Use of vitisin compounds for production of hematopoietic cells

Abstract

The present invention relates to the use of vitisin compounds for expansion and / or differentiation of hematopoietic cells. The invention also relates to a method for the in vitro production of erythrocyte progenitor cells, said method comprising contacting hematopoietic cells with a cell culture medium comprising a vitisin compound.

Description

Technical Field

[0001] The present invention belongs to the field of medicine. More specifically, it relates to a novel method for producing erythroid progenitor cells and erythrocytes. Background Art

[0002] Maintaining a constant supply of oxygen to tissues is essential for the survival of many organisms, especially humans. Red blood cells transport oxygen throughout the body via the bloodstream. This transport is ensured by hemoglobin, a protein unique to red blood cells that is able to bind oxygen. When red blood cells reach tissues, oxygen diffuses through the capillary walls. The role of red blood cells is therefore fundamental.

[0003] In emergency situations (bleeding) or pathological conditions (blood disorders, cancer, etc.), transfusion of red blood cells is necessary. In 2016, more than 100 million blood bags were collected and distributed worldwide to meet the transfusion needs. Until now, transfusions have relied entirely on blood from donors. However, since the health crisis related to Covid-19, blood bag reserves have been decreasing and donors are no longer coming to donate blood on a regular basis.

[0004] In adults, red blood cell production, or erythropoiesis, occurs in the bone marrow, known as the hematopoietic marrow, which is found in the ends of flat and long bones. In the bone marrow, multipotent stem cells, known as hematopoietic stem cells, sequentially differentiate into different types of red blood cell progenitors and ultimately mature red blood cells.

[0005] Culture media for hematopoietic cell expansion and differentiation are known to allow the production of erythrocytes in vitro. Various erythrocyte production tests have been performed over time, particularly as reviewed in Yoojin Seo et al., Stem Cells International, vol. 2019, article ID 9281329. In particular, different cell sources have been tested and different protocols have been developed involving different cytokine mixtures, sequential steps adapted to different stages of cell differentiation, modified culture times (18 to 60 days) or the use or non-use of bioreactors.

[0006] Current methods present many difficulties for large-scale production, such as low red blood cell yields at the end of culture, inability to achieve efficient transfusion volumes, and complex production systems involving culture in several consecutive and distinct steps, relatively long culture times to obtain mature red blood cells, and occasional overly rapid directed maturation into red blood cells or differentiation into cells that cannot properly perform the enucleation step (Akimov S. et al., 2005, Stem cells, 23(9): 1423-1433; Hirose SI et al., 2013, Stem Cell Reports, 1(6): 499-508; Huang X., 2013, Mol. Ther., 22(2): 451-463). Finally, other methods resort to co-culture with feeder cells (Kurita R. et al., 2013, PloS One, 8(3): e59890), which is both complex and expensive to perform.

[0007] It can therefore be seen that developing new, more efficient methods of producing red blood cells will have the potential to meet the demand for blood cell supply.

[0008] The invention described herein is directed to meeting these needs. Summary of the invention

[0009] According to a first aspect, the present invention relates to an in vitro method for producing red blood cells, the method comprising:

[0010] a) providing multipotent hematopoietic cells, preferably of human origin, selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, and optionally expanding the multipotent hematopoietic cells;

[0011] b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells;

[0012] c) inducing the unipotent erythroid progenitor cells to differentiate into erythroid precursor cells; and

[0013] d) inducing the erythroid precursor cells to mature into erythroid cells, and

[0014] e) optionally recovering the obtained red blood cells;

[0015] In particular, when step a) comprises a cell expansion step, in step a), step b), step c) and / or step d) the cells are contacted with a vitisin compound.

[0016] The present invention also relates to an in vitro method for producing unipotent erythroid progenitor cells, the method comprising:

[0017] a) providing multipotent hematopoietic cells, preferably of human origin, selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, and optionally expanding the multipotent hematopoietic cells;

[0018] b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells; and

[0019] c) optionally recovering the obtained unipotent erythroid progenitor cells;

[0020] In particular, when step a) comprises a cell expansion step, in step a) and / or step b) the cells are contacted with a vitisin compound.

[0021] The present invention also relates to an in vitro method for producing red blood cells, the method comprising:

[0022] a) providing unipotent erythroid progenitor cells, which are preferably obtained by the method for producing unipotent erythroid progenitor cells according to the present invention;

[0023] b) inducing the unipotent erythroid progenitor cells to differentiate into unipotent erythroid precursor cells;

[0024] c) inducing the erythroid precursor cells to mature into erythroid cells, and

[0025] d) optionally recovering the obtained red blood cells,

[0026] wherein in step b) and / or step c), the cells are contacted with a vitisin compound.

[0027] In the method according to the invention, the cells are contacted with the vitisin compound in a medium suitable for the nutritional requirements of hematopoietic cells, in particular a medium suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells. The hematopoietic cells are preferably selected from HSC, MPP, CMP, MEP cells, unipotent erythroid progenitor cells, erythroid precursor cells and combinations thereof, preferably HSC or unipotent erythroid progenitor cells.

[0028] According to one embodiment, said cells are contacted with said vitisin compound during i) the expansion and / or differentiation of multipotent hematopoietic cells or ii) the expansion and / or differentiation of unipotent erythroid progenitor cells.

[0029] According to another embodiment, the cells are contacted with the vitisin compound during expansion and / or differentiation of multipotent hematopoietic cells and during expansion and / or differentiation of unipotent erythroid progenitor cells, and optionally during maturation of erythroid precursor cells.

[0030] In particular, the cells are contacted with the vitisin compound for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the expansion and / or differentiation of multipotent hematopoietic cells), and / or for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the expansion and / or differentiation of unipotent erythroid progenitor cells).

[0031] In the method according to the invention, said vitisin compound is present in said culture medium at a concentration between 1 μM and 500 μM, preferably between 5 μM and 100 μM, when brought into contact with said cells.

[0032] According to a second aspect, the present invention relates to a cell culture medium which is suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells and comprises a vitisin compound.

[0033] Specifically, the vitisin compound is present in the culture medium at a concentration between 1 μM and 500 μM, preferably between 5 μM and 100 μM.

[0034] The present invention also relates to the in vitro use of such a cell culture medium for (i) the expansion and / or differentiation of hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) or unipotent erythroid progenitor cells, and / or (ii) the differentiation and / or maturation of erythroid precursor cells, and / or (iii) the production of unipotent erythroid progenitor cells and / or erythroid cells.

[0035] According to a third aspect, the present invention relates to the use of a vitisin compound for supplementing a cell culture medium suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells.

[0036] The present invention also relates to the use of a vitisin compound for (i) stimulating the expansion and / or differentiation of hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) or unipotent erythroid progenitor cells, and / or (ii) stimulating the differentiation and / or maturation of erythroid precursor cells, and / or (iii) stimulating the production of unipotent erythroid progenitor cells and / or erythroid cells.

[0037] In the methods, culture media and uses contemplated by the present invention, the vitisin compound is selected from vitisin B and its derivatives.

[0038] Preferably, the vitisin B derivative is a vitisin B isomer, preferably a vitisin B stereoisomer.

[0039] Specifically, the vitisin B derivative has the following formula:

[0040] (II)

[0041] or

[0042] (III).

[0043] Preferably, the vitisin compound is vitisin B. DETAILED DESCRIPTION

[0044] The present invention relates to the production of red blood cells using compounds derived from plants of the genus Vitis. Vitisin B is a stilbene compound belonging to the polyphenol family, which can be extracted in particular from grape branches. Compared with other antioxidant molecules such as resveratrol, Vitisin B has surprising advantages.

[0045] This molecule is expressed in human hematopoietic cells, especially CD34 + Use on cells showed two important and reproducible effects:

[0046] 1) Vitisin B increases CD34 + Expansion of multipotent hematopoietic cells to promote their commitment to unipotent erythroid progenitors, and

[0047] 2) Vitisin B can also accelerate the differentiation of progenitor cells into erythroid precursor cells and their enucleation to produce mature cells: erythrocytes.

[0048] Therefore, the method according to the invention allows a more efficient production of red blood cells.

[0049] The in vitro culture method according to the present invention not only has the advantages of being simple and economical, but also paves the way for large-scale industrial production of erythrocyte progenitor cells and erythrocytes. This makes it possible to reduce the risk of blood shortages or transfusion bottlenecks while providing optimal safety for transfusion patients.

[0050] Erythropoiesis and hematopoietic cells

[0051] Hematopoiesis is a continuous biological process that allows the renewal and maintenance of all blood cells: erythrocytes, polymorphonuclear cells, platelets, monocytes and lymphocytes. Hematopoiesis is defined as a set of mechanisms that occur in the bone marrow, starting from hematopoietic stem cells (HSCs), ensuring the continuous and regulated renewal of blood cells, namely erythrocytes (RBCs), leukocytes (WBCs, neutrophils, eosinophils, monocytes, lymphocytes) and platelets (Neildez-Nguyen et al., 2002).

[0052] Erythropoiesis is the biological process that allows the renewal of red blood cells. During erythropoiesis, hematopoietic stem cells (HSCs) differentiate into multipotent progenitors (MPPs), common myeloid progenitors (CMPs), bipotent progenitors MEPs (megakaryocyte and erythroid progenitors), then into BFU-E (burst-forming unit erythroid) and CFU-E (colony-forming unit erythroid) progenitors, the latter two of which are irreversibly committed to the erythroid lineage (unipotent), and finally into erythroid precursors (Iwasaki and Akashi, Immunity. 2007 Jun;26(6):726-40). The terminal differentiation stage of erythropoiesis allows the formation of reticulocytes and then erythrocytes from erythroid precursors after enucleation (expulsion of the nucleus).

[0053] The process of erythropoiesis is well known to those skilled in the art and can be monitored by the expression dynamics of various specific markers according to the differentiation stage of the cells. Specifically, the CD34 surface antigen (to select the original CD34 + hematopoietic cell populations), transferrin receptor (CD71), a marker of early erythroid differentiation, and glycophorin A (GPA), a marker of terminal differentiation.

[0054] Glycophorin A (GPA) is a 10 kDa protein expressed at the membrane level of erythroid precursors (proerythroblasts) and erythrocytes. It is therefore a marker of terminal erythroid differentiation, as it is absent in committed progenitors (BFU-E and CFU-E), becomes progressively visible on basophilic erythroblasts, and remains stably expressed on erythrocytes.

[0055] Transferrin receptor 1 (CD71) is a 95 kDa protein expressed on the membrane of progenitor cells and erythroid precursor cells. Unlike GPA, the expression of CD71 decreases as differentiation proceeds, as erythroid cells do not express CD71. Therefore, it is a marker of early erythroid differentiation.

[0056] The differentiation of erythrocytes is associated with the reduction and disappearance of CD71 and the acquisition of GPA, which ultimately leads to the production of CD71-based - GPA + Phenotypically mature cells (erythrocytes and reticulocytes).

[0057] Specifically, erythroid differentiation can be observed by flow cytometry on the basis of specific expression markers such as membrane surface antigens (clusters of differentiation (DC)) such as CD34, CD123, CD45RA, CD71 and GPA markers.

[0058] The various erythrocytes can also be observed under the microscope by staining with May-Grünwald Giemsa (MGG). Specifically, at each differentiation step, a reduction in the size of the cell and the nucleus is observed, which ultimately leads to the formation of mature anucleated erythrocytes. This cytological technique allows, in particular, the analysis of the maturation of erythroid precursor cells, thereby detecting and quantifying the various precursor cells and mature erythrocytes.

[0059] CFC (colony forming cell) colony formation assays also allow the identification of various erythroid cells. This assay quantifies the number of hematopoietic progenitor cells (HP) that are able to form mature erythroid colonies when cultured in semisolid culture media (e.g., culture media containing methylcellulose). These culture media are preferably supplemented with cytokines required for erythropoietic differentiation. These colony formation assays allow the detection and quantification of progenitor cells with erythroid potential without the need for phenotypic markers.

[0060] These techniques are well known to those skilled in the art.

[0061] The terms "hematopoietic cells", "erythroid lineage cells" and "hematopoietic lineage cells" as used herein are equivalent and can be used interchangeably. These terms refer to any cell that allows the formation of red blood cells. These terms include multipotent hematopoietic cells, unipotent erythroid progenitor cells and erythroid precursor cells as defined below. In particular, hematopoietic cells include HSC, MPP, CMP, MEP, BFU-E, CFU-E, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts. The hematopoietic cells according to the present invention are preferably obtained from a donor subject, in particular a healthy donor subject. In particular, hematopoietic cells, in particular HSC, can be obtained or prepared from a biological sample of the donor, such as a blood sample (e.g., peripheral blood, umbilical cord blood or placental blood) or a bone marrow sample.

[0062] Preferably, the cells used in the present invention are cells of human origin.

[0063] As used herein, the term "multipotent hematopoietic cell" refers to a multipotent CD34+ cell that is capable of differentiating to produce at least two cell types of various blood cells, namely white blood cells (immune cells), red blood cells, and platelets. + Cells. In the context of the present invention, the term "multipotent hematopoietic cells" includes HSC, MPP, CMP and MEP. Preferably, MPP has the phenotype CD34 + LIN neg CD123 + CD45RA neg , CMPs have the phenotype CD34 + LIN neg CD123 + CD45RAneg , and MEPs have the phenotype CD34 + LIN neg CD123 neg CD45RA neg Preferably, the multipotent hematopoietic cells used in the present invention are selected from HSC, MPP, CMP and MEP and combinations thereof. More particularly preferably, the multipotent hematopoietic cells used in the present invention are HSC, optionally in combination with MPP, CMP and / or MEP.

[0064] The term "hematopoietic stem cell" or "HSC" as used herein refers to a multipotent stem cell that is capable of differentiating to produce all blood cells, i.e., white blood cells (immune cells), red blood cells, and platelets. According to a preferred embodiment, the HSC is a human HSC. HSC carries specific markers, such as high expression of the primitive marker CD34, lack of expression of the marker CD45RA (an isoform of the phosphotyrosine phosphatase CD45), and expression of the optional marker CD133. Preferably, HSC also expresses the IL3 receptor (CD123). The term "HSC" particularly includes cells known as "side population" (SP) cells. The term "SP cells" combines all cells that can be demonstrated by flow cytometry to have the ability to efflux vital dyes, such as Hoescht or circulating violet dye (DCV). Thus, preferably, SP cells have CD34 + DCV low Phenotype.

[0065] Multipotent hematopoietic cells can be obtained from various sources according to methods known to those skilled in the art. They can be isolated, in particular, from bone marrow, cell apheresis, whole blood or even umbilical cord blood (or placental blood), for example using an immunomagnetic system or a sorting system based on the presence of specific membrane receptors (e.g., CD133, CD123, CD45 and / or CD34).

[0066] Pluripotent hematopoietic cells can also be obtained by differentiating embryonic stem cells or induced pluripotent stem (iPS) cells, preferably induced pluripotent stem cells. The techniques for differentiating pluripotent stem cells into pluripotent hematopoietic cells are well known to those skilled in the art. Several protocols have been published, especially the protocol of Lengerke C. et al. (2009, Ann. NY Acad. Sci., 1176: 219-27), which includes 17 days of differentiation through an intermediate step of embryoid bodies and a combination of the following cytokines: SCF, Flt-3 ligand, IL-3, IL-6, G-CSF and BMP-4.

