Method for the production of polyploid megakaryocytes, and method for the production of IPSC-derived platelet-like particles using the same

Abstract

The invention pertains to methods for the production of polyploid megakaryocytes using diploid megakaryocytes derived from induced pluripotent stem cells (iPSCs), the polyploid megakaryocytes so produced, and their use for the production of iPSC-derived platelet-like particles. The invention further pertains to the iPSCs-derived platelet-like particles produced using the polyploid megakaryocytes of the invention, and their use as a medicament.

Description

[0001] METHOD FOR THE PRODUCTION OF POLYPLOID MEGAKARYOCYTES, AND METHOD FOR THE PRODUCTION OF IPSC-DERIVED PLATELET-LIKE PARTICLES USING THE SAME

[0002] FIELD OF THE INVENTION

[0003] The invention pertains to methods for the production of polyploid megakaryocytes using diploid megakaryocytes preferably derived from a megakaryocyte progenitor, yet preferably derived from induced pluripotent stem cells (iPSCs), the polyploid megakaryocytes so produced, and their use for the production of iPSC-derived platelet-like particles. The invention further pertains to the iPSCs- derived platelet-like particles produced using the polyploid megakaryocytes of the invention, and their use as a medicament.

[0004] BACKGROUND OF THE INVENTION

[0005] Thrombocytopenia, that is to say abnormally low levels of platelets (also known as thrombocytes) in the blood of a patient, is common and found in many pathologies, particularly in hematological malignancies and solid cancers. Thrombocytopenia frequency has increased with the use of heavy chemotherapies or certain targeted therapies, and with it the risk of hemorrhagic complications (Vanderschueren et al. 2000). Platelet transfusion has become an essential element in the treatment of these pathologies, the demand for which in Europe is on average 5 units per 1 ,000 inhabitants per year, each unit containing 200-300 billion platelets.

[0006] Blood platelets, or thrombocytes, are irregular, disc shaped cell fragments that circulate in the blood and are essential for hemostasis, angiogenesis, and innate immunity. In vivo, in the human body, platelets are formed and released into the bloodstream by precursor cells called megakaryocytes that reside primarily in the bone marrow but are also found in the lung and peripheral blood. Before a megakaryocyte has the capacity to release platelets, it enlarges considerably to an approximate diameter of 100 pm and fills with high concentrations of ribosomes that facilitate the production of platelet-specific proteins (Long M, et al.). Cellular enlargement is mediated by multiple rounds of endomitosis, a process that amplifies the DNA by as much as 64-fold (Ebbe S. et al.). Polyploidy of the megakaryocytes plays an important role in their ability to produce platelets: it has been suggested that a single megakaryocyte after 3 cycles of endomitoses (16N) may yield approximately 125 times more platelets than 8 megakaryocytes derived from 3 standard mitoses (2N) (Mazzi et al., 2018).

[0007] Currently, platelets are sourced uniquely from donations, and their storage time is limited only to a few days. Indeed, the necessity to store platelet concentrates at room temperature to minimize loss of platelets functional integrity, poses a major risk for bacterial contamination. In this context, efforts have been made to supplement donor-derived platelets, and in particular to find alternatives sources of platelets, in particular via the in vitro production of platelets using purified megakaryocytes obtained from umbilical cord blood. However, a unit of cord blood contains only approximately 1 million hematopoietic progenitors (Avanzi et al.) whose maintenance and expansion remain very limited in vitro.

[0008] In an effort to find new sources of platelets, several authors have suggested the use of induced pluripotent stem cells (iPSCs) as a source of megakaryocytes for the purpose of in vitro platelet production. However megakaryocytes derived from CD34+progenitor cells induced into the megakaryocyte lineage have a limited capacity for polyploidization, compared to those derived from human embryonic stem cells (hESCs) (Bluteau et al.), which impacts their ability to produce platelets in large volumes.

[0009] There is therefore still a need for processes and cell lines that would allow for the production of large numbers of polyploid megakaryocytes, preferably derived from a megakaryocyte progenitor, yet preferably derived from iPSCs, which could then be used for the production of platelets and plateletlike particles.

[0010] SUMMARY OF THE INVENTION

[0011] As disclosed herein and in particular details in the experimental part, the inventors have identified several molecules which expression level or activity plays an important role in the regulation of the ploidy of megakaryocytes obtained from megakaryocyte progenitors, yet preferably iPSCs. Indeed, the inventors have shown that that decreasing the level or activity of either one of TP53 or p21 , or of the combination of p21 and p19, in the megakaryocytes of the invention provides a critical background that facilitates the generation of polyploid cells from these megakaryocytes.

[0012] The inventors have further determined that suppressing the expression or activity of either one of MPS1 , DYRK1A, and ROCK, in a megakaryocyte preferably derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, where the level or activity of either one of TP53 or p21 , or of the combination of p21 and p19 has been suppressed lead to the formation of a very large number of multinucleated megakaryocytes and a consequent increase in the quantity of platelets or iPSC-derived platelet-like particles that can be obtained in vitro. The inventors have put forth a method for the production of polyploid megakaryocytes, based on diploid megakaryocyte preferably derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, that stems from these findings.

[0013] The invention pertains to a method for the production of polyploid megakaryocytes comprising the steps of: (a) providing a diploid megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, and

[0014] (b) inducing polyploidy of the diploid megakaryocyte by: i. suppressing the expression or activity of a protein chosen in the list consisting of TP53 or p21 , preferably suppressing the expression or activity of the combination of p21 and p19, and; ii. suppressing the expression or activity of a protein chosen in the list consisting of MPS1 and DYRK1 , preferably suppressing the expression or activity of MPSI and DYRK1 ; and

[0015] Hi. optionally, suppressing the expression or activity of ROCK, thereby producing a polyploid megakaryocyte.

[0016] Preferably, in the method of the invention

[0017] (a) when present, suppressing the expression or activity of TP53 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of TP53;

[0018] (b) when present, suppressing the expression or activity of p21 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p21 ;

[0019] (c) when present, suppressing the expression or activity of p19 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p19, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p19;

[0020] (d) when present, suppressing the expression or activity of MPS1 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 , preferably reversine;

[0021] (e) when present, suppressing the expression or activity of DYRK1 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 , preferably harmine; and (f) when present, suppressing the expression or activity of ROCK is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK, preferably Y27632.

