Methods and media for the production of haematopoietic cells

Abstract

The present invention relates to processes for the in vitro production of hematopoietic cells, comprising contacting hematopoietic progenitors with a DNTM3A inhibitor, hematopoietic cells comprising said DNTM3A inhibitor and uses thereof.

Description

[0001] METHODS AND MEDIA FOR THE PRODUCTION OF HAEMATOPOIETIC CELLS

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to the field of medicine. More specifically, it relates to new methods for the production of hematopoietic cells.

[0004] BACKGROUND OF THE INVENTION

[0005] Blood and / or platelets transfusions can be a lifesaving procedure, but may involve risks, including infectious and non-infectious complications. There is debate in the medical literature concerning the appropriate use of blood and blood products.

[0006] Blood is composed of blood cells suspended in blood plasma. Blood cells develop from hematopoietic stem cells in the bone marrow through a highly regulated process called haematopoiesis. The main blood cells are red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes).

[0007] Erythrocytes are the most abundant type of blood cells and are primarily responsible for transporting oxygen from the lungs to tissues throughout the body.

[0008] On the other hand, platelets are small fragments of megakaryocytes (MKs) that play a critical role in thrombosis, haemostasis and maintenance of vascular function. They do so by adhering to exposed extracellular matrix proteins at sites of vascular injury, where they become activated and form a haemostatic plug, preventing excessive blood loss and stimulating wound repair. Platelets also have important roles in angiogenesis and inflammation.

[0009] In blood centres, patients are able to donate either whole blood or platelets, that will be used in various clinical purposes and set ups, such as acute blood loss, anaemia, oncology, thrombocytopenia or coagulopathies.

[0010] However, many countries are facing critical shortages of blood and platelets. In January 2024, the American Red Cross announced an emergency blood shortage, with the lowest number of blood donors in 20 years. These shortages are putting significant strain on the healthcare system and potentially compromising patient care across various medical scenarios. The situation underscores the urgent need for increased blood and / or platelets supplies to meet the ongoing demand for lifesaving treatments.

[0011] The invention seeks to meet these needs. SUMMARY OF THE INVENTION

[0012] The inventors have shown that culturing hematopoietic progenitor cells in the presence of a DNMT3 A inhibitor, considerably increases the yield of production of hematopoietic cells. The inventors have also demonstrated that these hematopoietic progenitors are still capable of differentiating efficiently into platelets or erythrocytes.

[0013] The methods, uses and engineered hematopoietic cells according to the invention allow the amplification, i.e., the multiplication, of hematopoietic progenitors while maintaining their ability to further differentiate later into cells of the erythrocyte or platelet lineage, in particular into erythrocytes and platelets.

[0014] The processes of the invention not only have the advantage of being simple and economical, but also pave the way for large-scale industrial production of hematopoietic cells, especially erythrocytes and platelets. This will help to reduce the risk of blood shortages or transfusion bottlenecks, while offering optimal safety for transfused patients.

[0015] In a first aspect, the invention concerns an in vitro method for amplifying hematopoietic progenitor cells, said method comprising culturing hematopoietic progenitor cells in the presence of a DNMT3 A inhibitor, preferably in an amplification culture medium.

[0016] In a second aspect, the invention relates to an in vitro method for producing hematopoietic cells, wherein said method comprises the steps of: i. Amplifying hematopoietic progenitor cells in the presence of a DNMT3A inhibitor, preferably in an amplification culture medium; ii. Differentiating the amplified hematopoietic progenitor cells into megakaryocytes or erythroid precursors, in particular in the absence of the DNMT3 A inhibitor, preferably in a differentiation culture medium; iii. Optionally, recovering the megakaryocytes or erythroid precursors; iv. Optionally, maturing the megakaryocytes into platelets or the erythroid precursors into erythrocytes; v. Optionally, recovering the platelets or erythrocytes.

[0017] Particularly, the DNMT3 A inhibitor is a nucleic acid molecule or a protein encoded by a nucleic acid molecule, said DNMT3 A inhibitor being expressed by the hematopoietic progenitor cells.

[0018] Preferably, expression of the DNMT3 A inhibitor is placed under the control of an inducible system or promoter.

[0019] In particular, the inducible system is a doxycycline-inducible TetO / TetR system. Typically, the DNMT3A inhibitor is a nucleic acid molecule, said nucleic acid molecule being a shRNA, a siRNA or a miRNA, preferably a shRNA.

[0020] In a third aspect, the invention concerns a genetically modified hematopoietic progenitor cell comprising a DNMT3 A inhibitor, preferably as defined herein. Preferably, the DNMT3 A inhibitor is an interfering nucleic acid molecule, such as a shRNA, placed under the control of an inducible system or promoter.

[0021] In a fourth aspect, the invention relates to the use of a hematopoietic progenitor cell as disclosed herein, for the in vitro production of haematopoietic cells selected from the group consisting of unipotent erythroid progenitors such as BFU-E, CFU-E; erythroid precursors such as proerythroblasts, basophil erythroblasts, polychromatophilic erythroblasts, orthochromatic erythroblasts; reticulocytes, erythrocytes, megakaryoblasts, megakaryocytes and platelets, preferably platelets or erythrocytes.

[0022] In a fifth aspect, the invention relates to an in vitro method for producing platelets, said method comprising differentiating the hematopoietic progenitor cells as disclosed herein into megakaryocytes, preferably in a differentiation culture medium such as disclosed herein, and maturing the megakaryocytes into platelets.

[0023] Alternatively, the invention concerns an in vitro method for producing erythrocytes, said method comprising differentiating the hematopoietic progenitor cells as disclosed herein into erythroid precursors, preferably in a differentiation culture medium such as disclosed herein, and maturing the erythroid precursors into erythrocytes.

[0024] In the methods and uses disclosed herein, the hematopoietic progenitor cell is preferably a megakaryocyte-erythroid progenitor (MEP).

[0025] In a sixth aspect, the invention concerns amplification and / or differentiation media that are suitable for the culture of the hematopoietic cells.

[0026] The amplification culture medium particularly comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), insulin; heparin; transferrin; serum, plasma or serum pool; optionally a glucocorticoid hormone; an autophagy inducer; erythropoietin (EPO); and glutamine. Preferably, the glucocorticoid hormone is selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazol, preferably is dexamethasone; and / or the autophagy inducer is selected from the group consisting of SMER-28, SMER-10 and SMER 18, preferably is SMER-28. In particular, the amplification culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), glutamine at a concentration of between about 1 ng / ml and about 15 ng / ml; insulin at a concentration between about 1 pg / mL and about 50 pg / mL; heparin at a concentration between about 0.5 U / mL and about 5 U / mL; transferrin at a concentration between about 200 pg / mL and about 400 pg / mL; plasma or serum pool, at a concentration between about 1% and about 10%; optionally dexamethasone at a concentration between 0.01 mM and 10 mM; SMER- 28 at a concentration between 0,1 pM and 10 pM; and erythropoietin (EPO) at a concentration of between about 0.5 lU / mL and about 10 lU / mL.

[0027] The differentiation culture medium typically comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), insulin and / or transferrin; glutamine, preferably L-glutamine; an antagonist of the aryl hydrocarbon receptor (AHR), preferably StemRegenin 1 (SRI); an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28; an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021; a growth factor of the TGF-P family, preferably Activin A; a protein kinase inhibitor, preferably midostaurin, foetal calf serum (FBS) or platelet lysate; thrombopoietin (TPO) or eltrombopag. In particular, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), Insulin- Transferrin-Selenium-Ethanolamine (ITS-X), foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin.

[0028] For example, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X at a concentration of between about 0,5% and about 5%; optionally transferrin at a concentration of between about 100 pg / ml and about 500 pg / ml; glutamine at a concentration of between about 1 ng / ml and about 10 ng / mL; SRI at a concentration of between about 50 nM and about 150 nM; SMER-28 at a concentration between 0,1 pM and 10 pM; CHIR99021 at a concentration between about 0,1 pM and about 5 pM; Activin A at a concentration between about 200 ng / ml and about 300 ng / ml; midostaurin at a concentration between about 1 pM and about 5 pM; foetal bovine serum (FBS) or platelet lysate at a concentration between about 0,1% and about 5; and eltrombopag or thrombopoietin (TPO) at a concentration of between about 20 ng / ml and about 40 ng / ml.

[0029] Alternatively, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X; transferrin; optionally hemin, glutamine, preferably L-glutamine; foetal bovine serum or platelet lysate; an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28; an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021; a growth factor of the TGF-P family, preferably Activin A; erythropoietin; an indole alkaloid, preferably hirsutine; a JAK2 inhibitor, especially JAK2-IN-6 and / or pacritinib, an inhibitor of Bcl-XL, preferably WEHI- 539; and optionally a FLU inhibitor, preferably TK216 and / or anagrelide. Preferably, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X, transferrin, hemin, foetal bovine serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, hirsutine, CHIR99021, Activin A, and a) WEHI-53, JAK2IN6, pacritinib, and optionally TK216 and / or anagrelide or b) TK216 and anagrelide and optionally hemin. Especially the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X at a concentration of between about 0,5% and about 5%; transferrin at a concentration of between about 100 pg / ml and about 500 pg / ml; glutamine at a concentration of between about 1 ng / ml and about 10 ng / ml; SMER-28 at a concentration between 0,1 pM and 10 pM; CHIR99021 at a concentration between about 0, 1 pM and about 5 pM; Activin A at a concentration between about 200 ng / ml and about 300 ng / ml; foetal calf serum (FBS) at a concentration of between about 5% and about 25%; erythropoietin (EPO at a concentration of between about 1 U / ml and about 10 U / ml; hirsutine at a concentration of between about 20 pM and about 100 pM; JAK2-IN-6 at a concentration of between about 1 pg / mL and about 15 pg / mL; pacritinib, at a concentration of between about 1 nM and about 15 nM; WEHI-539 at a concentration of between about 0.1 nM and about 10 nM; optionally TK216, at a concentration of between about 20 ng / ml and about 40 ng / ml, and optionally anagrelide, at a concentration of between about 1 pg / ml and about 100 pg / ml. Alternatively, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X at a concentration of between about 0,5% and about 5%; transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml; glutamine at a concentration of between about 1 ng / ml and about 10 ng / ml; SMER-28, at a concentration between 0, 1 pM and 10 pM; CHIR99021 at a concentration between about 0,1 pM and about 5 pM; Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml; foetal calf serum (FBS), at a concentration of between about 5% and about 25%; optionally hemin at a concentration between about 1 pM and about 100 pM; erythropoietin (EPO) at a concentration of between about 1 U / ml and about 10 U / ml ; hirsutine, at a concentration of between about 20 pM and about 100 pM; TK216, at a concentration of between about 20 ng / ml and about 40 ng / ml, and anagrelide, at a concentration of between about 1 pg / ml and about 100 pg / ml. In a seventh aspect, the invention concerns the use of a differentiation culture medium of the invention for the differentiation of hematopoietic progenitor cells into megakaryocytes, optionally into platelets. Alternatively, the invention concerns the use of a differentiation culture medium of the invention, for the differentiation of hematopoietic progenitor cells into erythroid precursors, optionally into erythrocytes.

[0030] Finally, the invention relates to a bioreactor comprising the genetically modified hematopoietic progenitor cells of the invention. Typically, said bioreactor further comprises an amplification culture medium or a differentiation culture medium of the invention.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Definitions

[0033] In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.

[0034] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.

[0035] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

[0036] The terms “polynucleotide”, “nucleic acid molecule”, “nucleic acid” and “nucleic acid sequence” are equivalent and refer to a polymeric form of nucleotides of any length, for example RNA or DNA or analogues thereof. Nucleic acids (e.g., components, or portions, of the nucleic acids) of the present invention may be naturally occurring, modified or engineered. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. Preferably, this term refers to an isolated nucleic acid.

[0037] As used herein, the term "isolated" indicates that the recited material (i.e, is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an "isolated" nucleic acid molecule is one which has been identified and separated and / or recovered from a component of its natural environment.

[0038] As used herein, “homology”, “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 70% identity, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The term “percentage of identity” in relation to sequences designates the level of identity or homology between said sequences and may be determined by techniques known per se in the art. Typically, the percentage of identity between two nucleic acid sequences is determined by means of computer programs such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443-453). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences are those described in Current Protocols in Molecular Biology (Ausubel et al., eds.1987) Supplement 30, section 7.7.18, Table 7.7.1. or the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Sequence identity between nucleotides or amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties. These terms also include sequences that have deletions and / or additions, as well as those that have substitutions, particularly conservative substitutions.

[0039] As used herein, the term "a", "an", "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. The term “and / or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.

[0040] As used herein, the terms "including", "containing" and "comprising" are used herein in their open, non-limiting sense.

[0041] As used herein, the term “consist essentially of’ refers to those elements required for a given embodiment. This term indicates the inclusion of any recited characteristics and permits the optional presence of elements that do not materially affect nor change the characteristics or functions of said embodiment.

[0042] The term “at least one” means “one or more” or “one or several”. For instance, it refers to one, two, three or more.

[0043] The term “about” as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to + / - 10% (e.g., + / - 0.5%, + / -1 %, + / -1.5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / -9.5%). The use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).

[0044] Hematopoietic cells

[0045] The hematopoietic cells useful in the invention are particularly described here below.

[0046] As used here, the term “hematopoietic progenitors” refers to progenitor cells obtained by differentiation of hematopoietic stem cells during haematopoiesis, especially erythropoiesis or thrombopoiesis. These progenitor cells are nucleated cells that have the ability to divide and later differentiate, in particular in cells of the erythrocyte or platelet lineage. Preferably, the hematopoietic progenitors envisioned herein are CD34+ cells.

[0047] In some instances, the hematopoietic progenitors derive from CD34+ hematopoietic stem cells, that have especially been previously isolated, in particular from a blood sample, especially cord blood or by apheresis.

[0048] Preferably, the hematopoietic cells envisioned herein are isolated cells. In some embodiments, the hematopoietic cells are isolated from a patient blood sample with or without mobilisation, umbilical cord blood or placenta, from a bone marrow sample or collection or from cytapheresis. Preferably, the hematopoietic stem cells are isolated from apheresis or cord blood. In some aspects, the hematopoietic progenitors used in the methods and uses of the invention can be obtained from blood donation or transfusion.

[0049] Preferably, the hematopoietic cells envisioned herein are mammalian cells, even more preferably human cells. In particular, the hematopoietic cells envisioned herein are not murine hematopoietic cells.

[0050] Particularly, the hematopoietic progenitors envisioned herein are not hematopoietic stem cells (HSCs) or pluripotent stem cells, especially not human embryonic stem cells. Preferably, the hematopoietic progenitors envisioned herein are not induced pluripotent stem (iPS) cells. Particularly, the hematopoietic progenitors envisioned herein are not monocytes.

[0051] Preferably, the hematopoietic progenitors envisioned herein, especially the genetically modified hematopoietic progenitors are not hematopoietic stem cells, such as murine hematopoietic stem cells.

[0052] Preferably, the hematopoietic progenitors envisioned herein, especially the genetically modified hematopoietic progenitors are not multipotent. Preferably, the hematopoietic progenitors envisioned herein, especially the genetically modified hematopoietic progenitors are bipotent.

[0053] Preferably, the hematopoietic progenitor is a megakaryocyte-erythroid progenitor (MEP). As used herein, the term “megakaryocyte-erythroid progenitor” or “MEP”, refers to a type of hematopoietic stem cell-derived progenitor cells that give rise to both megakaryocytes and erythroid cells. MEPs are considered to be bipotent progenitors committed to the megakaryocytic and erythroid lineages, and they play a crucial role in the production of both platelets and red blood cells.

[0054] Native MEP are generally characterized by the expression of specific surface markers, such as CD34, CD71, and CD110. Alternatively, the MEP envisioned herein, especially the genetically modified MEP can be characterized by the expression of specific surface markers, such as CD164+, CD47+, CD71+, CD135; CD36; CD123’ and CD110+.

[0055] In some embodiments, the hematopoietic progenitor of the invention, preferably the MEP, leads to cells of the platelet lineage, especially platelets.

[0056] As used herein, the term “cells of the platelet lineage” refer to cell types involved in the development and production of platelets. In the context of the invention, it refers to cells that are committed progenitors that specifically give rise to megakaryocytes, especially downstream of MEP. This term encompasses unipotent cells of the platelet lineage, especially megakaryocyte Progenitors (MkPs), megakaryocytes and platelets.

[0057] As used herein, the term “megakaryocytes progenitor” or “MKP” refers to a type of hematopoietic stem cell-derived progenitor cells. They are characterized by the expression of specific surface markers, such as CD41, CD151, and CD 110, and are committed to the megakaryocytic lineage. MKPs give rise to mature megakaryocytes, which are responsible for the production of blood platelets.

[0058] As used herein, the term “megakaryocyte” denotes a bone marrow cell responsible for the production of blood platelets necessary for haemostasis. Megakaryocytes are the precursor of platelets. Preferably, this term encompasses megakaryoblasts, promegakaryocytes and megakaryocytes.

[0059] Megakaryocytes are known to be responsible for generating platelets through a series of cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Subsequently, they release platelets through a process involving the formation of proplatelets and preplatelets, which undergo subsequent fission (Machlus et al., J Cell Biol. 2013 Jun 10; 201(6): 785-796). The production of platelets by megakaryocytes requires an intricate series of remodelling events, and abnormalities in this process can lead to clinically significant disorders such as thrombocytopenia or thrombocythemia.

[0060] As used herein, the term “mature megakaryocytes” refers to a population of megakaryocytes which express in a stable way GPIb and allbp3 surface markers. As used herein, a “stable way expression” of a surface marker denotes that in a cellular population, at least 70% of cells express these surface markers.

[0061] Typically, mature MK cells are identified by the expression of CD41, CD42 and CD62, preferably CD41 and CD42, for example by flow cytometry. In some aspect, the genetically modified MK of the invention has a ploidy level of 2N, 4N 6N, 8N or 16N.

[0062] As used herein, the term “platelet” or “thrombocyte” refers to anucleated cytoplasmic bodies derived from megakaryocytes that are involved in the cellular mechanisms of primary haemostasis leading to the formation of blood clots.

[0063] The process of platelet production involves the conversion of preplatelets into proplatelets. As used herein, “preplatelets” refer to platelet precursors that are discoid in shape. They are characterized by a cortical microtubule band that rims the cytoplasmic surface of the membrane on the disc face. Preplatelets also contain organelles such as secretory granules, invaginated membranes, and mitochondria. As used herein, the term “proplatelets” denotes any structural form of a megakaryocyte or its fragments, such as cytoplasmically-linked platelet-like particles, that could result in platelet formation. The structural forms include, but are not limited to, cells with long cytoplasmic extensions, projections or pseudopodia that contain swellings encompassing platelet bodies in various stages of formation, such as, nodules, blebs, and the like. In particular, proplatelets are characterized by their elongated shape and are filled with microtubule bundles, which are the major structural component of proplatelets.

[0064] In some embodiments, the hematopoietic progenitor, preferably the MEP, leads to cells of the erythroid lineage, especially erythrocytes.

[0065] As used herein, the term “cells of the erythroid lineage” refer to cell types involved in the development and production of erythrocytes. In the context of the invention, it refers to cells that are committed progenitors, especially downstream of MEP. This term especially encompasses unipotent cells of the erythroid lineage, especially unipotent erythroid progenitors such as Burst Forming Unit-Erythroid (BFU-E) and Colony Forming Unit-Erythroid (CFU-E); erythroid precursors such as proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts; reticulocytes and erythrocytes.

[0066] As used herein, the term “unipotent erythroid progenitors” encompasses BFU-E (Burst Forming Unit - E) and CFU-E (Colony Forming Unit - E) cells, which are hematopoietic cells irreversibly committed to the erythrocyte lineage.

[0067] As used herein, the term “erythroid precursors” to refer to cells obtained by differentiation of unipotent erythroid progenitors and capable of producing reticulocytes. The term includes proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts and orthochromatic erythroblasts. It is possible to distinguish between these cells on a cytological basis by staining the cells with the May-Grunwald-Giemsa (MGG) stain and by analysing the staining and the nuclear / cytoplasmic ratio using techniques known to those skilled in the art.

[0068] As used herein the term “reticulocytes” refers to cells that are immature red blood cells (RBCs). Reticulocytes must go through a process of maturation to form mature RBCs.

[0069] As used here, the terms ‘red blood cell’, ‘mature red blood cell’, ‘haematocyte’, ‘erythrocyte’ and ‘mature erythrocyte’ are equivalent and may be used interchangeably. The term ‘erythrocyte’ refers to an enucleated cell displaying markers characteristic of erythrocyte maturation. In particular, erythrocytes express glycophorin A (CD235a) but do not express CD36. Their identification can be based on cytological criteria well known to those skilled in the art, such as the size of the cell and the absence of a cell nucleus. The man skilled in the art is aware of techniques to distinguish the different populations of hematopoietic cells, such as flow cytometry, gene expression profiling (i.e., determination of specific gene signature), CFC (colony-forming cell) clonogenic assays, histology techniques such as using May-Grunwald Giemsa (MGG) staining, imaging or functional assays.

[0070] In particular, the hematopoietic cells can be identified by flow cytometry, by a range of surface markers and proteins. Suitable antibodies fused to fluorochromes against hematopoietic markers are known in the art and are particularly described in the Example section.

[0071] Megakaryocyte-erythroid progenitor (MEP) can particularly be identified by commonly associated markers, such as CD164, CD38, CD71, CD110 and / or CD123

[0072] Megakaryocytes progenitors (MKP) can particularly be identified by commonly associated markers, such as CD 164, CD41 (Glycoprotein Ilb / IIIa), CD 110, CD 117, CD 150, CD151 and / or CD42.

[0073] Megakaryocytes can particularly be identified by commonly associated markers, such as Von Willebrand Factor (VWF), CD41 (Glycoprotein Ilb / IIIa), CD42 (Glycoprotein lb), CXCR4 and / or Thrombopoietin Receptor (Tpo R).

[0074] Platelets can particularly be identified by commonly associated markers, such as CD31 / PECAM-1, CD63, CD42b (GPIb alpha), CD41 (Integrin alpha 2b) and / or CD62P (P- Selectin).

[0075] BFU-E cells typically have a CD34+CD36negGPAnegCD123negCD71lowphenotype. CFU-E cells have a CD34negCD36negGPAnegCD123negCD71high.

[0076] Erythroid precursors, especially orthochromatic erythroblasts, can particularly be identified by commonly associated markers, such as CD235a, CD49d, CD36, CD71, Band3, CD34 and CD105, preferably CD235a, CD71, CD36, Band3 and CD49d.