[0067] The term "embryonic stem cell" as used herein refers to a cell derived from the internal cell mass of the blastocyst, which has the ability to cause the formation of all body tissues (mesoderm, endoderm, ectoderm) including germline cells. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4 and NANOG and surface markers SSEA3 / 4, Tra-1-60 and Tra-1-81. Embryonic stem cells can be obtained without destroying the embryo from which they are derived, for example, by the technology described by Chung et al. (Cell stem Cell, 2008, 2(2): 113-117). In a specific embodiment, and for legal or ethical reasons, the embryonic stem cell is a non-human embryonic stem cell. In another specific embodiment, the embryonic stem cell used in the present invention is a human embryonic stem cell, preferably obtained without destroying the embryo from which they are derived. The embryo used is preferably an unnecessary embryo obtained under the background of a birth plan after obtaining supervision and ethical authorization according to current law.

[0068] The term "induced pluripotent stem cell" (iPSC, iPSc or iPS) used herein refers to a pluripotent stem cell obtained by genetic reprogramming of differentiated somatic cells, which has a morphology similar to that of embryonic stem cells and the potential for self-renewal and pluripotency. These cells are particularly positive for pluripotency markers, particularly alkaline phosphatase staining and the expression of NANOG, SOX2, OCT4 and SSEA3 / 4 proteins. The method for producing induced pluripotent stem cells is well known to those skilled in the art, and is particularly described in articles such as Yu (Science, 2007, 318(5858): 1917-1920), Takahashi (Cell, 2007, 131(5): 861-872) and Nakagawa (Nat. Biotechnol., 2008, 26(1): 101-106).

[0069] The term "unipotent erythroid progenitor" as used herein refers to a progenitor cell obtained by differentiation of HSC during erythropoiesis, which has the ability to divide and subsequently differentiate into erythroid precursor cells, and then differentiate into erythroid cells by denucleation. Specifically, after the MEP stage (the last multipotent stage), the cells gradually acquire markers specific to erythroid progenitors. First, the CD71 marker (transferrin receptor) is observed, followed by the appearance of glycophorin A (GPA or CD235a). Unipotent erythroid progenitors include BFU-E (burst forming unit-E) and CFU-E (colony forming unit-E) cells, which are irreversibly committed to the erythroid lineage. BFU-E (burst forming unit-E) cells have CD34 + CD36 neg GPAneg CD123 neg CD71 low Phenotype. CFU-E (colony forming unit-E) cells have CD34 neg CD36 neg GPA neg CD123 neg CD71 high Phenotype.

[0070] As used herein, the term "erythroid precursor cell" defines a cell that is obtained by differentiation of a unipotent erythroid progenitor cell and is capable of producing reticulocytes. The term encompasses proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts. These cells can be distinguished on a cytological basis by staining the cells with May-Grünwald Giemsa (MGG) stain, analyzing the staining and the nuclear / cytoplasmic ratio, by techniques well known to those skilled in the art.

[0071] As used herein, the terms "erythrocyte", "mature erythrocyte" and "erythrocyte" are equivalent and can be used interchangeably. The term "erythrocyte" refers to an enucleated cell with characteristic markers of erythrocyte maturation. Erythrocytes express, inter alia, glycophorin A (CD235a), but do not express the CD36 marker. Their identification can be based on cytological criteria known to those skilled in the art, such as cell size and absence of the cell nucleus.

[0072] According to one embodiment, the multipotent hematopoietic cells used to generate unipotent erythrocyte progenitor cells or to generate erythrocytes in the method according to the invention are obtained by sampling from a donor, or derived from cells obtained from a donor. Specifically, the erythrocyte progenitor cells or erythrocytes thus obtained can be used for transplantation into a recipient patient, especially by blood transfusion.

[0073] The donor and the recipient can be the same or different individuals. In one embodiment, 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, particularly an individual without a blood disease.

[0074] Vitisin compounds

[0075] The present invention includes the use of vitisin compounds in the in vitro or in vitro production of multipotent hematopoietic cells and / or unipotent erythroid progenitor cells and / or erythroid cells, in particular to promote the expansion and / or differentiation of hematopoietic cells. Specifically, the vitisin compound allows multipotent hematopoietic cells (especially HSCs) to undergo faster and greater erythroid differentiation into unipotent erythroid progenitor cells. In addition, it allows unipotent erythroid progenitor cells to undergo faster and greater erythroid differentiation into erythroid precursor cells and / or erythroid precursor cells to undergo erythroid differentiation into erythroid cells.

[0076] As used herein, the term "vitisin compound" refers to a class of stilbenes belonging to the polyphenol family, which can be extracted, for example, from grape branches. The term refers to vitisin B or a derivative thereof.

[0077] Preferably, the term "vitisin compound" does not include compounds selected from resveratrol, pallidol, ε-viniferin, ampelopsis, oxy-resveratrol, piceatannol and / or gennethol.

[0078] According to a preferred embodiment, the vitisin compound is vitisin B.

[0079] Vitisin B is a tetramer of resveratrol found in plants of the genus Vitis, such as wine grapes (Vitis vinifera).

[0080] Vitisin B is also known as

[0081] - 1,3-benzenediol, 5-[(2R,2′S,3R,3′S)-5-[(1E)-2-[(2S,3S)-3-(3,5-dihydroxyphenyl)-2,3-dihydro-6-hydroxy-2-(4-hydroxyphenyl)-4-benzofuranyl]vinyl]-2,2′,3,3′-tetrahydro-6′-hydroxy-2,2′-bis(4-hydroxyphenyl)[3,4′-bibenzofuran]-3′-yl]-(9CI, ACI);

[0082] - 1,3-benzenediol, 5-[5-[2-[3-(3,5-dihydroxyphenyl)-2,3-dihydro-6-hydroxy-2-(4-hydroxyphenyl)-4-benzofuranyl]vinyl]-2,2′,3,3′-tetrahydro-6′-hydroxy-2,2′-bis(4-hydroxyphenyl)[3,4′-bibenzofuran]-3′-yl]-,[2R-[2α,3β(2′S ,3′S ),5[E(2S ,3S )]]]-(ZCI); or

[0083] -5-[(2R,2′S,3R,3′S)-5-[(1E)-2-[(2S,3S)-3-(3,5-dihydroxyphenyl)-2,3-dihydro-6-hydroxy-2-(4-hydroxyphenyl)-4-benzofuranyl]vinyl]-2,2′,3,3′-tetrahydro-6′-hydroxy-2,2′-bis(4-hydroxyphenyl)[3,4′-bibenzofuran]-3′-yl]-1,3-benzenediol (ACI), and specifically has CAS number 142449-90-9.

[0084] Specifically, vitisin B used according to the present invention has the following formula (I):

[0085] (I)

[0086] Vitisin B may especially be in the form of a salt or a hydrate.

[0087] According to other embodiments, the vitisin compound is a vitisin B derivative. Preferably, the vitisin derivative is a vitisin B isomer, preferably a vitisin B stereoisomer. Specifically, the vitisin B stereoisomer includes an R or S stereoisomer at any stereochemical site of vitisin B.

[0088] Examples of isomers may be, but are not limited to, compounds having CAS numbers selected from CAS 165883-77-2, CAS 180580-73-8, CAS 142507-86-6, CAS 1373138-12-5, CAS 1807631-15-7, CAS 879897-47-9, CAS 879897-46-8 and CAS 681004-56-8 or compounds of formula (I), (II) and (III) described below and combinations thereof.

[0089] Specifically, the vitisin B derivative can be, for example, a stereoisomer of vitisin B described in patent application WO 2015 / 126129. Preferably, the vitisin B derivative is a stereoisomer that takes the Z position (compound 20 of patent application WO 2015 / 126129) or the E position (compound 22 of patent application WO 2015 / 126129) according to the double bond.

[0090] The vitisin B derivative may in particular have the following formula (II) or (III):

[0091] (II)

[0092] or

[0093] (III).

[0094] According to certain specific embodiments, the vitisin compound is selected from compounds of formula (I), (II) and (III) and combinations thereof.

[0095] Therefore, according to certain embodiments, the vitisin compound can be selected from vitisin B and vitisin B derivatives having CAS numbers selected from CAS142449-90-9, CAS165883-77-2, CAS180580-73-8, CAS142507-86-6, CAS1373138-12-5, CAS1807631-15-7, CAS879897-47-9, CAS879897-46-8 and CAS681004-56-8, or compounds of formula (I), (II) and (III) described above, and combinations thereof.

[0096] Contacting the vitisin compound with the cell can be carried out by adding the vitisin compound to a culture medium, in particular a culture medium suitable for growing hematopoietic cells. The culture medium to which the vitisin compound is added is preferably suitable for the nutritional requirements of hematopoietic cells, and is particularly suitable for the growth and / or differentiation of hematopoietic cells. Such culture media are known to those skilled in the art, and some of them are described in more detail below.

[0097] Preferably, the vitisin compound is present in an amount of about 0.5 µM to about 1 mM, about 0.5 µM to about 500 µM, about 0.5 µM to about 200 µM, about 0.5 µM to about 100 µM, about 0.5 µM to about 50 µM, about 0.5 µM to about 40 µM, about 0.5 µM to about 30 µM, about 0.5 µM to about 20 µM, about 0.5 µM to about 15 µM, or about 0.5 µM to about 10 µM, about 5 µM to about 1 mM, about 5 µM to about 500 µM, about 5 µM to about 200 µM, about 5 µM to about 100 µM, about 5 µM to about 75 µM, about 5 µM to about 50 µM, about 5 µM to about 40 µM, about 5 µM to about 30 µM, about 5 µM to about 20 µM, about 5 µM to about 15 µM, or about 5 µM to about 10 µM. In some embodiments, the present invention relates to a method for treating a vitisin-B-containing compound. The method comprises the steps of: using a concentration of about 10 μM to about 1 μM, preferably about 10 μM to about 1 mM, about 10 μM to about 500 μM, about 10 μM to about 200 μM, about 10 μM to about 100 μM, about 10 μM to about 75 μM, about 10 μM to about 50 μM, about 10 μM to about 40 μM, about 10 μM to about 30 μM, about 10 μM to about 20 μM, preferably a concentration of about 10 μM. Preferably, the vitisin compound is used at a concentration of 1 μM to 500 μM, preferably 5 μM to 100 μM. In particular, vitisin-B is used at a concentration of about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 80, 90, 95 or 100 μM.

[0098] Production method

[0099] The present invention relates to methods of producing or preparing erythroid lineage cells.

[0100] Specifically, the production method according to the present invention may include:

[0101] a) expansion, differentiation and / or maturation of said erythroid lineage cells, and

[0102] b) optionally recovering the propagated and / or differentiated and / or mature erythroid lineage cells, and

[0103] c) optionally storing the obtained / recovered erythroid lineage cells.

[0104] Preferably, the erythroid lineage cells are selected from HSC, MPP, CMP, MEP, BFU-E, CFU-E and erythroid precursor cells (i.e., proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, normochromatic erythroblasts), reticulocytes and / or erythrocytes and any combination thereof.

[0105] In the method according to the invention, said vitisin compound may be contacted with said cells of erythroid lineage, preferably in a suitable culture medium, during cell expansion, differentiation and / or maturation, preferably during cell expansion and / or differentiation.

[0106] In certain embodiments, the vitisin compound is contacted with multipotent hematopoietic cells, in particular cells selected from HSC, MPP, CMP and MEP cells, and combinations thereof, so as to expand the cells and / or obtain unipotent erythroid progenitor cells, erythroid precursor cells and / or erythroid cells through differentiation of the cells.

[0107] In other embodiments, the vitisin compound is contacted with unipotent erythroid progenitor cells in order to expand the progenitor cells and / or obtain erythroid precursor cells and / or erythroid cells by differentiation of the progenitor cells.

[0108] In other embodiments, the vitisin compound is contacted with erythroid precursor cells so that erythroid cells are obtained through differentiation and maturation of the precursor cells.

[0109] In other embodiments, the vitisin compound is combined with CD34 + Cells, i.e., cells selected from the group consisting of HSC, MPP, CMP, MEP and BFU-E cells, and combinations thereof, are contacted in order to expand the cells and / or obtain CFU-E progenitor cells, erythroid precursor cells and / or erythroid cells through differentiation of the cells.

[0110] In particular, the present invention relates to an in vitro method for producing red blood cells, the method comprising:

[0111] a) providing multipotent hematopoietic cells, preferably of human origin, selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, and optionally expanding the multipotent hematopoietic cells;

[0112] b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells;

[0113] c) inducing the unipotent erythroid progenitor cells to differentiate into erythroid precursor cells; and

[0114] d) inducing the erythroid precursor cells to mature into erythroid cells, and

[0115] The obtained red blood cells are optionally recovered.

[0116] In this method, in step a) (when this step comprises expansion of said multipotent hematopoietic cells), step b), step c) and / or step d), cells (multipotent hematopoietic cells, unipotent erythroid progenitor cells and / or erythroid precursor cells) are contacted with a vitisin compound.

[0117] Preferably, the present invention relates to an in vitro method for producing red blood cells, the method comprising:

[0118] a) providing multipotent hematopoietic cells selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, preferably of human origin, and expanding the multipotent hematopoietic cells;

[0119] b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells;

[0120] c) inducing the unipotent erythroid progenitor cells to differentiate into erythroid precursor cells; and

[0121] d) inducing the erythroid precursor cells to mature into erythroid cells, and

[0122] The obtained red blood cells are optionally recovered.

[0123] In this method, in step a), step b), step c) and / or step d), the cells (multipotent hematopoietic cells, unipotent erythroid progenitor cells and / or erythroid precursor cells) are contacted with a vitisin compound.

[0124] The present invention also relates to an in vitro method for producing unipotent erythroid progenitor cells, the method comprising:

[0125] a) providing multipotent hematopoietic cells, preferably of human origin, selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, and optionally expanding the multipotent hematopoietic cells; and

[0126] b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells; and

[0127] c) optionally recovering the obtained unipotent erythroid progenitor cells.

[0128] In this method, in step a) (when this step comprises expansion of said multipotent hematopoietic cells) and / or in step b), said cells (multipotent hematopoietic cells and / or unipotent erythroid progenitor cells) are contacted with a vitisin compound.

[0129] Preferably, the present invention also relates to an in vitro method for producing unipotent erythroid progenitor cells, the method comprising:

[0130] a) providing multipotent hematopoietic cells selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, preferably of human origin, and expanding the multipotent hematopoietic cells; and

[0131] b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells; and

[0132] c) optionally recovering the obtained unipotent erythroid progenitor cells.

[0133] In this method, in step a) and / or step b), the cells (multipotent hematopoietic cells and / or unipotent erythroid progenitor cells) are contacted with a vitisin compound.

[0134] The main purpose of this approach is to induce differentiation of multipotent hematopoietic cells into unipotent erythroid progenitors and to allow expansion of this progenitor population while maintaining its ability to differentiate into erythroid cells at a later stage, or to continue to differentiate this progenitor population into erythroid precursors and erythroid cells.

[0135] The present invention also relates to an in vitro method for producing erythroid precursor cells, the method comprising:

[0136] a) Provide unipotent erythroid progenitors;

[0137] b) inducing the unipotent erythroid progenitor cells to differentiate into erythroid precursor cells; and

[0138] The obtained erythroid precursor cells are optionally recovered.