[0022] Preferably in the method of the invention, step b) of inducing polyploidy of the diploid megakaryocyte is by suppressing the expression or activity of TP53 or of the combination of p21 and p19, and by suppressing the expression or activity of MPS1 , DYRK1 , and ROCK.

[0023] Preferably in the method of the invention, the diploid megakaryocyte is genetically modified so as to silence TP53, p21 or a combination of p21 and p19, and

[0024] (a) suppressing the expression or activity of MPS1 is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 ;

[0025] (b) suppressing the expression or activity of DYRK1 is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 ;

[0026] (c) suppressing the expression or activity of ROCK is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK.

[0027] Preferably in the method of the invention:

[0028] (a) suppressing the expression or activity of MPS1 is obtained by culturing the diploid megakaryocyte in the presence of reversine, preferably reversine at a concentration of between 1 and 5 pM, yet preferably reversine at a concentration of about 2.5 pM;

[0029] (b) suppressing the expression or activity of DYRK1A is obtained by culturing the diploid megakaryocyte in the presence of harmine, preferably harmine at a concentration of between 2 and 5 pM, yet preferably harmine at a concentration of about 2.5 pM;

[0030] (c) suppressing the expression or activity of ROCK is obtained by culturing the diploid megakaryocyte in the presence of Y27632, preferably Y27632 at a concentration of between 1 and 10 pM, yet preferably Y27632 at a concentration of about 10 pM; or

[0031] (d) any combination of (a) to (c).

[0032] Preferably in the method of the invention, the diploid megakaryocyte is derived from a human iPSC, yet preferably derived from a human HLA-universal induced pluripotent stem cell (iPSC) line.

[0033] Preferably in the method of the invention, the diploid megakaryocyte derived from an iPSC is obtained by

[0034] (a) inducing differentiation of an iPSC into an hematopoietic progenitor cell by: i. Culturing an iPSC in the presence of molecules chosen from the list consisting of BMP4, CHIR, VEGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 12 and 36 hours, preferably for about 24 hours; ii. Culturing a cell obtained from step a) i) in the presence of molecules chosen from the list consisting of BMP4, VEGF, bFGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 60 and 84 hours, preferably for about 72 hours;

[0035] Hi. Culturing a cell obtained from step a) ii) in the presence of molecules chosen from the list consisting of VEGF, bFGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno- free cell culture medium, for between 36 and 60 hours, preferably for about 48 hours; iv. Culturing a cell obtained from step a) iii) in the presence of molecules chosen from the list consisting of VEGF, bFGF, SCF, Flt3L and a combination thereof, v. preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 12 and 36 hours, preferably for about 24 hours; vi. Culturing a cell obtained from step a) iv) in the presence of molecules chosen from the list consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL6 and a combination thereof, preferably at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 132 and 156 hours, preferably for about 144 hours, thereby obtaining a hematopoietic progenitor cell; and

[0036] (b) inducing differentiation of the hematopoietic progenitor cell into a diploid megakaryocyte by culturing an hematopoietic progenitor cell in the presence of molecules chosen from the list consisting of SCF, TPO and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 324 and 348 hours, preferably for about 336 hours, thereby obtaining a diploid megakaryocyte.

[0037] The invention further pertains to a polyploid megakaryocyte produced by the method of the invention.

[0038] The invention further pertains to a population of megakaryocytes produced by the method of the invention, Preferably, the population of megakaryocytes comprises at least 30%, 40% 50%, 60% preferably 65% of polyploid megakaryocytes.

[0039] The invention further pertains to a composition comprising the polyploid megakaryocyte of the invention or the population of megakaryocytes produced by the method of the invention, and a cell culture medium, wherein the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of TP53, p21 , p19, MPS1 , DYRK1 , and ROCK. The invention further pertains to a megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, wherein said megakaryocyte is genetically modified so as to silence:

[0040] - a gene selected in the list consisting of the genes encoding p21 , p19, MPS1 , DYRK1 , and ROCK, and

[0041] - optionally, the gene encoding TP53.

[0042] The invention further pertains to a method for the production of iPSC-derived platelet-like particles comprising culturing the polyploid megakaryocyte of the invention or the population of megakaryocytes produced by the method of the invention in conditions appropriate for the production of platelet-like particles, wherein said conditions comprise culturing the megakaryocyte or the population of megakaryocytes in the presence of an inhibitor of the expression or activity of a protein chosen from the list consisting of TP53, p21 , p19, MPS1 , DYRK1 , and ROCK.

[0043] The invention further pertains to iPSC-derived platelet-like particles produced by the method for the production of iPSC-derived platelet-like particles of the invention described above.

[0044] The invention further pertains to iPSC-derived platelet-like particles produced by the method for the production of iPSC-derived platelet-like particles of the invention described above, for use as a medicament, in particular for the treatment or prevention of thrombocytopenia, preferably neonatal thrombocytopenia or thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL.

[0045] FIGURE LEGEND

[0046] Figure 1 . Sorting strategy of 2N and 8N MKs for scRNA seq experiment. (A) Dot plots (left panel) and ploidy plots (right panel) showing the CD41 + CD42+ MKs and the ploidy level, respectively derived from iPSC, cord blood and apheresis. (B) May-Grunwald-Giemsa (MGG) staining of 2N and 8N MKs after sorting.

[0047] Figure 2. Molecular signature of the different phases of the cell cycle through MK ontogeny. The cell cycle phases are shown through ontogeny according to the indicated MK ploidy levels (2N and 8N).

[0048] Figure 3. Differential gene expression through MK ontogenic stages. (A) Venn diagram showing the molecular signatures of 2N MKs that are progressively downregulated through ontogenic progression. (B) Venn diagram showing the molecular signatures of 8N MKs that are progressively enriched through ontogenic progression.