[0077] Erythrocytes can particularly be identified by commonly associated markers, such as CD235a+, BAND3+, CD36’ and CD7E.

[0078] In some embodiments, the hematopoietic progenitors of the invention are isolated from a patient blood sample with or without mobilisation, umbilical cord blood or placenta, from a bone marrow sample or collection or from cytapheresis. In some aspects, the hematopoietic stem cells are isolated from apheresis or cord blood.

[0079] Hematopoietic cells can be isolated by any technique known by the person skilled in the art, such as Immunomagnetic Cell Separation, Density Gradient Centrifugation, Fluorescence- Activated Cell Sorting (FACS), Magnetic-Activated Cell Sorting (MACS) or Aptamer-Based Cell Isolation. Preferably, the hematopoietic progenitors envisioned herein are isolated by FACS, typically using antibodies against specific membrane antigens of hematopoietic cells of interest.

[0080] According to one embodiment, the haematopoietic cells envisioned herein are derived from a sample taken from a subject or donor or are derived from cells obtained from a subject or donor. In particular, the haematopoietic cells, especially the platelets and / or erythrocytes, may be intended for transplantation into a subject or recipient subject, in particular by transfusion.

[0081] The terms ‘individual’, ‘host’, ‘subject’ and ‘patient’ are used interchangeably herein, and refer to an animal, preferably a mammal, more preferably a human for example a child or an adult. Preferably, the donor and / or recipient are both mammals, especially both humans.

[0082] The donor and the recipient of the haematopoietic cells envisioned herein may be the same individual or different individuals. Typically, the haematopoietic progenitor cells disclosed herein are from a donor or group of donors. The recipient particularly received the erythrocytes and / or platelets obtained from the donor’s haematopoietic progenitor cells.

[0083] In some embodiments, the donor and the recipient are the same individual. In another preferred embodiment, the donor is different from the recipient. In this case, the donor is preferably a healthy donor, in particular an individual free from haematological pathology.

[0084] In some aspect, the CD34+ cells that are genetically modified to express the DNMT3A inhibitor have been isolated from a healthy subject. Alternatively, the CD34+ cells that are genetically modified to express the DNMT3 A inhibitor have been isolated from a patient that has a blood condition or disease, in particular haematological disorder or disease, such as hemoglobinopathies, RBC enzymopathies, anaemia, thalassemia or drepanocytosis.

[0085] Preferably, the hematopoietic progenitors used in the methods and uses of the invention are obtained from a healthy subject or a population of healthy subjects.

[0086] DNMT3A inhibitors

[0087] The present application relates to in vitro processes for the production of hematopoietic cells comprising contacting hematopoietic progenitors with a DNMT3 A inhibitor. The purpose of this process is to allow the amplification, i.e., the multiplication, of this population of progenitors while maintaining their ability to eventually differentiate later into megakaryocytes and / or platelets. As used herein, the term "DNA methyltransferase" refers to a family of enzymes that catalyses the transfer of a methyl group to DNA. Three active DNA methyltransferases have been identified in mammals, including DNMT1, DNMT3A and DNMT3B.

[0088] As used herein “DNMT3 A” refers to a DNA methyltransferase enzyme that plays a key role in the establishment of DNA methylation patterns during development. In human, DNMT3A is encoded by the DNMT3 A gene located on chromosome 2. Alternative names to “DNMT3 A” may be, for example, DNA (cytosine-5-)-methyltransferase 3 alpha; DNA cytosine methyltransferase 3A2; DNA methyltransferase HsalllA; OTTHUMP000002011492; M.HsalllA; DNA (cytosine- 5)-methyltransferase 3 A; DNA MTase HsalllA; Dnmt3a; DNMT3A2; EC 2.1.1.37; or OTTHUMP000002011502. DNMT3 A is described in the art as a 130 kDa protein encoded by 23 exons. Exemplary DNMT3A GenBank® sequences, incorporated by reference herein, are as follows: BC023612.2, AAH23612.1, NM_022552.3 NMJ53759.2 NMJ75629.1

[0089] NM-175630.1, NM_001130823, NM_001318730, NM_001318731, NM_001379. Sequences of DNMT3A are also disclosed under the Uniprot reference Q6PJ37 or P26358.2.

[0090] As used herein a “DNMT3 A inhibitor” is a substance that can block or reduce the activity of the DNA methyltransferase 3A (DNMT3A) enzyme. This inhibition can be achieved through various mechanisms, such as binding to the enzyme in a covalent or non-covalent manner, or through the inhibition of DNMT3A expression.

[0091] In some aspects, the DNMT3A inhibitor is able to target / inhibit both isoforms of DNMT3A, i.e., DNMT3 Al and DNMT3 A2. As described in the art, DNMT3 Al is the full-length form of the protein, while DNMT3 A2 is a shorter isoform that lacks the first six exons of the amino-terminal domain. In some aspects, the DNMT3A inhibitor is able to specifically inhibit / target DNMT3A1. In some aspects, the DNMT3 A inhibitor is able to inhibit / target DNMT3 Al but not DNMT3 A2.

[0092] DNMT3 A inhibitors may be of any kind, such as nucleic acid, protein, a chemical compound or a small molecule.

[0093] The hematopoietic progenitor cells may be contacted with the DNTM3 A inhibitor on the first day of culture or after several days of culture, for example after 5 to 15 days, preferably after 2 days of culture.

[0094] Preferably, the hematopoietic progenitor cells are in contact with the DNMT3A inhibitor during 2 to 40 days, preferably between 15 to 40 days, more preferably between 20 to 40 days, most preferably between 25 to 35 days. Preferably, the hematopoietic progenitor cells are in contact with the DNMT3A inhibitor during 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 82, 29, 30, 31, 32, 33, 34, 35 days.

[0095] The concentrations of the DNTM3 A inhibitor may be constant or vary throughout the culture or contact. The duration of contact between the DNTM3A inhibitor may be constant or vary throughout the culture.

[0096] The hematopoietic cells can be brought into contact with the DNTM3A inhibitor once or several times during culture.

[0097] In some aspects, the DNMT3 A inhibitor is a small molecule or a chemical compound.

[0098] Preferably, the DNMT3 A inhibitor is a small molecule selected from the group consisting of DY-46-2 (e.g., CAS No.: 1105110-83-5), RG108 (e.g., CAS No.: 48208-26-0), DNMT3A-IN-1 (e.g., CAS No. : 1403598-56-0), azacitidine (e.g., CAS No.: 320-67-2, vidaza), decitabine ((e.g., CAS No.: 2353-33-5, dacogen), pyrazolone and pyridazine (e.g., such as described in Sandoval et al., J. Med. Chem. 2022, 65, 15, 10554-10566 and Huang et al., Bioorg Med Chem Lett. 2021 May 15:40: 127908, incorporated herein by reference), CM-272 (e.g., CAS No.: 1846570-31-7) and CM-579 (e.g., CAS No.: 1846570-40-8), preferably from the group consisting of DNMT3A- IN-1 (e.g., CAS No. : 1403598-56-0), azacitidine (e.g., CAS No.: 320-67-2, vidaza), decitabine ((e.g., CAS No.: 2353-33-5, dacogen), pyrazolone and pyridazine (e.g., such as described in Sandoval et al., J. Med. Chem. 2022, 65, 15, 10554-10566 and Huang et al., Bioorg Med Chem Lett. 2021 May 15:40: 127908, incorporated herein by reference), CM-272 (e.g., CAS No.: 1846570-31-7) and CM-579 (e.g., CAS No.: 1846570-40-8).

[0099] In some embodiments, the DNMT3 A inhibitor is not RG108.

[0100] In some aspects, the DNMT3 A inhibitor is a protein or a peptide, for example such as MeCP2 (methyl-CpG-binding protein 2).

[0101] Typically, when the DNMT3 A inhibitor is a small molecule, a chemical compound or a protein or peptide, such inhibitor may be added to the culture medium, such as the amplification medium described herein. Preferably, contact between the hematopoietic progenitor cells and the DNMT3A inhibitor may be achieved by adding the DNTM3A inhibitor to the hematopoietic progenitor cells culture medium, by placing the hematopoietic progenitor cells in a culture medium containing the DNTM3 A inhibitor. In some instances, wherein the DNMT3A inhibitor is a protein or peptide, such DNMT3A inhibitor can be encoded by a nucleic acid molecule to be expressed in the hematopoietic progenitor, preferably under conditions allowing expression of the DNMT3 A inhibitor.

[0102] In some aspects, the DNMT3 A inhibitor is a nucleic acid. Typically, the DNMT3 A inhibitor is an interfering nucleic acid molecule.

[0103] As used herein, the term “interfering nucleic acid molecule” refers to a DNA or RNA molecule designed to reduce, block, or modify the expression of a gene, such as DNMT3 A gene. Interfering nucleic acid molecules comprises a nucleic acid sequence complementary to the targeted gene, such as DNMT3 A gene.

[0104] In particular, when the DNMT3 A inhibitor is a nucleic acid molecule, such molecule targets 1, 2 or 3 exons of DNMT3A. Sequences of DNTM3A exons are described in the art. Particularly, the DNMT3A inhibitor targets exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22 or exon 23 of DNMT3A. Preferably, the DNMT3A inhibitor targets and / or inhibits exon 1, exon 2, exon 3, exon 4, exon 5 or exon 6 of DNMT3A. Even more preferably, the DNMT3 A inhibitor targets and / or inhibits DNMT3 A exon 2.

[0105] Exemplary nucleic acids include miRNAs and those for RNAi, including siRNAs, shRNAs and antisense RNAs.

[0106] In some embodiments of the invention, DNMT3A is inhibited with the employment of microRNAs (miRNAs). Exemplary microRNAs that can be used as DNTM3 A inhibitor herein can be selected from the group consisting of : hsa-miR-30c, hsa-miR-429, hsa-miR-29c, hsa-miR-29a, hsa-miR-30d, hsa-miR-218, hsa-miR-410, hsa-miR-132, hsa-miR-30a, hsa-miR-144, hsa-miR- 383, hsa-miR-212, hsa-miR-96, hsa-miR-370, hsa-miR- 200c, hsa-miR-182, hsa-miR-143, hsa- miR-101, hsa-miR-30b, hsa-miR- 194, hsa-miR-29b, and hsa-miR-30e (SABiosciences) and any combination thereof.

[0107] In some aspects, the DNTM3 A inhibitor is a small interfering RNA (siRNA) or an antisense polynucleotide. siRNAs are double-stranded RNA molecules that are non-coding and typically 15- 25 base pairs in length. siRNAs targeting DNMT3A are for example siRNA sc-37757.

[0108] In some aspects, the DNTM3 A inhibitor is a short hairpin RNA (shRNA). shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain aspects, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleave the shRNA to form siRNA. shRNAs targeting DNMT3 A are for example shRNA sc-37757-SH (Santa Cruz Biotechnology) or Origene Technologies 1SET DNMT3A Human shRNA Plasmid Kit (Locus ID 1788) (Origene), Human DNMT3A shRNA Plasmid (abbexa), pAPM-D4 miR30-DNMT3a tsl (Plasmid #115865) (Addgene).

[0109] Preferably, the DNTM3 A inhibitor is a shRNA having a nucleic acid sequence as set forth in SEQ ID NO: 1. Alternatively, the DNTM3A inhibitor is a shRNA having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity with SEQ ID NO: 1. Particularly, the DNTM3A inhibitor is a shRNA having a nucleic acid sequence having 1, 2 or 3 nucleic acid mutation(s) in SEQ ID NO: 1, said mutation being preferably selected from additions, deletions and / or substitutions.

[0110] The nucleic acid sequence of a DNMT3A inhibitor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof.

[0111] The nucleic acid sequence encoding for the DNMT3 A inhibitor can typically be comprised in an expression cassette and / or expression vector.

[0112] Therefore, in some embodiments of the invention, aspects include use of expression vectors to generate inhibition of DNMT3A in hematopoietic progenitor cells. Such vectors may particularly encode for any of the proteins, peptides and nucleic acid molecules described herein.

[0113] Particularly, the expression vector is used to transform a host cell, preferably a hematopoietic progenitor cell, and allow the expression of the nucleic acid of interest in said cell. Preferably, the hematopoietic progenitor cell comprises only one vector encoding for the DNMT3 A inhibitor.

[0114] As used herein, the terms “nucleic acid construct” and "vector" are equivalent and refer to a nucleic acid molecule that serves to transfer a passenger nucleic acid sequence, such as DNA or RNA, into a host cell. A vector may comprise an origin of replication, a selectable marker, and optionally a suitable site for the insertion of a nucleic acid sequence or gene. A vector can be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome. It can also comprise expression elements including, for example, a promoter, the correct translation initiation sequence such as a ribosomal binding site and a start codon, a termination codon, and a transcription termination sequence. A nucleic acid construct may also comprise other regulatory regions such as enhancers, silencers and boundary elements / insulators to direct the level of transcription of a given gene. Vectors capable of directing the expression of genes and / or nucleic acid sequences to which they are operatively linked can also be referred to herein as “expression vectors”. The vectors envisioned herein are particularly suitable for mammalian cell transformation, especially human cell transformation. The vector may be circular or linear, single- or doublestranded. The expression vector may include one or more nucleic acids or expression cassettes as described herein.

[0115] The vector is advantageously selected from plasmids, phages, phagemids, viruses, cosmids, artificial chromosomes. The vectors may be prepared from commercially available vectors or produced for example from baculoviruses, retroviruses, adenoviruses or lentiviruses according to techniques known in the art.

[0116] In some embodiments, the vector is a plasmid. Examples of suitable plasmid for expression in mammalian cells, in particular hematopoietic progenitor cells are pLKO-based vector, pLVTHM, pSico and pLL3.7.

[0117] In some embodiments, the vector is a viral vector. In particular, the vector is selected from the group consisting of an adenovirus (AV), an adeno-associated virus (AAV), a herpes simplex virus (HSV) and a lentiviral vector (LV). Such vectors typically comprise promoter(s) that is / are operative in a eukaryotic cell, such as hematopoietic progenitor cells. Preferably, the vector encoding for the DNMT3 A inhibitor is a lentiviral vector (LV).

[0118] Advantageously, the expression vector includes regulatory elements allowing the expression of the nucleic acid of interest. These elements may include, for example, origin of replication (ORI), transcription promoters, transcription activators, terminator sequences, start and stop codons. The expression vector may additionally comprise a reporter gene or a selection marker that permit easy selection of host cells comprising the vector, such as gene(s) which confer resistance to the selective antibiotics G418 / geneticin, blasticidin, hygromycin B, puromycin and / or zeocin.

[0119] Methods for selecting these elements are well known to the skilled person.

[0120] The vectors envisioned herein typically comprise an expression cassette that comprises a nucleic acid molecule encoding for a DNTM3A inhibitor. “An expression cassette” particularly comprises DNA sequences consisting of a gene and regulatory sequences, such as a promoter, the gene of interest (open reading frame, ORF), and a terminator.

[0121] In particular, the DNMT3A inhibitor nucleic acid sequence or the expression cassette comprising such is operably linked to the sequences necessary for its / their expression. The expression “operably linked” indicates that the elements are combined so that the expression of the coding sequence is under the control of a transcriptional promoter. Typically, the promoter’s sequence is placed upstream (5') of the one or more genes of interest. Spacer sequences may be present between the regulatory elements and the gene, provided they do not prevent expression by translation of the encoded protein. The expression cassette may also include at least one “enhancer” activating sequence operably linked to the promoter. In particular, the DNMT3A inhibitor is under the control of a promoter allowing its expression in a hematopoietic progenitor cell such as described herein.

[0122] As used herein, a "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription of a nucleic acid sequence. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and / or expression of that sequence.

[0123] A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30 110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control of a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA. The promoters may or may not be associated with enhancers, wherein the enhancers may be naturally associated with the particular promoter or associated with a different promoter.

[0124] The man skilled in the art is aware to choose suitable promoter depending on the type of nucleic acid to be expressed. Typically, known promoters for expressing shRNAs or siRNAs are type 3 RNA polymerase III promoter, such as the Hl promoter and the U6 small nuclear RNA (snRNA) promoter. Alternatively, known promoters for expressing miRNAs are typically RNA polymerases II promoters such as the CMV, SV40, or CMV enhancer / p-actin (CA) promoter.

[0125] In some aspects, the DNMT3A inhibitor can be placed under the control of constitutive or inducible promoters. Examples of constitutive promoters suitable to be used in the present invention, in particular for the expression of the DNMT3A inhibitor, are known in the art such as Human elongation factor- 1 alpha (EF-1 alpha), Adenovirus major late promoter, Human cytomegalovirus immediate early promoter (hCMV-IE), Simian virus 40 (SV40) promoter, Rous sarcoma virus long terminal repeat (RSV-LTR), Epstein-Barr virus immediate early promoter, Translation elongation factor la (EF-1 a) promoter, an actin promoter, a PGK promoter and Human ubiquitin C promoter.1

[0126] Preferably, the expression of the DNMT3 A inhibitor is placed under the control of an inducible promoter.

[0127] Examples of inducible systems and promoters suitable to be used in the present invention, in particular for the expression of the DNMT3A inhibitor, are known in the art such as an metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, an oestradiol promoter (Carlie & Amon, 2008, Cell, 133, 280-91), a methionine promoter (Care et al, 1999, Mol. Microbiology, 34, 792-798), a doxycycline-inducible TetO / TetR system, steroids inducible promoters and antibiotics inducible promoter.

[0128] Preferably, the nucleic acids encoding the DNMT3 A inhibitor are placed under the control of a tetracycline operator, also known as the doxycycline-inducible TetO / TetR system. The expression of the DNMT3A inhibitor may thus be regulated by the presence or absence of tetracycline or one of its derivatives such as anhydrotetracycline (ATc) doxycycline. The Tet system comprises two complementary circuits: the tTA dependent circuit (Tet-OFF system) and the rtTA dependent circuit (Tet-ON system). Such system is for example described in Smith et al. (Smith et al. 2016, Genome BioL, 17, 45). For example, the tetracycline repressor (TetR) and the DNMT3A inhibitor may be constitutively expressed whereas gRNA expression may be induced by addition of tetracycline, anhydrotetracycline (ATc) or doxycycline. Alternatively, the expression of the DNMT3 A inhibitor may be placed under the control of the tetracycline operator.

[0129] Particularly, the Tet-On system that can be used is based on the use of the tetracycline transactivator protein (tTA), which is created by fusing the tetracycline repressor (TetR) protein present in Escherichia coli bacteria with the activating domain of the VP 16 protein present in herpes virus. The rtTA protein is able to bind to DNA on a specific TetO operating sequence only if it is bound to a tetracycline. Several repeating TetO sequences may be placed under the control of a promoter such as the EFl alpha long promoter. TetO sequences coupled to the promoter are called a tetracycline response element (TRE) and respond to tetracycline transactivator protein (tTA) binding by causing an increase in the expression of the nucleic acid sequence under the promoter’s control. In some particular aspects, the vector of the invention comprises a long EFl alpha promoter and a Hl promoter that are regulated by a third-generation TET-ON system present in the vector and consisting of a tTR-KRAB unit, a fusion between the bacterial tetracycline transactivation repressor (tTR) protein and a DNA-binding domain present in potent gene expression transactivator proteins.

[0130] In some particular aspects, the vector of the invention is a lentiviral vector comprising a tetracycline operator as described above. Preferably, said vector allows the production of two copies of the DNMT3 A inhibitor.

[0131] In some particular aspects, the expression cassette or vector of the invention comprises a nucleic acid molecule which is a shRNA, a siRNA, a miRNA or an antisense RNA targeting DNMT3 A, said nucleic acid molecule being under the control of an inducible promoter, preferably a doxycycline-inducible TetO / TetR system.

[0132] Preferably, the expression cassette or vector of the invention comprises a nucleic acid molecule which is a shRNA targeting DNMT3A, preferably of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, under the control of an inducible promoter, preferably a doxycycline-inducible TetO / TetR system.

[0133] In some particular aspects, the vector of the invention is a lentiviral vector comprising a tetracycline operator as described above and two copies of a shRNA targeting DNMT3A, preferably of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto.

[0134] In some embodiments, expression vector, expression cassette or nucleic acid can typically be used to transform hematopoietic progenitor cell, in a stable or transient manner. Typically, the vector is a vector for stable or transient expression of a gene or sequence.

[0135] Therefore, the present invention also relates to a haematopoietic host cell, preferably a haematopoietic progenitor of the invention, comprising a DNTM3A inhibitor such as disclosed herein.

[0136] As used herein, the term "host cell" refers to a eukaryotic cell, preferably a human host cell and it includes any transformable organism that is capable of replicating a vector and / or expressing a heterologous nucleic acid molecule encoded by a vector. The host cell envisioned herein can be any hematopoietic cell described herein, preferably a hematopoietic progenitor such as described herein. A host cell may be "transfected" or "transformed" which refers to a process by which recombinant, exogenous or heterologous nucleic acid molecule(s) is / are transferred or introduced into the host cell.

[0137] Any transfection methods known in the art can be used to transfect the hematopoietic progenitors envisioned herein, such as virus mediated transfection, electroporation, biolistic, sonoporation or chemical transfection (e.g., using calcium phosphate or cationic lipid). Preferably, the hematopoietic progenitors envisioned herein are transfected with a lentiviral vector.

[0138] In some aspects, recombinant lentiviral vector and / or lentiviruses encoding for the DNMT3 A inhibitor are obtained by using transient transfection of human embryonic kidney (HEK) 293 T cells.

[0139] In some aspects, CD34+ hematopoietic cells or hematopoietic progenitors are transfected in a primary medium. Typically, the primary medium comprises component(s) that enable(s) the CD34+ hematopoietic cells or hematopoietic progenitors to be committed to the myeloid lineage.

[0140] Typically, the primary medium comprises or consists of a base culture medium such as IMDM or an equivalent medium, glutamine, plasma or pool serum, insulin, heparin, dexamethasone, SMER28, transferrin, EPO, stem cell factor (SCF) and IL-3. This medium can be supplemented with rapamycin and / or polybrene.