[0139] In this method, in step b) the cells (unipotent erythroid progenitor cells and / or erythroid precursor cells) are contacted with a vitisin compound.

[0140] Preferably, the unipotent erythroid progenitor cells provided in step a) are obtained by the method for producing unipotent erythroid progenitor cells according to the present invention.

[0141] The present invention also relates to an in vitro method for producing red blood cells, the method comprising:

[0142] a) Provide unipotent erythroid progenitors;

[0143] b) inducing the unipotent erythroid progenitor cells to differentiate into unipotent erythroid precursor cells;

[0144] c) inducing the erythroid precursor cells to mature into erythroid cells, and

[0145] optionally recovering the obtained red blood cells,

[0146] In this method, the cells (unipotent erythroid progenitor cells and / or erythroid precursor cells) are contacted with a vitisin compound in step b) and / or step c).

[0147] Preferably, the unipotent erythroid progenitor cells provided in step a) are obtained by the method for producing unipotent erythroid progenitor cells according to the present invention.

[0148] The present invention also relates to an in vitro method for producing red blood cells, the method comprising:

[0149] a) providing erythroid precursor cells; and

[0150] b) inducing the erythroid precursor cells to mature into erythroid cells, and

[0151] optionally recovering the obtained red blood cells,

[0152] In this method, in step b) erythroid precursor cells are contacted with a vitisin compound.

[0153] Preferably, the erythroid precursor cells provided in step a) are obtained by the method for producing erythroid precursor cells according to the present invention.

[0154] The multipotent hematopoietic cells provided in the method according to the present invention can be obtained by any method known to those skilled in the art. As described above, they can be isolated from bone marrow, cell apheresis, whole blood, umbilical cord or placental blood samples, for example using an immunomagnetic system or a sorting system based on the presence of specific membrane receptors. They can also be obtained by differentiation of embryonic stem cells or induced pluripotent stem (iPS) cells as described above. Optionally, before using them in one of the methods according to the present invention, these multipotent hematopoietic cells can be expanded by culturing in a culture medium suitable for expanding these cells. The multipotent hematopoietic cells provided include HSC, MPP, CMP and / or MEP, preferably HSC and optional MPP, CMP and / or MEP. The multipotent hematopoietic cells provided may also include any other cell type. Specifically, these cells may be CD34 + cells, and may also include BFU-E.

[0155] The unipotent erythroid progenitor cells provided in the method according to the present invention can be obtained by the method for producing unipotent erythroid progenitor cells according to the present invention or by any other method known to those skilled in the art. Specifically, these progenitor cells can be separated from bone marrow, cell apheresis, whole blood, umbilical cord or placental blood samples, for example, using an immunomagnetic system or a sorting system based on the presence of specific membrane receptors. They can also be obtained by differentiating multipotent hematopoietic cells using any method known to those skilled in the art. Optionally, before being used in one of the methods according to the present invention, these unipotent erythroid progenitor cells can be amplified by culturing in a culture medium suitable for amplifying these cells. The unipotent erythroid progenitor cells provided include BFU-E and / or CFU-E. They can also include other cell types, such as HSC, MPP, CMP and / or MEP.

[0156] The erythrocyte precursor cells provided in the method according to the present invention can be obtained by the method for producing erythrocyte precursor cells according to the present invention or by any other method known to those skilled in the art. Specifically, these erythrocyte precursor cells can be separated from bone marrow, cell apheresis, whole blood, umbilical cord or placental blood samples, for example, using an immunomagnetic system or a sorting system based on the presence of specific membrane receptors. They can also be obtained by differentiating multipotent hematopoietic cells or unipotent erythrocyte progenitor cells using any method known to those skilled in the art. Optionally, before being used in one of the methods of the present invention, these erythrocyte precursor cells can be amplified by culturing in a culture medium suitable for amplifying these cells.

[0157] In the method according to the invention, the cells may be contacted with the vitisin compound in a medium suitable for the nutritional requirements of hematopoietic cells. Depending on the step of carrying out such contact, such medium may be a medium suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells.

[0158] The culture medium suitable for the nutritional requirements of hematopoietic cells can be any culture medium known to those skilled in the art for meeting the requirements of these cells, more specifically for meeting the requirements of cells selected from HSC, MPP, CMP, MEP cells, unipotent erythroid progenitor cells, erythroid precursor cells and combinations thereof. This culture medium meets the minimum survival requirements of the cells to be cultured, and allows the maintenance, expansion, differentiation and / or maturation of hematopoietic cells, depending on the circumstances and the supplements provided. Many culture media suitable for the nutritional requirements of the hematopoietic cells of interest are known to those skilled in the art, and these culture media are commercially available, such as Iscove's Modified Dulbecco's Medium (IMDM culture medium), Stem Span SFEM II culture medium (Stemcell technologies) or "Human StemMACS HSC Expansion Medium XF" (Miltenyi Biotec). The composition of these culture media is well known.

[0159] Preferably, in the method according to the invention, the vitisin compound is contacted with the cells in a culture medium suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells. Such culture medium is well known to those skilled in the art. Examples of such culture medium are specifically mentioned in the "culture medium" section below. In general, culture medium suitable for cell expansion, differentiation and maturation can be obtained from the same basic culture medium suitable for the nutritional requirements of hematopoietic cells, such as IMDM culture medium or Stem Span SFEM II culture medium, and the substantial difference is their cytokine composition.

[0160] As used herein, the terms "growth", "propagation" or "expansion" refer to the propagation of cells. Thus, a culture medium suitable for the expansion of hematopoietic cells is a culture medium that allows the propagation of multipotent hematopoietic cells (i.e., HSC, MPP, CMP and / or MEP, unipotent erythroid progenitor cells and / or erythroid precursor cells). A culture medium suitable for the expansion of a certain cell type may also be suitable for the differentiation of this cell type. In contrast, when only expansion is required, for example, in order to increase the pool of multipotent hematopoietic cells or unipotent erythroid progenitor cells, the culture medium suitable for expansion hardly stimulates cell differentiation.

[0161] In the method according to the present invention, the multipotent hematopoietic cells can be expanded before differentiation. This expansion can be carried out in any culture medium suitable for expanding multipotent hematopoietic cells (especially HSC) and known to those skilled in the art. Specifically, the multipotent hematopoietic cells can be expanded in a culture medium suitable for the nutritional requirements of hematopoietic cells, such as IMDM culture medium or StemSpan SFEM II culture medium, which contains or is supplemented with G-SCF, SCF, FLT3-L and TPO (especially at the concentrations described in the "culture medium" section below).

[0162] In the method according to the invention, the erythroid progenitor cells can be expanded before differentiation. This expansion can be carried out in any culture medium suitable for expanding erythroid progenitor cells and known to those skilled in the art. Specifically, erythroid progenitor cells can be expanded in a culture medium suitable for the nutritional requirements of hematopoietic cells, such as IMDM culture medium or Stem Span SFEM II culture medium, which contains or is supplemented with G-SCF, SCF, FLT3-L and TPO (particularly at the concentrations described below in the "culture medium" section).

[0163] In the method according to the present invention, erythrocyte precursor cells can be expanded before maturation.This expansion can be carried out in any culture medium that is suitable for expanding erythrocyte precursor cells and well known to those skilled in the art.Specifically, erythrocyte precursor cells can be expanded in the culture medium that is suitable for the nutritional requirement of hematopoietic cells, for example IMDM culture medium or Stem Span SFEM II culture medium, comprising or being supplemented with EPO, hydrocortisone, suitable growth factor, transferrins, serum and / or blood plasma or its any combination (particularly the concentration described in " culture medium " part hereinafter) .

[0164] The term "differentiation" refers to the acquisition of characteristics by cultured cells that were not present in the cells originally used to seed the culture medium. In the present context, the term refers to the acquisition of cell characteristics that are more advanced in the process of erythropoiesis. Thus, a culture medium suitable for hematopoietic cell differentiation is a culture medium that allows multipotent hematopoietic cells (e.g., HSCs, MPPs, CMPs, and / or MEPs) to differentiate into unipotent erythroid progenitors and / or unipotent erythroid progenitors to differentiate into erythroid precursors and / or erythroid precursors to differentiate so as to obtain erythroid cells after maturation. Typically, a culture medium suitable for differentiation of a certain cell type is also suitable for cell expansion.

[0165] In the method according to the present invention, multipotent hematopoietic cells can be differentiated into unipotent erythroid progenitor cells. This differentiation can be carried out in any culture medium suitable for differentiating multipotent hematopoietic cells (especially HSC) and known to those skilled in the art. Specifically, multipotent hematopoietic cells can be differentiated in a culture medium suitable for the nutritional requirements of hematopoietic cells, such as IMDM culture medium or StemSpan SFEM II culture medium, comprising IL3, IL6, SCF, serum albumin (especially bovine albumin), insulin, transferrin and optionally EPO.

[0166] In the method according to the present invention, erythroid progenitor cells can be differentiated into erythroid precursor cells. This differentiation can be carried out in any culture medium that is suitable for differentiation erythroid progenitor cells and well known by persons skilled in the art. Specifically, erythroid progenitor cells can be differentiated in a culture medium that is suitable for the nutritional requirements of hematopoietic cells comprising IL3, SCF, hydrocortisone and optional EPO, for example in IMDM culture medium or Stem Span SFEM II culture medium.

[0167] The term "maturation" as used herein refers to the process by which erythrocyte precursor cells become erythrocytes, particularly involving enucleation of the cells. Therefore, a culture medium suitable for hematopoietic cell maturation is a culture medium that allows erythrocyte precursor cells to mature into erythrocytes. Typically, a culture medium suitable for the expansion and / or differentiation of hematopoietic cells does not promote such maturation. The maturation of erythrocyte precursor cells is particularly reflected in the expression of erythrocyte maturation markers such as CD235a and enucleation.

[0168] In the method according to the present invention, erythrocyte precursor cells can be matured to obtain erythrocytes. This maturation can be carried out in any culture medium suitable for the maturation of erythrocyte precursor cells and known to those skilled in the art. Specifically, erythrocyte precursor cells can be matured in a culture medium suitable for the nutritional requirements of hematopoietic cells, such as IMDM culture medium or Stem Span SFEM II culture medium, which contains or is supplemented with EPO, preferably 2 or 3 IU / ml EPO. Preferably, the culture medium does not contain any cytokine. Preferably, erythrocyte precursor cell maturation is induced at a high cell concentration (e.g., higher than 5 million cells / ml culture).

[0169] Therefore, in the method according to the invention,

[0170] - the expansion of the multipotent hematopoietic cells can be carried out by placing the multipotent hematopoietic cells in a culture medium suitable for the expansion of multipotent hematopoietic cells, in particular the culture medium described above or in the "culture medium" section, for a period of typically 3 to 15 days, preferably 5 to 8 days;

[0171] - expansion of erythroid progenitor cells can be performed by placing said erythroid progenitor cells in a culture medium suitable for erythroid progenitor cell expansion, in particular a culture medium as described above or in the section "culture medium", for a period of typically 3 to 15 days, preferably 4 to 12 days;

[0172] - expansion of erythroid precursor cells can be performed by placing said erythroid precursor cells in a culture medium suitable for expansion of erythroid precursor cells, in particular a culture medium as described above or in the section "culture medium", for a period of typically 2 to 6 days, preferably 2 to 4 days;

[0173] - the induction of differentiation of multipotent hematopoietic cells into erythroid progenitor cells can be carried out by placing the multipotent hematopoietic cells in a culture medium suitable for differentiation of multipotent hematopoietic cells, in particular the culture medium described above or in the "culture medium" section, for a period of usually 3 to 10 days, preferably 3 to 6 days;

[0174] - the induction of differentiation of erythroid progenitor cells into erythroid precursor cells can be performed by placing said erythroid progenitor cells in a culture medium suitable for erythroid progenitor cell differentiation, in particular a culture medium as described above or in the section "culture medium", for a period of usually 3 to 10 days, preferably 3 to 6 days;

[0175] - Induction of maturation of erythroid precursor cells can be carried out by placing said erythroid precursor cells in a culture medium suitable for maturation of erythroid precursor cells, in particular a culture medium as described above or in the "culture medium" section, for a period of usually 5 to 15 days, preferably 8 to 12 days.

[0176] Techniques for changing the composition of the culture medium are well known to those skilled in the art. Specifically, adding a molecule or increasing its concentration can be performed directly in a pre-existing culture medium, and removing a molecule or reducing its concentration can be performed by centrifuging the cells and resuspending them in a new culture medium or by diluting the culture medium.

[0177] The times given above for each step of the method are for guidance only. A person skilled in the art can easily define / adjust these times by observing the number and / or properties of cells in culture.

[0178] In the method according to the invention, the cells may be contacted with the vitisin compound during the following steps:

[0179] - a step of expanding multipotent hematopoietic cells, and / or

[0180] - a step of differentiating multipotent hematopoietic cells into unipotent erythroid progenitor cells, and / or

[0181] - a step of expanding unipotent erythroid progenitor cells, and / or

[0182] - a step of differentiating the unipotent erythroid progenitor cells into erythroid precursor cells; and / or

[0183] - a step of expanding erythroid precursor cells, and / or

[0184] - Steps that induce erythroid precursor cells to mature into erythroid cells.

[0185] According to certain specific embodiments, during each of these steps, the cell is contacted with the vitisin compound.

[0186] According to other specific embodiments, said cells are contacted with said vitisin compound during the step of expanding multipotent hematopoietic cells and / or the step of differentiating multipotent hematopoietic cells into unipotent erythroid progenitor cells.

[0187] According to other specific embodiments, said cells are contacted with said vitisin compound during the step of expanding unipotent erythroid progenitor cells and / or the step of differentiating unipotent erythroid progenitor cells into erythroid precursor cells.

[0188] According to other specific embodiments, said cells are contacted with said vitisin compound during the step of expanding unipotent erythroid progenitor cells and / or the step of differentiating unipotent erythroid progenitor cells into erythroid precursor cells and / or the step of differentiating unipotent erythroid progenitor cells into erythroid precursor cells.

[0189] According to other specific embodiments, the cells are contacted with the vitisin compound during (i) the step of expanding the multipotent hematopoietic cells and / or the step of differentiating the multipotent hematopoietic cells into unipotent erythroid progenitor cells and (ii) the step of expanding the unipotent erythroid progenitor cells and / or the step of differentiating the unipotent erythroid progenitor cells into erythroid precursor cells.

[0190] According to other specific embodiments, the cells are contacted with the vitisin compound during (i) the step of expanding multipotent hematopoietic cells and / or the step of differentiating multipotent hematopoietic cells into unipotent erythroid progenitor cells, (ii) the step of expanding unipotent erythroid progenitor cells and / or the step of differentiating unipotent erythroid progenitor cells into erythroid precursor cells, and (iii) the step of maturing erythroid precursor cells into erythroid cells.

[0191] According to other specific embodiments, the cells are contacted with the vitisin compound during the step of differentiating the multipotent hematopoietic cells into unipotent erythroid progenitors and the step of differentiating the unipotent erythroid progenitors into erythroid precursors, and optionally during the step of expanding the multipotent hematopoietic cells and / or the step of expanding the unipotent erythroid progenitors.