[0049] Figure 4. Pilot experiment for MK polyploidy induction. Dot plots and their corresponding ploidy plots of wild-type and TP53KO-like MK with or without DYRK1A and MPS1 inhibitors separately or in combination at the indicated concentrations. Figure 5. Assessment of MK yields from wild-type and invalidated TP53 hematopoietic progenitors. (A) Dot plots showing the percentage of CD41 + CD42+ MK with or without MPS1 / DYRK1A inhibitors in the indicated conditions. (B) Histogram showing the mean number of MKs in the indicated conditions. Data are presented as mean ± sem, n=3 (*, p<0.05).

[0050] Figure 6. MPS1 i / DYRK1 Ai cooperate with invalidated TP53 to increase the MK size and ploidy levels. (A) MK size (left panel, mean FSC) and MK granularity / complexity (right panel, mean SSC). (B) MK ploidy plots and their corresponding histograms showing the percentage of >4N MK (upper) and the mean ploidy (lower) in the indicated conditions. (C) MK morphology analyses after MGG coloration in the indicated conditions. Data are represented as mean ± sem, n=3 (*, p<0.05).

[0051] Figure 7. MPS1 i / DYRK1Ai / ROCKi cooperate with invalidated TP53 to increase platelet yields. (A) Venn diagram of the upregulated genes in 8N versus. 2N MKs according to the ontogenic stage. Molecular signatures enriched in the 8N embryonic genes are shown. (B) Dot plots of CD41 + CD42+ platelets in the indicated conditions. (C) Histogram showing the mean number of platelets generated per MK in the indicated conditions. Data are represented as mean ± sem (*, p<0.05).

[0052] Figure 8. MPS1 i / DYRK1 Ai / ROCKi cooperate with invalidated TP53 to increase platelet yields. (A) Dot plots of CD41 + CD42+ platelets in the indicated conditions. (B) Histogram showing the mean number of platelets generated per MK in the indicated conditions. These experiments were performed one or two times and are indicated in the figure.

[0053] Figure 9. MPS1 i / DYRK1 Ai cooperate with p21 KO and even more in combination with p19KO to dramatically increase MK ploidy levels. (A) MK dot plots and their corresponding ploidy level in the indicated conditions. (B) Histograms showing MK yields, the percentage of >4N MK and MK modal ploidy in the indicated conditions.

[0054] Figure 10. Invalidation of both p21 and p19 slightly increase platelet yields in presence of MPS1 i / DYRK1 Ai / ROCKi which is further enhanced by Palbociclib. (A) Histogram shows the mean percentage of MK bearing platelets (left) or the mean number of platelets per MK (right). (B) Images showing MK forming platelets according to increasing concentrations of Palbociclib (right panel). Histogram shows the total yield of platelets obtained from p19KO / p21 KO with DYRK1Ai / MPS1 i / ROCKi in the indicated Palbociclib concentrations. All data are presented as mean + / - SEM (**p<0.01).

[0055] Figure 11. MPS1 i alone in the context of p19KO / p21 KO recapitulates the ploidy level of TP53KD MKs with MPS1 i / DYRK1Ai. (A) Ploidy plots of shTP53 and p19KO / p21 KO MKs treated either with MPS1 i alone or in combination with DYRK1AL Histograms show the percentage of >4N MK (left) or modal ploidy (right) in the indicated conditions.

[0056] Figure 12. TP53KD with MPS1 i / DYRK1 Ai / ROCKi do not alter platelet function. (A) Gating strategy to assess GPIIbllla activation from CD42-positive platelets in absence or presence of thrombine agonist. (B) Histogram shows the mean percentage of PAC1 + platelets in the indicated conditions. (C) Left panel: images of fibrinogen-adhered platelets in absence or presence of thrombine. Platelets were detected using the CD41 marker and actin remodeling was assessed according to phalloidin aspect. Right panel: histogram shows the mean percentage of platelets exhibiting stress fibers in the indicated conditions. Data are represented as mean + / - sem (*, p<0.05).

[0057] DEFINITIONS

[0058] In the context of the invention, the term “diploid” or “diploid cell” should be construed as generally understood in the field, that is to say as referring to a cell or cells harboring two complete sets of chromosomes. Independently of the species considered, sets of chromosomes are typically designated as “N”, and diploid cells may therefore be designated as “2N” cells, to indicate that they harbor 2 sets of chromosomes. When referring to human cells, the term diploid refers to cells harboring 23 chromosome pairs, so 46 chromosomes in total.

[0059] In the context of the invention, the term “polyploid”, “polyploid cell”, “polyploidy” should be construed as generally understood in the field, that is to say as referring to a cell or cells harboring an abnormally high number of chromosomes, i.e. more than 2 sets of chromosomes (>2N). In particular, polyploid cells may be tetrapioid cells. The term “polyploid”, “polyploid cell” or “polyploidy” encompasses cells which number of chromosomes exceeds 2 sets of chromosomes, such as for instance 4 sets of chromosomes (4N), but may include 4 to 64 sets (4N to 64N), 4 to 32 sets (4N to 32N), 8 to 64 sets (8 to 64N), 16 to 32 sets (16 to 32N), or 16 to 64 sets (16 to 64N). The term “tetrapioid” or “tetrapioid cell” or “4N cells” refer to a cell harboring 4 sets of chromosomes (4N).

[0060] In the context of the invention, the terms “somatic cells” refer to differentiated adult cells.

[0061] In the context of the invention, the term “totipotent cell” should be construed as generally understood in the field, that is to say as a cell with an unlimited differentiation spectrum, that is to say capable of differentiating into any type of cell including both embryonic and placental cells.