[0141] Preferably, the amplification medium of the invention comprises or consists of a base medium, preferably an IMDM medium or an equivalent medium, supplemented with:

[0142] - insulin, preferably human insulin, at a concentration between about 1 pg / mL and about 50 pg / mL, preferably between about 5 pg / mL and about 20 pg / mL, more preferably at a concentration of about 10 pg / mL;

[0143] - heparin, preferably human heparin, at a concentration between about 0.5 U / mL and about 5 U / mL, preferably between about 1 U / mL and about 3 U / mL, more preferably at a concentration of about 2 U / mL;

[0144] - transferrin, preferably human transferrin, at a concentration between about 200 pg / mL and about 400 pg / mL, preferably between about 300 pg / mL and about 350 pg / mL, more preferably at a concentration of about 330 pg / mL;

[0145] - serum, plasma or serum pool, preferably human plasma, at a concentration between about 1% and about 10%, preferably between about 3% and about 7%, more preferably at a concentration of about 5%; - a glucocorticoid hormone, preferably selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazol, more preferably dexamethasone, at a concentration between 0.01 mM and 10 mM, preferably between 0.01 mM and 5 mM, more preferably between 0.05 mM and 1 mM, and particularly preferably of about 0.1 mM;

[0146] - an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0147] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 0.5 lU / mL and about 10 lU / mL, preferably between about 1 lU / mL and about 5 lU / mL, even more preferably at a concentration of about 3 lU / mL;

[0148] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 15 ng / ml, preferably between about 1 ng / mL and about 10 ng / mL, more preferably between about 3 ng / mL and about 7 ng / mL, most preferably about 5 ng / mL,

[0149] - stem cell factor (SCF) at a concentration of between about 10 ng / mL and about 500 ng / mL, preferably between about 25 ng / mL and about 250 ng / mL, more preferably between about 50 ng / mL and about 150 ng / mL, most preferably about 100 ng / mL; and

[0150] - interleukin 3 (IL-3) at a concentration of between about 1 ng / mL and about 15 ng / mL, preferably between about 1 ng / mL and about 10 ng / mL, more preferably between about 3 ng / mL and about 7 ng / mL, most preferably about 5 ng / mL.

[0151] In some aspect, this medium further comprises:

[0152] - rapamycin at a concentration between about 0,1 pg / mL and about 100 pg / mL, preferably between about 1 pg / mL and about 50 pg / mL, more preferably between about 5 pg / mL and about 15 pg / mL, most preferably at a concentration of about 10 pg / mL; and

[0153] - polybrene at a concentration between about 0,1 pg / mL and about 50 pg / mL, preferably between about 0, 1 pg / mL and about 20 pg / mL, more preferably between about 2 pg / mL and about 6 pg / mL, most preferably at a concentration of about 4 pg / mL;

[0154] In some very specific aspects, the primary medium comprises or consists of a base culture medium such as IMDM or an equivalent medium, 5ng / mL glutamine, 5% (v / v) human S / D AB plasma (EFS), 10 pg / mL insulin, 2 U / mL heparin (Panpharma), 0.1 mM dexamethasone, 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma), 3 U / mL EPO (Binocrit), 100 ng / mL stem cell factor (SCF) and 5. ng ml1IL-3 (all from Miltenyi Biotec). This medium can be supplemented with 10 pg / mL rapamycin and 4 pg / mL polybrene.

[0155] In some aspects, the hematopoietic cells are cultured between 1 and 10 days, preferably between 5 and 10 days, more preferably about 7 days in the primary medium.

[0156] Hematopoietic host cells containing the transformed nucleic acid molecule(s), vector(s) or expression cassette(s) encoding the DNMT3A inhibitor disclosed herein may be referred to as “transgenic” cells.

[0157] “Genetically altered cells”, “transgenic cells”, "engineered cells", "recombinant cells" or “genetically modified cells” refer to a cell into which an exogenous nucleic acid molecule such as, for example, a DNMT3A inhibitor or a vector encoding such, has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced nucleic acid molecule, especially encoding for a DNMT3A inhibitor. These terms particularly denote cells which have been modified by the introduction of recombinant or heterologous nucleic acids (e.g., one or more DNA constructs or their RNA counterparts, preferably encoding for a DNMT3 A inhibitor) and further includes the progeny of such cells which retain part or all of such genetic modification. A transformed host cell includes the primary subject cell and its progeny.

[0158] Particularly, the genetically modified hematopoietic progenitor cells envisioned herein have been transduced with or comprise any of the nucleic acid molecule(s), expression vector(s) or cassette(s), according to the invention. Particularly, the hematopoietic progenitor cells are transduced once or twice with the nucleic acid molecule(s), expression vector(s) or cassette(s), comprising the DNTM3 A inhibitor.

[0159] Particularly, the genetically modified hematopoietic progenitor cell is obtained by transduction of CD34+ hematopoietic cells with a nucleic acid molecule, expression cassette or vector encoding for the DNMT3A inhibitor. Preferably, the obtained genetically modified hematopoietic progenitor cell is a megakaryocyte-erythroid progenitor (MEP).

[0160] In some aspects, the invention concerns a genetically modified hematopoietic cell comprising a DNMT3A inhibitor, preferably a hematopoietic progenitor comprising a DNMT3A inhibitor. Preferably, the genetically modified hematopoietic progenitor cell is able to express the DNMT3 A inhibitor disclosed herein. Even more preferably, the genetically modified hematopoietic cell is able to transiently express the DNMT3 A inhibitor disclosed herein.

[0161] In some aspects, the invention concerns a genetically modified progenitor cell that has been modified to express a DNMT3 A inhibitor in a transitory or reversible manner. In some embodiments, the expression of the DNMT3A inhibitor, even transiently, may lead to a DNMT3A gene knock down or knock-out, especially in the hematopoietic progenitor cells envisioned herein.

[0162] Typically, the DNMT3 A is not DNMT3 A knock-out or knock-in.

[0163] In some embodiments, the DNMT3A is not obtained by inducing a mutation in DNMT3A protein, such as R878C mutation. In some embodiments, the DNTM3A inhibitor is not RG108.

[0164] In some instances, the inhibition of DNMT3 A in genetically modified hematopoietic cell leads to the immortalization of said hematopoietic progenitor cells (e.g., allowing them to eventually proliferate indefinitely). Therefore, the genetically modified hematopoietic cells described herein are typically immortal or immortalized genetically modified hematopoietic cells. Preferably, the genetically modified hematopoietic cells described herein are typically immortal or immortalized MEPs.

[0165] The immortal or immortalized hematopoietic cells are typically used to establish or obtain an immortal or immortalized hematopoietic cell line. As used herein, the term "immortal" or "immortalized" means that, based upon current observations, these cells, under the culture conditions described herein, have shown no tendency to undergo terminal differentiation or cell senescence, but rather retain the capacity to divide indefinitely.

[0166] In particular the immortal hematopoietic cells are stable / alive / able to proliferate after at least 20, 25, 30, 35, 40, 45, 50 rounds of proliferation, preferably 50 rounds of proliferation.

[0167] In particular the immortal hematopoietic cells are stable / alive after at least 100, 150 or 180 days, especially in the amplification medium.

[0168] In some aspects, the genetically modified hematopoietic cells of the invention expressed specific antigens on their membranes such as CD 164, NOTCH, CD71, CD47, CD110. Therefore, such genetically modified cells can be specifically characterized or isolated, for example using flow cytometry techniques.

[0169] In some embodiments, the genetically modified hematopoietic cells have a doubling time of between 20 and 30 hours, preferably between 25 and 28 hours, particularly about 26.5 hours.

[0170] In some embodiments, the invention concerns a genetically modified hematopoietic progenitor cell, preferably a MEP, that comprises a nucleic acid molecule encoding for a DNMT3 A inhibitor such as disclosed herein, wherein the expression of said DNMT3A inhibitor is preferably placed under the control of an inducible promoter or system, preferably a doxycycline-inducible TetO / TetR system. In some embodiments, the invention concerns a genetically modified hematopoietic progenitor cell that comprises an expression cassette or vector comprising a nucleic acid molecule encoding for a DNMT3A inhibitor such as disclosed herein, wherein the expression of said DNMT3A inhibitor is preferably placed under the control of an inducible promoter or system, preferably a doxycycline-inducible TetO / TetR system.

[0171] In some embodiments, the invention concerns a genetically modified hematopoietic progenitor cell, preferably a MEP, that comprises a vector comprising a nucleic acid molecule encoding for a shRNA targeting DNMT3A, preferably a shRNA of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, placed under the control of an inducible promoter or system, preferably a doxycycline-inducible TetO / TetR system.

[0172] Preferably, the genetically modified hematopoietic cell comprises only one copy of a nucleic acid molecule, expression cassette or vector encoding for a DNMT3 A inhibitor.

[0173] Preferably, the genetically modified hematopoietic cell comprises only one copy of a vector comprising a nucleic acid molecule encoding for a shRNA targeting DNMT3A, preferably a shRNA of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, placed under the control of an inducible promoter or system, preferably a doxycycline-inducible TetO / TetR system.

[0174] Preferably, the genetically modified hematopoietic cell comprises only one vector comprising two nucleic acid molecules encoding for a shRNA targeting DNMT3A, preferably a shRNA of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, placed under the control of an inducible promoter or system, preferably a doxycycline-inducible TetO / TetR system.

[0175] Alternatively, the genetically modified hematopoietic cell comprises a vector (preferably in a single copy) comprising an expression cassette allowing the expression of two copies of a nucleic acid molecule encoding for a shRNA targeting DNMT3 A, preferably a shRNA of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, placed under the control of an inducible promoter or system, preferably a doxycycline-inducible TetO / TetR system. Such system is particularly described in Szulc et al., Nature Methods, vol. 3 No.2 2006, the content of which is incorporated herein by reference.

[0176] Any of the hematopoietic cells, especially genetically modified hematopoietic cells and / or DNMT3 A inhibitor disclosed herein can be used in the methods and uses of the invention.

[0177] Methods and uses The invention also concerns methods and uses, especially for the production of hematopoietic cells such as hematopoietic progenitors, erythrocytes and platelets. Preferably, the methods envisioned herein are in vitro or ex vivo.

[0178] In some aspects, the process according to the invention is a process for the production and / or amplification of hematopoietic progenitors in the presence of a DNMT3A inhibitor such as described herein.

[0179] The hematopoietic cells described herein can typically be comprised or organized in a two- dimensional (2D) or three-dimensional (3D) cell culture. The present invention also relates to a 2D or 3D cell culture comprising or consisting of genetically modified hematopoietic cells as described herein.

[0180] For instance, 2D cell culture can be a monolayer culture of the genetically modified hematopoietic cells according to the invention, for example on a flat surface like plastic or glass (e.g., culture flask or petri dish) or on a particular scaffold.

[0181] Alternatively, the cell culture can be a 3D culture. 3D cell cultures typically include suspension cultures on non-adherent plates, cultures in concentrated medium, in gel-like substances and cultures on scaffold. Typically, the 3D culture is a suspension culture, preferably under gentle agitation, especially an agitation under 100 rpm, preferably between 30 and 80 rpm.

[0182] Any of the uses and methods disclosed herein may comprise an initial step of preparing the hematopoietic progenitor cells of the invention, in particular the preparation of genetically modified hematopoietic progenitor cells as envisioned herein. This initial step typically includes the culture of the hematopoietic progenitor cells of the invention in a primary medium such as described herein.

[0183] Typically, the process according to the invention comprises a step of amplifying hematopoietic progenitor cells in a culture medium, preferably in an amplification medium, and in the presence of a DNMT3 A inhibitor.

[0184] Preferably, the inhibition of DNMT3 A leads to an increase of amplification capacity of the hematopoietic progenitor cells by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%, in particular in comparison with hematopoietic progenitor cells that are not / have not been in contact with a DNMT3 A inhibitor.

[0185] The term “cell amplification”, “cell proliferation”, “cell multiplication”, “cell increase” or “cell expansion” are used interchangeably herein and refer to a process whereby the number of cells increases. It typically involves the process by which a cell grows and divides to produce two daughter cells, leading to an exponential increase in cell number.

[0186] The step of amplification of hematopoietic progenitors is typically performed in an amplification culture medium. The amplification culture medium can be any suitable medium for hematopoietic cell amplification. Preferably, the amplification medium is such as described in WO20 19 / 002625. Even more preferably, the amplification medium is as described hereafter under the section “Amplification and Differentiation Media”.

[0187] Preferably, the hematopoietic progenitor cells are cultured in a culture medium at a concentration between 0.05 et 0.1 x 106cellules / ml, preferably between 500 and 2000 cells / mL, and more preferably around 1000 cells / ml.

[0188] The culture medium is preferably changed twice a week, preferably about every 3 or 4 days, especially so that the cells do not exceed a concentration of about 106cells / ml.

[0189] In some embodiments, the hematopoietic progenitor cells are cultured in the amplification culture medium for at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks or at least 25 weeks.

[0190] Preferably, the hematopoietic progenitor cells are cultured in the amplification culture medium during 2 to 40 days, preferably between 15 to 40 days, more preferably between 20 to 40 days, most preferably between 25 to 35 days. Preferably, the hematopoietic progenitor cells are cultured in the amplification culture medium during at least 20, 21, 22, 23, 24, 25, 26, 27, 82, 29, 30, 31, 32, 33, 34 or 35 days.

[0191] In some instances, the hematopoietic progenitor cells are cultured in the amplification culture medium in the presence the inducer of the inducible system (e.g., doxycycline) during 2 to 40 days, preferably between 10 and 40 days, more preferably between 20 and 40 days, even more preferably between 20 and 30 days, most preferably between 20 and 25 days. Preferably, the hematopoietic progenitor cells are cultured in the amplification culture medium in the presence the inducer of the inducible system (e.g., doxycycline) during at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

[0192] 22, 23, 24, 25, 26, 27, 82, 29, 30, 31, 32, 33, 34 or 35 days. Preferably, the hematopoietic progenitor cells are cultured in the amplification culture medium in the presence the inducer of the inducible system (e.g., doxycycline) during no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

[0193] 23, 24, 25, 26, 27, 82, 29, 30, 31, 32, 33, 34 or 35 days. In some instances, the hematopoietic progenitor cells are cultured in the amplification culture medium in the presence the inducer of the inducible system (e.g., doxycycline) during at least or about three weeks.

[0194] Preferably, the amplification of hematopoietic progenitor cells is not performed in cocultivation with mesenchymal stem cells (MSCs).

[0195] In some embodiments, the invention also concerns an in vitro method for amplifying hematopoietic progenitor cells, said method comprising culturing genetically hematopoietic progenitor cells under conditions allowing the expression of a DNMT3A inhibitor, typically in a primary medium and / or an amplification medium such as described herein.

[0196] The “conditions allowing the expression” of a DNMT3 A inhibitor are conditions wherein the DNMT3 A inhibitor is expressed by a cell, preferably by the hematopoietic progenitors.

[0197] For example, in case where the DNMT3A inhibitor is under the control of an inducible promoter, the condition allowing the expression of the DNMT3A inhibitor is met when such promoter allows the expression of the DNMT3A inhibitor by the cell. For example, when the inducible system is Tet-On / Tet-off, the induction of the expression of the DNMT3A inhibitor is performed in the presence of doxycycline, typically added to the culture medium, preferably in an amplification culture medium such as described herein.

[0198] Such embodiments typically involve genetically modified hematopoietic progenitor cells as described above.

[0199] The invention therefore also concerns a method for amplifying / producing hematopoietic progenitors, wherein said method comprises culturing the hematopoietic progenitors, preferably in a primary medium and / or an amplification medium under conditions allowing the expression of a DNMT3 A inhibitor, preferably a shRNA as described herein.

[0200] The invention also concerns an in vitro method for producing immortalized hematopoietic progenitor cells, said method comprising producing genetically modified hematopoietic progenitor cells which are able to express a DNMT3 A inhibitor under the control of an inducible system or promoter.

[0201] In some aspects, the invention concerns an in vitro method for producing genetically modified hematopoietic progenitors, wherein said method comprises or consists of i. culturing CD34+ hematopoietic cells in a primary medium, preferably between 2 and 7 days, ii. introducing a nucleic acid molecule encoding a DNMT3 A inhibitor in the CD34+ hematopoietic cells, preferably a shRNA targeting DNMT3A as described herein, to obtain genetically modified hematopoietic progenitors; iii. inducing the expression of the DNMT3 A inhibitor, preferably the shRNA targeting DNMT3 A as described herein, preferably in a primary medium or amplification medium such as disclosed herein, wherein the expression of the DNMT3 A inhibitor is typically induced between 2 and 7 days; iv. culturing or amplifying the genetically modified hematopoietic progenitors, preferably in an amplification medium, typically between 20 and 25 days; v. optionally, recovering the amplified genetically modified hematopoietic progenitors. Optionally, said method comprises a first step or providing CD34+ hematopoietic cells, for example CD34+ cells that have been isolated or sorted from a blood sample by any techniques known to the person skilled in the art, such as flow cytometry.

[0202] In some aspects, the invention concerns an in vitro method for producing hematopoietic cells, wherein said method comprises or consists of: i) Amplifying genetically modified hematopoietic progenitors of the invention, preferably in an amplification culture medium; ii) Differentiating the amplified genetically modified hematopoietic progenitor cells into megakaryocytes or erythroid precursors, in particular in the absence of a DNMT3 A inhibitor or absence of induction of the expression of the DNMT3 A inhibitor, preferably in a differentiation culture medium, typically between 2 days and 7 days; iii) Optionally, recovering the megakaryocytes or erythroid precursors; iv) Optionally, maturing the megakaryocytes into platelets or the erythroid precursors into erythrocytes; v) Optionally, recovering the platelets or erythrocytes.

[0203] Preferably, the genetically modified hematopoietic progenitors are obtained from CD34+ hematopoietic cells, more preferably by a method such as described above.

[0204] In some aspects, the invention concerns an in vitro method for producing hematopoietic cells, wherein said method comprises or consists of: i) Differentiating genetically modified hematopoietic progenitors of the invention into megakaryocytes or erythroid precursors, in particular in the absence of a DNMT3 A inhibitor or absence of induction of the expression of the DNMT3 A inhibitor, preferably in a differentiation culture medium, typically between 2 days and 7 days; ii) Optionally, recovering the megakaryocytes or erythroid precursors; iii) Optionally, maturing the megakaryocytes into platelets or the erythroid precursors into erythrocytes; iv) Optionally, recovering the platelets or erythrocytes.

[0205] Preferably, the genetically modified hematopoietic progenitors are obtained from CD34+ hematopoietic cells, more preferably by a method such as described above. Typically, said genetically modified hematopoietic progenitors have been previously amplified in a suitable amplification medium.

[0206] In some aspects, the method comprises:

[0207] - culturing the genetically modified hematopoietic progenitors, preferably in a primary medium and / or amplification medium, typically between 2 and 7 days, preferably about 2 days;

[0208] - inducing the expression of the DNMT3A inhibitor, preferably the shRNA targeting DNMT3A as described herein, preferably in a primary medium and / or amplification medium, wherein the expression of the DNMT3A inhibitor is typically induced between 20 and 35 days;

[0209] - optionally, recovering the amplified hematopoietic progenitors.

[0210] Preferably, the method comprises:

[0211] - optionally, culturing the genetically modified hematopoietic progenitors, preferably in a primary medium, typically between 2 and 7 days;

[0212] - culturing the genetically modified hematopoietic progenitors, preferably in an amplification medium, typically between 2 and 7 days;

[0213] - inducing the expression of the DNMT3A inhibitor, preferably the shRNA targeting DNMT3 A as described herein preferably in the amplification medium, wherein the expression of the DNMT3A inhibitor is typically induced between 20 and 35 days; and

[0214] - optionally, recovering the amplified hematopoietic progenitors.

[0215] Alternatively, said method comprises:

[0216] - culturing the genetically modified hematopoietic progenitors, preferably in a primary medium, typically between 2 and 7 days;

[0217] - inducing the expression of the DNMT3A inhibitor, preferably the shRNA targeting DNMT3A as described herein preferably in an amplification medium, wherein the expression of the DNMT3A inhibitor is typically induced between 20 and 35 days; and

[0218] - optionally, recovering the amplified hematopoietic progenitors. Preferably, the hematopoietic progenitor cells are cultured in the amplification culture medium at a concentration between 0.05 et 0.1 x 106cellules / ml, preferably between 500 and 2000 cells / mL, and more preferably around 1000 cells / ml.

[0219] The culture medium is preferably changed twice a week, preferably about every 3 or 4 days, so that the cells do not exceed a concentration of about 106cells / ml.

[0220] In some aspects, the invention also concerns the use of genetically modified hematopoietic progenitor cells according to the invention for the production, preferably in vitro, of hematopoietic progenitors, in particular according to the processes described herein.

[0221] The invention also concerns the use of immortalized hematopoietic progenitor cells for the production, preferably in vitro, of hematopoietic progenitors in particular according to the processes described herein.

[0222] The invention also relates to an in vitro method for producing hematopoietic cells, wherein said method comprises the steps of: i. Amplifying hematopoietic progenitor cells in the presence of a DNMT3A inhibitor, preferably in an amplification culture medium; ii. Differentiating the amplified hematopoietic progenitor cells into megakaryocytes or erythroid precursors, in particular in the absence of the DNMT3 A inhibitor, preferably in a differentiation culture medium; iii. Optionally, recovering the megakaryocytes or erythroid precursors; iv. Optionally, maturing the megakaryocytes into platelets or the erythroid precursors into erythrocytes; v. Optionally, recovering the platelets or erythrocytes.

[0223] This method may comprise a first step of culturing the hematopoietic cells in a primary medium.

[0224] Accordingly, the invention allows the production of either i) cells of the platelet lineage, such as megakaryocytes and platelets or ii) cells of the erythroid lineage, such as erythroid precursors or erythrocytes.

[0225] Methods and uses applied to these two distinct cell lineages are provided herein.

[0226] In some aspects, the process according to the invention is a process for the production of cells of the platelet lineage, especially megakaryocytes and / or platelets. The invention particularly concerns a method, preferably an in vitro method, comprising the differentiation of hematopoietic progenitor cells in megakaryocytes and / or platelets.

[0227] The term “differentiation”, as used herein, refers to the acquisition by cultured cells of characteristics that were not present in the cells initially used to inoculate the medium. The term “cell differentiation”, particularly refers to a process whereby undifferentiated progenitor cells acquire a more specialized fate.

[0228] Preferably, the step of differentiation of hematopoietic progenitor cells is performed in the absence of a DNMT3 A inhibitor. Typically, in embodiments wherein the hematopoietic progenitor cells are genetically modified to transiently / inducibly express a DNTM3A inhibitor, said DNTM3 A inhibitor is not expressed or its expression is not induced during differentiation.

[0229] In the context of the platelet lineage, it refers to a process whereby the undifferentiated hematopoietic progenitor cells are differentiated to the specialized megakaryocyte and thrombocyte having a platelet-producing capacity.