[0192] According to other specific embodiments, the cells are contacted with the vitisin compound during the step of differentiating the multipotent hematopoietic cells into unipotent erythroid progenitors and the step of differentiating the unipotent erythroid progenitors into erythroid precursors, and optionally during the step of expanding the multipotent hematopoietic cells and / or the step of expanding the unipotent erythroid progenitors, and optionally during the step of maturing the erythroid precursors into erythroid cells.

[0193] Preferably, said cells are contacted with said vitisin compound at least during the step of expanding unipotent erythroid progenitor cells and / or the step of differentiating unipotent erythroid progenitor cells into erythroid precursor cells.

[0194] When the cell is contacted with the vitisin compound during a step of the method, the vitisin compound may be present in the culture medium during the entire step or a portion of the step. Preferably, the vitisin compound is present throughout the step. Alternatively, the vitisin compound may be present for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days during the step.

[0195] In particular, the cells may be contacted with a vitisin compound for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the expansion and / or differentiation of multipotent hematopoietic cells), and / or for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the expansion and / or differentiation of unipotent erythroid progenitor cells).

[0196] Preferably, the cells are contacted with the vitisin compound for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the expansion of multipotent hematopoietic cells), at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the differentiation of multipotent hematopoietic cells), at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the expansion of unipotent erythroid progenitors), and at least 1 day, preferably at least 3, 5, 7, 10 or 12 days (during the differentiation of unipotent erythroid progenitors).

[0197] Specifically, the cells may be contacted with the vitisin compound for 2 to 15 days, particularly 3 to 12 days, especially 3 to 10 days.

[0198] According to a particular embodiment, the method according to the invention may comprise

[0199] - expanding the multipotent hematopoietic cells for a period of typically 3 to 15 days, preferably 5 to 8 days, in a medium suitable for the expansion of multipotent hematopoietic cells, preferably IMDM medium supplemented with G-SCF, SCF, FLT3-L and TPO and further comprising a vitisin compound, preferably vitisin B;

[0200] and

[0201] - Inducing differentiation of multipotent hematopoietic cells into erythroid progenitor cells in a medium suitable for differentiation of multipotent hematopoietic cells, preferably IMDM medium supplemented with IL3, IL6, SCF, serum albumin (particularly bovine albumin), insulin, transferrin and optionally EPO and further comprising a vitisin compound, preferably vitisin B, for a period of typically 3 to 10 days, preferably 3 to 6 days.

[0202] According to a particular embodiment, the method according to the invention may comprise

[0203] - expanding the erythroid progenitors for a period of typically 3 to 15 days, preferably 4 to 12 days, in a medium suitable for the expansion of erythroid progenitors, preferably IMDM or Stem Span SFEM II medium supplemented with G-SCF, SCF, FLT3-L and TPO and further comprising a vitisin compound, preferably vitisin B; and

[0204] - Inducing differentiation of erythroid progenitor cells into erythroid precursor cells for a period of typically 3 to 10 days, preferably 3 to 6 days, in a medium suitable for differentiation of erythroid progenitor cells, preferably IMDM or Stem Span SFEM II medium supplemented with IL3, SCF, hydrocortisone and optionally EPO and further comprising a vitisin compound, preferably vitisin B.

[0205] According to a specific embodiment, the method according to the invention may comprise inducing the maturation of erythroid precursor cells into erythroid cells for a period of typically 5 to 15 days, preferably 8 to 12 days, in a medium suitable for maturation of erythroid precursor cells, preferably EPO-supplemented IMDM or Stem Span SFEM II medium, preferably comprising 2 or 3 IU / ml EPO and also comprising a vitisin compound, preferably vitisin B. Preferably, this maturation medium does not comprise any cytokines.

[0206] According to certain embodiments, the hematopoietic cells are cultured in a medium suitable for the nutritional requirements of the hematopoietic cells for less than 50 days, less than 45 days, less than 40 days, less than 35 days, or less than 30 days. Specifically, the hematopoietic cells are cultured in a medium suitable for the nutritional requirements of the hematopoietic cells for 3 to 35 days, 7 to 35 days, 15 to 35 days, 25 to 35 days, 28 to 35 days, 7 to 28 days, 15 to 28 days, 20 to 28 days, preferably about 28 or 35 days.

[0207] In a specific embodiment, when the method comprises producing red blood cells from unipotent progenitor cells, the hematopoietic cells are cultured in a culture medium suitable for the nutritional requirements of the hematopoietic cells for 25 to 30 days, in particular for about 28 days.

[0208] In a specific embodiment, when the method comprises producing red blood cells from multipotent hematopoietic cells, in particular from HSCs, the hematopoietic cells are cultured in a medium suitable for the nutritional requirements of the hematopoietic cells for 30 to 40 days, in particular for about 35 days.

[0209] In the method according to the invention, the cell culture step, ie the expansion, differentiation and / or maturation step, is preferably carried out at a temperature suitable for the propagation and / or differentiation of hematopoietic cells. Preferably, the temperature is about 37°C.

[0210] During the cell culture step, the CO2 and / or O2 content is preferably controlled. Preferably, the CO2 content is between 2% and 10%, 3% and 7%, or 4% and 6%, and preferably about 5%. The O2 content is preferably between 1% and 30%, 5% and 25%, 10% and 25%, or 15% and 25%, and preferably about 20%.

[0211] In a particularly preferred manner, the cells are cultured at about 37° C., a CO 2 content of 5% and / or an O 2 content of 20%.

[0212] Preferably, the hematopoietic cells of interest in the present invention are cultured at a concentration of 200 to 10,000 cells / ml, preferably 500 to 2,000 cells / ml, even more preferably about 1,000 cells / ml.

[0213] Preferably, during the culturing step, the hematopoietic cells are contacted with mesenchymal cells (MSCs) or stromal cells. The terms "stromal cells", "MSCs" or "mesenchymal cells" refer to non-hematopoietic bone marrow cells, such as endothelial cells, non-striated vascular cells (fibrotic cells), adipocytes and macrophages. Mesenchymal stem cells are present in various tissues of the adult body, mainly in the bone marrow, but also in adipose tissue. They can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes, thereby producing and / or repairing bone and adipose tissue. In the medullary niche, mesenchymal cells and hematopoietic cells come into contact. Specifically, multipotent hematopoietic cells and hematopoietic progenitor cells are extrinsically regulated, particularly in the medullary hematopoietic niche, involving cell-to-cell contact and diffusible factors. Mesenchymal stem cells secrete growth factors that control the proliferation and differentiation of these hematopoietic cells.

[0214] Preferably, the stromal or mesenchymal cells are grown under suitable culture conditions such that they adhere or do not adhere to the substrate surface.

[0215] Hematopoietic cells can be co-cultured with mesenchymal cells (MSCs) to form fused cell clusters. A person skilled in the art can use any known technique to obtain fused MSC cell clusters.

[0216] According to certain specific embodiments, in the method according to the present invention, the step of differentiating the multipotent hematopoietic cells into unipotent erythroid progenitor cells and / or the step of differentiating the unipotent erythroid progenitor cells into erythroid precursor cells and / or the step of maturing the erythroid precursor cells are performed in co-culture with MSC cells. Preferably, the step of differentiating the multipotent hematopoietic cells into unipotent erythroid progenitor cells and the step of differentiating the unipotent erythroid progenitor cells into erythroid precursor cells, and optionally the step of maturing the erythroid precursor cells, are performed in co-culture with MSC cells.

[0217] When the method according to the present invention comprises a step of co-culturing with MSCs, the culture medium may further comprise fetal calf serum (FCS), in particular at a concentration of about 1% to about 10%, preferably about 1% to about 5%.

[0218] The method according to the invention may also comprise one or more additional steps.

[0219] The method according to the invention may in particular further comprise the step of collecting blood, preferably umbilical cord blood, placental blood and / or peripheral blood, from the individual.

[0220] The method according to the invention may also comprise one or more cell sorting steps, in particular a cell selection step based on the expression of markers such as CD34, CD36, CD123, CD235a, CD117, CD71 and / or GPA or any combination thereof. This preliminary step may in particular be carried out by immunomagnetic techniques using antibodies against various phenotypic markers of the cells of interest, in particular phenotypic markers as described in the section "erythropoiesis and hematopoietic cells".

[0221] The method for producing erythrocytes according to the invention may also comprise a nucleated cell removal step. This step in particular allows obtaining a homogenous population comprising only mature erythrocytes.

[0222] The production method according to the present invention may further comprise a step of recovering the obtained cells. This step may be performed by any technique known to those skilled in the art (especially filtration, centrifugation and removal of the culture medium).

[0223] The production method according to the present invention may also include a step of washing the obtained / recovered cells. This step can be performed by any technique known to those skilled in the art (especially a series of filtration, centrifugation and resuspension steps).

[0224] The production method according to the present invention may further comprise a step of storing the obtained / recovered cells. This step may be performed by any technique known to those skilled in the art (especially freezing). Specifically, the recovered cells may be mixed with a solution containing decomplemented fetal calf serum (FCS) and a cryoprotectant in a certain volume ratio before freezing.

[0225] In the method according to the invention, the vitisin compound, preferably vitisin-B, may be added to any culture medium suitable for the expansion, differentiation and / or maturation steps.

[0226] Specifically, the vitisin compound (preferably vitisin B) can be present in an amount of about 0.5 µM to about 1 mM, about 0.5 µM to about 500 µM, about 0.5 µM to about 200 µM, about 0.5 µM to about 100 µM, about 0.5 µM to about 50 µM, about 0.5 µM to about 40 µM, about 0.5 µM to about 30 µM, about 0.5 µM to about 20 µM, about 0.5 µM to about 15 µM, or about 0.5 µM to about 10 µM, about 5 µM to about 1 mM, about 5 µM to about 500 µM, about 5 µM to about 200 µM, about 5 µM to about 100 µM, about 5 µM to about 75 µM, about 5 µM to about 50 µM, about 5 µM to about 40 µM, about 5 µM to about 30 µM, about 5 µM to about 20 µM, about 5 µM to about 15 µM, or about 5 µM to about 10 µM. In some embodiments, the vitisin compound is present in the culture medium at a concentration of about 1 μM to about 10 μM, preferably about 10 μM to about 1 mM, about 10 μM to about 500 μM, about 10 μM to about 200 μM, about 10 μM to about 100 μM, about 10 μM to about 75 μM, about 10 μM to about 50 μM, about 10 μM to about 40 μM, about 10 μM to about 30 μM, about 10 μM to about 20 μM, preferably at a concentration of about 10 μM. Preferably, the vitisin compound is used at a concentration of 1 μM to 500 μM, preferably 5 μM to 100 μM. Specifically, vitisin-B can be used at a concentration of about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 80, 90, 95 or 100 μM.

[0227] Culture medium

[0228] The present invention also relates to a cell culture medium comprising a vitisin compound, in particular a cell culture medium suitable for the nutritional requirements of hematopoietic cells, in particular multipotent, pluripotent or unipotent hematopoietic cells (preferably selected from HSC, MPP, CMP, MEP cells, unipotent erythroid progenitor cells, erythroid precursor cells and combinations thereof) and comprising a vitisin compound. Specifically, the cell culture medium according to the present invention can be a culture medium suitable for the maintenance, expansion, differentiation and / or maturation of hematopoietic cells, preferably cells selected from HSC, MPP, CMP, MEP cells, unipotent erythroid progenitor cells, erythroid precursor cells and combinations thereof. This culture medium can be particularly useful in the various steps of the method according to the present invention.

[0229] The culture medium according to the invention can be any basal culture medium known to those skilled in the art that is suitable for the nutritional requirements of hematopoietic cells, in particular multipotent hematopoietic cells, unipotent progenitor cells and / or erythroid precursor cells, to which a vitisin compound is added. Many culture media suitable for the nutritional requirements of hematopoietic cells are known to those skilled in the art and are commercially available, such as Iscove's Modified Dulbecco's Medium (IMDM medium) and Stem SpanSFEM II medium (Stemcell technologies) or "Human StemMACS HSC Expansion Medium XF" (MiltenyiBiotec).

[0230] The cell culture medium is preferably selected from Iscove's Modified Dulbecco's Medium (IMDM medium) and StemSpan SFEM II medium (Stemcell technologies). Preferably, the culture medium is Iscove's Modified Dulbecco's Medium (IMDM_PAN BIOTECH). The IMDM medium is especially obtained from Dulbecco's Modified Eagle's Medium (DMEM), which is not only rich in sodium selenite, but also rich in vitamins and amino acids. The composition of IMDM and Stem Span SFEM II medium is well known in the literature. The IMDM medium especially contains amino acids, vitamins, salts, D-glucose, HEPES and sodium pyruvate.

[0231] The culture medium according to the invention may be liquid, semi-solid or solid. A person skilled in the art knows how to solidify a culture medium, in particular by adding agar.

[0232] The culture medium according to the present invention may contain one or more amino acids, antibiotics, vitamins, salts, minerals or lipids. The culture medium according to the present invention may also contain other compounds that are beneficial to the expansion and / or differentiation of hematopoietic cells, especially growth or survival factors, such as described in patent application WO 2010 / 096746. Specifically, the cell culture medium may be supplemented with erythropoietin (EPO), stem cell factor (SCF), hydrocortisone (HC), growth factors, interleukin 3 (IL3), interleukin 6 (IL6), interleukin 11 (IL11), transferrin, heparin, insulin, thrombopoietin, FMS type tyrosine kinase 3 ligand (Flt3-L), granulocyte colony stimulating factor (G-CSF), L-glutamine, inositol, antibiotics such as penicillin and / or streptomycin, folic acid, monothioglycerol, iron nitrate, iron sulfate, BIT 9500 supplement (Stem Cell Technologies), serum albumin, serum and / or plasma, or any combination thereof. The concentration of these various compounds is readily determined by one skilled in the art based on the supplier's recommendations or general knowledge in the art. One skilled in the art knows how to adjust the presence of such compounds to be suitable for hematopoietic cell culture.

[0233] The culture medium according to the present invention may optionally contain antibiotics, such as penicillin and / or streptomycin, in a concentration between about 0.1% and about 10%, preferably between about 0.5% and about 5%, more preferably about 1%.

[0234] The compounds in the culture medium according to the invention are preferably human compounds obtained by recombinant or purified techniques.