[0062] In the context of the invention, the term “a pluripotent cell” or “PC” should be construed as generally understood in the field, that is to say as a cell capable of differentiating into any of the three germ layers: endoderm (gut, lungs and liver), mesoderm (muscle, skeleton, blood vascular, urogenital, dermis), or ectoderm (nervous, sensory, epidermis), but not into extra-embryonic tissues like the placenta or yolk sac. Examples of pluripotent cells include embryonic stem cells and induced pluripotent stem cells (iPSCs). Human pluripotent stem cells (hPSCs) are characterized by specific cell-surface markers, such as the glycolipid antigens SSEA3 and SSEA4, as well as the glycoprotein antigens TRA-1-60 and TRA-1-81 . The pluripotency status of a cell can be verified by detecting the presence of these biological markers, which are surface antigens, on the external membrane of the cells. In the context of the invention, the term “embryonic stem cells” or “ESC” should be construed as generally understood in the field, that is to say as referring to stem cells obtained from fertilized embryos at the blastocyst stage, for instance fertilized embryos grown in vitro for 5 days as described by Thomson JA, et al., which also describes how to establish embryonic stem cell lines from embryonic tissues. Embryonic stem cells are typically considered as totipotent cells, but can also be described as pluripotent cells in the sense that they can differentiate according to a differentiation spectrum at least as large as other known pluripotent stem cells (such as adult stem cells, referred to as somatic stem cells, or induced pluripotent stem cell).

[0063] In the context of the invention, the term “megakaryocyte progenitor” refers to cells or cell lines from which megakaryocytes can be derived. In the context of the invention, the term “megakaryocyte progenitor” encompasses:

[0064] - undifferentiated iPSC expressing pluripotency markers , in particular expressing the markers POU5F1 , NANOG, SOX2, KLF4, SSEA3, SSEA4, TRA-1-60 and TRA-1-81

[0065] - cells obtained from iPSC expressing CD34+CD43+, CD34+CD43+GPA+CD41 +,

[0066] - iPSC reprogrammed into the MK lineage by overexpression of GATA1 , FLI1 , TAL1 as described by Moreau et al. 2016,

[0067] - Immortalized cells obtained from iPSC as described by Nakamura et al. 2014,

[0068] - CD34+ progenitors isolated from cord blood, from leukapheresis, or from bone marrow, such as hematopoietic progenitor cells (HPCs) or hematopoietic stem cells (HSCs) as defined herein, including CD34+CD41 + CD42+ / - MK progenitors derived from CD34+ cells isolated from cord blood, from leukapheresis, or from bone marrow, and- Immortalized cell lines (CD34+CD41 + / -) derived from CD34+ progenitors and able to be differentiated toward MK lineage.

[0069] In the context of the invention, the term “induced pluripotent stem cells” or “iPSC” should be construed as generally understood in the field, that is to say as referring to pluripotent stem cells obtained from a non-pluripotent cell, typically an adult somatic cell (a cell of the body, rather than gametes or an embryo), by controlled expression of certain genes (the products of these gene are often referred to in the literature as «defined reprogramming factors »), such as overexpressing Oct4, Sox2, Klf4 and c-Myc in mice cells (but other genes combinations may be used). With respect to their potential of differentiation, iPSCs are believed to be similar to natural pluripotent stem cells, such as embryo stem cells (ESCs) in many respects. iPSCs are not adult stem cells, but reprogrammed cells given pluripotent capabilities. iPSCs do not refer to cells as they are found in nature. Preferably, iPSC are not derived from embryonic cells (EC).

[0070] Processes to obtain iPSC from mouse cell cultures were described by Takahashi, K., et al. (2006). Processes to obtain iPSC from human cell cultures were described by Takahashi, K., et al.. (2007), Yu, J., et al. (2007). Most techniques and methods to obtain iPSCs have been reviewed by Lewandowski J, et al. In the context of the invention, the iPSC are preferably of mammalian origin, preferably of human origin. In other terms, the iPSCs are preferably derived from mammalian cells, that is to say they have been obtained using mammalian cells, yet preferably the iPSCs are derived from human cell, that is to say they have been obtained using human cells. In a preferred embodiment, the iPSCs are preferably derived from mammalian somatic cells, that is to say they have been obtained using mammalian somatic cells, yet preferably the iPSCs are derived from human somatic cells, that is to say they have been obtained using human somatic cells.

[0071] Induced pluripotent stem cells can be obtained by induction of pluripotency in cells from different sources. Tipically, cells such as skin fibroblasts, keratinocytes, adipose-derived stem cells, dermal papilla cells, blood cells, in particular hematopoietic stem cells from blood cells (including hematopoietic stem cells from bone marrow, from umbilical cord blood and placenta, or from peripheral blood, are known in the art and can be used as somatic cells for the induction of iPSCs, as reviewed by Li J, et al.

[0072] In the context of the invention, the term “a multipotent cell” should be construed as generally understood in the field, that is to say as a cell capable of differentiating into specific cell types within a particular primary germ layer (ectoderm, endoderm, or mesoderm). Multipotent cells include progenitor cells such as for instance hematopoietic progenitor cells, also referred to as hematopoietic stem cells.

[0073] In the context of the invention, the terms “hematopoietic progenitor cells” (HPCs) or “hematopoietic stem cells” (HSCs) should be construed as generally understood in the art, that is to say as referring to multipotent cells present in blood and bone marrow. HPCs are capable of forming mature blood cells, such as red blood cells, megakaryocytes and white blood cells. HPCs can be found in bone marrow, peripheral blood (which includes peripheral blood progenitor cells (PBPCs)), umbilical cord blood.

[0074] In the context of the invention, the term “megakaryocyte” should be construed as generally understood in the art and refers to cells of the hematopoietic lineage and which express on their surface, that is to say on the external surface of their cell membrane, at least the receptor c-MpI (also herein referred to as MPL receptor or c-Mpl-receptor) and the biological markers CD41 and CD42. Megakaryocytes may be sub-divided into immature (CD41 +CD42-) and mature megakaryocytes (CD41 +CD42+). Megakaryocytes may also be sub-divided into diploid and polyploid megakaryocytes. Immature megakaryocytes are typically diploid megakaryocytes. During maturation, a part of megakaryocytes become polyploid, giant, and specialized in the massive production of blood platelets that can be identified by their size (diameter of about 50-150 pm), and their large, poly-lobed nucleus.