[0230] Preferably, in the context of the platelet lineage, the differentiation of the hematopoietic progenitor cells leads to functional platelet, having the capacity of clotting. Functional testing of platelets relies on testing their activation, aggregation, adhesion, secretion, and / or clotting performance, for example using flow cytometry, aggregation systems, flow-based assays, and clot models. The functionality of platelets can particularly be investigated using an aggregation assay in presence of calcium, thrombin and fibrinogen.

[0231] Preferably, the differentiation of hematopoietic progenitor cells in megakaryocytes and optionally platelets is performed in a differentiation culture medium, especially a differentiation culture medium adapted to the differentiation of cell of the platelet lineage, typically as described below.

[0232] Preferably, the hematopoietic progenitor cells are cultured in the differentiation culture medium at a concentration between 0.05 et 0.5 x 106cellules / ml, preferably between 50000 and 400000 cells / mL, and more preferably around 200000 cells / ml.

[0233] Preferably, the hematopoietic progenitor cells are cultured in the differentiation culture medium during 2 to 7 days, preferably between 4 to 8 days, more preferably between 6 and 8 days, particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0234] In some embodiments, for obtaining platelet, the hematopoietic progenitor cells of the invention are cultured in the differentiation culture medium of the invention during at least 4, 6, 7, 8, 9 or 10 days. Preferably, for obtaining platelet, the hematopoietic progenitor cells of the invention are cultured in the differentiation culture medium of the invention during no more than 10, 9, 8 or 7 days. More preferably, for obtaining platelet, the hematopoietic progenitor cells of the invention are cultured in the differentiation culture medium of the invention between 3 and 8 days, preferably between 4 and 7 days.

[0235] In some embodiments, for obtaining reticulocytes, the hematopoietic progenitor cells of the invention are cultured in the differentiation culture medium of the invention during at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, especially at least about 4 days. Preferably, for obtaining reticulocytes, the hematopoietic progenitor cells of the invention are cultured in the differentiation culture medium of the invention during no more than 8, 7, 6 or 5 days. Preferably, for obtaining reticulocytes, the hematopoietic progenitor cells of the invention are cultured in the differentiation culture medium of the invention between 2 and 8 days, between 2 and 6 days, preferably between 2 and 5 days.

[0236] Preferably, the hematopoietic progenitor cells and / or megakaryocytes are cultured in the differentiation culture medium in the absence of mesenchymal stem cells (MSCs).

[0237] In some aspects, the invention concerns an in vitro method for producing megakaryocytes and / or platelets, wherein the method comprises: i) Amplifying hematopoietic progenitor cells in the presence of a DNMT3A inhibitor, preferably in an amplification culture medium, ii) Differentiating the amplified hematopoietic progenitor cells into megakaryocytes, preferably in a differentiation culture medium, in particular in the absence of a DNMT3 A inhibitor; iii) Optionally, maturing the megakaryocytes into platelets; iv) Optionally, recovering the megakaryocytes and / or platelets.

[0238] The invention particularly concerns an in vitro method for producing megakaryocytes and / or platelets, said method comprising differentiating the genetically modified hematopoietic progenitor cells into megakaryocytes in a differentiation culture medium, and optionally maturing the megakaryocytes into platelets.

[0239] Particularly, the invention concerns an in vitro method for producing megakaryocytes and / or platelets, wherein the method comprises: i) Amplifying genetically modified hematopoietic progenitor cells of the invention, preferably in an amplification culture medium, by inducing the expression of a DNMT3A inhibitor, ii) Differentiating the amplified hematopoietic progenitor cells into megakaryocytes, preferably in the absence of the expression of the DNMT3A inhibitor, in particular in a differentiation culture medium; iii) Optionally, maturing the megakaryocytes into platelets; iv) Optionally, recovering the megakaryocytes and / or platelets.

[0240] Preferably, in step i) the genetically modified hematopoietic progenitor cells are cultured under conditions allowing expression of the DNMT3A inhibitor, e.g., by adding doxycycline in the amplification medium.

[0241] Preferably, in step ii) the absence of DNMT3A inhibitor expression is performed by stopping or repressing the expression of DNMT3 A or by stopping the induction of the expression of DNMT3 A, e.g., without / in the absence of doxycycline in the amplification medium.

[0242] Typically, when the inducible system in the Tet-On / Tet-Off system, the amplification step is performed in the presence of doxycycline; and the differentiation step is performed in the absence of doxycycline.

[0243] Preferably, the invention concerns an in vitro method for producing megakaryocytes and / or platelets, wherein the method comprises: i) Amplifying genetically modified hematopoietic progenitor cells, said genetically modified hematopoietic progenitor cells comprising a vector encoding for a shRNA that inhibits DNMT3 A under the control of an inducible promoter or system, by inducing the expression of said shRNA, preferably in an amplification culture medium, ii) Differentiating the amplified hematopoietic progenitor cells into megakaryocytes, without the induction of the expression of the shRNA, preferably in a differentiation culture medium; iii) Optionally, maturing the megakaryocytes into platelets; iv) Optionally, recovering the megakaryocytes and / or platelets.

[0244] Typically, when the inducible system in the Tet-On / Tet-Off system, the amplification step is performed in the presence of doxycycline; and the differentiation step is performed in the absence of doxycycline.

[0245] In particular embodiments, the invention concerns an in vitro method for producing megakaryocytes and / or platelets, wherein the method comprises: i) Amplifying genetically modified hematopoietic progenitor cells, said genetically modified hematopoietic progenitor cells comprising a vector encoding for a shRNA of SEQ ID NO: 1 or of a sequence having at least 80% identity thereto, said shRNA being under the control of a Tet-On system, preferably in an amplification culture medium, in the presence of doxycycline for inducing the expression of the shRNA; ii) Differentiating the amplified hematopoietic progenitor cells into megakaryocytes, preferably in a differentiation culture medium and in the absence of doxycycline; iii) Optionally, maturing the megakaryocytes into platelets; iv) Optionally, recovering the megakaryocytes and / or platelets.

[0246] In particular embodiments, the invention concerns an in vitro method for producing megakaryocytes and / or platelets, wherein the method comprises: i) Differentiating hematopoietic progenitor cells into megakaryocytes, preferably in a differentiation culture medium and typically in the absence of doxycycline; said genetically modified hematopoietic progenitor cells comprising a nucleic acid sequence encoding for a shRNA targeting DNMT3A, preferably of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, said shRNA being under the control of an inducible promoter or system such as a Tet-On / Tet Off system; ii) Optionally, maturing the megakaryocytes into platelets; iii) Optionally, recovering the megakaryocytes and / or platelets.

[0247] In some aspects, the methods disclosed herein further comprise a step of maturation of the megakaryocytes into platelets. Maturing or inducing maturation of megakaryocytes into platelets are used interchangeably. As used herein “maturation of megakaryocytes” defines the process by which megakaryocytes will become platelets, which involves, in particular, the formation of proplatelets and preplatelets. Preferably, maturation of megakaryocytes occurs in a maturation medium. Said maturation medium may have a similar or identical composition that the differentiation medium.

[0248] In some embodiments, in the methods of the invention, the maturation step of megakaryocytes is comprised in the differentiation step.

[0249] Typically, mature megakaryocytes or platelets are CD41+CD42+ cells. In some aspects, maturation of MK cells can be monitored by the expression of particular genes. Typically, during MK maturation, the cells overexpress MPL, VWF and FLU mRNA.

[0250] In some aspects, the method further comprises a step of recovering the megakaryocytes and / or platelets.

[0251] In some aspects, the method further comprises a step of testing the functionality of the obtained platelets, for example a thrombin activation assay. In such test, functionality of the platelets can be assessed by measuring the expression level of CD62p on their membranes. Platelets expressing CD62p on their membranes are functional.

[0252] In some aspects, the methods of the invention further comprise a first step of providing hematopoietic stem cells (CSH) or hematopoietic progenitors cells, preferably from a subject.

[0253] The processes according to the invention may also include a step of washing the hematopoietic progenitors, megakaryocytes and / or platelets obtained / recovered. This step may be carried out by any technique known to the skilled person, in particular by a succession of filtration, centrifugation and resuspension steps.

[0254] According to some embodiments, the process for the production of platelets according to the invention further includes a step of eliminating nucleated cells. This step particularly results in a homogeneous population comprising only mature platelets.

[0255] The process for the production of megakaryocytes and / or platelets may further include a step of recovering the megakaryocytes and / or platelets obtained. This step may be carried out by any technique known to the skilled person, in particular by filtration, centrifugation and removal of the culture medium.

[0256] Accordingly, the invention also relates to a population of hematopoietic progenitors obtained or obtainable by any one of the processes of the invention. According to another aspect, the present invention relates to a population of megakaryocytes and / or platelets obtained or obtainable by the process of the invention.

[0257] According to another aspect, the invention relates to the use of hematopoietic progenitors obtained or obtainable by the process according to the invention for the production of megakaryocytes and / or platelets.

[0258] In some aspects, the process according to the invention is a process for the production of cells of the erythroid lineage, especially erythroid precursors and / or red blood cells.

[0259] In another aspect, the process according to the invention is a process for the production of cells of the erythrocyte lineage, especially erythroid precursors and / or erythrocytes.

[0260] The invention particularly concerns a method, preferably an in vitro method, comprising the differentiation of hematopoietic progenitor cells in erythroid precursors and / or erythrocytes.

[0261] In the context of the erythroid lineage, the term “differentiation” further refers to a process whereby the undifferentiated hematopoietic progenitor cells are differentiated to the specialized erythroid precursors and reticulocytes having a RBC-producing capacity. Preferably, the step of differentiation of hematopoietic progenitor cells is performed in the absence of a DNMT3 A inhibitor.

[0262] Preferably, the differentiation of hematopoietic progenitor cells in erythroid precursors and optionally erythrocytes is performed in a differentiation culture medium, especially a differentiation culture medium adapted to the differentiation of cell of the erythroid lineage, typically as described below.

[0263] Preferably, the hematopoietic progenitor cells are cultured in the differentiation culture medium at a concentration between 0.05 et 0.1 x 106cellules / ml, preferably between 500 and 2000 cells / mL, and more preferably around 1000 cells / ml.

[0264] The culture medium is preferably changed twice a week, preferably about every 3 or 4 days, so that the cells do not exceed a concentration of about 106cells / ml.

[0265] Preferably, the hematopoietic progenitor cells are cultured in the differentiation culture medium during 2 to 10 days, preferably between 3 to 6 days, particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0266] Preferably, the hematopoietic progenitor cells and / or erythroid precursors are cultured in the differentiation culture medium in the absence of mesenchymal stem cells (MSCs).

[0267] In some aspects, the invention concerns an in vitro method for producing erythroid precursors and / or erythrocytes, wherein the method comprises: i) Amplifying hematopoietic progenitor cells, preferably in an amplification culture medium, in the presence of a DNMT3 A inhibitor, ii) Differentiating the amplified hematopoietic progenitor cells into erythroid precursors, preferably in a differentiation culture medium, in particular in the absence of a DNMT3 A inhibitor; iii) Optionally, maturing the erythroid precursors into erythrocytes; iv) Optionally, recovering the erythroid precursors and / or erythrocytes.

[0268] The invention particularly concerns an in vitro method for producing erythroid precursors and / or erythrocytes, said method comprising differentiating the genetically modified hematopoietic progenitor cells into erythroid precursors in a differentiation culture medium, and optionally maturing the erythroid precursors into erythrocytes.

[0269] Particularly, the invention concerns an in vitro method for producing erythroid precursors and / or erythrocytes, wherein the method comprises: i) Amplifying genetically modified hematopoietic progenitor cells of the invention, preferably in an amplification culture medium, by inducing the expression of a DNMT3A inhibitor, ii) Differentiating the amplified hematopoietic progenitor cells into erythroid precursors, preferably in the absence of the expression of the DNMT3A inhibitor, in particular in a differentiation culture medium; iii) Optionally, maturing the erythroid precursors into erythrocytes; iv) Optionally, recovering the erythroid precursors and / or erythrocytes.

[0270] Preferably, in step i) the genetically modified hematopoietic progenitor cells are cultured under conditions allowing expression of the DNMT3A inhibitor, e.g., by adding doxycycline in the amplification medium.

[0271] Preferably, in step ii) the absence of DNMT3A inhibitor expression is performed by stopping or repressing the expression of DNMT3A or by stopping the induction of the expression of DNMT3A, e.g., without / in the absence of doxycycline in the amplification medium.

[0272] Typically, when the inducible system in the Tet-On / Tet-Off system, the amplification step is performed in the presence of doxycycline; and the differentiation step is performed in the absence of doxycycline.

[0273] Preferably, the invention concerns an in vitro method for producing erythroid precursors and / or erythrocytes, wherein the method comprises: i) Amplifying genetically modified hematopoietic progenitor cells, said genetically modified hematopoietic progenitor cells comprising a vector encoding for a shRNA that inhibits DNMT3 A under the control of an inducible promoter or system, by inducing the expression of said shRNA, preferably in an amplification culture medium, ii) Differentiating the amplified hematopoietic progenitor cells into erythroid precursors, without the induction of the expression of the shRNA, preferably in a differentiation culture medium; iii) Optionally, maturing the erythroid precursors into erythrocytes; iv) Optionally, recovering the erythroid precursors and / or erythrocytes.

[0274] Typically, when the inducible system in the Tet-On / Tet-Off system, the amplification step is performed in the presence of doxycycline; and the differentiation step is performed in the absence of doxycycline. In particular embodiments, the invention concerns an in vitro method for producing erythroid precursors and / or erythrocytes, wherein the method comprises: i) Amplifying genetically modified hematopoietic progenitor cells, said genetically modified hematopoietic progenitor cells comprising a vector encoding for a shRNA of SEQ ID NO: 1 or of a sequence having at least 80% identity thereto, said shRNA being under the control of a Tet- On / Tet-Off system, preferably in an amplification culture medium, in the presence of doxycycline for inducing the expression of the shRNA; ii) Differentiating the amplified hematopoietic progenitor cells into erythroid precursors, preferably in a differentiation culture medium and in the absence of doxycycline; iii) Optionally, maturing the erythroid precursors into erythrocytes; iv) Optionally, recovering the erythroid precursors and / or erythrocytes.

[0275] In particular embodiments, the invention concerns an in vitro method for producing erythroid precursors and / or erythrocytes, wherein the method comprises: i) Differentiating genetically modified hematopoietic progenitor cells into erythroid precursors, preferably in a differentiation culture medium and especially in the absence of doxycycline; said genetically modified hematopoietic progenitor cells comprising a nucleic acid sequence encoding for a shRNA targeting DNMT3 A, preferably of SEQ ID NO: 1 or of a sequence having at least 80% sequence identity thereto, said shRNA being under the control of an inducible promoter or system such as a Tet-On / Tet-Off system; ii) Optionally, maturing the erythroid precursors into erythrocytes; iii) Optionally, recovering the erythroid precursors and / or erythrocytes.

[0276] In some aspects, the methods disclosed herein further comprise a step of maturation of the erythroid precursors into erythrocytes. Maturing or inducing maturation of erythroid precursors into erythrocytes are used interchangeably. As used herein “maturation of erythroid precursors,” defines the process by which erythroid precursors will become erythrocytes and which involves, in particular, the formation of reticulocytes. Typically, maturation of erythroid precursors results in the enucleation of the hematopoietic cells.

[0277] In some embodiments, in the methods of the invention, the maturation step of erythroid precursors is comprised in the differentiation step. Preferably, maturation of erythroid precursors occurs in a maturation medium. Said maturation medium may have a similar or identical composition than the differentiation medium. Particularly, said maturation medium may additionally comprise TK216.

[0278] Typically, CD235a, CD71, CD36, BAND3, and / or CD49d markers or expression levels can be used to characterize the kinetics of erythroid maturation. In particular, reticulocytes are CD235a+BAND3+CD71low / 'CD36low / 'CD49dlow / ' and red blood cells are CD235a+BAND3+CD7T CD36'CD49d'.

[0279] In some aspects, the methods of the invention further comprise a step of recovering the erythroid precursors and / or erythrocytes.

[0280] In some aspects, the methods of the invention further comprise a first step of providing hematopoietic stem cells (CSH) or hematopoietic progenitor cells, preferably from a subject.

[0281] The processes according to the invention may also include a step of washing the hematopoietic progenitors, erythroid precursors and / or red blood cells obtained / recovered. This step may be carried out by any technique known to the skilled person, in particular by a succession of filtration, centrifugation and resuspension steps.

[0282] According to some embodiments, the process for the production of erythrocytes according to the invention further includes a step of eliminating nucleated cells. This step particularly results in a homogeneous population comprising only mature erythrocytes.

[0283] The process for the production of erythroid precursors and / or erythrocytes may further include a step of recovering the erythroid precursors and / or erythrocytes obtained. This step may be carried out by any technique known to the skilled person, in particular by filtration, centrifugation and removal of the culture medium.

[0284] Accordingly, the invention also relates to a population of hematopoietic progenitors obtained or obtainable by any one of the processes of the invention. According to another aspect, the present invention relates to a population of erythroid precursors and / or erythrocytes obtained or obtainable by the processes of the invention.

[0285] According to another aspect, the invention relates to the use of hematopoietic progenitors obtained or obtainable by the processes according to the invention for the production of erythroid precursors and / or erythrocytes.

[0286] According to another aspect, the present invention relates to a pharmaceutical composition comprising hematopoietic cells, especially hematopoietic progenitors, platelets or erythrocytes, obtained by the process of the invention and a pharmaceutically acceptable carrier. As used here, the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable support” are equivalent and refer to any substance other than an active ingredient present in a pharmaceutical composition. Its addition is intended in particular to facilitate the preservation and administration of cells, without modifying their properties. The pharmaceutically acceptable carrier used for the formulation of compositions containing hematopoietic progenitors, megakaryocytes and / or platelets according to the invention may be, for example, selected from the group consisting of saline, PBS solution with human serum albumin added, and mixtures thereof, or any other saline solution having an osmolarity suitable for the preservation of hematopoietic progenitors, megakaryocytes and / or platelets and, preferably, which can be directly administered to the subject. For the formulation of compositions containing platelets, a saline-adenine-glucose- mannitol (SAGM) medium may also be used as a pharmaceutically acceptable carrier such as an injection or storage solution, alone or in combination with the other pharmaceutically acceptable carriers listed above.

[0287] The invention also relates to the use of hematopoietic cells obtained or obtainable according to the invention, or a pharmaceutical composition comprising said cells, for the preparation of a medicament, in particular a biological medicament, for the treatment of a haematological disease or disorder, especially in a subject in need thereof.

[0288] As used here, the term “biological medicament” refers to a medicinal product whose active substance is produced by or extracted from a biological source.

[0289] In some embodiments, any of the hematopoietic cells disclosed herein can be used to produce platelets or red blood cells comprising a cargo of interest. Typically, such platelets or red blood cells are loaded with drugs. This constitutes a unique drug delivery system as a biologic or hybrid carrier capable of greatly enhancing pharmacokinetics, altering pharmacodynamics, and modulating immune responses to appended cargoes. Drugs and probes can either be encapsulated into the hematopoietic cells inner volume or coupled to the cell surface. These natural carriers can be employed for drug delivery, for example, via loading drugs into donor or autologous RBCs or platelets prior to transfusion to a patient. Strategies for drug delivery by hematopoietic cells are particularly disclosed in Villa et al., Adv Drug Deliv Rev. 2016 Nov 15;106(Pt A):88-103 and Magnani M, et al. Biotechnol Appl Biochem. 1998;28(Pt 1): 1— 6, their content being incorporated herein by reference.

[0290] Accordingly, the hematopoietic progenitor cells of the invention or the RBC or platelets obtained therefrom can further comprise a cargo. In some embodiment, the invention therefore concerns the use of hematopoietic progenitor cells of the invention or the RBC or platelets obtained therefrom for drug delivery.

[0291] Any of the hematopoietic cells or pharmaceutical composition disclosed herein may be useful in the treatment of haematological diseases or disorder or may be used for transfusion, in particular in a subject in need thereof.

[0292] In some embodiments, the subject in need envisaged by the invention is in need of a red blood cells and / or platelets transfusion or suffer from a haematological pathology or another pathology inducing an insufficiency in the production of haematopoietic cells, such as red blood cells or platelets.

[0293] In some embodiments, the subject in need thereof suffers from a platelet disorder or disease, such as thrombopenia, haemorrhage or platelet refractoriness. Platelet refractoriness is defined as a repeated suboptimal response to platelet transfusions with lower-than-expected posttransfusion count increments. Refractoriness can be caused by immune and nonimmune factors.

[0294] In some embodiments, the subject has a low platelet count. In particular, the subject has a platelet count of less than 140 x 109cells / L, 100 x 109cells / L or less, 50 x 109cells / L or less or 25 x 109cells / L or less.

[0295] In some embodiments, the subject in need thereof suffers from a haematological disorder or disease, such as hemoglobinopathies, RBC enzymopathies, anaemia, thalassemia or drepanocytosis.

[0296] As used here, the term “treatment” refers to any action aimed at improving or eliminating symptoms, slowing the progression of the disease, stopping the progression of the disease or eliminating the disease. This term refers more specifically to an increase in the level of healthy platelets and circulating platelets, preferably to reach normal values for the subject’s age. This term includes both preventive and curative treatment. The term “therapeutically effective amount” as used here refers to an amount sufficient to have an effect on at least one symptom of the condition, and more specifically to increase the level of healthy platelets and circulating haemoglobin in the treated subject.

[0297] The invention particularly relates to megakaryocytes and / or platelets obtained or obtainable by the process of the invention or a pharmaceutical composition comprising such, for use in the treatment of a platelet disease or disorder, such as thrombopenia, Fetal and Neonatal Alloimmune Thrombocytopenia, post-transfusion purpura, haemorrhage or platelet refractoriness. The invention also relates to the use of megakaryocytes and / or platelets obtained by the process of the invention or a pharmaceutical composition comprising such, for platelet transfusion.

[0298] The invention also relates to erythroid precursors and / or erythrocytes obtained by the process of the invention or a pharmaceutical composition comprising such, for use in the treatment of a hemoglobinopathies, RBC enzymopathies, anaemia, haemorrhage, erythroleukemia, thalassemia, or drepanocytosis.

[0299] The invention also relates to the use of erythroid precursors and / or erythrocytes obtained by the process of the invention or a pharmaceutical composition comprising such, for blood transfusion, especially in the context of acute blood loss, such as haemorrhage or surgical procedures.

[0300] The invention further relates to a method for treating a patient suffering from a haematological disease or disorder, comprising administering to said patient a therapeutically effective amount or number of hematopoietic cells of the invention, especially obtained by the processes of the invention.