[0235] Preferably, the culture medium according to the invention comprises, in particular:

[0236] - IL-3, preferably human IL-3, at a concentration between about 1 ng / mL and about 20 ng / mL, preferably between about 5 ng / mL and about 15 ng / mL, more preferably about 5 or 10 ng / mL; and / or

[0237] - IL-6, preferably human IL-6, at a concentration between about 1 ng / mL and about 20 ng / mL, preferably between about 5 ng / mL and about 15 ng / mL, more preferably about 10 ng / mL; and / or

[0238] - SCF at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably between about 50 ng / mL and about 100 ng / mL, most preferably about 100 ng / mL; and / or

[0239] - EPO, preferably human EPO, at a concentration between about 0.5 IU / mL and about 10 IU / mL, preferably between about 1 IU / mL and about 5 IU / mL, more preferably about 2 or 3 IU / mL; and / or

[0240] - FLT3-L at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably 100 ng / mL; and / or

[0241] - TPO at a concentration between about 0.1 ng / mL and about 200 ng / mL, preferably between about 1 ng / mL and about 100 ng / mL, preferably between about 10 ng / mL and about 50 ng / mL, preferably 20 ng / mL; and / or

[0242] - G-CSF at a concentration between about 0.1 ng / mL and about 200 ng / mL, preferably between about 1 ng / mL and about 100 ng / mL, preferably between about 5 ng / mL and about 25 ng / mL, preferably 10 ng / mL, and / or

[0243] - transferrin, preferably human transferrin, at a concentration between about 50 μg / mL and about 400 μg / mL, preferably between about 50 μg / mL and about 200 μg / mL, preferably between 100 μg / mL and 150 μg / mL, more preferably about 120 μg / mL; and / or

[0244] - Insulin, preferably human insulin, in a concentration between about 1 μg / mL and about 50 μg / mL, preferably between about 5 μg / mL and about 20 μg / mL, more preferably about 10 μg / mL; and / or

[0245] - heparin, preferably human heparin, in a concentration between about 0.5 U / mL and about 10 U / mL, preferably between about 1 U / mL and about 5 U / mL, more preferably about 3 U / mL; and / or

[0246] - L-glutamine at a concentration between about 0.1 mM and about 50 mM, preferably between about 1 mM and about 15 mM, preferably between about 1 mM and about 5 mM, most preferably about 4 mM; and / or

[0247] - inositol in a concentration between about 10 μg / mL and about 200 μg / mL, preferably between about 20 μg / mL and about 100 μg / mL, preferably between 20 μg / mL and 60 μg / mL, more preferably about 40 μg / mL; and / or

[0248] - folic acid in a concentration between about 0.1 μg / mL and about 100 μg / mL, preferably between about 1 μg / mL and about 100 μg / mL, preferably between 5 μg / mL and 20 μg / mL, more preferably about 10 μg / mL; and / or

[0249] - Monothioglycerol, with a concentration of about 10 -5 M to about 10 -3 M, preferably between about 0.1×10 -4 M is about 10×10 -4 M, preferably between 10 -4 M is about 5×10 -4 M, more preferably about 1.6×10 -4 M; and / or

[0250] - Hydrocortisone (HC), with a concentration of about 10 -7 M to about 10 -8 M, preferably about 10 -6 M; and / or

[0251] - fetal bovine serum at a concentration between about 1% and about 10%, preferably between about 3% and about 7%, more preferably about 5%; and / or

[0252] - ferric nitrate at a concentration between about 1 ng / mL and about 200 ng / mL, preferably between about 10 ng / mL and about 150 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, more preferably about 90 ng / mL;

[0253] - ferric sulfate at a concentration between about 100 ng / mL and about 1500 ng / mL, preferably between about 500 ng / mL and about 1500 ng / mL, preferably between about 800 ng / mL and about 1000 ng / mL, more preferably about 900 ng / mL;

[0254] - human or bovine serum albumin at a concentration between about 1 mg / mL and about 50 mg / mL, preferably between about 5 mg / mL and about 20 mg / mL, more preferably about 10 mg / mL;

[0255] - albumin in a concentration between about 1% and about 10%, preferably between about 3% and about 7%, even more preferably about 4%, and / or

[0256] - BIT 9500 supplement (Stem Cell Technologies) at a concentration between about 1% and about 30%, preferably between about 10% and about 20%, more preferably about 15%; and / or

[0257] - serum, plasma or a pool of serum, preferably of human origin, in a concentration of between about 1% and about 10%, preferably between about 3% and about 7%, more preferably about 5%, and / or platelet lysate, preferably of human origin, in a concentration of between about 0.05% and about 0.5%, preferably between about 0.1% and about 0.5%, more preferably about 0.3%;

[0258] or any combination thereof.

[0259] According to certain embodiments, the culture medium according to the present invention is a culture medium comprising:

[0260] - L-glutamine at a concentration between about 0.1 mM and about 50 mM, preferably between about 1 mM and about 15 mM, preferably between about 1 mM and about 5 mM, most preferably about 4 mM; and / or

[0261] - inositol in a concentration between about 10 μg / mL and about 200 μg / mL, preferably between about 20 μg / mL and about 100 μg / mL, preferably between 20 μg / mL and 60 μg / mL, more preferably about 40 μg / mL; and / or

[0262] - folic acid in a concentration between about 0.1 μg / mL and about 100 μg / mL, preferably between about 1 μg / mL and about 100 μg / mL, preferably between 5 μg / mL and 20 μg / mL, more preferably about 10 μg / mL; and / or

[0263] - Monothioglycerol, with a concentration of about 10 -5 M to about 10 -3 M, preferably between about 0.1×10 -4 M is about 10×10 -4 M, preferably between 10 -4M is about 5×10 -4 M, more preferably about 1.6×10 -4 M; and / or

[0264] - transferrin, preferably human transferrin, at a concentration between about 50 μg / mL and about 400 μg / mL, preferably between about 50 μg / mL and about 200 μg / mL, preferably between 100 μg / mL and 150 μg / mL, more preferably about 120 μg / mL; and / or

[0265] - Insulin, preferably human insulin, in a concentration between about 1 μg / mL and about 50 μg / mL, preferably between about 5 μg / mL and about 20 μg / mL, more preferably about 10 μg / mL; and / or

[0266] - ferric nitrate at a concentration between about 1 ng / mL and about 200 ng / mL, preferably between about 10 ng / mL and about 150 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, more preferably about 90 ng / mL;

[0267] - ferric sulfate at a concentration between about 100 ng / mL and about 1500 ng / mL, preferably between about 500 ng / mL and about 1500 ng / mL, preferably between about 800 ng / mL and about 1000 ng / mL, more preferably about 900 ng / mL;

[0268] - human serum albumin at a concentration between about 1 mg / mL and about 50 mg / mL, preferably between about 5 mg / mL and about 20 mg / mL, more preferably about 10 mg / mL; and / or

[0269] - IL-3, preferably human IL-3, at a concentration between about 1 ng / mL and about 20 ng / mL, preferably between about 5 ng / mL and about 15 ng / mL, more preferably about 5 ng / mL; and / or

[0270] - SCF at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably between about 50 ng / mL and about 100 ng / mL, most preferably about 100 ng / mL; and / or

[0271] - EPO, preferably human EPO, at a concentration between about 0.5 IU / mL and about 10 IU / mL, preferably between about 1 IU / mL and about 5 IU / mL, more preferably about 3 IU / mL; and / or

[0272] - Hydrocortisone (HC), with a concentration of about 10 -7 M to about 10 -8 M, preferably about 10 -6 M.

[0273] According to certain embodiments, the culture medium is an expansion medium particularly suitable for expanding multipotent hematopoietic cells or unipotent erythroid progenitor cells, such as IMDM or Stem Span II medium, and further comprises:

[0274] - G-CSF at a concentration between about 0.1 ng / mL and about 200 ng / mL, preferably between about 1 ng / mL and about 100 ng / mL, preferably between about 5 ng / mL and about 25 ng / mL, preferably 10 ng / mL, and

[0275] - SCF at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably 100 ng / mL, and

[0276] - FLT3-L at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably 100 ng / mL, and

[0277] - TPO at a concentration between about 0.1 ng / mL and about 200 ng / mL, preferably between about 1 ng / mL and about 100 ng / mL, preferably between about 10 ng / mL and about 50 ng / mL, preferably 20 ng / mL.

[0278] According to certain embodiments, the culture medium is an expansion medium particularly suitable for expanding erythroid precursor cells, such as IMDM or Stem Span II medium, and further comprises:

[0279] - SCF, in particular at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably between about 50 ng / mL and about 100 ng / mL, most preferably about 100 ng / mL;

[0280] - Hydrocortisone (HC), particularly in a concentration of about 10 -7 M to about 10 -8 M, preferably about 10 -6 M; and / or optional

[0281] - transferrin (BIT), preferably human transferrin, in a concentration between about 50 μg / mL and about 400 μg / mL, preferably between about 50 μg / mL and about 200 μg / mL, preferably between 100 μg / mL and 150 μg / mL, more preferably about 120 μg / mL;

[0282] - EPO, preferably human EPO, in a concentration between about 0.5 IU / mL and about 10 IU / mL, preferably between about 1 IU / mL and about 5 IU / mL, more preferably about 2 IU / mL, and / or

[0283] - serum, plasma or a pool of serum, preferably of human origin, at a concentration between about 1% and about 10%, preferably between about 3% and about 7%, more preferably about 5%; and / or platelet lysate, preferably of human origin, at a concentration between about 0.05% and about 0.5%, preferably between about 0.1% and about 0.5%, more preferably about 0.3%.

[0284] According to certain other embodiments, the culture medium is a differentiation medium that is particularly capable of differentiating multipotent hematopoietic cells into unipotent erythroid progenitor cells, such as IMDM or Stem Span II medium, and further comprises:

[0285] - IL-3, preferably human IL-3, at a concentration between about 1 ng / mL and about 20 ng / mL, preferably between about 5 ng / mL and about 15 ng / mL, more preferably about 10 ng / mL;

[0286] - IL-6, preferably human IL-6, at a concentration between about 1 ng / mL and about 20 ng / mL, preferably between about 5 ng / mL and about 15 ng / mL, more preferably about 10 ng / mL;

[0287] - SCF at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 25 ng / mL and about 100 ng / mL, preferably about 50 ng / mL,

[0288] - bovine serum albumin (BSA) at a concentration between about 1 mg / mL and about 50 mg / mL, preferably between about 5 mg / mL and about 20 mg / mL, more preferably about 10 mg / mL;

[0289] - transferrin (BIT), preferably human transferrin, in a concentration between about 50 μg / mL and about 400 μg / mL, preferably between about 50 μg / mL and about 200 μg / mL, preferably between 100 μg / mL and 150 μg / mL, more preferably about 120 μg / mL;

[0290] - Insulin, preferably human insulin, in a concentration between about 1 μg / mL and about 50 μg / mL, preferably between about 5 μg / mL and about 20 μg / mL, more preferably about 10 μg / mL;

[0291] - optionally EPO, preferably human EPO, at a concentration between about 0.5 IU / mL and about 10 IU / mL, preferably between about 1 IU / mL and about 5 IU / mL, more preferably about 2 IU / mL, and

[0292] - Optionally fetal calf serum (FCS), in particular in a concentration between about 1% and about 10%, preferably between about 1% and about 5%.

[0293] BSA, insulin and transferrin may be provided, inter alia, by using BIT 9500 (Stem Cell Technologies) at a concentration between about 1% and about 30%, preferably between about 10% and about 20%, more preferably about 15%.

[0294] According to certain embodiments, the culture medium is a differentiation medium particularly suitable for differentiating unipotent progenitor cells into erythroid progenitor cells, such as IMDM or Stem Span II medium, and further comprises:

[0295] - IL-3, preferably human IL-3, in particular at a concentration between about 1 ng / mL and about 20 ng / mL, preferably between about 5 ng / mL and about 15 ng / mL, more preferably about 5 ng / mL; and

[0296] - SCF, in particular at a concentration between about 1 ng / mL and about 500 ng / mL, preferably between about 10 ng / mL and about 200 ng / mL, preferably between about 50 ng / mL and about 150 ng / mL, preferably between about 50 ng / mL and about 100 ng / mL, most preferably about 100 ng / mL; and

[0297] - Hydrocortisone (HC), particularly in a concentration of about 10 -7 M to about 10 -8 M, preferably about 10 -6 M; and / or optionally

[0298] - optionally EPO, preferably human EPO, particularly at a concentration between about 0.5 IU / mL and about 10 IU / mL, preferably between about 1 IU / mL and about 5 IU / mL, more preferably about 3 IU / mL; and

[0299] - Optionally fetal calf serum (FCS), in particular in a concentration between about 1% and about 10%, preferably between about 1% and about 5%.

[0300] According to certain other embodiments, the culture medium is a maturation medium, such as IMDM or Stem Span II medium without cytokines. Preferably, the maturation medium does not contain any cytokines but is supplemented with erythropoietin (about 2 or 3 IU / mL).

[0301] According to a very particular embodiment, the culture medium according to the invention comprises the vitisin compound and the compound of Table 1 (preferably in the concentrations mentioned in Table 1).

[0302]

[0303] According to another embodiment, the culture medium according to the invention is a culture medium comprising the compounds of Table 1, preferably in the concentrations mentioned in Table 1, as well as BSA, human insulin, human transferrin and optionally 2-mercaptoethanol, said culture medium further comprising a vitisin compound.

[0304] The vitisin compound, preferably vitisin-B, may be added to any of the culture media described above, such as expansion, differentiation and / or maturation media. Specifically, the vitisin compound, preferably vitisin-B, can be present in an amount of about 0.5 µM to about 1 mM, about 0.5 µM to about 500 µM, about 0.5 µM to about 200 µM, about 0.5 µM to about 100 µM, about 0.5 µM to about 50 µM, about 0.5 µM to about 40 µM, about 0.5 µM to about 30 µM, about 0.5 µM to about 20 µM, about 0.5 µM to about 15 µM, or about 0.5 µM to about 10 µM, about 5 µM to about 1 mM, about 5 µM to about 500 µM, about 5 µM to about 200 µM, about 5 µM to about 100 µM, about 5 µM to about 75 µM, about 5 µM to about 50 µM, about 5 µM to about 40 µM, about 5 µM to about 30 µM, about 5 µM to about 20 µM, about 5 µM to about 15 µM, or about 5 µM to about 10 µM. The vitisin compound is present in the culture medium according to the invention in a concentration of 1 μM to about 10 μM, preferably about 10 μM to about 1 mM, about 10 μM to about 500 μM, about 10 μM to about 200 μM, about 10 μM to about 100 μM, 10 μM to about 75 μM, about 10 μM to about 50 μM, about 10 μM to about 40 μM, about 10 μM to about 30 μM, about 10 μM to about 20 μM, preferably about 10 μM. Preferably, the vitisin compound is used in a concentration of 1 μM to 500 μM, preferably 5 μM to 100 μM. Specifically, the vitisin compound, preferably vitisin B, is added to the culture medium at a concentration of about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 80, 90, 95 or 100 μM.

[0305] The culture medium containing the vitisin compound according to the present invention can be advantageously used in methods for preparing hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP), unipotent erythroid progenitor cells, erythroid precursor cells and erythrocytes. In particular, the culture medium containing the vitisin compound according to the present invention can be used in any method according to the present invention, in particular the method as described in the "Cultivation Method" section above.

[0306] Therefore, the present invention also relates to the use of a cell culture medium according to the present invention, in particular a culture medium suitable for the expansion, differentiation and / or maturation of hematopoietic cells and comprising a vitisin compound, in particular a culture medium as described in any of the various embodiments above, for (i) the expansion and / or differentiation of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), megakaryocyte-erythroid progenitors (MEPs) or unipotent erythroid progenitors, and / or (ii) the differentiation and / or maturation of erythroid precursors, and / or (iii) the production of unipotent erythroid progenitors and / or erythrocytes, in particular by the method according to the present invention described below, more particularly for stimulating the differentiation of HSCs, MPPs, CMPs and / or MEPs into unipotent erythroid progenitors, the differentiation of unipotent erythroid progenitors into erythroid precursors and / or the stimulation of the maturation of erythroid precursors into erythrocytes.