[0075] In the context of the invention, the terms “thrombopoietin receptor c-MpI”, also herein called “MPL”, refer to the receptor c-MpI of human origin and encoded by the gene MPL, and preferably of peptide sequence the sequence NP_005364.1 In the context of the invention, the terms “biological marker CD41”, also herein called “CD41 ” or “Integrin alpha 2b”, refer to the protein that in humans is encoded by the ITGA2B gene (NCBI Gene ID: 3674), and preferably of peptide sequence the NCBI Reference Sequence: NP_000410.2. In the context of the invention, the terms “cell expressing CD41 ”, “platelet expressing CD41 ”, "iPSC- derived platelet-like particles expressing CD41” or “CD41 cell” refer to a cell, platelet or iPSC- derived platelet-like particles, which comprises on its surface the protein CD41. The presence of CD41 on the surface of a cell can easily be measured by usual methods known in the art such as ELISA or flow cytometry, using anti-CD41 antibodies.

[0076] In the context of the invention, the terms “biological marker CD42a”, also called “GP9” or “glycoprotein IX platelet”, refer to the protein that in humans is encoded by the GP9 gene (NCBI Gene ID: 2815), and preferably of peptide sequence the NCBI Reference Sequence: NP_000165.1 .

[0077] In the context of the invention, the terms “cell expressing CD42a”, “platelet expressing CD42a”, "iPSC-derived platelet-like particles expressing CD42a” or “CD42a cell” refer to a cell, platelet or iPSC-derived platelet-like particles, which comprises on its surface the protein CD42a. The presence of CD42a on the surface of a cell can easily be measured by usual methods known in the art such as ELISA or flow cytometry, using anti- CD42a antibodies.

[0078] In the context of the invention, the term “CD34” refers to the human transmembrane protein CD34 encoded by the gene (NCBI Gene ID: 947).

[0079] In the context of the invention, the terms “CD43”, “leukosialin” or“sialophorin” refer to the human transmembrane cell surface protein encoded by the SPN (sialophorin) (NCBI Gene ID: 6693).

[0080] In the context of the invention, the terms “POU5F1 ”, refer to the protein disclosed in the Uniprot database under reference Q01860.

[0081] In the context of the invention, the terms “NANOG”, refer to the protein disclosed in the Uniprot database under reference Q9H9S0.

[0082] In the context of the invention, the terms “SOX2”, refer to the protein disclosed in the Uniprot database under reference P48431 .

[0083] In the context of the invention, the terms “KLF4”, refer to the protein disclosed in the Uniprot database under reference 043474.

[0084] In the context of the invention, the terms “GPA”, referto the protein disclosed in the Uniprot database under reference P02724. In the context of the invention, the terms “GATA1”, refer to the protein disclosed in the Uniprot database under reference P15976.

[0085] In the context of the invention, the terms “FLI1”, referto the protein disclosed in the Uniprot database under reference Q01543.

[0086] In the context of the invention, the terms “TAL1”, refer to the protein disclosed in the Uniprot database under reference P17542Q01543Q01543.

[0087] In the context of the invention, the terms “SSEA3”, refer to the protein disclosed in the PubChem database under reference CID 53262342.

[0088] In the context of the invention, the terms “SSEA4”, refer to the protein disclosed in the PubChem database under reference CID 172642848.

[0089] In the context of the invention, the terms “TRA-1-60” and “TRA-1-81” refer to epitopes of podocalyxin, wherein podocalyxin, is the protein disclosed in the Uniprot database under reference 000592.

[0090] In the context of the invention, the terms “cell expressing CD34” or “CD34+ cell” refer to a cell which comprises on its cell surface the protein CD34. The presence of CD34 on the surface of a cell can easily be measured by usual methods known in the art such as ELISA or flow cytometry, using anti-CD34 antibodies.

[0091] In the context of the invention, the terms “cell expressing CD43” or “CD43+ cell” refer to a cell which comprises on its cell surface the protein CD43 The presence of CD43 on the surface of a cell can easily be measured by usual methods known in the art such as ELISA or flow cytometry, using anti-CD43 antibodies.

[0092] In the context of the invention, the term “platelets”, “blood platelets” or “thrombocytes” should be construed as generally understood in the art, that is to say as referring to anucleated cytoplasmic bodies produced by megakaryocytes and leading to the formation of blood clots. The cytoplasm of polyploid megakaryocytes emits extensions called proplatelets. Platelets are then formed by fragmentation of the proplatelet ends. Platelets generally measure between about 1.5 and 3.5 pm in diameter and generally have a lenticular or discoid shape.

[0093] In the context of the invention, the terms "iPSC-derived platelet-like particles” and “megakaryocyte progenitor-derived platelet-like particles" refer respectively to cytoplasmic bodies produced by a megakaryocyte derived from an IPSC or from a megakaryocyte progenitor, respectively; or their fragments, including platelets and cytoplasmically-linked platelet-like particles that could result in platelet formation. The structural forms include, but are not limited to, cells with long cytoplasmic extensions, projections or pseudopodia that contain swellings encompassing platelet bodies in various stages of formation, such as, nodules, blebs, and the like. iPSC-derived platelet-like particles ma present with a larger size than platelet particle derived from autolmogous platelets, i.e. platelets of a patient,

[0094] In the context of the invention, the terms “platelets”, “megakaryocyte progenitor-derived plateletlike particles" and “iPSC-derived platelet-like particles” preferably refer to cytoplasmic bodies produced by megakaryocytes which express CD41 and CD42a, as assessed by flow cytometry and which fulfill at least one of the following criteria:

[0095] • possess platelet functionality as measured by assessing activation in response to collagen, thrombin, ADP. In the context of the invention, activation is assessed by measuring degranulation of the platelet or platelet-like particle, for instance via detection of P-selectin expression by flow cytometry, or

[0096] • capacity forthrombus formation, as assessed in a microfluidic chamber coated with collagen. Yet preferably, both the term “platelets” and the term “iPSC-derived platelet-like particles” refer to cytoplasmic bodies produced by megakaryocytes which express CD41 and CD42a, as assessed by flow cytometry and which fulfill at least one of the following criteria:

[0097] • possess platelet functionality as measured by assessing activation in response to collagen, thrombin, ADP. In the context of the invention, activation is assessed by measuring degranulation of the platelet or platelet-like particle, for instance via detection of P-selectin expression by flow cytometry, or

[0098] • capacity forthrombus formation, as assessed in a microfluidic chamber coated with collagen, and further possess the capacity for in vivo hemostasis after platelet or platelet-like particle transfusion in NSG mice rendered thrombocytopenic.