[0301] In a particular embodiment, the invention relates to a method for treating a subject in need thereof, wherein the method comprises: a) providing a biological sample from the subject, the biological sample comprising hematopoietic stem cells (CSH) or hematopoietic progenitors; b) genetically modifying the hematopoietic progenitors of the subject, in particular by introducing a DNMT3 A inhibitor into the hematopoietic progenitors as envisioned herein; c) amplifying the hematopoietic progenitors, by an amplification method such as disclosed herein; and d) administering the amplified hematopoietic progenitors to the subject.

[0302] In a particular embodiment, the invention relates to a method for treating a subject in need thereof, wherein the method comprises: a) providing a biological sample from the subject, the biological sample comprising hematopoietic stem cells (CSH) and / or hematopoietic progenitors; b) genetically modifying the hematopoietic progenitors of the subject, in particular by introducing a vector encoding a DNMT3A inhibitor, especially a shRNA DNMT3A inhibitor as disclosed herein, preferably under the control of an inducible promoter or system into the hematopoietic progenitors; c) amplifying the hematopoietic progenitors, by an amplification method such as disclosed herein; and d) administering the amplified hematopoietic progenitors to the subject.

[0303] Preferably, the amplified hematopoietic progenitors are isolated or purified before administration to the subject.

[0304] In a particular embodiment, the invention relates to a method for treating a subject in need thereof, wherein the method comprises: a) providing a biological sample from the subject, the biological sample comprising hematopoietic stem cells (CSH) and / or hematopoietic progenitors; b) genetically modifying the hematopoietic progenitors of the subject, in particular by introducing a DNMT3 A inhibitor into the hematopoietic progenitors as envisioned herein; c) amplifying the hematopoietic progenitors, preferably by an amplification method such as disclosed herein; d) differentiating the amplified hematopoietic progenitors into megakaryocytes or platelets, preferably by a differentiation method such as disclosed herein; e) administering the megakaryocytes or platelets to the subject.

[0305] Preferably, the megakaryocytes or platelets are isolated or purified before administration to the subject.

[0306] In a particular embodiment, the invention relates to a method for treating a subject in need thereof, wherein the method comprises: a) providing a biological sample from the subject, the biological sample comprising hematopoietic stem cells (CSH) and / or hematopoietic progenitors; b) genetically modifying the hematopoietic progenitors of the subject, in particular by introducing a DNMT3 A inhibitor into the hematopoietic progenitors as envisioned herein; c) amplifying the hematopoietic progenitors, preferably by an amplification method such as disclosed herein; d) differentiating the amplified hematopoietic progenitors into erythroid progenitors or erythrocytes, preferably by a differentiation method such as disclosed herein; e) administering the erythroid progenitors or erythrocytes to the subject.

[0307] Preferably, the erythroid progenitors or erythrocytes are isolated or purified before administration to the subject. In the processes according to the invention, the cell culture steps, namely the amplification, differentiation and / or maturation steps, are preferably performed at a temperature suitable for the multiplication and / or differentiation of hematopoietic cells. Preferably, the temperature is about 37°C.

[0308] The CO2 and / or O2 content is preferably controlled during the cell culture steps. Preferably, the CO2 content is between 2% and 10%, between 3% and 7% or between 4% and 6%, and is preferentially about 5%. The O2 content is preferably between 1% and 30%, between 5% and 25%, between 10% and 25% or between 15% and 25%, and is preferentially about 20%.

[0309] In a particularly preferred manner, the cells are cultured at about 37°C, with a CO2 content of 5% and / or an O2 content of 20%.

[0310] Preferably, the hematopoietic cells of interest in the present invention are cultured at a concentration (or cell density) of between 100 and 10 0000 cells / ml, preferably between 1000 and 10000 cells / ml, and even more preferably at about 10000 cells / ml.

[0311] In some aspects, the hematopoietic cells are cultured under gentle agitation, especially between 30 and 80 rpm, preferably about 30 rpm.

[0312] In some embodiments, the amplification step and / or differentiation step occurs in a bioreactor.

[0313] Amplification and Differentiation media

[0314] According to still another aspect, the present invention relates to a cell culture medium adapted to the nutritional requirements of hematopoietic progenitors, and in particular adapted to the amplification and / or differentiation hematopoietic cells.

[0315] The culture medium according to the invention may be liquid, semisolid or solid. A person skilled in the art knows how to solidify a culture medium, notably by adding agar.

[0316] As used herein the terms “base medium”, “base culture medium” and “basal cell culture medium” are used interchangeably and refer to a base of the cell culture medium adapted to the growth, amplification and / or differentiation of cells of the hematopoietic line may be any base known by the skilled person to meet the needs of hematopoietic cells, such as cells of the erythroid or platelet lineage.

[0317] The base of the cell culture medium may include Iscove’s modified Dulbecco’s medium (IMDM) or an equivalent medium adapted to the nutritional requirements of progenitor cells, such as Minimal essential medium (MEM), Dulbecco’s modified Eagle's medium (DMEM), Glasgow's MEM and DMEM / F-12, StemSpan SFEM (STEMCELL Technologies) StemMACS progenitor cell (Expansion Media XF, human, Invitrogen).

[0318] Further compounds can be added to the base medium, such as insulin, transferrin, stem cell factor (SCF), heparin, IL-3, erythropoietin (EPO), thrombopoietin (TPO), growth factors, serum, GM-CSF, IL-3, IL-6, IL-11, plasma, platelet lysate, serum pool, an autophagy inducer, a glucocorticoid hormone, glutamine, ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), StemRegenin 1 (SRI), CHIR99021, activin A or midostaurine or any combination thereof.

[0319] Compounds added to the culture medium are preferably human compounds obtained by recombinant or purification techniques.

[0320] As used herein, the term “serum pool” refers to a mixture of human AB plasmas (most often a mixture of more than 100 different plasmas) that are viro-attenuated. To do this, AB plasmas from transfusion centres are mixed, viro-attenuated and finally aliquoted. The serum pool can then be used fresh or kept frozen.

[0321] As used herein, the term “platelet lysate” refers to a product rich in growth factors that is obtained as a result of the lysis of platelet concentrates. To do this, platelet concentrates from transfusion centres can be mixed before being lysed. There are different lysis methods well known to the skilled person, in particular ultrasound lysis, the use of solvents and / or detergents, or cryolysis. Platelet concentrates are preferably lysed by cryolysis. Cryolysis consists of freeze / thaw cycles, usually two cycles, causing the platelets to rupture and the growth factors they contain to be released into the plasma. Optionally, lysis may be followed by centrifugation and / or filtration. The platelet lysate may then be used fresh or kept frozen, preferably at -20°C or -80°C.

[0322] In particular embodiments, the culture medium comprises an autophagy inducer. As used herein, the terms “autophagy,” “autolysis” and “autophagocytosis” are equivalent and can be used interchangeably. Autophagy refers to the degradation of part of the cell’s cytoplasm by its own lysosomes. In particular, the autophagy inducer is capable of inhibiting mTOR pathway, such as metformin, rapamycin, perifosine, everolimus, resveratrol or tamoxifen, autophagosome formation activators such as the compound MG- 132 (a 26S proteasome inhibitor), the compound SAHA (a pan-histone deacetylase inhibitor), trichostatin A or valproic acid, or small molecules acting independently of the mTOR pathway such as SMER-28, SMER-10 or SMER 18.

[0323] Preferably, the culture medium is supplemented with an autophagy inducer selected from the group consisting of Small Molecule Enhancer of Rapamycin 28 (SMER-28), SMER 10 and SMER 18, and a combination thereof. According to a preferred embodiment, the autophagy inducer is SMER-28. In particular embodiments, the culture medium comprises a glucocorticoid hormone.

[0324] As used here, the terms “glucocorticoid hormone,” “glucocorticoid,” “corticosteroid,” or “corticoid” are equivalent and can be used interchangeably. These terms refer to natural or synthetic steroid hormones with a pregnane nucleus and an action on protein and carbohydrate metabolism.

[0325] According to an embodiment, the glucocorticoid hormone is selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazol, and derivatives and mixtures thereof.

[0326] According to a preferred embodiment, the glucocorticoid hormone is dexamethasone.

[0327] According to a very particular embodiment, the autophagy inducer is SMER-28 and the glucocorticoid hormone is dexamethasone.

[0328] Optionally, the culture cell medium can further comprise an activator of the HIF pathway. As used here, the term “HIF pathway” refers to the signalling pathway initiated by hypoxia-inducible factor (HIF) which stimulates EPO secretion and thus activates the EPO-R / JAK2 / STAT5 / BCL- XL pathway.

[0329] Preferably, the HIF pathway activator according to the invention is a prolyl hydroxylase (PHIS) inhibitor selected from the group consisting of dimethyloxalylglycine (DMOG), N- oxalylglycine (NOG), desferrioxamine (DFO), FG-4383, F-0041, FG-2216, FG-4592, S956711, ethyl-3,4 dihydroxy-benzoate (EDHB), TM6089, TM655, TM6008, 8-hydroxyquinoline, and derivatives thereof. Preferably, the HIF pathway activator is DMOG.

[0330] In some aspects, the invention concerns an amplification culture medium. As used herein, the terms “amplification culture medium”, “amplification medium”, “expansion medium” and “expansion culture medium” are used interchangeably and refer to a culture medium in which the hematopoietic cells, preferably the hematopoietic progenitors of the invention are cultured or multiplied. It typically comprises a culture base medium suitable for the amplification of hematopoietic cells.

[0331] Particularly, the amplification culture medium envision herein comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), glutamine, insulin, heparin, transferrin, plasma pool, dexamethasone, SMER28 and EPO. Particularly, the amplification culture medium of the invention comprises or consists of a base culture medium such as an IMDM medium, insulin, heparin, transferrin, serum or plasma, dexamethasone, SMER28, EPO and glutamine.

[0332] Preferably, the amplification medium of the invention comprises or consists of a base medium, preferably an IMDM medium or an equivalent medium, supplemented with:

[0333] - insulin, preferably human insulin, at a concentration between about 1 pg / mL and about 50 pg / mL, preferably between about 5 pg / mL and about 20 pg / mL, more preferably at a concentration of about 10 pg / mL;

[0334] - heparin, preferably human heparin, at a concentration between about 0.5 U / mL and about 5 U / mL, preferably between about 1 U / mL and about 3 U / mL, more preferably at a concentration of about 2 U / mL;

[0335] - transferrin, preferably human transferrin, at a concentration between about 200 pg / mL and about 400 pg / mL, preferably between about 300 pg / mL and about 350 pg / mL, more preferably at a concentration of about 330 pg / mL;

[0336] - serum, plasma or serum pool, preferably human, at a concentration between about 1% and about 10%, preferably between about 3% and about 7%, more preferably at a concentration of about 5%;

[0337] - optionally a glucocorticoid hormone, preferably selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazol, more preferably dexamethasone, at a concentration between 0.01 mM and 10 mM, preferably between 0.01 mM and 5 mM, more preferably between 0.05 mM and 1 mM, and particularly preferably of about 0.1 mM;

[0338] - an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0339] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 0.5 lU / mL and about 10 lU / mL, preferably between about 1 lU / mL and about 5 lU / mL, even more preferably at a concentration of about 2 or 3 lU / mL; and

[0340] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 15 ng / ml, preferably between about 1 ng / ml and about 10 ng / ml, more preferably between about 3 ng / ml and about 7 ng / ml, most preferably about 5 ng / ml. Preferably, the amplification medium of the invention comprises or consists of a base medium, preferably an IMDM medium or an equivalent medium, supplemented with:

[0341] - insulin, preferably human insulin, at a concentration between about 1 pg / mL and about 50 pg / mL, preferably between about 5 pg / mL and about 20 pg / mL, more preferably at a concentration of about 10 pg / mL;

[0342] - heparin, preferably human heparin, at a concentration between about 0.5 U / mL and about 5 U / mL, preferably between about 1 U / mL and about 3 U / mL, more preferably at a concentration of about 2 U / mL;

[0343] - transferrin, preferably human transferrin, at a concentration between about 200 pg / mL and about 400 pg / mL, preferably between about 300 pg / mL and about 350 pg / mL, more preferably at a concentration of about 330 pg / mL;

[0344] - human plasma, at a concentration between about 1% and about 10%, preferably between about 3% and about 7%, more preferably at a concentration of about 5%;

[0345] - dexamethasone at a concentration between 0.01 mM and 10 mM, preferably between about 0.01 mM and about 5 mM, more preferably between about 0.05 mM and about 1 mM, and particularly preferably of about 0.1 mM;

[0346] - SMER-28 at a concentration between 0,1 pM and 10 pM, preferably between about 0.5 pM and about 5 pM, more preferably between about 1 pM and about 3 pM, and particularly preferably of about 2,27 pM;

[0347] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 0.5 lU / mL and about 10 lU / mL, preferably between about 1 lU / mL and about 5 lU / mL, even more preferably at a concentration of about 2 or 3 lU / mL; and

[0348] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 15 ng / ml, preferably between about 1 ng / ml and about 10 ng / ml, more preferably between about 3 ng / ml and about 7 ng / ml, most preferably about 5 ng / ml.

[0349] In some embodiments, the amplification medium of the invention comprises or consists of a base medium, preferably an IMDM medium or an equivalent medium, supplemented with:

[0350] - insulin, preferably human insulin, at a concentration between about 5 pg / mL and about 15 pg / mL,

[0351] - heparin, preferably human heparin, at a concentration between about 1 U / mL and about 5 U / mL; - transferrin, preferably human transferrin, at a concentration between about 300 pg / mL and about 400 pg / mL;

[0352] - human plasma, at a concentration between about 3% and about 7%;

[0353] - optionally, dexamethasone at a concentration between about 0.05 mM and about 0.5 mM;

[0354] - SMER-28 at a concentration between about 1 pM and about 3.5 pM, and particularly preferably of about 2,27 pM;

[0355] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 lU / mL and about 5 lU / mL; and

[0356] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml.

[0357] In some embodiments, the amplification medium of the invention comprises or consists of a base medium, preferably an IMDM medium or an equivalent medium, supplemented with:

[0358] - insulin, preferably human insulin, at a concentration between about 8 pg / mL and about 12 pg / mL,

[0359] - heparin, preferably human heparin, at a concentration between about 1 U / mL and about 3 U / mL;

[0360] - transferrin, preferably human transferrin, at a concentration between about 300 pg / mL and about 350 pg / mL;

[0361] - human plasma, at a concentration between about 4% and about 6%;

[0362] - optionally, dexamethasone at a concentration between about 0.05 mM and about 0.3 mM;

[0363] - SMER-28 at a concentration between about 1.5 pM and about 3 pM;

[0364] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 2 lU / mL and about 4 lU / mL; and

[0365] - glutamine, preferably L-glutamine, at a concentration of between about 3 ng / ml and about 7 ng / ml.

[0366] Preferably, the expansion medium comprises or consists of a culture base medium such as IMDM, about 5 ng / mL glutamine, about 5% (v / v) human S / D AB plasma, about 10 pg / mL insulin, about 2 U / mL heparin, about 0.1 mM dexamethasone, about 2.27pM SMER28, about 330 pg / mL transferrin and about 3 U / mL EPO. The expansion medium of the invention can typically be supplemented with doxycycline, at a concentration that allows the expression of the DNMT3A inhibitor. Typically, the doxycycline concentration in the medium is of between 10 ng / ml and 200 ng / ml, preferably 10 and 100 ng / ml, more preferably between 25 ng / ml and 75 ng / ml. The expansion medium of the invention can typically be supplemented with doxycycline, preferably at a concentration of 50 ng / mL. This supplementation typically allows the expression of the DNMT3 A inhibitor of the invention.

[0367] The present invention also concerns the use of an amplification culture medium, preferably such as described here above, for the amplification of hematopoietic progenitor cells, in particular genetically modified hematopoietic progenitor cells such as described herein.

[0368] In some aspects, the invention relates to a differentiation culture medium, in particular adapted for cells of the platelet lineage. A medium adapted to the growth and / or differentiation of cells of the platelet line is a medium allowing the differentiation of hematopoietic progenitor cells into megakaryocytes and / or platelets as well as the multiplication of megakaryocytes and / or platelets. This differentiation culture medium may be called herein “differentiation medium”; “megakaryocytes differentiation culture medium” or “MK differentiation culture medium” or “MK differentiation medium”.

[0369] In some aspects, the differentiation culture medium comprises or consists of a base culture medium, a thrombopoietin agonist, an aryl hydrocarbon receptor (AHR) antagonist, an autophagy inducer, a glucocorticoid hormone, an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), a growth factor of the TGF-P family and a protein kinase inhibitor.

[0370] Particularly, the invention concerns a differentiation culture medium comprising or consisting essentially of a culture base medium preferably IMDM, ITS-X (Insulin-Transferrin- Selenium-Ethanolamine), optionally transferrin, foetal bovine or calf serum or platelet lysate, glutamine, a thrombopoietin agonist, an aryl hydrocarbon receptor (AHR) antagonist, an autophagy inducer, an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), a growth factor of the TGF-P family and a protein kinase inhibitor.

[0371] Particularly, the invention concerns a differentiation culture medium comprising or consisting essentially of a culture base medium preferably IMDM, ITS-X (Insulin-Transferrin- Selenium-Ethanolamine), foetal bovine or calf serum or platelet lysate, glutamine, a thrombopoietin agonist, an aryl hydrocarbon receptor (AHR) antagonist, an autophagy inducer, an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), a growth factor of the TGF-P family and a protein kinase inhibitor. In some embodiments, the MK differentiation culture medium comprises ITS-X (Insulin- Transferrin-Selenium-Ethanolamine) and further additional transferrin. Alternatively, the MK differentiation culture medium comprises ITS-X without the need of additional transferrin. These two media can be used interchangeably for the differentiation of hematopoietic cells of the platelet lineage.

[0372] Particularly, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), Insulin-Transferrin- Selenium -Ethanolamine (ITS-X), foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin. Said differentiation medium is particularly suitable for the differentiation of megakaryocytes into platelets.

[0373] Particularly, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), Insulin-Transferrin- Selenium -Ethanolamine (ITS-X), transferrin, foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin. Said differentiation medium is particularly suitable for the differentiation of megakaryocytes into platelets.

[0374] Alternatively, the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), Insulin-Transferrin- Selenium-Ethanolamine (ITS-X), optionally transferrin, optionally hemin, foetal bovine serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, hirsutine, CHIR99021, Activin A, and a) WEHI-53; JAK2IN6 and pacritinib; and optionally TK216 and / or anagrelide or b) TK216 and anagrelide and optionally hemin, for the differentiation of hematopoietic progenitor cells into erythroid precursors, optionally into erythrocytes. Said differentiation medium is particularly suitable for the differentiation of erythroid precursors into erythrocytes.

[0375] In some embodiments, the differentiation medium, especially the MK differentiation medium comprises or consists of a base medium, such as an IMDM or an equivalent medium, supplemented with ITS-X (Insulin-Transferrin-Selenium-Ethanolamine); glutamine, preferably L-glutamine; an inhibitor / antagonist of the aryl hydrocarbon receptor (AHR), preferably StemRegenin 1 (SRI); an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28; an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021; a growth factor of the TGF-P family, preferably Activin A; a protein kinase inhibitor, preferably midostaurin, foetal calf serum (FBS) or platelet lysate; thrombopoietin (TPO) or eltrombopag, preferably human TPO. In some aspects, the invention concerns a differentiation medium, especially a MK differentiation culture medium comprising or consisting of a base culture medium preferably IMDM, insulin, transferrin, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin.

[0376] Preferably, the differentiation medium, especially the MK differentiation culture medium, comprises or consists of a base medium, such as IMDM, supplemented with ITS-X (Insulin- Transferrin-Selenium -Ethanolamine), optionally Transferrin, foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin.

[0377] Preferably, the differentiation medium, especially the MK differentiation culture medium comprises or consists of a base medium, such as IMDM, supplemented with ITS-X (Insulin- Transferrin-Selenium -Ethanolamine), foetal bovine serum (FBS), glutamine, eltrombopag or thrombopoietin, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin.

[0378] In some embodiments, the differentiation medium, especially the MK differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with:

[0379] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0380] - optionally transferrin, at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0381] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 and about 7,5 ng / ml , preferably of about 5 ng / ml;

[0382] - an inhibitor / antagonist of the aryl hydrocarbon receptor (AHR), preferably StemRegenin 1 (SRI), at a concentration of between about 50 nM and about 150 nM of preferably between about 75 nM and about 125 nM, preferably of about 100 nM;

[0383] - an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0384] - an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021, at a concentration between about 0, 1 pM and about 5 pM, preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM; - a growth factor of the TGF-P family, preferably Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml;

[0385] - a protein kinase inhibitor, preferably midostaurin, preferably between about 1 pM and about 5 pM, preferably between about 2 pM and about 3 pM, preferably of about 2,5 pM;

[0386] - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%; and

[0387] - thrombopoietin (TPO) or eltrombopag, preferably human TPO, at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0388] In some embodiments, the differentiation medium, especially the MK differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with:

[0389] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine) at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0390] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 and about 7,5 ng / ml , preferably of about 5 ng / ml;

[0391] - optionally transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0392] - StemRegenin 1 (SRI), at a concentration of between about 50 nM and about 150 nM of preferably between about 75 and about 125 nM, preferably of about 100 nM;

[0393] - SMER-28 at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and

[0394] 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0395] - CHIR99021 at a concentration between about 0,1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM;

[0396] - Activin A at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml;

[0397] - midostaurin at a concentration between about 1 pM and about 5 pM, preferably between about 2 pM and about 3 pM, preferably of about 2,5 pM; - foetal bovine serum (FBS) or platelet lysate, preferably foetal bovine serum (FBS), at a concentration between about 0,1% and about 5%, preferably between about 0,5% and about 1,5%, preferably of about 1%; and

[0398] - eltrombopag or thrombopoietin (TPO), preferably human TPO, at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0399] In some embodiments, the differentiation medium, especially the MK differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with:

[0400] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine) at a concentration of between about 0,1% and about 10%,

[0401] - optionally transferrin at a concentration of between about 100 pg / ml and about 500 pg / ml;

[0402] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 15 ng / ml;

[0403] - StemRegenin 1 (SRI), at a concentration of between about 10 nM and about 500 nM;

[0404] - SMER-28 at a concentration between 0,1 pM and 15 pM;

[0405] - CHIR99021 at a concentration between about 0,1 pM and about 10 pM;

[0406] - Activin A at a concentration between about 100 ng / ml and about 500 ng / ml;

[0407] - midostaurin at a concentration between about 1 pM and about 15 pM;

[0408] - foetal bovine serum (FBS) at a concentration between about 0,1% and about 10%; and

[0409] - thrombopoietin (TPO), preferably human TPO, at a concentration of between about 10 ng / ml and about 100 ng / ml.