[0307] The present invention also relates to the use of vitisin compounds for supplementing cell culture media, in particular cell culture media suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells, more particularly cell culture media suitable for (i) the expansion and / or differentiation of hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), megakaryocyte-erythroid progenitor cells (MEPs) or unipotent erythroid progenitor cells and / or (ii) the maturation of erythroid precursor cells and / or (iii) the production of unipotent erythroid progenitor cells and / or erythroid cells.

[0308] The present invention also relates to the use of vitisin compounds for (i) stimulating the expansion and / or differentiation of hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), megakaryocyte-erythroid progenitor cells (MEPs) or unipotent erythroid progenitor cells, and / or (ii) stimulating the maturation of erythroid precursor cells, and / or (iii) stimulating the production of unipotent erythroid progenitor cells and / or erythroid cells.

[0309] The vitisin compound can simply be added to a basal medium, preferably a basal medium suitable for the needs of hematopoietic cells. The vitisin compound is preferably added to a medium selected from an expansion medium, a differentiation medium and a maturation medium, in particular as defined above. In particular, the vitisin compound can be added so that it is present in the supplemented medium at a concentration as defined above.

[0310] Compositions based on vitisin compounds and uses thereof

[0311] According to another aspect, the present invention relates to the use of a vitisin compound for promoting or stimulating the production of hematopoietic cells, in particular red blood cells, in an individual, in particular an individual suffering from a blood disorder.

[0312] The present invention also relates to the vitisin compounds as drugs for treating blood diseases. The term "drug" as used herein includes drugs for human and animal use in human and veterinary medicine and refers to any pharmacologically acceptable substance that produces a therapeutic and / or beneficial effect.

[0313] The present invention also relates to a composition, in particular a pharmaceutical composition, comprising a vitisin compound for use in treating a blood disease or treating a patient suffering from blood loss.

[0314] As used herein, "pharmaceutical composition" refers to a preparation of an active agent with other optional chemical components (e.g., physiologically suitable carriers and / or excipients). The pharmaceutical composition according to the present invention encompasses pharmaceutical compositions for human medicine and pharmaceutical compositions for animal medicine (i.e., veterinary compositions).

[0315] The term "treatment" refers herein to producing a desired pharmacological and / or physiological effect. The effect may be preventive in terms of complete or partial prevention of a disease or symptom, and / or may be therapeutic in terms of partial or complete cure of a disease and / or an adverse reaction attributable to the disease. The term "treatment" as used herein encompasses any treatment of a disease in a subject, particularly a human, to: reduce the incidence and / or risk of recurrence of the disease during a symptom-free period; alleviate or mitigate the symptoms of the disease; prevent the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; inhibit the disease, i.e., stop its development (e.g., reduce the rate of progression); reduce the frequency of onset of the disease; and alleviate the disease, i.e., cause complete or partial regression of the disease.

[0316] The terms "blood disease", "blood disorders" or "blood pathology" are interchangeable and refer herein to diseases that affect blood components (blood cells such as red blood cells, white blood cells and platelets) or other components (hemoglobin, blood proteins) or that affect blood production or its mechanisms (coagulation, etc.). In particular, the blood diseases contemplated by the present invention are diseases associated with damage to red blood cells or insufficient production of hematopoietic cells, preferably red blood cells.

[0317] The blood disease may be selected in particular from sickle cell disease, myeloproliferative syndrome (leukemia, Vaquez's disease), aplastic anemia and anemia of other etiologies.

[0318] The pharmaceutical composition or vitisin compound may also be used to treat insufficient production of hematopoietic cells, preferably red blood cells, caused by another disease, such as cancer.

[0319] The pharmaceutical composition or vitisin compound can also be used to treat patients who suffer from severe blood loss, especially in the case of patients who need blood transfusion. Specifically, blood transfusion occurs in patients whose hemoglobin levels are less than 10. Blood transfusion includes intravenous injection of blood or blood components. Therefore, in addition to blood transfusion, the pharmaceutical composition or vitisin compound can also be used.

[0320] The pharmaceutical composition according to the present invention comprises a vitisin compound as an active ingredient.

[0321] In one embodiment, the pharmaceutical composition may further contain at least one additional pharmaceutically active ingredient.The term "active pharmaceutical ingredient" means any compound or substance, the administration of which has a therapeutic effect or beneficial effect on the health or general condition of the patient or subject to which it is administered.

[0322] The formulation of such pharmaceutical compositions may vary depending on the route of administration and the dosage intended for use of the composition.

[0323] The compositions according to the present invention may also include a pharmaceutically acceptable carrier. The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" or "pharmaceutically acceptable solvent" as used herein are interchangeable and include any compound or combination of compounds known to those skilled in the art to be useful in the formulation of pharmaceutical or veterinary compositions. Those skilled in the art know how to select the most suitable solvent and excipient to prepare a given type of formulation.

[0324] The vitisin compound or pharmaceutical composition can be used for administration to a subject. The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to an animal, preferably a mammal, more specifically a human. Preferably, the patient to be treated is a human, such as a child or an adult. In particular, the subject contemplated by the present invention is affected by a blood pathology or other pathology (such as cancer) that induces insufficient production of hematopoietic cells (preferably red blood cells).

[0325] The present invention also relates to a method for treating a blood pathology or other pathology that induces insufficient production of hematopoietic cells. The treatment method according to the present invention generally involves administering an effective amount of the vitisin compound or a composition comprising a vitisin compound to an individual in need thereof. Specifically, the treatment is for therapeutic or preventive purposes to limit or delay the onset of a blood pathology or another pathology that induces insufficient production of hematopoietic cells or symptoms associated with the disease.

[0326] The therapeutically effective amount or sufficient amount of the vitisin compound or the pharmaceutical composition comprising the compound is an amount capable of achieving the desired effect (ie, the effect of promoting the production of hematopoietic cells, especially red blood cells, in the patient).

[0327] In a particular embodiment, conventional treatments known to those skilled in the art for the treatment of hematopathologies can be supplemented by the administration of a vitisin compound according to the invention. For example, the administration of the vitisin compound can be combined with the administration of erythropoietin or a blood transfusion. The administration of the vitisin compound can be performed before, during and / or after the conventional treatment envisioned for the disease to be treated.

[0328] Reagent test kit

[0329] In a final aspect, the present invention also relates to a kit comprising compounds for producing a cell culture medium according to the invention, in particular as described above in the "culture medium" section, said culture medium not comprising any vitisin compound, said kit further comprising a vitisin compound.

[0330] Specifically, the present invention relates to a kit comprising:

[0331] - a cell culture medium, in particular a culture medium for the expansion, differentiation and / or maturation of hematopoietic cells, preferably as described above in the section "Culture medium", said culture medium not comprising any vitisin compound; and

[0332] - vitisin compounds, and

[0333] - optionally hematopoietic cells as described above in the section "Erythropoiesis and hematopoietic cells", in particular HSCs, MPPs, CMPs, MEPs, unipotent erythroid progenitors and / or erythroid precursors; and

[0334] - optionally a guide containing instructions for use of the kit.

[0335] Preferably, the kit comprises:

[0336] - a cell culture medium, in particular as described above under the section "Culture medium", preferably IMDM medium and / or Stem Span II medium, said culture medium not comprising any vitisin compound;

[0337] - one or more compounds selected from erythropoietin (EPO), stem cell factor (SCF), hydrocortisone (HC), growth factors, interleukin 3 (IL3), interleukin 6 (IL6), interleukin 11 (IL11), transferrin, heparin, insulin, thrombopoietin, FMS type tyrosine kinase 3 ligand (Flt3-L), granulocyte colony stimulating factor (G-CSF), L-glutamine, inositol, antibiotics such as penicillin and / or streptomycin, folic acid, monothioglycerol, iron nitrate, Iron sulfate, human serum albumin, serum or plasma or any combination thereof, preferably selected from erythropoietin (EPO), stem cell factor (SCF), hydrocortisone (HC), growth factors, interleukin 3 (IL3), interleukin 6 (IL6), interleukin 11 (IL11), BIT9500, FMS type tyrosine kinase 3 ligand (Flt3-L), granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), antibiotics such as penicillin and / or streptomycin or any combination thereof:

[0338] - vitisin compounds;

[0339] - optionally hematopoietic cells as described above in the section "Erythropoiesis and hematopoietic cells", in particular HSCs, MPPs, CMPs, MEPs, unipotent erythroid progenitors and / or erythroid precursors; and

[0340] - optionally a guide containing instructions for use of the kit.

[0341] Alternatively, the kit according to the present invention may comprise:

[0342] - a cell culture medium as defined above in the "Culture Medium" section and comprising a vitisin compound; and

[0343] - optionally hematopoietic cells as described above in the section "Erythropoiesis and hematopoietic cells", in particular HSCs, MPPs, CMPs, MEPs, unipotent erythroid progenitors and / or erythroid precursors; and

[0344] - optionally a guide containing instructions for use of the kit.

[0345] In particular, the kit of the invention may comprise, in a common container or in separate containers, any components necessary to produce a dehydrated form of the culture medium.

[0346] The kit according to the invention may also comprise the various materials and reagents for the use according to the invention in suitable containers and packaging materials, in particular pipettes, test tubes or bottles.

[0347] The kit may also include any means for collecting a biological sample (eg, blood), such as a syringe.

[0348] The present invention also relates to the use of a kit according to the invention for producing erythroid progenitor cells, erythroid precursor cells and / or erythrocytes, in particular according to the above-described method of the invention.

[0349] As used herein, the term "comprising" is used to refer to substances, compounds, or methods that are essential to the invention, but is open to the inclusion of non-specific elements, whether essential or not. The use of "comprising" means inclusion rather than limitation.

[0350] The term "and / or" as used herein should be considered as a specific description of each of the two specified features or components, whether or not the other is present. For example, "A and / or B" should be considered as specifically disclosing each of the following elements: (i) A, (ii) B, and (iii) A and B, as if each were presented individually.

[0351] As used herein, the term "about" in connection with all values ​​(including the lower and upper limits of the numerical range) means that the acceptable variation is 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 list of values ​​modifies each of the values ​​therein (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). In addition, when describing a list of values ​​(e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54% or 85.4%).

[0352] All references cited in this patent application, including journal articles or abstracts, published patent applications, issued patents, or any other references, are hereby incorporated by reference in their entirety, including all results, tables, figures, and text presented in said references.

[0353] Other characteristics and advantages of the invention will emerge more clearly on reading the examples which follow, given as non-limiting illustrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0354] Figure 1 :Using peripheral blood CD34 + During the expansion of cells, vitisin B has an important effect on hematopoietic progenitor cells, especially CD34 + CD133neg The data represent the absolute number of cell subsets identified for each phenotype at the end of the protocol. The data represent the average of four experiments.

[0355] Figure 2 :Effects of vitisin B and other resveratrol derivatives / analogs on erythroid potential CD34 + CD133 neg Comparison of the effects of 100 μg / ml on the production of progenitor cells in vitro. Data represent CD34 + CD133 neg Comparison of the proportion (A) and absolute number (B) of progenitor cells as a function of the molecules tested. The data represent the mean of five experiments.

[0356] Figure 3 :In peripheral blood CD34 + Effect of vitisin B on erythroid differentiation in cells starting with a simplified erythroid differentiation procedure (ERYTHRO 1 protocol, detailed in the experimental section). Data represent the level of CD71 membrane expression (mean fluorescence intensity, relative arbitrary value) (A) and the relative proportion of cells that acquired GPA expression on their membrane (B) as a function of the dose of vitisin B in the culture medium. The data represent the mean of six experiments. The indicated amounts of vitisin B correspond to concentrations of 5, 10, 15, 20 and 30 µM, respectively.

[0357] Figure 4 :In the isolated peripheral blood CD34 + Effect of vitisin B on erythroid differentiation in a simplified erythroid differentiation procedure (ERYTHRO 1 protocol, detailed in the experimental section) starting with SP, CMP and MEP subsets of cells. Data represent the evolution of the relative proportion of cells expressing GPA and CD71 on their membrane (A) and the evolution of the level of CD71 membrane expression (mean fluorescence intensity, relative to arbitrary values) during culture in the presence or absence of vitisin B (10 µM) in the culture medium (B). The data represent the mean of three experiments.

[0358] Figure 5 According to the optimized three-stage production process (ERYTHRO 2 protocol, described in detail in the experimental section), vitisinB is activated against placental blood CD34 +Effect of vitisin B on the total expansion of cells. The graphs represent the absolute number of cells obtained and counted during the culture in the absence or presence of vitisin B (10 µM). CTRL: absence of vitisin B / vitisin B in culture; I: presence of vitisin B / vitisin B in phase I; II: presence of vitisin B / vitisin B in phases I and II; III: presence of vitisin B in all three phases of the protocol. The data represent the average of two experiments.

[0359] Figure 6 :Effects of vitisin B on placental and peripheral blood CD34 + Effect of cell differentiation into erythroid cells. The expression dynamics of glycophorin A (GPA) and transferrin receptor 1 (CD71) during erythroid differentiation were analyzed in the presence or absence of vitisin B molecules (10 µM). Figures A and B: CD34 + The data represent the average of two experiments. neg GPA + Figure 3 Evolution of the relative proportion (A) and amount (B) of cells with different phenotypes. Figure 3 Evolution of the relative proportion (A) and amount (B) of cells with different phenotypes. + The data represent the average of five experiments. neg GPA + Phenotypic evolution of cells.

[0360] Figure 7 :Vitisin B affects CD34 from placental blood + Effect of the expansion of BFU-E and CFU-E erythroid progenitors of cells. The number of primitive (BFU-E, Figure A) and mature (CFU-E, Figure B) erythroid progenitors was estimated in the background of colony formation assays performed on D4, D8 and D11 of erythroid culture. The data represent the average of four experiments.

[0361] Example

[0362] Introduction – Main steps

[0363] 1. First step – Molecular testing of hematopoietic cell expansion

[0364] Vitisin B was tested in a hematopoietic cell expansion protocol and compared with seven other stilbenoid molecules.

[0365] In this study, CD34 derived from steady-state peripheral blood was used.+ cell.

[0366] A single cell culture protocol specifically for the bulk expansion of hematopoietic progenitor cells was used. This is the "Expansion Protocol" described in Section B.1. of the Materials and Methods section below.

[0367] The results obtained in this step made it possible to identify the molecule "Vitisin B" as one that spontaneously promotes the expansion of the subpopulation with erythroid potential.

[0368] 2. The second step in the study – investigating the effect of vitisin B on red blood cell production

[0369] Vitisin B was tested to determine its ability to optimize erythroid progenitor and erythroid production in a culture system dedicated to erythroid differentiation.

[0370] Various cell populations of interest were tested:

[0371] ○ Total CD34 + cell.

[0372] ○ Different CD34 + Subpopulations: side population (SP), CMP (common myeloid progenitor), MEP (megakaryocyte and erythroid progenitor), and BFU-E (erythroid burst-forming unit).

[0373] All these different cell populations are physiologically organized along the hierarchy of SP->CMP->MEP->BFU-E->erythroblasts / reticulocytes / erythrocytes on the one hand and SP->CMP->other blood cells on the other hand. Therefore, these different populations contribute to the production of mature blood cells.

[0374] Therefore, the potential of vitisin B to target a cell population with erythroid potential (CD34 + , SP, CMP, MEP and BFU-E).

