individual's sample can serve as a model to study the efficacy and/or toxicity of a drug candidate for this particular individual. This type of analysis can be carried out in particular in the context of personalized medicine, to define the most suitable treatment for an individual.

By “testing physical and/or mechanical conditions”, we mean studying the impact of these conditions on the reconstitution of bone marrow. In this embodiment, at least one physical or mechanical condition is applied to the reconstitution according to the invention. After an exposure or incubation time, the reconstitution is analyzed in order to determine the changes that have been caused by the said at least one physical or mechanical condition tested. These changes may in particular concern cell morphology, cell growth and death, the formation or disappearance of vessels, the expression of certain proteins, cell differentiation, etc. The result of adding of the physical or mechanical condition by comparing it to a reconstruction on which the condition has not been applied.

By “physical condition”, we mean in particular the use of waves such as magnetic, electromagnetic or ultrasound waves.

By “mechanical condition” is meant in particular pressure, contraction, stretching, gravity, weightlessness, shear. The invention relates to the use of a reconstitution of bone marrow according to the invention, as a model for the long-term study of hematopoiesis and/or the differentiation of mesenchymal stem cells.

By “long term”, we mean over more than 3 days, more than 10 days, more than 15 days, more than 20 days and preferably up to 21 days.

By “study of hematopoiesis”, we mean the study of the proliferation and differentiation of blood cells.

In the context of the present application, the term "comprising" must be interpreted as covering all the characteristics specifically mentioned, as well as possibly certain additional non-specified. Furthermore, the use of the term "comprising" also describes the embodiment in which no characteristic other than the specific ones mentioned is present (i.e. "consisting of").

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

FIGURES

Figure 1 presents the relative expression of genes involved in osteoblastic, adipocyte and vascular (endothelial cells) lineages present in bone marrow reconstructions after culture in 2D models. After selection and amplification in EGM2 medium, the cells are cultured without passage, in the same culture container, for 14 days in 2D in the medium according to the invention (with FCS and intralipids). The gene expression is quantified by Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR) and calculated after normalization by a reference gene using the 2° ^t method, n=5.

Figure 2 presents the relative expression of genes involved in the osteoblastic, adipocyte and vascular (endothelial cells) lineages present in the bone marrow reconstructions after culture in the 2D models. After selection and amplification in EGM2 medium, the cells are cultured without passage, in the same culture container, for 14 days in 2D in either a medium with FCS and intralipids, or a medium with LP without intralipid, or in their medium of amplification (EGM2 medium), corresponding to the CTRL condition. Gene expression is quantified by Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR) and calculated after normalization by a reference gene using the 2° ^t method, n=3. Figure 3 presents the relative expression of genes corresponding to the prohaematopoietic factors present in the bone marrow reconstructions after culture in the 2D models. After selection and amplification in EGM2 medium, the cells are cultured without passage, in the same culture container, for 14 days in 2D in either a medium with FCS and intralipids, or a medium with LP without intralipid, or in their medium of amplification (EGM2 medium), corresponding to the CTRL condition. Gene expression is quantified by Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR) and calculated after normalization by a reference gene using the 2° ^T method, n=3.

Figure 4 shows the number of CD34CD38 cells, the most immature hematopoietic cells, obtained after co-culture for 14 days of hematopoietic stem cells (HSC) with bone marrow reconstitutions obtained according to the method described in the invention.

Example:

Example 1:

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

After 14 days of culture, these cells are separated and used to generate different models of bone marrow, in 2 and in 3 dimensions. For this purpose, the cells are cultured in a medium allowing the differentiation in the same culture of a part of the MSCs into osteoblasts (bone compartment), of another part into adipocytes (adipose compartment), while allowing the organization of 'a vascular network The medium used here is composed of a base of MEMa medium supplemented with 2% fetal calf serum, 50uM ascorbic acid, 0.04% (v/v) intralipids, 50ng/mL of Bone Morphogenetic Protein 7. 5 pg/mL insulin, 10 pg/mL apotransferrin and 10 ng/mL VEGF.

For 3D models, two technologies are used: the generation of spheroids based on cellular self-organization and the use of a cell/biomaterial complex infused into a bioreactor in which the biomaterial is bTΰR. The inventors have demonstrated the presence of the different compartments by gene analyzes (RT-qPCR). Figure 1 presents these results on the 2D model.

The inventors have also demonstrated the presence of the different compartments by fluorescence analyzes (immunofluorescence and fluorescent probes), targeting markers specific to each compartment (Osterix for the osteoblasts, CD31 for the endothelial cells, Bodipy for the adipocytes). These different markers were highlighted in the 2D model, the spheroid model (3D model) as well as in the model on bTOR biomaterial in a perfused bioreactor (3D model).