[0410] In some embodiments, the differentiation medium, especially the MK differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with:

[0411] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine) at a concentration of between about 0,5% and about 5%, preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0412] - optionally transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml; - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml, preferably between about 2,5 and about 7,5 ng / ml , preferably of about 5 ng / ml;

[0413] - StemRegenin 1 (SRI), at a concentration of between about 50 nM and about 150 nM, preferably between about 75 nM and about 125 nM, preferably of about 100 nM;

[0414] - SMER-28 at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0415] - CHIR99021 at a concentration between about 0, 1 pM and about 5 pM, preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM;

[0416] - Activin A at a concentration between about 200 ng / ml and about 300 ng / ml, preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml;

[0417] - midostaurin at a concentration between about 1 pM and about 5 pM, preferably between about 2 pM and about 3 pM, preferably of about 2,5 pM;

[0418] - foetal bovine serum (FBS) at a concentration between about 0,1% and about 5%, preferably between about 0,5% and about 1,5%, preferably of about 1%; and

[0419] - thrombopoietin (TPO), preferably human TPO, at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0420] In some embodiments, the differentiation medium, especially the MK differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with:

[0421] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine) at a concentration of between about 0,5% and about 2%;

[0422] - optionally transferrin; at a concentration of between about 300 pg / ml and about 400 pg / ml;

[0423] - glutamine, preferably L-glutamine, at a concentration of between about 3 and about 7 ng / ml;

[0424] StemRegenin 1 (SRI), at a concentration of between about 50 nM and about 150 nM;

[0425] SMER-28 at a concentration of between about 1 pM and about 5 pM;

[0426] CHIR99021 at a concentration between about 1 pM and about 3 pM; - Activin A at a concentration between about 200 ng / ml and about 300 ng / ml, preferably of about 250 ng / ml;

[0427] - midostaurin at a concentration between about 1 pM and about 5 pM;

[0428] - foetal bovine serum (FBS) at a concentration between about 10% and about 15%; and

[0429] - thrombopoietin (TPO) or eltrombopag, preferably human TPO, at a concentration of between about 20 ng / ml and about 40 ng / ml.

[0430] In some very particular embodiments, the differentiation medium, especially the MK differentiation medium of the invention comprises or consists of a base medium, preferably IMDM, supplemented with about 5 ng / mL glutamine, about 15% (v / v) fetal bovine serum, about 1% ITSX, about 2.27pM SMER28, about 330 pg / mL transferrin, about 30 ng / mL TPO, about 250 ng / mL activin A, about 1.5 pM CHIR 99021, about lOOnM SRI and about 2.5 pM midostaurin.

[0431] In some very particular embodiments, the differentiation medium, especially the MK differentiation medium of the invention comprises or consists of a base medium, preferably IMDM, supplemented with about 5 ng / mL glutamine, about 15% (v / v) fetal bovine serum, about 1% ITSX, about 2.27pM SMER28, about 30 ng / mL TPO, about 250 ng / mL activin A, about 1.5 pM CHIR 99021, about lOOnM SRI and about 2.5 pM midostaurin.

[0432] The present invention also relates to the use of the cell culture media according to the invention and as described above for the production and / or amplification of hematopoietic progenitors and / or the production of cells of the platelet lineage, such as megakaryocytes and platelets, in particular according to the processes of the invention described above.

[0433] The present invention also concerns the use of a differentiation culture medium such as described above for the differentiation of hematopoietic progenitor cells into megakaryocytes, and optionally platelets.

[0434] The present invention particularly concerns the use of a differentiation culture medium such as described herein for the differentiation of genetically modified hematopoietic progenitor cells into megakaryocytes, and optionally platelets.

[0435] The invention especially concerns the use of a culture medium comprising ITS-X (Insulin- Transferrin-Selenium -Ethanolamine), optionally Transferrin, foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin, for the differentiation of hematopoietic progenitor cells of the invention into megakaryocytes. In some aspects, the invention relates to a differentiation culture medium, in particular adapted for cells of the erythroid lineage. A medium adapted to the growth and / or differentiation of cells of the erythroid line is therefore a medium allowing the differentiation of hematopoietic progenitor cells into erythroid progenitors, erythroid precursors and erythrocytes as well as the multiplication of such cells. This differentiation culture medium may be called herein “erythroid differentiation culture medium”, “erythrocytes differentiation culture medium” or “Ery differentiation culture medium” or “Ery differentiation medium”.

[0436] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with insulin; transferrin; optionally hemin, glutamine, preferably L-glutamine; foetal bovine serum (FBS) or platelet lysate; an autophagy inducer, an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), a growth factor of the TGF-P family, erythropoietin, an indole alkaloid, one or more JAK2 inhibitor(s), an inhibitor of Bcl-XL, and optionally one or more FLU inhibitor(s).

[0437] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, Insulin-Transferrin-Selenium-Ethanolamine (ITS-X), transferrin; optionally hemin, glutamine, preferably L-glutamine; foetal bovine serum (FBS) or platelet lysate; an autophagy inducer, an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), a growth factor of the TGF-P family, erythropoietin, an indole alkaloid, one or more JAK2 inhibitor(s), an inhibitor of Bcl-XL, and optionally one or more FLU inhibitor(s).

[0438] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium, such as an IMDM medium or an equivalent medium, supplemented with ITS-X (Insulin-Transferrin-Selenium-Ethanolamine); transferrin; optionally hemin, glutamine, preferably L-glutamine; foetal bovine serum (FBS) or platelet lysate; an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28; an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021; a growth factor of the TGF-P family, preferably Activin A; erythropoietin; an indole alkaloid, preferably hirsutine; a JAK2 inhibitor, especially JAK2-IN-6 and / or pacritinib, an inhibitor of Bcl-XL, preferably WEHI- 539; and optionally a FLU inhibitor, preferably TK216 and / or anagrelide.

[0439] In some aspects, the invention concerns a differentiation medium, especially the Ery differentiation culture medium comprising or consisting essentially of a base medium such as an IMDM medium or an equivalent medium, ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), Transferrin, foetal calf serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, Activin A, CHIR99021, hirsutine, and a) WEHI-53; JAK2IN6 and pacritinib; and optionally TK216 and / or anagrelide or b) TK216 and anagrelide and optionally hemin.

[0440] In some embodiments, the invention concerns a differentiation medium, especially the Ery differentiation culture medium comprising or consisting essentially of a base medium such as an IMDM medium or an equivalent medium, ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), Transferrin, foetal calf serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, Activin A, CHIR99021, hirsutine, and WEHI-53; JAK2IN6 and pacritinib. This medium can typically be further supplemented with TK216 and / or anagrelide.

[0441] In some embodiments, the invention concerns a differentiation medium, especially an Ery differentiation culture medium comprising or consisting essentially of a base medium such as an IMDM medium or an equivalent medium, ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), Transferrin, foetal calf serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, Activin A, CHIR99021, hirsutine, TK216, anagrelide and optionally hemin.

[0442] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium such as an IMDM medium or an equivalent medium, supplemented with:

[0443] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,1% and about 10%;

[0444] - transferrin, at a concentration of between about 100 pg / ml and about 500 pg / ml,

[0445] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 50 ng / ml;

[0446] - an autophagy inducer, preferably SMER-28, at a concentration between 0, 1 pM and 20 pM;

[0447] - CHIR99021, at a concentration between about 0,1 pM and about 10 pM;

[0448] - Activin A, at a concentration between about 50 ng / ml and about 500 ng / ml;

[0449] - optionally hemin, at a concentration between about 1 pM and about 100 pM;

[0450] - foetal calf serum (FBS), at a concentration of between about 5% and about 30%; and

[0451] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml; - an indole alkaloid, preferably hirsutine, at a concentration of between about 10 pM and about 100 pM;

[0452] - one or more JAK2 inhibitor, especially JAK2-IN-6, at a concentration of between about 1 pg / mL and about 15 pg / mL and / or pacritinib, at a concentration of between about 1 nM and about 15 nM;

[0453] - a Bcl-XL inhibitor, preferably WEHI-539, at a concentration of between about 0.1 nM and about 10 nM; and

[0454] - optionally one or more FLU inhibitor, especially TK216 at a concentration of between about 10 ng / ml and about 100 ng / ml and / or anagrelide at a concentration of between about 1 pg / ml and about 50 pg / ml.

[0455] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium such as an IMDM medium or an equivalent medium, supplemented with:

[0456] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0457] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0458] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml, preferably between about 2,5 and about 7,5 ng / ml, preferably of about 5 ng / ml;

[0459] - an autophagy inducer, preferably SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0460] - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, more preferably of about 1,5 pM;

[0461] - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, more preferably of about 250 ng / ml;

[0462] - optionally hemin, at a concentration between about 1 pM and about 100 pM, preferably between about 10 pM and about 50 pM, more preferably between about 10 pM and about 30 pM, particularly of about 20pM ;

[0463] - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%; and - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0464] - an indole alkaloid, preferably hirsutine, at a concentration of between about 20 pM and about 100 pM, between about 40 pM and about 60 pM, preferably between about 20 pM and about 40 pM, preferably about 30 pM;

[0465] - one or more JAK2 inhibitor, especially JAK2-IN-6, at a concentration of between about 1 pg / mL and about 15 pg / mL, preferably between about 4 pg / mL and about 8 pg / mL, preferably about 6.3 pg / mL and / or especially pacritinib, at a concentration of between about 1 nM and about 15 nM, preferably between about 4 nM and about 8 nM, preferably about 6 nM;

[0466] - a Bcl-XL inhibitor, preferably WEHI-539, at a concentration of between about 0.1 nM and about 10 nM, preferably between about 0.5 nM and about 5 nM, preferably about 1.1 nM; and

[0467] - optionally one or more FLU inhibitor, preferably TK216 at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml and / or anagrelide at a concentration of between about 1 pg / ml and about 50 pg / ml.

[0468] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium such as an IMDM medium or an equivalent medium, supplemented with:

[0469] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0470] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 and about 7,5 ng / ml, preferably of about 5 ng / ml;

[0471] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0472] - an autophagy inducer, preferably SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0473] - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, more preferably of about 1,5 pM;

[0474] - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, more preferably of about 250 ng / ml; - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%; and

[0475] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0476] - an indole alkaloid, preferably hirsutine, at a concentration of between about 20 pM and about 100 pM, between about 40 pM and about 60 pM, preferably between about 20 pM and about 20 pM, preferably about 30 pM;

[0477] - one or more JAK2 inhibitor, especially JAK2-IN-6, at a concentration of between about 1 pg / mL and about 15 pg / mL, preferably between about 4 pg / mL and about 8 pg / mL, preferably about 6.3 pg / mL and / or especially pacritinib, at a concentration of between about 1 nM and about 15 nM, preferably between about 4 nM and about 8 nM, preferably about 6 nM;

[0478] - a Bcl-XL inhibitor, preferably WEHI-539, at a concentration of between about 0.1 nM and about 10 nM, preferably between about 0.5 nM and about 5 nM, preferably about 1.1 nM; and

[0479] - optionally one or more FLU inhibitor, preferably TK216 or anagrelide at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0480] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium such as an IMDM medium or an equivalent medium, supplemented with:

[0481] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0482] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0483] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 and about 7,5 ng / ml, preferably of about 5 ng / ml;

[0484] - an autophagy inducer, preferably SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0485] - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, more preferably of about 1,5 pM; - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, more preferably of about 250 ng / ml;

[0486] - hemin, at a concentration between about 1 pM and about 100 pM, preferably between about 10 pM and about 50 pM, more preferably between about 10 pM and about 30 pM, particularly of about 20pM ;

[0487] - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%; and

[0488] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0489] - an indole alkaloid, preferably hirsutine, at a concentration of between about 20 pM and about 100 pM, between about 40 pM and about 60 pM, preferably between about 20 pM and about 40 pM, preferably about 30 pM;

[0490] - one or more FLU inhibitor, preferably TK216 and / or anagrelide at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0491] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium such as an IMDM medium or an equivalent medium, supplemented with:

[0492] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0493] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0494] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 ng / ml and about 7,5 ng / ml, preferably of about 5 ng / ml;

[0495] - SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and

[0496] 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0497] - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM;

[0498] - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml; - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20% , preferably of about 15%;

[0499] - optionally hemin, at a concentration between about 1 pM and about 100 pM, preferably between about 10 pM and about 50 pM, more preferably between about 10 pM and about 30 pM, particularly of about 20pM ;

[0500] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0501] - hirsutine, at a concentration of between about 20 pM and about 100 pM, or between about 40 pM and about 60 pM, preferably between about 20 pM and about 40 pM, preferably about 30 pM;

[0502] - JAK2-IN-6, at a concentration of between about 1 pg / mL and about 15 pg / mL, preferably between about 4 pg / mL and about 8 pg / mL, preferably about 6.3 pg / mL;

[0503] - pacritinib, at a concentration of between about 1 nM and about 15 nM, preferably between about 4 nM and about 8 nM, preferably about 6 nM;

[0504] - WEHI-539, at a concentration of between about 0.1 nM and about 10 nM, preferably between about 0.5 nM and about 5 nM, preferably about 1.1 nM; and

[0505] - optionally a FLU inhibitor, preferably TK216 or anagrelide, preferably TK216, at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0506] In some embodiments, the differentiation medium, especially the Ery differentiation medium does not comprise hemin.

[0507] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base culture medium such as an IMDM medium or an equivalent medium, supplemented with:

[0508] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0509] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0510] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 ng / ml and about 7,5 ng / ml , preferably of about 5 ng / ml; - SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM;

[0511] - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM;

[0512] - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml;

[0513] - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%;

[0514] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0515] - hirsutine, at a concentration of between about 20 pM and about 100 pM, preferably between about 40 pM and about 60 pM, preferably about 30 pM;

[0516] - JAK2-IN-6, at a concentration of between about 1 pg / mL and about 15 pg / mL, preferably between about 4 pg / mL and about 8 pg / mL, preferably about 6.3 pg / mL;

[0517] - pacritinib, at a concentration of between about 1 nM and about 15 nM, preferably between about 4 nM and about 8 nM, preferably about 6 nM; and

[0518] - WEHI-539, at a concentration of between about 0.1 nM and about 10 nM, preferably between about 0.5 nM and about 5 nM, preferably about 1.1 nM.

[0519] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base culture medium such as an IMDM medium or an equivalent medium, supplemented with:

[0520] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0521] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0522] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 and about 7,5 ng / ml, preferably of about 5 ng / ml;

[0523] - SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM; - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM;

[0524] - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml;

[0525] - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%;

[0526] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0527] - hirsutine, at a concentration of between about 20 pM and about 100 pM, preferably between about 40 pM and about 60 pM, preferably about 30 pM;

[0528] - JAK2-IN-6, at a concentration of between about 1 pg / mL and about 15 pg / mL, preferably between about 4 pg / mL and about 8 pg / mL, preferably about 6.3 pg / mL;

[0529] - pacritinib, at a concentration of between about 1 nM and about 15 nM, preferably between about 4 nM and about 8 nM, preferably about 6 nM; and

[0530] - WEHI-539, at a concentration of between about 0.1 nM and about 10 nM, preferably between about 0.5 nM and about 5 nM, preferably about 1.1 nM.

[0531] This medium can typically be further supplemented with TK216, especially at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml.

[0532] In some embodiments, the differentiation medium, especially the Ery differentiation medium comprises or consists of a base medium such as an IMDM medium or an equivalent medium, supplemented with:

[0533] - ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), at a concentration of between about 0,5% and about 5% of preferably between about 0,5% and about 1,5%, preferably of about 1%;

[0534] - transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml, preferably between about 250 pg / ml and about 400 pg / ml, preferably about 330 pg / ml;

[0535] - glutamine, preferably L-glutamine, at a concentration of between about 1 ng / ml and about 10 ng / ml , preferably between about 2,5 and about 7,5 ng / ml , preferably of about 5 ng / ml;

[0536] - SMER-28, at a concentration between 0,1 pM and 10 pM, preferably between 0.5 pM and 5 pM, more preferably between 1 pM and 3 pM, and particularly preferably of about 2,27 pM; - CHIR99021 , at a concentration between about 0, 1 pM and about 5 pM , preferably between about 1 pM and about 2 pM, preferably of about 1,5 pM;

[0537] - Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml , preferably between about 225 ng / ml and about 275 ng / ml, preferably of about 250 ng / ml;

[0538] - foetal calf serum (FBS), at a concentration of between about 5% and about 25%, preferably between about 10% and about 20%, preferably of about 15%;

[0539] - optionally hemin, at a concentration between about 1 pM and about 100 pM, preferably between about 10 pM and about 50 pM, more preferably between about 10 pM and about 30 pM, particularly of about 20pM ;

[0540] - erythropoietin (EPO), preferably human EPO, at a concentration of between about 1 U / ml and about 10 U / ml, preferably between about 2 U / ml and about 5 U / ml, preferably about 3 U / ml;

[0541] - hirsutine, at a concentration of between about 20 pM and about 100 pM, or between about 40 pM and about 60 pM, preferably between about 20 pM and about 40 pM, preferably about 30 pM;

[0542] - TK216, at a concentration of between about 20 ng / ml and about 40 ng / ml, preferably between about 25 ng / ml and about 35 ng / ml, preferably about 30 ng / ml; and

[0543] - anagrelide, at a concentration of between about 1 pg / ml and about 100 pg / ml, preferably between about 1 pg / ml and about 20 pg / ml, preferably about 10 ng / ml.

[0544] In some very particular embodiments, the differentiation medium, especially the Ery differentiation medium of the invention comprises or consists of a base medium, preferably IMDM, supplemented with about 5ng / mL glutamine, about 15% (v / v) fetal bovine serum, about 1% ITSX, about 2.27pM SMER28, about 330 pg / mL transferrin, about 3 U / mL EPO, about 250 ng / mL activin A, about 1.5 pM CHIR 99021, about 50 pM hirsutine, about 6.3 pg / mL JAK2IN6, about 1.1 nM WEHI-539 and about 6 nM pacritinib.

[0545] In some very particular embodiments, the differentiation medium, especially the Ery differentiation medium of the invention comprises or consists of a base medium, preferably IMDM, supplemented with about 5ng / mL glutamine, about 15% (v / v) fetal bovine serum, about 1% ITSX, about 2.27pM SMER28, about 330 pg / mL transferrin, about 3 U / mL EPO, about 250 ng / mL activin A, about 1.5 pM CHIR 99021, about 50 pM hirsutine, about 6.3 pg / mL JAK2IN6, about 1.1 nM WEHI-539, about 6 nM pacritinib, and optionally, preferably after at least two days , about 30 nM TK216. Preferably, the differentiation medium, especially the Ery differentiation medium is supplemented with TK216 or anagrelide to induce maturation of erythroid precursor into erythrocytes. Preferably, this supplementation occurs after 2, 3, 4 or 5 days of culture in the differentiation medium.

[0546] In some very particular embodiments, the differentiation medium, especially the Ery differentiation medium of the invention comprises or consists of a base medium, preferably IMDM, supplemented with about 5ng / mL glutamine, about 15% (v / v) fetal bovine serum, about 1% ITSX, about 2.27pM SMER28, about 330 pg / mL transferrin, about 3 U / mL EPO, about 250 ng / mL activin A, about 1.5 pM CHIR 99021, about 50 pM hirsutine, about 30 nM TK216, about 10 pg / mL anagrelide and about 20 pM hemin.

[0547] The present invention also relates to the use of the cell culture media according to the invention and as described above for the production and / or amplification of hematopoietic progenitors and / or the production of cells of the erythroid lineage, especially erythrocytes, in particular according to the processes of the invention described above.

[0548] The present invention also concerns the use of a differentiation medium, especially an Ery differentiation culture medium such as described above for the differentiation of hematopoietic progenitor cells into erythroid progenitors, such as erythroblasts, especially orthochromatic erythroblasts, and optionally erythrocytes.

[0549] The present invention particularly concerns the use of a differentiation medium, especially an Ery differentiation culture medium such as described herein for the differentiation of genetically modified hematopoietic progenitor cells into erythroid progenitors, such as erythroblasts, especially orthochromatic erythroblasts, and optionally erythrocytes.

[0550] The invention especially concerns the use of a culture medium comprising or consisting of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X (Insulin-Transferrin-Selenium-Ethanolamine), Transferrin, foetal calf serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, Activin A, CHIR99021, hirsutine, and a) WEHI-53; JAK2IN6 and pacritinib; and optionally TK216 and / or anagrelide or b) TK216 and anagrelide and optionally hemin, , for the differentiation of hematopoietic progenitor cells into erythroid progenitors, such as erythroblasts, especially orthochromatic erythroblasts.

[0551] Especially, the invention relates to the use of: a culture medium comprising or consisting of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X (Insulin-Transferrin-Selenium- Ethanolamine), Transferrin, foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin, for the differentiation of hematopoietic progenitor cells into megakaryocytes optionally into platelets; or a culture medium comprising or consisting of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X (Insulin-Transferrin-Selenium- Ethanolamine), Transferrin, foetal calf serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, Activin A, CHIR99021, hirsutine, and a) WEHI-53; JAK2IN6 and pacritinib; and optionally TK216 and / or anagrelide or b) TK216 and anagrelide and optionally hemin, for the differentiation of hematopoietic progenitor cells into erythroid precursors, optionally into erythrocytes.

[0552] Preferably, the culture medium envisioned herein (amplification and / or differentiation medium) does not include vascular endothelial growth factor (VEGF), IL-6, bone morphogenetic protein (BMP), cysteine, Rho kinase inhibitor, FLT3-ligand and / or hydrocortisone.

[0553] Preferably, the amplification medium and / or differentiation medium do(es) not comprise mesenchymal stem cells (MSCs).

[0554] In some aspect, the amplification and / or differentiation media further comprises a DNTM3 A inhibitor. In some embodiments, the DNTM3A inhibitor is not RG108.

[0555] Alternatively, especially when the invention uses a genetically modified hematopoietic cell as described herein, the amplification and / or differentiation media does not comprise a DNTM3A inhibitor, in particular RG108.

[0556] In some embodiments, the amplification medium or differentiation medium is comprised in a bioreactor.

[0557] Kit

[0558] Any of the DNMT3 A inhibitors, hematopoietic cells and culture media described herein may be comprised in a kit.