[0375] These cell populations of interest are derived from two different cell sources, steady state peripheral blood (SSBP) and / or placental blood (USP). Thus, the cell population of interest studied is derived from one of these two sources, or optionally from both.

[0376] Specifically, CD34 + The cells are derived from peripheral blood and / or placental blood, SP, CMP and MEP cells are derived from peripheral blood, and BFU-E cells are derived from placental blood.

[0377] Two different culture protocols were used that allowed the cell population of interest to differentiate into erythroid cells:

[0378] ○ Simplified two-stage protocol (described in Materials and Methods Section B.2.b)

[0379] ○ Optimized three-stage protocol (described in Materials and Methods Section B.2.c)

[0380] Table 2 below summarizes the study organizations.

[0381]

[0382] I. Materials and Methods

[0383] A. Isolation of Populations of Interest

[0384] Table 3 below summarizes the + The phenotypes of the different populations of interest from which the cells were isolated (phenotypes described in II.A.1 below) are cross-referenced with the corresponding sections of the Materials and Methods.

[0385]

[0386] 1. CD34 + Isolation of cells

[0387] This separation is based on the immunomagnetic sorting technique. Hematopoietic lineage cells with the CD34 surface antigen are selected by previously separating the mononuclear blood cell fraction from peripheral blood or placental blood leukocytes. Cells are separated from the blood by density gradient (d = 1.077) using isotonic Ficoll solution. The mononuclear cells are then cleared of residual platelets by density gradient (d = 1.040), and the cell pellet is washed with sucrose solution (d = 1.0721) and then centrifuged (430 × g, 10 min, 4 ° C). After the removal of platelets, the pellet containing mononuclear cells is recovered and rinsed twice with selection buffer (PBS pH 7.2, EDTA 200 mM and human albumin (20%)) by centrifugation (430 × g, 10 min, 4 ° C). The pellet is then resuspended in 2 mL of selection buffer supplemented with DNase and Tegelin (0.5 g / mL).

[0388] The obtained CD34 was then isolated by immunomagnetic positive selection using a selection kit (Indirect Kit CD34 Microbead Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). + cells (Peytour et al., 2010).

[0389] 2. Different CD34 + Isolation of cell subpopulations

[0390] a. Isolation of side population cells (SP cells)

[0391] SP cells can be identified by their ability to efflux certain dyes, such as Hoescht or circulating violet dye (DCV). This efflux is carried out using the calcium-dependent ATP-binding cassette (ABCG2) pump (Brunet de la Grange et al., 2013). To identify previously isolated CD34 + To culture SP cells, incubate them with DCV dye (5 µM) in DMEM + The cells were incubated in Dulbecco's modified Eagle's medium (DMEM), 2% FCS, 1% HEPES at 37°C in the dark for 2 h. After incubation, all operations until final cell sorting were performed on ice and in PBS. + The PBS was used to culture the cells. + The culture medium is a glucose and calcium depleted medium to stop the efflux pumps. + The cells were rinsed and centrifuged (430 × g, 10 min, 4°C). The pellet was resuspended in PBS. + After incubation for 15 min, cells were incubated with the viability marker 7AAD (7-aminoactinomycin-D) (1:100,000), which allowed exclusion of any dead cells, and anti-CD34-APC antibody (1:100,000) (Beckman Coulter). + Rinse in PBS, centrifuge (430 × g, 10 min, 4°C) and resuspend in PBS + Medium. CD34 with low fluorescence intensity for DCV marker + Cells (CD34 + SP cells) were analyzed (UV 350 nm or violet 405 nm), sorted and recovered using an ARIA cell sorter (Becton Dickinson).

[0392] b. Isolation of CMP and MEP Progenitors

[0393] CD34 +Cells were labeled for 15 min with anti-CD34-BV421 (1:100,000), anti-CD45RA-BB517 (1:100,000), anti-CD123-Alexa647 (1.5:100,000) antibodies (BD Pharmingen) and anti-LIN-PE antibody cocktail (2:100,000). The LIN cocktail antibodies are directed against mature cell-specific markers (CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a). Thus, this LIN cocktail allows enrichment of “LIN” by depletion of mature cells. neg "Immature cells.

[0394] After incubation with these antibodies, cells were washed with PBS. + The cells were rinsed with PBS + 2% fetal calf serum (FCS) solution and centrifuged (430 × g, 10 min, 4°C).

[0395] Resuspend the cells in PBS + After that, cells with the phenotype of CD34 were sorted and harvested using ARIA cell sorter. + LIN neg CD123 + CD45RA neg CMPs and phenotypes for CD34 + LIN neg CD123 neg CD45RA neg MEPs (Manz et al., 2002).

[0396] c. Isolation of BFU-E and CFU-E Progenitor Cells

[0397] Placental blood CD34 + Cells were labeled with antibodies specific for surface antigens (i.e., anti-CD34, anti-GPA, anti-CD123, and anti-CD36 antibodies) that allow targeting and differentiation of BFU-E primitive erythroid progenitors. Primitive erythroid progenitors (BFU-E) are defined by the following phenotypes: CD34 + CD123 neg CD36 neg GPA neg Mature erythroid progenitor cells (CFU-E) are defined by the following phenotypes: CD34 neg CD123 neg CD36 + GPA neg CD71 high .

[0398] For immunolabeling, the cells were incubated with blocking solution at 4°C for 10 minutes to saturate Fc receptors as nonspecific antibody binding sites. Antibodies were added and the suspension was incubated at room temperature in the dark for 15 minutes. Excess antibodies were removed by washing the cells with 40 mL PBS / BSA buffer and centrifuging at 300×g for 10 minutes at 4°C, and the cell pellet was centrifuged at 15×10 6 The cells were resuspended at a concentration of 10 cells / mL. The cell suspension was incubated with the fluorescent viability marker 7-AAD (7-aminoactinomycin D) on ice for 10 minutes to highlight cell viability and exclude dead cells.

[0399] After phenotypic labeling, primitive erythroid progenitors (BFU-E) were isolated by cell sorting technology (ARIA BD).

[0400] After cell sorting of the population enriched for BFU-E primitive erythroid progenitors, they were washed in PBS buffer and centrifuged at 300 × g for 10 minutes at 4° C. The cell pellet was resuspended.

[0401] The enrichment of the cell population with BFU-E was verified by performing a CFC (colony forming cell) colony formation assay.

[0402] B. Cell Culture Strategies

[0403] The cells were cultured under sterile conditions in a microbiological safety cabinet. The cells were grown in an incubator at 37°C with 5% CO2.

[0404] Depending on the goal and the starting cell population, several culture strategies are used:

[0405] 1. About the first research step: “Amplification plan”

[0406] This is a strategy for expanding hematopoietic progenitor cells. The cultures were established by isolating CD34 + Cells (as described in Section II.A.1) were cultured for 12 days in Stem Span II medium (Stem Cell Technologies) supplemented with the following cytokines: SCF 100 ng / mL, FLT3-L 100 ng / mL, TPO 20 ng / mL, G-CSF 10 ng / mL. Proliferation was quantified (counted on Malassez slides) and CD34 expression by flow cytometry. + Phenotypic analysis of cell subsets Amplification products were analyzed. Vitisin B was used at different concentrations, specifically 10 and 50 µM. Vitisin B was also compared with the following stilbenes at a concentration of 10 µM:

[0407] Resveratrol, Pallidol, E-grapevine, ampelopsis, oxyresveratrol, piceatannol, gennetol.

[0408] 2. About the second study step: "ERYTHRO 1 and ERYTHRO 2 programs"

[0409] This is a strategy for erythroid differentiation of various hematopoietic cells of interest.

[0410] a. Expansion / pre-stimulation of SP cells

[0411] This step involves only SP cells. Due to the low number of SP cells that can be isolated (essentially rare cells) and their very immature state, it is preferred to expand and pre-stimulate this subpopulation before proceeding to the erythroid differentiation protocol. Therefore, SP cells are pre-cultured for seven days in liquid cell culture medium (Stem Span medium) supplemented with granulocyte colony stimulating factor (G-SCF) (10 ng / mL), SCF (100 ng / µL), FLT3-L (100 ng / µL), and thrombopoietin (TPO) (100 ng / µL). After this step, the SP cells are subjected to the erythroid differentiation protocol.

[0412] b. ERYTHRO 1 protocol: simplified ex vivo erythroid differentiation protocol (two-stage)

[0413] This protocol is applicable to CD34 cells from peripheral blood and placental blood. + Cells, pre-stimulated SP cells, CMP cells and MEP cells. For this culture, the cell population of interest was isolated as previously described and cultured in IMDM medium supplemented with the following substances:

[0414] 15% BIT 9500 (Stem Cell Technologies). BIT is a mixture of BSA (bovine serum albumin), insulin, and transferrin.

[0415] Cytokines: 10 ng / mL IL3, 10 ng / mL IL6, 50 ng / mL SCF.

[0416] The antibiotic mixture penicillin-streptomycin (100 U / mL) was especially used to avoid any bacterial contamination.

[0417] For CD34 +For SP cells, all cells recovered after the "expansion / pre-stimulation" step (i.e., 5,000-50,000 cells) were cultured in 24-well plates.

[0418] The training consists of two stages:

[0419] Phase 1: D0 to D6, culture without EPO

[0420] Phase 2: Starting from D6, add 2 IU / ML EPO and extend the culture to D21 (minimum) and D28 (maximum).

[0421] To test the influence of vitisin B in this context, the cultivation was performed in the absence and presence of vitisin B:

[0422] ● CTRL condition: vitisin B is absent in the entire regimen.

[0423] ● Test conditions: Presence of 5, 10, 15, 20 or 30 µM vitisin B during the entire protocol (Phase 1 + Phase 2).

[0424] During the culture protocol, differentiation products were analyzed by quantifying proliferation (counting) and analyzing erythroid differentiation markers by flow cytometry using the markers CD71 and CD235a.

[0425] c. ERYTHRO 2 regimen: three-stage erythroid differentiation regimen

[0426] This protocol is suitable for CD34 + Cells. Stage 1 of this protocol is specifically for BFU-E cells. This protocol was adapted from Vlaski et al. (Experimental Hematology 2009; 37:573-584).

[0427] The ex vivo erythroid production is established after 28 days of cell culture and involves three stages, namely 1) expansion of primitive erythroid progenitors, followed by 2) differentiation of the progenitors into erythroid precursors, and finally 3) a terminal stage of maturation with enucleation of the cells and the development of mature erythroid cells.

[0428] i. Detailed description of the three phases of the program

[0429] Phase I of the protocol begins at D0 with the inoculation of 104 On day 4, the cultures were diluted by withdrawing 3 mL and replacing it with an equal volume of fresh medium. On day 8, to allow for the survival and differentiation of these distinct cytokine-dependent erythroid progenitors, cells from all conditions were plated at 3 × 10 5 / mL was resuspended in 5 mL of IMDM medium containing cytokines (human interleukin 3 (hIL3), stem cell factor (SCF), hydrocortisone (HC), and erythropoietin (EPO)) and incubated for another 3 days.

[0430] Phase II of the protocol begins on day 11 and corresponds to the development of erythroid precursor cells. From day 11 to the end of the process, the cells are placed on a layer of mesenchymal stromal cells (MSCs) (co-culture), allowing their final maturation into functional enucleated cells by means of contact between stromal cells and erythroid precursor cells.

[0431] The suspended red blood cells were plated at 3x10 5 The cells were seeded at a concentration of 1:100 μg / ml and co-cultured from day 11 to day 15 in fresh culture medium containing the cytokines required by MSCs (SCF, IL-3, EPO, and HC) and 5% fetal calf serum (FCS).

[0432] On day 15, the suspended erythrocytes were removed from the culture, washed, and then re-seeded onto a new MSC-attached layer and cultured for an additional four days in 2 mL of fresh medium containing only EPO.

[0433] Phase III of the protocol begins on day 19. During this phase, cells complete maturation by enucleation of orthochromatic erythroblasts to form reticulocytes and thus erythrocytes. On day 19, cells are washed and plated on a new layer of MSCs in 2 mL of fresh cytokine-free medium. On day 23, cultures are fed with fresh medium until day 28, corresponding to the last day of culture in the erythropoiesis protocol.

[0434] ii. Description of the culture medium

[0435] The cells were incubated in Iscove's Modified Dulbecco's Medium (IMDM_PAN BIOTECH) supplemented with various agents to obtain a medium that promotes the growth of cells committed to the erythroid lineage, namely:

[0436] L-Glutamine (4 mM),

[0437] Streptomycin and penicillin (1%),

[0438] Inositol (40 µg / mL),

[0439] Folic acid (10 µg / mL),

[0440] Monothioglycerol (1.6×10 -4 M),

[0441] Transferrin (120 µg / mL),

[0442] Insulin (10 µg / mL),

[0443] Ferric nitrate (90 ng / mL),

[0444] Ferrous sulfate (900 ng / mL),

[0445] Human serum albumin (10 mg / ml).

[0446] After the medium is prepared, cytokines are extemporaneously added to the medium:

[0447] 100 ng / mL of stem cell factor (SCF),

[0448] 5 ng / mL interleukin-3 (IL-3),

[0449] 3 IU / mL of erythropoietin (EPO),

[0450] 10 -6 M of hydrocortisone (HC).

[0451] CD34 + The cells were incubated at 37°C under 20% O2 and 5% CO2 for 28 days.

[0452] iii. Description of the culture conditions used

[0453] In order to test the role of vitisin B in this context, various conditions were defined.

[0454] For CD34 + Cell

[0455] ● CTRL condition: vitisin B is absent in the entire regimen.

[0456] ● Condition I: Presence of vitisin B (10 µM) in phase I of the protocol.

[0457] ● Condition II: vitisin B (10 µM) was present in both phases I and II of the protocol.

[0458] ● Condition III: vitisin B (10 µM) was present during all three phases of the protocol.

[0459] Cells from each condition were analyzed. Erythroid differentiation and maturation were monitored by quantifying progenitor cells, analyzing the expression of GPA and CD71 markers, and also by morphological analysis.

[0460] For BFU-E

[0461] ● CTRL conditions: cells were cultured in the absence of vitisin B for 7 days.

[0462] ● Test conditions: Cells were cultured in the presence of 10 μM vitisin B for 7 days.

[0463] C. Strategies for analyzing cell cultures

[0464] a. Expansion analysis strategy (Phase 1 of the study)

[0465] At the end of the hematopoietic cell expansion protocol, cell proliferation and expansion of both immature and committed cell populations were analyzed.

[0466] Cell proliferation was assessed by counting cells on Malassez slides by exclusion of dead cells using trypan blue.

[0467] Expansion of the cell population is based on phenotypic analysis by flow cytometry related to the evolution of the expression of the membrane markers CD34 and CD133. These markers allow the definition of more or less immature cell populations organized hierarchically as follows: CD34 + CD133 + -> CD34 + CD133 neg -> CD34 neg CD133 negTo this end, the cells were incubated with anti-CD34 (1:100,000) (BD Pharmingen) and anti-CD133 (1:100,000, BD Pharmingen) antibodies for 15 minutes. The cells were then rinsed with PBS solution and centrifuged (430 × g, 10 min, 4°C). After resuspending the cell pellet in PBS, the cells were analyzed by flow cytometry (FACSCantoII, Becton Dickinson, San Jose, CA).