Example 2;

The bone marrow reconstitutions were carried out as in example 1 but different compositions of the differentiation medium are tested.

The cells recovered after a primary culture of bone marrow in EGM2 are seeded at 20,000 cells/cm ² in EGM2 medium and left in this medium for 4 to 6 days, at 37° C. and 5% C0 ₂ . The EGM2 medium is then replaced by one of the two differentiation media (SVF or LP) described below. The differentiation medium is renewed at the rate of 2 changes per week. After 14 days in differentiation medium, the culture is stopped to carry out a gene analysis.

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

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

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

The results show that there is no significant difference between the two differentiation media tested. Each of the two media allows the in vitro differentiation of part of the MSCs into osteoblasts, of another part into adipocytes while allowing the maintenance of the CD31 positive cells corresponding to the endothelial compartment (FIG. 2). Furthermore, the inventors have observed a trend. The SVF-based medium would make it possible to differentiate more MSCs into adipocytes and the LP-based medium more MSCs into osteoblasts. These results suggest an adaptability/flexibility of the process. Indeed, several studies show that the differentiation of MSCs is modified by physical and mechanical constraints, which should be taken into consideration for 3D models in which these constraints are different from standard 2D models (variable stiffness of biomaterials, variable contraction spheroids/organoids, etc.). The production process could therefore make it possible to adapt to these constraints by adapting the differentiation medium.

On the other hand, the older the bone marrow, the more the adipose tissue increases in proportion and loses functionality with a drop in vascularization, conversely, the younger the bone marrow, the greater the bone density with a more vascularity. Thus, the modulation of the composition of the culture medium could allow the study of aging at the level of the hematopoietic niche.

In addition, the results show an increase in the expression of the main pro-haematopoietic factors in the bone marrow reconstructions generated in vitro using the method according to the invention, compared with the CTRL condition (FIG. 3). These factors are secreted or expressed in vivo by the marrow microenvironment at the 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.

Example 3;

In order to reinforce the functional study of bone marrow reconstitutions in vitro, the inventors tested the addition of hematopoietic stem cells to these bone marrow reconstitutions, in order to evaluate hematopoiesis in vitro (FIG. 4).

Formation of bone marrow reconstitutions: After selection and amplification in EGM2 medium, the mesenchymal stem cells and/or the mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells are cultured without passage, in the same culture container, for 14 days in 2D in either a medium with FCS and intralipids, or 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 FCS, the mesenchymal stem cells and/or the 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 FCS. This medium being a reference medium used in most publications for the culture of mesenchymal stem cells, it corresponds to the “MES” condition.

Coculture with hematopoietic stem cells (HSCs): HSCs are sorted from placental blood units based on the positive CD34 marker. They are then placed in coculture with the previously generated 2D bone marrow reconstructions, for 14 days in IMDM medium + 10% FCS + 1 mM hydrocortisone. At the end of the coculture, all the cells are recovered, counted, then analyzed by flow cytometry (n=3). The first results show better maintenance and proliferation of the most immature hematopoietic cells (CD34VCD38) with the bone marrow reconstitutions according to the invention than under the control conditions. Indeed, in many models, most of these cells differentiate quickly and are poorly maintained.

Claims

1) Culture medium making it possible to obtain 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 stem cells cells and/or a mixture of mesenchymal stem cells, endothelial progenitors and endothelial cells, comprising: a) fetal bovine serum (FCS) and/or platelet lysate (LP), b) 10 - 100 mM acid 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) intralipids.

2) Process 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 consisting in: 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) Culture method according to claim 2, in which 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 medium and the same container of culture, without assembly of cells a posteriori.

4) Culture method according to one of claims 2 to 3, wherein endothelial progenitors and endothelial cells are organized into vessels in the culture medium, at the same time as said mesenchymal stem cells differentiate. 5) Method of culturing according to any one of claims 2 to 4, wherein the cells are primary cells.

6) Culture method according to any one of claims 2 to 5, in which the cells are all derived from the same sample from a single subject.

7) 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 of producing a reconstitution of bone marrow comprising the method of in vitro culture 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 method of claim 8.

10) Reconstruction of bone marrow comprising osteoblasts, adipocytes and vessel-forming endothelial cells.

11) Composition comprising cells obtained by the method of any one of claims 2 to 6.

12) Reconstitution of bone marrow according to any one of claims 9 to 10 or composition according to claim 11 for its use for 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, the testing of compounds, and/or the testing of physical and mechanical conditions.

14) Use of a reconstitution of bone marrow as defined in claim 9 or claim 10, in a prosthesis or a medical device.