[0099] These criteria and the different assays known and routinely used in the art for assessing them have been thoroughly reviewed and discussed by Sugimoto et al. (2022) and Nakamura et al. (2014).

[0100] In the context of the invention, the term “BMP4”, refers to the protein BMP4, also called bone morphogenetic protein 4, of human origin and encoded by the gene BMP4 (NCBI Gene ID: 652) and preferably of peptide sequence the sequence NP_001193.2.

[0101] In the context of the invention, the term “CHIR”, refers to CHIR 99021 , which is known as a potent and highly selective inhibitor of glycogen synthase kinase 3 (GSK-3). The chemical name of this inhibitor is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1 H-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile. In the context of the invention, the term “VEGF”, refers to the vascular endothelial growth factor of human origin and encoded by the gene VEGFA (NCBI Gene ID: 7422) and preferably of peptide sequence the sequence NP_003367

[0102] In the context of the invention, the term “bFGF”, refers to the fibroblast growth factor 2 of human origin and encoded by the gene FGF2 (NCBI Gene ID: 2247) and preferably of peptide sequence the sequence NP_001348594.1

[0103] In the context of the invention, the term “SCF”, refers to Stem Cell Factor of human origin, also known as SCF, KIT-ligand, KL, or steel factor, and encoded by the gene KITLG (NCBI Gene ID: 4254) and preferably of peptide sequence the sequence NP_000890.1

[0104] In the context of the invention, the term “Flt3L”, refers to the protein named “fms related receptor tyrosine kinase 3 ligand” of human origin and encoded by the gene FLT3LG (NCBI Gene ID: 2323) and preferably of peptide sequence the sequence NP_001450.2

[0105] In the context of the invention, the term “TPO”, refers to the thyroid peroxidase of human origin and encoded by the gene TPO (NCBI Gene ID: 7173) and preferably of peptide sequence the sequence NP_000451 .1

[0106] In the context of the invention, the term “IL6”, refers to the interleukin 6 of human origin and encoded by the gene IL6 (NCBI Gene ID: 3569) and preferably of peptide sequence the sequence NP_000591 .1

[0107] In the context of the invention, the term “TP53”, refers to the tumor protein p53 of human origin and encoded by the gene TP53 (NCBI Gene ID: 7157) and preferably of peptide sequence the sequence NP_000537.3

[0108] In the context of the invention, the term “p21”, refers to cyclin dependent kinase inhibitor 1A of human origin, also known as CDKN1 , WAF1 , p21 CIP1 or CIP1 , and encoded by the gene CDKN1A (NCBI Gene ID: 1026) and preferably of peptide sequence the sequence NP_000380.1

[0109] In the context of the invention, the term “p19”, refers to cyclin dependent kinase inhibitor 2D of human origin, also known as p19, INK4D p19-INK4D, and encoded by the gene CDKN2D (NCBI Gene ID: 1032) and preferably of peptide sequence the sequence NP_001791.1 In the context of the invention, the term “MPS1 ”, refers to TTK protein kinase of human origin, also known as TTK, ESK, PYT, CT96, MPH1 , MPS1 , MPS1 L1 and encoded by the gene TTK (NCBI Gene ID: 7272) and preferably of peptide sequence the sequence NP_0003309.2

[0110] In the context of the invention, the term “DYRK1A”, or term “DYRK1”, refers to the protein named “dual specificity tyrosine phosphorylation regulated kinase 1A”, also known as MNB, DYRK, HP86, MNBH, MRD7, DYRK1 and encoded by the gene DYRK1A (NCBI Gene ID: 1859) and preferably of peptide sequence the sequence NP_001334650.1

[0111] In the context of the invention, the term “ROCK”, refers to Rho associated coiled-coil containing protein kinase 1 of human origin, also known as ROCK-I or P160ROCK and encoded by the gene ROCK1 (NCBI Gene ID: 6093) and preferably of peptide sequence the sequence NP_005397.1

[0112] In the context of the invention, the term “CDK4”, refers to cyclin dependent kinase 4 of human origin, encoded by the gene CDK4 (NCBI Gene ID: 1019) and preferably of peptide sequence the sequence NP_000066.1.

[0113] In the context of the invention, the term “CDK6”, refers to cyclin dependent kinase 6 of human origin, encoded by the gene CDK6 (NCBI Gene ID: 1021) and preferably of peptide sequence the sequence NP_001250.

[0114] In the context of the invention, the term “reversine” refers to the compound 2-(4-Morpholinoanilino)- 6-cyclohexylaminopurine identified under CAS number 656820-32-5.

[0115] In the context of the invention, the term “harmine” refers to the compound 7-Methoxy-1-methyl-9H- pyrido[3,4-b]indole identified under CAS number 442-51-3.

[0116] In the context of the invention, the term “Y-27632” or “Y27632” refers to the compound R)- +)-trans- 4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride identified under CAS number 129830-38-2.

[0117] In the context of the invention the term “Chroman 1 ” refers to the chemical compound referred under CAS number 1273579-40-0.

[0118] In the context of the invention the term “Fasudil Hydrochloride” refers to the chemical compound referred under CAS number 105628-07-7.

[0119] In the context of the invention the term “Belumosudil” refers to the chemical compound referred under CAS number 911417-87-3. In the context of the invention the term “Thiazovivin” refers to the chemical compound referred under CAS number 1226056-71-8.

[0120] In the context of the invention the term “Afuresertib” refers to the chemical compound referred under CAS number 1047644-62-1.

[0121] In the context of the invention the term “GSK269962A” refers to the chemical compound referred under CAS number 850664-21-0.

[0122] In the conext of the invention the term “UC2288” refers to 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3- ((1 r,4r)-4-(5-(trifluoromethyl)pyridin-2-yloxy)cyclohexyl)urea, trans-1 -(4-Chloro-3 trifluoromethyl- phenyl)-3-(4-hydroxy-cyclohexyl)-urea.