[0559] In an aspect, the present invention relates to a kit comprising: a nucleic acid molecule, an expression cassette and / or a vector according to the invention; preferably a vector encoding a shRNA that is a DNMT3 A inhibitor optionally, hematopoietic cells, preferably hematopoietic progenitor cells; optionally, an amplification culture medium and / or differentiation medium according to the invention, or suitable compounds for preparing such media; and optionally, a guide containing instructions for using such a kit, especially instructions for transforming the hematopoietic progenitor cells with the nucleic acid molecule, an expression cassette and / or a vector, culturing the hematopoietic cells, amplifying the genetically modified hematopoietic progenitor cells and / or differentiating such cells into cells of the platelet or erythroid lineage.

[0560] In another aspect, the present invention relates to a kit comprising: genetically modified progenitor cells according to the invention; optionally, an amplification culture medium and / or differentiation medium according to the invention; or suitable compounds for preparing such medium, optionally, a guide containing instructions for using such a kit, especially instructions for culturing the hematopoietic cells, amplifying the genetically modified hematopoietic progenitor cells and / or differentiating such cells into cells of the platelet or erythroid lineage.

[0561] Where appropriate, the kit may comprise suitably aliquoted components. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also may generally contain a second, third or other additional container into which the additional component(s) may be separately placed.

[0562] The present invention also relates to the use of a kit according to the invention to produce hematopoietic cells, preferably selected from the group consisting of MEP, BFU-E, CFU-E, proerythroblasts, basophil erythroblasts, polychromatophil erythroblasts, orthochromatic erythroblasts, reticulocytes, erythrocytes, megakaryoblasts, megakaryocytes and platelets, preferably platelets or erythrocytes., in particular according to the processes of the invention described above.

[0563] The present invention also relates to the use of a kit according to the invention to produce cells of the platelet lineage, especially megakaryocytes and / or platelets said kit comprising: optionally, genetically modified hematopoietic progenitor cells according to the invention or means for obtaining such cells;

[0564] - optionally, an amplification medium as described herein; or suitable compounds for preparing such medium,

[0565] - a “megakaryocyte differentiation medium” as described herein; or suitable compounds for preparing such medium, optionally, a guide containing instructions for using such a kit, especially instructions for culturing the hematopoietic cells, amplifying the genetically modified hematopoietic progenitor cells and / or differentiating such cells into cells of the platelet lineage, preferably megakaryocytes and / or platelets.

[0566] The present invention also relates to the use of a kit according to the invention to produce cells of the erythroid lineage, especially erythroid precursors and / or erythrocytes, said kit comprising: optionally, genetically modified hematopoietic progenitor cells according to the invention or means for obtaining such cells;

[0567] - optionally, an amplification medium as described herein; or suitable compounds for preparing such medium,

[0568] - a “erythrocytes differentiation medium” as described herein; or suitable compounds for preparing such medium, optionally, a guide containing instructions for using such a kit, especially instructions for culturing the hematopoietic cells, amplifying the genetically modified hematopoietic progenitor cells and / or differentiating such cells into cells of the platelet erythroid, preferably erythroid precursors and / or erythrocytes.

[0569] In particular, the present kit may comprise any ingredients required to produce the culture medium in dehydrated form, in a common container or in separate containers.

[0570] The kit according to the invention may also comprise various materials and reagents for use in accordance with the present invention in suitable containers and packaging materials, notably pipettes, tubes or bottles.

[0571] The kit may also comprise any means for collecting a biological sample such as blood, for example a syringe.

[0572] Bioreactor

[0573] The invention also relates to a bioreactor comprising hematopoietic progenitor cells in the presence of a DNMT3 A inhibitor.

[0574] Typically, the bioreactor comprises the genetically modified hematopoietic cells or the immortal cell line described herein, optionally with a suitable culture medium. Particularly, the invention also relates to a bioreactor comprising the amplification medium or differentiation medium described herein.

[0575] The invention also relates to the use of a bioreactor for the culture of hematopoietic progenitor cells in the presence of a DNMT3 A inhibitor. The invention also concerns to the use of a bioreactor for the culture of genetically modified hematopoietic cells of the invention in an amplification medium or differentiation medium of the invention.

[0576] As used herein, "bioreactor" means a device or system that supports the growth of cells or of tissues; more particularly it enables the amplification differentiation and maintenance of hematopoietic cells.

[0577] In some embodiments, the total cell density of the genetically modified cells of the invention in the bioreactor is at least 10000 cells / mL; 25000 or 50000 cells / mL.

[0578] Preferably, the hematopoietic cell culture in the bioreactor is a 3D cell culture.

[0579] A bioreactor system may comprise at least one bioreactor, bioreactor tank, or reactor chamber. For example, a bioreactor system may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 reactor chambers. For example, the bioreactor is a PBS-MINI MagDrive bioreactor (Stemcells technologies). Alternatively, the bioreactor is a hollow-fiber bioreactor.

[0580] A bioreactor chamber may have an internal volume suitable for large-scale cell culture. A reactor chamber may have an internal volume of at least about 500 ml, 1 L, 10L, 50L, 100L, 500L, lOOOL, 5000L or 10000L.

[0581] In some embodiments, in the bioreactor or methods of the invention, the concentration of dissolved O 2 and CO 2 are monitored in the cell culture. The CO2 and / or O2 content is preferably controlled during the cell culture steps. Preferably, the CO2 content is between 2% and 10%, between 3% and 7% or between 4% and 6%, and is preferentially about 5%.

[0582] In some embodiments, in the bioreactor, the cell culture steps, namely the amplification, differentiation and / or maturation steps, are preferably performed at a temperature suitable for the multiplication and / or differentiation of hematopoietic cells. Preferably, the temperature is about 37°C.

[0583] In some embodiments, in the bioreactor or methods of the invention, the culture medium of the hematopoietic cells is exchanged about every other day if needed.

[0584] All references cited in this application, including journal articles or abstracts, published patent applications, granted patents or any other reference, are fully incorporated here by reference, which includes all results, tables, figures and texts presented in these references. BRIEF DESCRIPTION OF THE DRAWINGS

[0585] Figure 1. A. Cell cycle kinetic: Day 1, Day 2 and Day 3. B. MGG staining of the immortalized cells at different time points (D46, D62, D84, D125). C. Amplification curves of shDNMT3A cells cultured in amplification medium, cells transduced with a shScramble and un-transduced CD34+ cells. D. Heatmap comparing shDNMT3A cells cultured in amplification medium with HSC, CMP, MEP, MK and Ery cells. E. Pictures of hematopoietic assay after 8 days of culture; CFU- MK like and progenitor colonies. F. Hematopoietic assays outcome in terms of total CFU in function of the addition of EPO, TPO or EPO and TPO. G. Hematopoietic assays outcome in terms of CFUMK like in function of the addition of EPO, TPO or EPO and TPO. H. Flow cytometry analyses of the hematopoietic assays in function of the addition of EPO, TPO or EPO and TPO. CD41a positive cells are engage in megakaryopoiesis while CD235A+CD41A+ cells are more MEPs. I. Over-expressed membrane markers of shDNMT3A cells cultured in amplification medium.

[0586] Figure 2. Functional in vivo assessment was conducted by injecting undifferentiated cells in irradiated NSG mice in retroorbital cavity. After 10 days, bone marrow (and blood) was (were) harvested and analysed through cytometry markers discriminating for human and mice megakaryopoiesis (h CD41a and h+m CD42a) and human erythropoiesis (h CD235a and h CD71).

[0587] Figure 3. A. MGG staining of shDNMT3A cells cultured in MK differentiation medium at D4. B. Immunofluorescence staining of shDNMT3 A cells cultured in MK differentiation medium at day 3. Left panel: CD41a in green and DAPI in blue, Right panel: CD41a in green, a -tubulin in red and DAPI in blue. C. This histogram shows the relative DNA content of shDNMT3A cells (PRIME cells) cultured in MK differentiation medium on day four. This was determined using flow cytometry with immunoperoxidase (IP) staining. Five distinct ploidy classes, or peaks, were identified. The ploidy values are exact doublings of the modal 2N value. D. Flow cytometry analyses of shDNMT3A cells cultured in MK differentiation medium. CD41a positive cells are MK while CD42a+CD41A+ cells are matured MKs. E. Platelets are obtained through fragmentation of fully mature megakaryocytes by repeated up and down pipetting. Size and shape, in addition to CD41a and CD42a markers are used to discriminate platelets from intact megakaryocytes and debris. Representative dot plot after MK fragmentation procedure, in blue CD42a+CD41a+ platelets, in red CD42a-CD41a- debris, in orange counting beads. F. Kinetics of platelet generation per seeded cell at DO. The counting beads allow platelets to be counted by flow cytometry. G. P-selectin (CD62P) is a platelet activation marker. After addition of thrombin in the differentiation medium, platelet CD62P expression was assessed by a flow cytometer. In orange: control cells cultured without thrombin and stained with isotype antibodies, in blue: cells cultured without thrombin and stained with CD62P antibodies, in bright red: cells cultured with thrombin and stained with CD62P antibodies.

[0588] Figure 4. A. Cell cycle kinetic: day 1, day 2, day 3 and day 4. B. Kinetics of enucleation percentages C. Diameters of the produced reticulocytes. D. Erythroid waterfall cells stained with CD235a, CD71, CD35, CD49d and BAND3 antibodies. Plots of SSC-A versus CD235a. Plots of CD71 versus CD36 of all CD235a cells. Plots of CD49d versus Band3 of all CD235a cells, positivity and then analysed. E. MGG staining at Day 1, Day 2, Day 3 and Day 4 of erythroid differentiation.

[0589] Figure 5. Kinetics of MK differentiation of UT7 EPO cells between Day 4 and Day 7. (A) Histogram representing the evolution of cell populations during MK differentiation, obtained by manual counting on May-Grunwald Giemsa-stained cytospins (B). (C) Representative diagram showing the characterization of the membrane proteins CD42a and CD41a after 7 days of differentiation. Results are expressed as mean percentage ± standard deviation, n=5 (D4), n=6 (D5), n=4 (D6) et n=6 (D7).

[0590] Figure 6. Kinetics of erythroid differentiation of UT7 EPO cells between Day 1 and Day 4. (A) Histogram representing the evolution of cell populations during erythroid differentiation UT7 EPO, obtained by manual counting on May-Grunwald Giemsa-stained cytospins (B). (C) Representative diagram showing the characterization of the membrane proteins CD235a and CD41a after 4 days of differentiation. Results are expressed as mean percentage ± standard deviation, n=4 (DI to D3) and n=7 (D4).

[0591] Figure 7. A. Doubling time of cells amplificated in 2D or 3D; B. Kinetics of MGG staining of PRIME cells cultured in MK differentiation medium from DI to D6.

[0592] Figure 8. A. Flow cytometry analyses of PRIME cells cultured in MK differentiation medium. CD41a positive cells are MK while CD42a+CD41A+ cells are matured MKs. B. Histogram of relative DNA content in PRIME cells cultured static or bioreactor in MK differentiation medium at day 3, determined by FCM. Three distinct peaks or ploidy classes of cells are identified. Ploidy values are exact doublings of the modal 2N value; C. MGG staining of MK and platelets obtained through fragmentation of fully mature megakaryocytes by repeated up and down pipetting; D. Histogram of the expression of the CD41a marker after MK fragmentation procedure; E. Aggregation assay. After addition of thrombin, fibrinogen and calcium, the platelets were incubated at 37°C for 5min. The clot appeared after 5 min (n=6).

[0593] EXAMPLES Other features and advantages of the invention will appear more clearly upon reading the following illustrative and non-limiting examples.

[0594] Example 1. Generation of an immortalized cell line for the production of platelets and

[0595] Material and Methods

[0596] Lentiviral vector

[0597] The inventors used the vector pLVUTHshGATAl-tTR-KRAB a gift from Patrick Aebischer & Didier Trono (Addgene). The inventors replaced shGATAl by inserting between Xmal-Mlul a shRNA targeting DNMT3 A (shDNMT3 A) of SEQ ID NO: 1.

[0598] Vector production

[0599] The inventors produced recombinant lentiviruses using transient transfection of human embryonic kidney (HEK) 293T cells (ATCC CRL-11268). HEK 293T cells were cultured in DMEM (Invitrogen, USA) supplemented with 10% FCS (Brunschwig, Switzerland). For transfection, the inventors plated 2.5xl06cells per 75 cm2 T-flasks. Next day, the 293T cells were transfected with 20 pg of vector DNA, 15 pg of psPAX2 and 5 pg of pMD2G using lipofectamine LTX plus reagent (Invitrogen). After 12-16 hrs, the medium was changed and the inventors harvested medium containing recombinant lentivectors 36-40 hrs later. Cellular debris were removed by centrifugation (2000rpm for 5min) and filtration through 0.45pm filter unit (Milliopore), LVs were concentrated by centrifugation into Amicon® Ultra Centrifugal Filter, 10 kDa MWCO (Merck). The concentrated LVs were used fresh.

[0600] Generation of immortalized line

[0601] Human CD34+ hematopoietic stem cells isolated from apheresis or cord blood were sorted with CD34 magnetic microbeads using MACS columns according to the manufacturer’s protocol (Miltenyi Biotec). The CD34+cells (104cells / ml) were cultured in a primary medium, IMDM (Sigma) containing 5ng / mL glutamine, 5% (v / v) human S / D AB plasma (EFS), 10 pg / mL insulin, 2 U / mL heparin (Panpharma), 0.1 mM dexamethasone, 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma), 3 U / mL EPO (Binocrit), lOO ng / mL stem cell factor (SCF) and S.ng mU1IL-3 (all from Miltenyi Biotec) for 48 hours.

[0602] They were then concentrated to 105cells / ml in primary medium supplemented with 10 pg / mL rapamycin and 4 pg / mL polybrene and transduced twice (two rounds of 24 hours) with the lentiviral vector pLVUTHshDNMT3a-tTR-KRAB. The cells were washed thoroughly and seeded at 104cells / ml in an expansion medium supplemented with 50 ng.ml-1 doxycycline (Sigma) to induce expression of shDNMT3A. From day 7 to day 30 they were maintained at a density of 1 * 105cells / mL at 37 °C, 5% CO2 in the expansion medium supplemented with doxycycline. Subsequently the cells were maintained in the expansion medium without doxycycline, seeded at a density of 0.5-1.5 x io5cells. ml'1at 37°C, 5% CO2 and split twice a week.

[0603] The genetically modified cell line comprising the Tet-inducible shDNMT3A expression system was named PRIME for Paris line of Megakaryocyte-Erythroid progenitors. PRIME cells described herein are from this genetically modified cell line.

[0604] Primary medium

[0605] The primary medium typically comprises or consists of IMDM (Sigma) containing 5ng / mL glutamine, 5% (v / v) human S / D AB plasma (EFS), lO pg / mL insulin, 2 U / mL heparin (Panpharma), 0.1 mM dexamethasone, 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma), 3 U / mL EPO (Binocrit), lOO ng / mL stem cell factor (SCF) and 5. ng ml1IL-3 (all from Miltenyi Biotec). This medium can be supplemented with 10 pg / mL rapamycin and 4 pg / mL polybrene. This medium can be supplemented with 50ng / mL doxycycline (Sigma).

[0606] Expansion medium

[0607] The expansion medium typically comprises or consists of IMDM (Sigma) containing 5ng / mL glutamine (Invitrogen), 5% (v / v) human S / D AB plasma (EFS), 10 pg / mL insulin, 2 U / mL heparin (Panpharma), 0.1 mM dexamethasone, 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma), 3 U / mL EPO (Binocrit). This medium can be supplemented with 50 ng / mL doxycycline (Sigma).

[0608] Differentiation in megakaryocytes (MK)

[0609] Transfected cells were seeded at 2.105c / ml in a differentiation medium adapted for hematopoietic cells of the platelet lineage and cultured during 7 days.

[0610] “MK medium” or “MK differentiation medium” typically comprises or consists of IMDM (Sigma) containing 5ng / mL glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma), 30 ng / mL TPO (Miltenyi), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021, lOOnM SRI, 2.5 pM midostaurin (all from Medchem).

[0611] “MK medium” or “MK differentiation medium” typically comprises or consists of IMDM (Sigma) containing 5ng / mL glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 30 ng / mL TPO (Miltenyi), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021, lOOnM SRI, 2.5 pM midostaurin (all from Medchem).

[0612] These two media can be used interchangeably for the differentiation of hematopoietic cells of the platelet lineage.

[0613] Differentiation in erythroid precursors (Ery)

[0614] Immortalized cells were seeded at 2.105c / ml in a differentiation medium for hematopoietic cells of the erythroid lineage and cultured during 4 days. “Ery medium” or “Ery differentiation medium” typically comprises or consists of IMDM (Sigma) containing 5ng / mL glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma), 3 U / mL EPO (Binocrit), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021 (Sigma), 50 pM hirsutine, 6.3 pg / mL JAK2IN6, 1.1 nM WEHI-539, 6 nM pacritinib, supplemented with 30 nM TK216 on day

[0615] 2 (all from Medchem).

[0616] Alternatively, the “Ery medium” or “Ery differentiation medium” comprises or consists of IMDM (Sigma) containing 5ng / mL glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 330 pg / mL transferrin (all from Sigma),

[0617] 3 U / mL EPO (Binocrit), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021 (Sigma), 50 pM hirsutine, 30 nM TK216, 10 pg / mL anagrelide and 20 pM hemin (all from Medchem).

[0618] These two media can be used interchangeably for the differentiation of hematopoietic cells of the erythroid lineage.

[0619] Flow cytometry

[0620] A sample of 3 x io5cells was incubated with antibodies in 70 pl of phosphate-buffered saline (PBS) (Sigma- Aldrich) containing 1% (v / v) human serum albumin (HSA) (LFB) (PBSA) for 1 h. The cells were washed twice with PHSA and then analysed on a Cytoflex flow cytometer using FloJow X software (Beckman Coulter).

[0621] Ery cells: CD235a-PC7 (Beckmann Coulter A71564) 1 / 100, CD49d-BV421 (Biolegend 304322) 5 / 200 CD36-KO525 (BD Optibuild 744986) 1 / 100, CD71-BV786 (BD horizon 563768) 1 / 100, Band3-PE (IBGRL 9439) 1 / 200, CD34-APC (Beckmann Coulter IM2472) 1 / 100 and CD105-PE Dazzle (Biolegend 323223) 1 / 200.

[0622] MK cells: CD41a APC (BD 15819778), CD42a PE (15899628) CD42b BV421 (15856459), CD62P FITC (10189912)

[0623] Legendscreen (700007) according to the manufacturer’s protocol (Biolegend).

[0624] Cell cycle: Hoechst 33342 (Medchem) was added to the culture medium of the cells at lOpg / ml. The cells were then incubated at 37°C. After 30 to 60 minutes, they were analysed without washing the media containing Hoechst.

[0625] PCR analysis

[0626] RNA (400 ng) was reverse transcribed into cDNA using reverse transcriptase kit (4374966 Invitrogen). Taqman fast advanced mix (4444558 Invitrogen) was used along Taqman expression assays used were : EPOR (Hs00959427_ml), TALI (Hs01097987_ml), GATA2 (Hs00231119_ml), MYB (Hs00920556_ml), LM02 (Hs00153473_ml), KIT (Hs00174029_ml), DNMT3A (Hs01027162_ml), BMI1 (Hs00995519_gl), KLF1 (Hs00610592_ml), Bcl211 (Cg04423839_ml), CDK2 (Hs01548894_ml), RUNX1T1 (Hs00231702_ml), FLU (Hs00956709_ml), GABPA (Hs01022023_ml), MPL

[0627] (Hs00180489_ml), GATA1 (Hs01085823_ml), NFE2 (Hs00232351_ml), HBB

[0628] (Hs00758889_sl), HBE (Hs00362216_ml), HBF (Hs0036113 l_gl), GAPDH (Hs02786624_gl), HPRT (Hs02800695_ml), TBP (Hs00427620_ml).

[0629] In vivo studies

[0630] N0D.Cg-PrkdcscidI12rgtmlWjl / SzJ (NSG) (Charles River, L’Abresle, France) were housed in the IRSN animal care facility. All experiments and procedures were performed in compliance with the French Ministry of Agriculture regulations for animal experimentation and approved by the local ethics committee (APAFIS approval number # 17559-2018111613396032v2 (2024)).

[0631] Mice, 5-8 weeks old and raised under sterile conditions, were sublethally irradiated with 2.4- 3.5 Grays from a 137Cs source (2.115 Gy / min) 24 h before cell injection. To ensure consistency between experiments, only female mice were used. Prior to transplantation, the mice were temporarily sedated with an intraperitoneal injection of ketamine and xylazine. Cells (4 xl06 per mouse were transplanted by retro-orbital injection in a volume of 100 pL using a 28.5 Gauge insulin needle). A total of 22 mice were used in this study. Animals were analysed 10 days after injection.

[0632] Results

[0633] Immortalized MEP

[0634] The inventors set out to generate immortalised hematopoietic progenitors, especially MEPs, using a Tet-inducible shDNMT3 A expression system. Apheresis or cord blood sorted CD34+ cells were cultured in amplification medium for 3 days, transduced with the shDNMT3A lentiviral suspension on day 4 and maintained in primary medium for 1 day. The following day, the cells were transferred to expansion medium containing doxycycline to induce shDNMT3 A expression and maintained in this medium. In expansion medium, the cells proliferated continuously for more than 180 days, well beyond the Hayflick limit, and cell cycle analyses were performed and showed that even after 3 days in culture, there were still cycling cells, i.e. G2-M phase (Fig. 1 A). Samples were frozen at regular intervals during this period. All samples were efficiently restored to culture after thawing. The genetically modified cell line comprising the Tet-inducible shDNMT3A expression system was named PRIME for Paris line of Megakaryocyte-Erythroid progenitors. The mean doubling time of the cells (Fig. IB) from day 30 to day 180 in continuous culture was 26.4+ / - 0.6h (n=68). Morphological analysis of PRIME cells showed no change in morphology over time (Fig. 1C), the immortalised cells expressed specific antigens on their membranes such as CD 164, NOTCH, CD71, CD47, CD110 (Fig. ID). The inventors performed scRNA sequencing of the shDNMT3 A cell line and compared the resulting data with a public scRNAseq database for HCS, CMP, MEP, Ery and MK (Fig. ID). The inventors performed haematopoietic assays by adding EPO, TPO or EPO and TPO, after 8 days of culture. There were two types of colonies: progenitor colonies and CFU-Mk (Fig. IE). There was no significant difference between the different conditions in terms of colony types (Fig. IF and 1G), but there were more CD235a+CD41a+ cells in the EPO-only condition and more cells expressing CD41a+CD235a- in the TPO-only condition.