[0468] The observed proportions of each phenotypic population and the total number of cells allowed calculation of the most immature cells (CD34 + CD133 + ) and more committed progenitor cells (CD34 + CD133 neg and CD34 neg CD133 neg ) compared to the amplification ratio.

[0469] b. Strategies to analyze erythroid differentiation (Phase 2 of the study)

[0470] i. Cell counting and phenotyping (for both ERYTHRO 1 and ERYTHRO 2 protocols)

[0471] During and at the end of the various erythroid differentiation protocols, cell proliferation and cell differentiation were analyzed. Cell proliferation was assessed by counting cells on Malassez slides with exclusion of dead cells using trypan blue. Different populations and culture conditions were also analyzed by flow cytometry to observe the appearance of erythroid differentiation markers and thus the kinetics of cell differentiation. To this end, cells were incubated for 15 minutes with erythroid differentiation-specific antibodies: anti-CD71 (1:100,000) (BD Pharmingen) and anti-CD235 (1:100,000) (BD Pharmingen) antibodies. Labeling with anti-CD34 antibody (1:100,000) (Beckman Coulter) was also performed along with other antibodies to monitor the loss of CD34, which demonstrates cell maturation. The cells were then rinsed with PBS solution and centrifuged (430 × g, 10 min, 4°C). After resuspending the cell pellet in PBS, the cells were analyzed by flow cytometry (FACSCantoII, Becton Dickinson, San Jose, CA).

[0472] The proportion of each phenotypic population and the total amount of cells thus observed allowed calculation of the amount of cells that had reached different stages of differentiation.

[0473] ii. Analysis of Erythroid Progenitor Generation (ERYTHRO 2 regimen only)

[0474] During the differentiation phase to erythrocytes, the population of interest produces progenitor cells committed to the erythroid pathway, which can be directly demonstrated by their colony-forming capacity, i.e. their individual ability to produce differentiated, morphologically recognizable colonies of cells during culture. For this purpose, the CFC (colony forming cell) test is used. Committed progenitor cells are able to produce colonies after cultivation in a semisolid medium consisting of methylcellulose and supplemented with cytokines. Thus, the committed progenitor cells produced during the erythroid culture (here BFU-E and CFU-E) can be counted.

[0475] This analysis of BFU-E and CFU-E production was performed only in Phase 1 of Protocol 2. Cells grown and harvested on days 4, 8, and 11 of Phase 1 were plated at concentrations of 150 cells / 250 μL H4034 (for day 4) and 300 cells / 250 μL in a ready-to-use cytokine-supplemented methylcellulose H4034 kit (Stem Cell Technology, Meylan, France).

[0476] II. Results

[0477] 1. Effects of vitisin B and various antioxidant molecules on the expansion of hematopoietic progenitor cells ( Figure 1 and 2 )

[0478] In the context of hematopoietic progenitor cell expansion (expansion protocol), vitisin B induced CD34 + Up to 250% expansion of the total number of phenotypic progenitor cells ( Figure 1 It was also observed that vitisin B induced the expression of specific subsets of CD34 + CD133 neg The erythroid potential of the subpopulation has been demonstrated by the inventors' team (Lapostolle et al., 2018 Hematologica Vol. 103 No. 10 (2018)). Therefore, these CD34 + CD133 neg Progenitor cells have the potential to generate mature erythrocytes. + The population is still CD34 + CD133 neg Overall, the effects of vitisin B occurred already at 10 µM, whereas increasing the dose to 50 µM did not induce additional effects.

[0479] Comparison of vitisin B with other polyphenols (stilbenes) derived from resveratrol (resveratrol, pallidol, E-viniferin, ampelopsis, oxyresveratrol, piceatannol, and genistein) at a dose of 10 µM showed that only vitisin B had a significant effect on CD34 + CD133 neg The proportion and total amount of cells have a convincing effect ( Figure 2 ). This means that in CD34 + In the context of cell expansion, vitisin B was able to expand a subset enriched in erythroid progenitors. This result suggests that vitisin B has a specific effect in the context of CD34 expansion, whereas other well-known antioxidant molecules such as resveratrol have no effect.

[0480] Thus, only vitisin B allowed expansion of an erythroid progenitor-enriched population relative to the other molecules tested, demonstrating the uniqueness of vitisin B's action.

[0481] 2. Effect of vitisin B on erythrocyte differentiation

[0482] a. In the context of the ERYTHRO 1 erythroid differentiation protocol

[0483] i. From peripheral blood CD34 + cell( Figure 3 )

[0484] In peripheral blood CD34 + The effect of vitisin B was observed at the level of erythroid cell differentiation and was assessed according to a simplified protocol (ERYTHRO 1 protocol, presented in Section B.2.b of Materials and Methods).

[0485] Under these conditions, the inventors observed that vitisin B induced CD34 + Cells differentiate more rapidly and to a greater extent into erythroid progenitor cells ( Figure 3 This effect was measured by flow cytometry on the basis of the expression of the membrane antigens CD71 and CD235a (glycophorin A, GPA) according to the following differentiation order: CD71 + GPA neg -> CD71 + GPA + ->CD71 low GPA + The inventors observed that the earlier acquisition of CD235a was accompanied by a more rapid decline in CD71 expression (CD71 low ), indicating differentiation.

[0486] Thus, after completion of both protocols (EXPANSION and ERYTHRO 1), two distinct and complementary effects of vitisin B were observed: expansion of progenitor cells with erythroid potential and acceleration of differentiation into erythroid precursor cells.

[0487] ii. From SP, CMP and MEP cells ( Figure 4 )

[0488] For sorted CD34 + Subpopulations (SP, CMP, and MEP) were observed to be similar to those of peripheral blood CD34 + These results show that for both CMPs and MEPs, erythroid differentiation (CD71 + GPA + The acquisition of phenotype) is accelerated ( Figure 4 A), and for SP, CMP, and MEP, CD71 membrane expression was reduced by up to 40% relative to controls as early as D7 of culture ( Figure 4 B).

[0489] b. In the context of the ERYTHRO 2 three-stage erythroid differentiation protocol

[0490] i. Placental blood CD34 + Total cell proliferation ( Figure 5 )

[0491] The effect of the vitisin B molecule on erythrocyte development was also investigated in a three-stage ex vivo erythrocyte production protocol according to an optimized protocol (ERYTHRO 2 protocol, presented in Section B.2.c of Materials and Methods). Figure 5 Represents the number of erythrocytes in culture during the erythropoietic protocol under the different conditions tested.

[0492] ● CTRL condition: vitisin B is absent in the entire regimen.

[0493] ● Condition I: Presence of vitisin B in Phase I of the protocol.

[0494] ● Condition II: Presence of vitisin B in both phases I and II of the protocol.

[0495] ● Condition III: Presence of vitisin B during all three phases of the regimen.

[0496] During phase I (days 0 to 11), cell proliferation was similar in each condition tested. Starting from the transition to phase II (day 11), a dramatic increase in cell proliferation was observed for the three conditions in the presence of vitisin B, reaching a peak proliferation at day 21. Specifically, the number of cells in condition I was 1.5 times that of the control condition (CTRL). These results were confirmed using placental blood CD34 + cells and using peripheral blood CD34 + Cells were observed in both conditions.

[0497] Erythroid cell expansion was significantly increased in the presence of vitisin B in culture. Vitisin B stimulates the production of progenitor cells, leading to the development of precursor cells and mature erythroid cells.

[0498] ii. Using CD34 from peripheral blood and placental blood + Erythroid differentiation of cells ( Figure 6 ).

[0499] Erythroid differentiation was observed by the expression of CD71 (transferrin receptor 1) and GPA (glycophorin A) markers to confirm the effect of vitisin B on differentiation during the ex vivo erythropoiesis protocol (ERYTHRO 2 protocol).

[0500] The presence of Vitisin B molecules increases the expression of CD71 neg GPA + The percentage of cells with phenotype ( Figure 6 A). Specifically, at day 8 of culture, less than 20% of cells expressed GPA. In the presence of vitisin B molecules, GPA expression was visible at day 15 and increased more rapidly until day 28 (more than 50% of cells expressed it) compared to CTRL conditions (40%).

[0501] For CD71 neg GPA + This effect was even more pronounced in terms of the absolute number of cells with the phenotype ( Figure 6 B). The presence of vitisin B molecules increased the expression of CD71 neg GPA + For peripheral blood CD34 + CD71 produced by cells neg GPA + The same effect was observed for the absolute number of cells ( Figure 6 C).

[0502] These results suggest that the presence of the vitisin B molecule allows red blood cells to differentiate faster and more efficiently, which ultimately reflects a more efficient production of red blood cells.

[0503] 3. Effects of vitisin B on BFU-E and CFU-E erythroid progenitor cells ( Figure 7 )

[0504] The inventors have also focused their attention on specific progenitor cell populations, namely BFU-E and CFU-E. These progenitor cells are physiologically generated by differentiation of more immature cells and are also generated ex vivo if culture conditions permit. They are detected by methylcellulose assay (see section Cbii in Materials and Methods), during which they generate colonies of mature red blood cells (reticulocytes and erythrocytes). The inventors have observed that the presence of vitisin B accelerates the differentiation of primitive erythroid progenitors (BFU-E; Figure 7 A), but also accelerates the emergence of more differentiated progenitor cells derived from BFU-E (CFU-E; Figure 7 B) emergence.

[0505] These results show the advantage of vitisin B on primitive hematopoietic cell populations, thereby improving the production of mature enucleated red blood cells (i.e., reticulocytes and erythrocytes).

[0506] III. Conclusion

[0507] The study of erythrocytes throughout this study made it possible to evaluate the effects of vitisin B on the expansion and differentiation of hematopoietic cells.

[0508] Specifically, vitisin B binds to CD34 + It exerts its stimulatory effect in the first developmental step associated with the commitment of cells to the erythroid lineage and the expansion of progenitor cells. + Proliferation of hematopoietic cells to promote their commitment to erythroid progenitors.

[0509] Vitisin B also allows accelerated differentiation of progenitor cells into erythroid precursors and their enucleation, giving rise to mature cells: reticulocytes and erythrocytes.

[0510] Thus, contacting hematopoietic cells with vitisin B allows for higher yields of ex vivo red blood cell production and speeds up the production process.

[0511] Thus, specifically, these two effects combined could overcome some of the bottlenecks observed in ex vivo erythrocyte production, particularly low yield and production time.

Claims

1. An in vitro method for producing red blood cells, the method comprising: a) providing multipotent hematopoietic cells, preferably of human origin, selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, and optionally expanding the multipotent hematopoietic cells; b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells; c) inducing the unipotent erythroid progenitor cells to differentiate into erythroid precursor cells; and d) inducing the erythroid precursor cells to mature into erythroid cells, and e) optionally recovering the obtained red blood cells; Wherein when step a) comprises a cell expansion step, in step a), step b), step c) and / or step d), the cells are contacted with a vitisin compound.

2. An in vitro method for producing unipotent erythroid progenitor cells, the method comprising: a) providing multipotent hematopoietic cells, preferably of human origin, selected from hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) and combinations thereof, and optionally expanding the multipotent hematopoietic cells; b) inducing differentiation of the multipotent hematopoietic cells into unipotent erythroid progenitor cells, and optionally expanding the unipotent erythroid progenitor cells; as well as c) optionally recovering the obtained unipotent erythroid progenitor cells; Wherein when step a) comprises a cell expansion step, in step a) and / or step b), the cells are contacted with a vitisin compound.

3. An in vitro method for producing red blood cells, the method comprising: a) providing unipotent erythroid progenitor cells, preferably obtained by the method according to claim 2; b) inducing the unipotent erythroid progenitor cells to differentiate into unipotent erythroid precursor cells; c) inducing the erythroid precursor cells to mature into erythroid cells, and d) optionally recovering the obtained red blood cells, wherein in step b) and / or step c), the cells are contacted with a vitisin compound.

4. The method according to any one of claims 1 to 3, wherein the cells are contacted with the vitisin compound in a culture medium suitable for the nutritional requirements of hematopoietic cells, in particular a culture medium suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells.

5. The method according to any one of claims 1 to 4, wherein the cells are contacted with the vitisin compound during i) expansion and / or differentiation of multipotent hematopoietic cells or ii) expansion and / or differentiation of unipotent erythroid progenitor cells.

6. The method according to any one of claims 1 to 5, wherein the cells are contacted with the vitisin compound during expansion and / or differentiation of multipotent hematopoietic cells and during expansion and / or differentiation of unipotent erythroid progenitor cells, and optionally during maturation of erythroid precursor cells.

7. The method according to any one of claims 1 to 6, wherein the cells are contacted with a vitisin compound for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days during the expansion and / or differentiation of multipotent hematopoietic cells and / or the cells are contacted with a vitisin compound for at least 1 day, preferably at least 3, 5, 7, 10 or 12 days during the expansion and / or differentiation of unipotent erythroid progenitor cells.

8. The method according to any one of claims 1 to 7, wherein the vitisin compound is present in the culture medium at a concentration between 1 μM and 500 μM, preferably between 5 μM and 100 μM, when contacting with the cells.

9. A cell culture medium suitable for the expansion and / or differentiation and / or maturation of hematopoietic cells and comprising a vitisin compound.

10. The cell culture medium according to claim 9, which is selected from the group consisting of HSC, MPP, CMP, MEP cells, unipotent erythroid progenitor cells, erythroid precursor cells, and combinations thereof. The cell culture medium according to claim 9 , wherein the hematopoietic cells are HSCs or unipotent erythroid progenitor cells.

12. The cell culture medium according to claim 9 or 10, wherein the vitisin compound is present in the culture medium at a concentration between 1 μM and 500 μM, preferably between 5 μM and 100 μM.

13. Use in vitro of a cell culture medium according to any one of claims 9 to 12 for (i) expansion and / or differentiation of hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), megakaryocyte-erythroid progenitor cells (MEP) or unipotent erythroid progenitor cells, and / or (ii) differentiation and / or maturation of erythroid precursor cells, and / or (iii) production of unipotent erythroid progenitor cells and / or erythroid cells.

14. Use of a vitisin compound for supplementing a cell culture medium suitable for expansion and / or differentiation and / or maturation of hematopoietic cells.

15. Use of a vitisin compound for (i) stimulating the expansion and / or differentiation of hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), megakaryocyte-erythroid progenitor cells (MEPs) or unipotent erythroid progenitor cells, and / or (ii) stimulating the differentiation and / or maturation of erythroid precursor cells, and / or (iii) stimulating the production of unipotent erythroid progenitor cells and / or erythroid cells.

16. The method according to any one of claims 1 to 8, the culture medium according to any one of claims 9 to 12 or the use according to any one of claims 13 to 15, wherein the vitisin compound is vitisin B or a derivative thereof.

17. The method according to claim 16, the culture medium according to claim 16 or the use according to claim 16, wherein the vitisin B derivative is a vitisin B isomer, preferably a vitisin B stereoisomer.

18. The method according to any one of claims 1 to 8, the culture medium according to any one of claims 9 to 12, or the use according to any one of claims 13 to 15, wherein the vitisin compound is a vitisin derivative of the formula: (II) or (III)。 19. The method according to any one of claims 1 to 8, the culture medium according to any one of claims 9 to 12 or the use according to any one of claims 13 to 15, wherein the vitisin compound is vitisin B.

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