[0123] In the context of the invention the term “BAY1217389” refers to the chemical compound referred under CAS number 1554458-53-5.

[0124] In the context of the invention the term AZ3146 refers to the chemical compound referred under CAS number 1124329-14-1.

[0125] In the context of the invention the term “NMS-P715” refers to the chemical compound referred under CAS number 1202055-32-0.

[0126] In the context of the invention the term “Luvixasertib hydrochloride” refers to the chemical compound referred under CAS number 1610677-37-6.

[0127] In the context of the invention the term “MPI-0479605” refers to the chemical compound referred under CAS number 1246529-32-7.

[0128] In the context of the invention the term “Mps1-IN-8” refers to the chemical compound referred under CAS number 2259843-95-1 .

[0129] In the context of the invention the term “BOS-172722” refers to the chemical compound referred under CAS number 1578245-44-9.

[0130] In the context of the invention the term “Empesertib” refers to the chemical compound referred under CAS number 1443763-60-7. In the context of the invention the term “Mps1-IN-1 ” refers to the chemical compound referred under CAS number 1125593-20-5.

[0131] In the context of the invention the term “Mps1-IN-2” refers to the chemical compound referred under CAS number 1228817-38-6.

[0132] In the context of the invention the term “Mps1-IN-7” refers to the chemical compound referred under CAS number 1578244-34-4.

[0133] In the context of the invention the term “Mps1-IN-3” refers to the chemical compound referred under CAS number 1609584-72-6.

[0134] In the context of the invention the term “CCT251455” refers to the chemical compound referred under CAS number 1400284-80-1 .

[0135] In the context of the invention the term “Mps-BAY2a” refers to the chemical compound referred under CAS number 1382477-96-4.

[0136] In the context of the invention the term “RMS-07” refers to the chemical compound referred under CAS number 2645409-78-3.

[0137] In the context of the invention the term “TC-Mps1-12” refers to the chemical compound referred under CAS number 1206170-62-8.

[0138] In the context of the invention the term “Mps1-IN-5” refers to the chemical compound referred under CAS number 2890819-31-3.

[0139] In the context of the invention the term “Mps1-IN-1 ” dihydrochloride refers to the chemical compound referred under CAS number 1883548-93-3.

[0140] In the context of the invention the term “Mps1-IN-4” refers to the chemical compound referred under CAS number 1263423-94-4.

[0141] In the context of the invention the term “AZ191 ” refers to the chemical compound referred under CAS number 1594092-37-1.

[0142] In the context of the invention the term “GSK-626616” refers to the chemical compound referred under CAS number 1025821-33-3. In the context of the invention the term “T025” refers to the chemical compound referred under CAS number 2407433-00-3.

[0143] In the context of the invention the term “Protein kinase inhibitor 1 hydrochloride” refers to the chemical compound referred under CAS number 2321337-71-5.

[0144] In the context of the invention the term “Dyrk1A-IN-8” refers to the chemical compound referred under CAS number 101578-13-6.

[0145] In the context of the invention the term “GNF2133” refers to the chemical compound referred under CAS number 2561414-56-8.

[0146] In the context of the invention the term “DYRK1-IN-“1 refers to the chemical compound referred under CAS number 2814486-79-6.

[0147] In the context of the invention the term “ILK-IN-3” refers to the chemical compound referred under CAS number 6975-75-3.

[0148] In the context of the invention the term “AZ-Dyrk1 B-33” refers to the chemical compound referred under CAS number 1679330-37-0.

[0149] In the context of the invention the term “ML167” refers to the chemical compound referred under CAS number 1285702-20-6.

[0150] In the context of the invention the term “Dyrk1A-IN-5” refers to the chemical compound referred under CAS number 1685235-41-9.

[0151] In the context of the invention the term “EHT 5372” refers to the chemical compound referred under CAS number 1425945-63-6.

[0152] In the context of the invention the term “GNF4877” refers to the chemical compound referred under CAS number 2041073-22-5.

[0153] In the context of the invention the term “Dyrk1A-IN-4” refers to the chemical compound referred under CAS number 2091883-59-7.

[0154] In the context of the invention the term “ON 108600” refers to the chemical compound referred under CAS number 1585246-23-6. In the context of the invention, the term “culturing”, when used in reference to cells or cell lines, should be construed as generally known in the art, that is to say as cultivating said cells outside of their natural environment, in vitro, under controlled conditions appropriate for their survival and the growth of the cell line considered. Conditions appropriate for maintenance or cells and the growth of the cell line considered encompass sterility, appropriate ranges of temperature, humidity levels and concentration in atmospheric gases such as, nitrogen, oxygen and CO2. Conditions that are appropriate for the survival and growth of cell lines are generally known in the art and can be adapted according to the organism of origin or cell type of the cell line considered based on the knowledge in the field. Culturing cells or cell lines may involve changing cell culture medium and passaging cell lines regularly, at a frequency that may depend on the cell type or cell species.

[0155] In the context of the invention, culturing a cell “in the presence of an inhibitor” should be construed as generally understood in the art, that is to say as indicating that said inhibitor is added to the cell culture conditions, preferably to the cell culture medium. The inhibitor may be added to the cell culture medium, either permanently or transiently. Permanent presence of an inhibitor may for instance correspond to situations where the inhibitor is added to cell culture medium from the first steps of the cell culture and be consistently added to the fresh cell culture medium used when the cell culture medium is changed and cells are passaged. Transient presence of an inhibitor may for instance correspond to situations where the inhibitor is added to cell culture medium at a specific point in time during cell culturing, and later eliminated for example when the cell culture medium is changed and cells are passaged.

[0156] In the context of the invention, the term “cell culture medium”, should be construed as generally known in the art, that is to say as a composition which may be used for the purpose of in vitro cell culture, and which comprises at least the nutrients necessary for survival or growth of cells or cell lines. Typically, cell culture media usually contain or consist essentially of salts, amino acids, vitamins, a carbon source, and may further contain growth factors. As used herein, the terms "a medium consisting essentially of’ refer to a medium that contains the specified ingredients and, optionally, other ingredients that do not materially affect its basic characteristics. Preferably the cell culture is a defined cell culture medium. In the context of the invention the terms "defined cell culture medium", also referred to as "defined culture medium" or "defined medium", mean that the identity and quantity of each cell culture medium ingredient is known.