[0635] After injection to sublethally immunocompromised mice (NSG), blood and bone marrow were analysed after 10 days, hCD41a+hCD42a+ and hCD235a+hCD71- were found (Fig 2) (n=22).

[0636] Megakaryopoiesis

[0637] To induce the development of the MK lineage, a specific MK differentiation medium was used containing several small molecules such as activin A, CHIR99021, TPO, SRI and midaustorin.

[0638] May-Grunwald-Giemsa stained cytospins from day 1 to day 7 (Fig. 3A) showed different levels of maturation, with immature MK (left) compared to mature MK (right). Immunofluorescence staining of MK with CD41a antibody (green) and DAPI (left) and with CD41a antibody (green), a-tubulin antibody (red) and DAPI (left). Ploidy analysis of PRIME- derived MKs (Fig. 3C) showed that they were of high ploidy levels with a maximum of 32N, noting that MKs derived from cord blood or adult CD34+ reached ploidy up to 16N and 32N, respectively (n=12). MK maturation was associated with an appearance and an increased expression of CD41a and of CD42a (Fig. 3D), leading to the presence of a CD41+CD42+ cell population, a phenotype defining mature MKs.

[0639] Proplatelet formation (Fig. 3B) was observed, after several up and down and filtration, cytometry analyses were performed and the data were analysed with platelet settings and counting beads (Fig 3E), the platelets CD41highCD42+ appeared in blue. The higher amount of platelet production was reached at day 6 with an average of 10 platelets per seeded cells at day 0 (n=3).

[0640] The functionality of these platelets was investigated using a thrombin activation assay. When thrombin is added to human platelets in vitro, it causes the platelets to secrete the contents of their storage granules, among other things, and the activated platelets therefore expressed CD62P on their membranes. As shown in Fig 3F, after thrombin activation, platelets expressed higher levels of CD62p on their membranes, indicating that they are functional (n=3). Platelets were therefore successfully produced from PRIME cells.

[0641] Erythropoiesis

[0642] To induce the development of the Ery lineage, a specific Ery differentiation medium was used containing several small molecules activin A, CHIR99021, SMER28, EPO, hirsutin, JAK2-IN-6, WEHI-539, pacritinib and optionally later supplemented with TK216. The inventors performed cell cycle analyses (Fig. 4A) and showed that after 4 days in culture, only cells in G0-G1 and sub-GO phases (i.e., reticulocytes) were present. The combination of CD235a, CD71, CD36, band 3, and a4 integrin (CD49d) expression levels can be used to characterize the kinetics of erythroid maturation. The inventors focused on these markers to demonstrate PRIME erythroid maturation. From day 0 to day 4, the level of CD235a increased, while the levels of CD36 and CD71 decreased dramatically. From day 2 to day 4, the expression of CD49d decreased and that of Band3 increased (Fig 4D). May-Grunwald-Giemsa stained cytospins were consistent with flow cytometry showing a huge increase in the number of reticulocytes at day 4 (Fig. 4E). Enucleation (Fig. 4B) reached up to 32.0+ / -6.1 % at day 4 (n=7) while the average diameter of the reticulocytes was around 13.6 + / -0.2 pm (n=436) (Fig 4C).

[0643] In conclusion, the inventors generated an immortalized, bipotent megakaryocyte-erythroid progenitor (MEP) cell line from genetically modified hematopoietic stem cells. Using a DNMT3A-targeting shRNA strategy combined with an inducible TET-ON lentiviral system, we achieved long-term expansion while preserving lineage plasticity. These MEPs have a robust capacity for bipotent differentiation and can efficiently differentiate into the erythroid or megakaryocytic lineage by modulating culture conditions.

[0644] Example 2. The differentiation medium can be used on other hematopoietic cell lines

[0645] Material and Methods

[0646] UT-7 / EPO

[0647] The UT-7 cell line (Komatsu et al., 1997) was derived from a 64-year-old male patient with acute megakaryoblastic leukaemia. This multipotent cell line is dependent on cytokines and growth factors. These factors include IL-3, GM-CSF, and EPO.

[0648] UT7 EPO amplification

[0649] UT7 EPO cells are amplified in an EPO-enriched culture medium, which is necessary for their proliferation. Since they are leukemia cells, they are naturally immortalized. The medium is changed twice a week, and the cells are cultured in flasks (37°C, 5% CO2).

[0650] To prepare 5 mL of the amplification medium for UT7 EPO cells, 4.5 mL of MEM were supplemented with glutamine and FBS, as well as 2 units / mL of EPO.

[0651] Cell differentiation

[0652] The amplified cells are deposited in a 24-well plate at a concentration of 200,000 cells per millilitre of differentiation medium. For erythroid differentiation, the cells are observed from days one to four. For megakaryocytic differentiation, the kinetics are studied from days four to seven.

[0653] Differentiation in megakaryocytes (MK) Transfected cells were seeded at 2.105 c / ml until day 7 in a differentiation medium adapted for hematopoietic cells of the platelet lineage.

[0654] “MK medium” or “MK differentiation medium” typically comprises or consists of IMDM (Sigma) containing 5ng.ml-l glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 30 ng.ml-l TPO (Miltenyi), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021, lOOnM SRI, 2.5 pM midostaurin (all from Medchem).

[0655] Differentiation in erythroid precursors (Ery)

[0656] Immortalized cells were seeded at 2.105 c / ml until day 4 in a differentiation medium for hematopoietic cells of the erythroid lineage.

[0657] “Ery medium” or “Ery differentiation medium” typically comprises or consists of IMDM (Sigma) containing 5ng.ml-l glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 330 pg.ml-1 transferrin (all from Sigma), 3 U.ml-1 EPO (Binocrit), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021 (Sigma), 50 pM hirsutine, 6.3 pg / mL JAK2IN6, 1.1 nM WEHI-539, 6 nM pacritinib, supplemented with 30 nM TK216 on day 2 (all from Medchem).

[0658] Alternatively, the “Ery medium” or “Ery differentiation medium” comprises or consists of IMDM (Sigma) containing 5ng.ml-l glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 330 pg.ml-1 transferrin (all from Sigma), 3 U.ml-1 EPO (Binocrit), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021 (Sigma), 50 pM hirsutine, 30 nM TK216, 10 pg / mL anagrelide and 20 pM hemin (all from Medchem).

[0659] These two media can be used interchangeably for the differentiation of hematopoietic cells of the erythroid lineage.

[0660] Results

[0661] Megakaryopoiesis

[0662] To induce megakaryocyte lineage development, the inventors used a differentiation medium specific to megakaryocytes containing activin A, CHIR99021, thrombopoietin (TPO), SRI, and midaustorin.

[0663] On day 7, May-Grunwald-Giemsa-stained cytospins revealed various stages of maturation, including immature and mature megakaryocytes (Figure 5). The number of cells was as follows: n = 5 on day 4; n = 6 on day 5; n = 4 on day 6; and n = 6 on day 7. Megakaryocyte maturation was associated with the appearance of CD41a and CD42a, resulting in a CD41+CD42+ cell population, which is a defining characteristic of mature megakaryocytes.

[0664] Erythropoiesis To induce erythroid lineage development, the inventors used the differentiation medium specific to erythroid cells. As mentioned above, this medium contained several small molecules, including activin A, CHIR99021, SMER28, EPO, hirsutin, JAK2-IN-6, WEHI-539, and pacritinib. They later supplemented this medium with TK216.

[0665] On day 4, the inventors examined a large population of CD235a+CD41a- cells. May- Grunwald-Giemsa-stained cytospins were consistent with flow cytometry results, indicating a significant increase in reticulocytes by day 4 (see Fig. 4E). Enucleation reached 13.0% ± 6.0% by day 4 (n = 7) (Figure 6).

[0666] In conclusion, these differentiation protocols consistently produce reticulocytes or megakaryocytes when applied to UT7 cells. This is a breakthrough, as previous attempts with this cell line had failed to produce these cells. These results demonstrate the protocol's reliability and reproducibility in various cellular contexts.

[0667] Example 3. Scaling up in Bioreactor

[0668] Material and Methods

[0669] To produce platelets in larger quantities, the inventors improved the culture method of PRIME cells by moving from 2D (culture Flasks of 15ml, or well plates, without agitation) to 3D (Bioreactor, 300-500 ml, with agitation) culture. For 3D culture, a PBS-MINI MagDrive Bioreactor (PBS) was used. This bioreactor has a small magnetic propeller that keeps the cells in suspension (30 rpm). The cells were cultured in the same medium (i.e., amplification and differentiation media) as in 2D culture. After differentiation, the cells were analysed by cytology, flow cytometry.

[0670] The same protocols and parameters (i.e., medium, cells concentration, 37°C, 5% CO2) were used to culture the cells in 2D and 3D. This enabled to compare the bioreactor results with those of the 2D culture and determine if the characteristics were similar.

[0671] Expansion medium

[0672] The expansion medium typically comprises or consists of IMDM (Sigma) containing Sng.mE1glutamine (Invitrogen), 5% (v / v) human S / D AB plasma (EFS), lO pg.mE1insulin, 2U.mf1heparin (Panpharma), 0.1 mM dexamethasone, 2.27pM SMER28, 330 pg. ml1transferrin (all from Sigma), 3 U.mE1EPO (Binocrit). This medium can be supplemented with SOng.mE1doxycycline (Sigma). The cells were seeded at 25000 or 50000 cells / ml.

[0673] Differentiation in megakaryocytes (MK)

[0674] Transfected cells were seeded at 2.105c / ml until day 7 in a differentiation medium adapted for hematopoietic cells of the platelet lineage. “MK medium” or “MK differentiation medium” typically comprises or consists of IMDM (Sigma) containing 5ng.ml1glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, SO ng.mE1TPO (Miltenyi), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021, lOOnM SRI, 2.5 pM midostaurin (all from Medchem). The cells were seeded at 200000 cells / ml.

[0675] Differentiation in erythroid precursors (Ery)

[0676] Immortalized cells were seeded at 2.105c / ml until day 4 in a differentiation medium for hematopoietic cells of the erythroid lineage.

[0677] “Ery medium” or “Ery differentiation medium” typically comprises or consists of IMDM (Sigma) containing Sng.mE1glutamine (Invitrogen), 15% (v / v) fetal bovine serum (Hyclone), 1% ITSX (Invitrogen), 2.27pM SMER28, 330 pg.mE1transferrin (all from Sigma), 3 U.mE1EPO (Binocrit), 250 ng / mL activin A (Proteintech), 1.5 pM CHIR 99021 (Sigma), 50 pM hirsutine, 30 nM TK216, 10 pg / mL anagrelide and 20 pM hemin (all from Medchem). The cells were seeded at 200000 cells / ml.

[0678] These two media can be used interchangeably for the differentiation of hematopoietic cells of the erythroid lineage.

[0679] Flow cytometry

[0680] A sample of 3 x io5cells was incubated with antibodies in 70 pl of phosphate-buffered saline (PBS) (Sigma- Aldrich) containing 1% (v / v) human serum albumin (HSA) (LFB) (PBSA) for 1 h. The cells were washed twice with PHSA and then analysed on a Cytoflex flow cytometer using FloJow X software (Beckman Coulter).

[0681] MK cells: CD41a APC (BD 15819778), CD42a PE (15899628) CD42b BV421 (15856459), CD62P FITC (10189912)

[0682] Cell cycle: Hoechst 33342 (Medchem) was added to the culture medium of the cells at lOpg / ml. The cells were then incubated at 37°C, after 30 to 60 minutes they were analyzed without washing the media containing Hoechst. Propidium Oidide (Medchem) was also used, a permeabilization was performed with 70% ethanol at 0° Celsius during more than 30 min. The cells were then whased and incubated at 37°C, after 30 to 60 minutes with Propidium iodide, they were analyzed without washing the media containing Propidium iodide.

[0683] Results

[0684] The mean doubling time of the cells (Fig. 7) in static culture was 29.5 + / -3.6 h (n=4) while it was 26.1 + / -8.0 h (n=4) in the bioreactor. Morphological analysis of PRIME cells showed no change in morphology.

[0685] To induce the development of the MK lineage, the MK differentiation medium was used. May-Grunwald-Giemsa stained cytospins from day 1 to day 6 (Fig. 8C) showed different levels of maturation, with immature MK (left) compared to mature MK (right). At D3, ploidy analysis of PRIME-derived MKs from static or bioreactor culture (Fig. 8B) showed that they were of high ploidy levels with a maximum of 8N (n=4). MK maturation was associated with an appearance and an increased expression of CD41a and of CD42a (Fig 8D), leading to the presence of a CD41+CD42+ cell population, a phenotype defining mature MKs.

[0686] Proplatelet formation (Fig 8B) was observed, after several up and down and filtration, cytometry analyses were performed (Fig. 8A).

[0687] The functionality of these platelets was investigated using an aggregation assay in presence of calcium, thrombin and fibrinogen. As shown in Fig 8F, the platelets clotted in presence of calcium, thrombin and fibrinogen.

[0688] Platelets were therefore successfully produced from PRIME cells cultured in bioreactor.

Claims

86Claims1. An in vitro method for amplifying hematopoietic progenitor cells, said method comprising culturing hematopoietic progenitor cells in the presence of a DNMT3 A inhibitor, preferably in an amplification culture medium.

2. An in vitro method for producing hematopoietic cells, wherein said method comprises or consists of the steps of: i) Amplifying hematopoietic progenitor cells in the presence of a DNMT3A inhibitor, preferably in an amplification culture medium; ii) Differentiating the amplified hematopoietic progenitor cells into megakaryocytes or erythroid precursors, in particular in the absence of the DNMT3 A inhibitor, preferably in a differentiation culture medium; iii) Optionally, recovering the megakaryocytes or erythroid precursors; iv) Optionally, maturing the megakaryocytes into platelets or the erythroid precursors into erythrocytes; v) Optionally, recovering the platelets or erythrocytes.

3. The method of claim 1 or 2, wherein the DNMT3A inhibitor is a nucleic acid molecule or a protein encoded by a nucleic acid molecule, said DNMT3 A inhibitor being expressed by the hematopoietic progenitor cells.

4. The method of claim 3, wherein expression of the DNMT3A inhibitor is placed under the control of an inducible system or promoter.

5. The method according to claim 4, wherein the inducible system is a doxycycline-inducible TetO / TetR system.

6. The method of any one of claims 1-5, wherein the DNMT3A inhibitor is a nucleic acid molecule, said nucleic acid molecule being a shRNA, a siRNA or a miRNA, preferably a shRNA.

7. A genetically modified hematopoietic progenitor cell comprising a DNMT3A inhibitor, preferably as defined in any one of claims 3-6.

8. The genetically modified hematopoietic progenitor cell of claim 7, wherein the DNMT3A inhibitor is an interfering nucleic acid molecule placed under the control of an inducible system or promoter.

879. Use of a hematopoietic progenitor cell according to claim 7 or 8, for the in vitro production of haematopoietic cells selected from the group consisting of unipotent erythroid progenitors such as BFU-E, CFU-E; erythroid precursors such as proerythroblasts, basophil erythroblasts, polychromatophil erythroblasts, orthochromatic erythroblasts; reticulocytes, erythrocytes, megakaryoblasts, megakaryocytes and platelets, preferably platelets or erythrocytes.

10. An in vitro method for producing platelets, said method comprising differentiating the hematopoietic progenitor cells according to claim 7 or 8 into megakaryocytes, preferably in a differentiation culture medium, and maturing the megakaryocytes into platelets.

11. An in vitro method for producing erythrocytes, said method comprising differentiating the hematopoietic progenitor cells according to claim 7 or 8 into erythroid precursors, preferably in a differentiation culture medium, and maturing the erythroid precursors into erythrocytes.

12. The method of any one of claims 1-6 and 10-11, the genetically modified hematopoietic progenitor cell of claim 7 or 8 or the use of claim 9, wherein the hematopoietic progenitor cell is a megakaryocyte-erythroid progenitor (MEP).

13. The method of any one of claims 1-6, wherein the amplification culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), insulin; heparin; transferrin; serum, plasma or serum pool; optionally a glucocorticoid hormone; an autophagy inducer; erythropoietin (EPO); and glutamine.

14. The method of claim 13, wherein the glucocorticoid hormone is selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazol, preferably is dexamethasone; and / or the autophagy inducer is selected from the group consisting of SMER- 28, SMER-10 and SMER 18, preferably is SMER-28.

15. The method of claim 12 or 13, wherein the amplification culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), glutamine at a concentration of between about 1 ng / ml and about 15 ng / ml; insulin at a concentration between about 1 pg / mL and about 50 pg / mL; heparin at a concentration between about 0.5 U / mL and about 5 U / mL; transferrin at a concentration between about 200 pg / mL and about 400 pg / mL; plasma or serum pool, at a concentration between about 1% and about 10%; optionally dexamethasone at a concentration between 0.01 mM and 10 mM; SMER-28 at a concentration between 0,1 pM and 10 pM; and erythropoietin (EPO) at a concentration of between about 0.5 lU / mL and about 10 lU / mL.8816. The method of any one of claims 1-6, 9, 10 and 12-15, wherein the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), insulin and / or transferrin; glutamine, preferably L-glutamine; an antagonist of the aryl hydrocarbon receptor (AHR), preferably StemRegenin 1 (SRI); an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28; an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021; a growth factor of the TGF-P family, preferably Activin A; a protein kinase inhibitor, preferably midostaurin, foetal calf serum (FBS) or platelet lysate; thrombopoietin (TPO) or eltrombopag.

17. The method of claim 16, wherein the differentiation culture medium comprises or consists of abase culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), Insulin- Transferrin-Selenium-Ethanolamine (ITS-X), optionally transferrin, foetal bovine serum (FBS) or platelet lysate, glutamine, thrombopoietin or eltrombopag, StemRegenin 1 (SRI), SMER28, CHIR99021, Activin A and midostaurin.

18. The method of claim 16 or 17, wherein the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X at a concentration of between about 0,5% and about 5%; optionally transferrin at a concentration of between about 100 pg / ml and about 500 pg / ml; glutamine at a concentration of between about 1 ng / ml and about 10 ng / mL; SRI at a concentration of between about 50 nM and about 150 nM; SMER-28 at a concentration between 0,1 pM and 10 pM; CHIR99021 at a concentration between about 0,1 pM and about 5 pM; Activin A at a concentration between about 200 ng / ml and about 300 ng / ml; midostaurin at a concentration between about 1 pM and about 5 pM; foetal bovine serum (FBS) or platelet lysate at a concentration between about 0,1% and about 5; and eltrombopag or thrombopoietin (TPO) at a concentration of between about 20 ng / ml and about 40 ng / ml.

19. The method of any one of claims 1-6, 9, 11-15, wherein the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X; transferrin; optionally hemin, glutamine, preferably L-glutamine; foetal bovine serum or platelet lysate; an autophagy inducer, preferably SMER-28, SMER-10 or SMER 18, more preferably SMER-28; an inhibitor of glycogen synthase kinase 3 beta (GSK-3P), preferably CHIR99021; a growth factor of the TGF-P family, preferably Activin A; erythropoietin; an indole alkaloid, preferably hirsutine; a JAK2 inhibitor, especially JAK2-IN- 6 and / or pacritinib, an inhibitor of Bcl-XL, preferably WEHI-539; and optionally a FLU inhibitor, preferably TK216 and / or anagrelide..8920. The method of claim 19, wherein the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X, transferrin, hemin, foetal bovine serum (FBS) or platelet lysate, glutamine, erythropoietin, SMER28, hirsutine, CHIR99021, Activin A, and a) WEHI-53, JAK2IN6, pacritinib, and optionally TK216 and / or anagrelide or b) TK216 and anagrelide and optionally hemin.

21. The method of claim 19 or 20, wherein the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X at a concentration of between about 0,5% and about 5%; transferrin at a concentration of between about 100 pg / ml and about 500 pg / ml; glutamine at a concentration of between about 1 ng / ml and about 10 ng / ml; SMER-28 at a concentration between 0,1 pM and 10 pM; CHIR99021 at a concentration between about 0,1 pM and about 5 pM; Activin A at a concentration between about 200 ng / ml and about 300 ng / ml; foetal calf serum (FBS) at a concentration of between about 5% and about 25%; erythropoietin (EPO at a concentration of between about 1 U / ml and about 10 U / ml; hirsutine at a concentration of between about 20 pM and about 100 pM; JAK2-IN-6 at a concentration of between about 1 pg / mL and about 15 pg / mL; pacritinib, at a concentration of between about 1 nM and about 15 nM; WEHI-539 at a concentration of between about 0.1 nM and about 10 nM; optionally TK216, at a concentration of between about 20 ng / ml and about 40 ng / ml, and optionally anagrelide, at a concentration of between about 1 pg / ml and about 100 pg / ml.

22. The method of claim 19 or 20, wherein the differentiation culture medium comprises or consists of a base culture medium, preferably an Iscove's Modified Dulbecco's Medium (IMDM), ITS-X at a concentration of between about 0,5% and about 5%; transferrin; at a concentration of between about 100 pg / ml and about 500 pg / ml; glutamine at a concentration of between about 1 ng / ml and about 10 ng / ml; SMER-28, at a concentration between 0,1 pM and 10 pM; CHIR99021 at a concentration between about 0,1 pM and about 5 pM; Activin A, at a concentration between about 200 ng / ml and about 300 ng / ml; foetal calf serum (FBS), at a concentration of between about 5% and about 25%; optionally hemin at a concentration between about 1 pM and about 100 pM; erythropoietin (EPO) at a concentration of between about 1 U / ml and about 10 U / ml ; hirsutine, at a concentration of between about 20 pM and about 100 pM; TK216, at a concentration of between about 20 ng / ml and about 40 ng / ml, and anagrelide, at a concentration of between about 1 pg / ml and about 100 pg / ml.

23. Use of a culture medium as defined in any one of claims 16-18 for the differentiation of hematopoietic progenitor cells into megakaryocytes, optionally into platelets.9024. Use of a culture medium as defined in any one of claims 19-22, for the differentiation of hematopoietic progenitor cells into erythroid precursors, optionally into erythrocytes.

25. A bioreactor comprising the genetically modified hematopoietic progenitor cell according to claim 7 or 8.

26. The bioreactor of claim 25, wherein said bioreactor further comprises an amplification culture medium as defined in any one of claims 13-15 or a differentiation culture medium as defined in any one of claims 16-22.

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