[0157] In the context of the invention, the term “serum-free”, when referring to a cell culture medium, refers to cell culture medium which does not comprise serum media additives, preferably does not comprise bovine serum albumin (BSA) or fetal calf serum (FCS). In the context of the invention the terms "essentially serum-free" mean that a medium contains essentially no serum media additives, preferably contains essentially no bovine serum albumin (BSA) or fetai calf serum (FCS). In the context of the invention, the term "xeno-free”, when referring to a cell culture medium, refers to cell culture medium which free of any cell or cell product of species other than that of the cultured cell. For instance, when the cell or cell lien which is culture is human or from a human origin, the culture medium used for culturing such cell or cell line will be considered as xeno-free if it does not comprise any cell or cell product, such as proteins and growth factors, other than from human origin.

[0158] In the context of the invention, the terms “suppressing the expression”, when referring to a protein, should be construed as generally understood in the art, that is to say as using specific means, such as molecular tools or cell culture conditions, so as to decrease the expression level of a protein compared to the expression level of said protein in reference conditions (e.g. without genetic modifications or without inhibitor in culture medium) The expression level of a protein can be detected by any conventional means enabling the measurement of protein levels, such as western blot.

[0159] In the context of the invention, the terms “suppressing the activity”, when referring to a protein, should be construed as generally understood in the art, that is to say as using specific means, such as molecular tools or cell culture conditions, so as to decrease the activity of said protein, compared to the activity of said protein in reference conditions (e.g. without genetic modifications or without inhibitor in culture medium). The activity of a protein may refer to a specific binding activity in which case the activity of the protein (and variations of the activity) can be detected and measured by any conventional means enabling the measurement of protein binding. The activity of a protein may refer to an enzymatic activity in which case the activity of the protein (and variations of the activity) can be detected by known means and methods enabling the measurement of this enzymatic activity.

[0160] In the context of the invention, the terms “genetically modified so as to silence a gene”, when referring to a cell of interest should be construed as generally understood in the art, that is to say as methods and means of molecular biology which modify the genetic material of the cell and which effect is to inhibit the expression of a functional protein. The amount of protein encoded by said gene may be decreased or even null. As is well known in the art, this may in some embodiments be obtained by knocking out of said gene, or by additions, substitutions or deletions within the target gene or its regulatory sequences (in particular its promoter) that impedes target gene expression. Alternatively, a non-functional protein may be produced as a result of the genetic modification. In other embodiments, the inactivation of a gene can be achieved indirectly by introducing into the cell of interest a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene of interest, and where the sequence encoding the shRNA is operably linked to a promoter allowing for stable expression of the shRNA molecule by the cell of interest, thereby inhibiting the expression of a functional protein.

[0161] As used herein, “operably linked” means that the elements being linked are arranged so that they function in concert for their intended purposes. For example, a promoter operably linked to a polynucleotide initiates and mediates transcription of this polynucleotide Operably linked sequences include both expression control sequences that are contiguous with a polynucleotide and expression control sequences that act in trans or at a distance to control a polynucleotide. The polynucleotide having a sequence encoding a shRNA, may be introduced into the cell using molecular tools known in the art such as expression vectors.

[0162] In the context of the invention, the term “vector” refers to a vehicle, preferably a nucleic acid molecule or a viral particle that contains the elements necessary to allow delivery, propagation and / or expression of any of the polynucleotide described herein within a host cell or subject. This term encompasses vectors for maintenance (cloning vectors) or vectors for expression in various host cells or subjects (expression vectors), extrachromosomal vectors (e.g. multicopy plasmids) or integration vectors (e.g. designed to integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates) as well as shuttle vectors (e.g. functioning in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g. for transferring nucleic acid molecule(s) in a viral genome). For the purpose of the invention, the vectors may be of naturally occurring genetic sources, synthetic or artificial, or some combination of natural and artificial genetic elements.

[0163] In the context of the invention, the term “expression vector” has to be understood broadly as including mRNA, plasmid and viral vectors. Expression vectors which are appropriate in the context of the present invention, include plasmid and viral vectors for expression in higher eukaryotic cells or subjects. Typically, such vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them. The present invention also encompasses vectors (e.g. plasmid DNA and mRNA) complexed to lipids or polymers to form particulate structures such as liposomes, lipoplexes or nanoparticles. Preferably, the expression vector is a viral vector.

[0164] The term "viral vector" as used herein refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle. The terms “virus”, “virions”, “viral particles” and “viral vector particle” are used interchangeably to refer to viral particles that are formed when the nucleic acid vector is transduced into an appropriate cell or cell line according to suitable conditions allowing the generation of viral particles. In the context of the present invention, the term “viral vector” has to be understood broadly as including nucleic acid vector (e.g. DNA viral vector) as well as viral particles generated thereof. The term “infectious” refers to the ability of a viral vector to infect and enter into a host cell or subject. Examples of suitable viral vectors include vectors generated from a variety of different viruses (e.g. poxviruses, adenoviruses, herpes viruses, paramyxoviruses, rhabdoviruses, lentiviruses, retroviruses, parvoviruses, alphaviruses, etc). Typically, such vectors are commercially available or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them.

[0165] In the context of the invention, the term “treating” or “treatment” means an improvement of the patient’s disease, disorder, or condition, which may be observed at the clinical, histological, and / or biochemical level. The terms “treating” or “treatment” notably include improving a clinical, histological, and / or biochemical symptom or parameter associated with the patient’s disease, disorder, or condition or inhibiting, reducing, or delaying progression or exacerbation of the patient’s disease, disorder, or condition (including secondary damage caused by the disease, disorder, or condition) to either a statistically significant degree or to a degree detectable to one skilled in the art. In some embodiments, treatment is assessed on a population basis such that a therapy is considered to “treat” a particular disease, disorder or condition if a statistically significant improvement of the patient’s disease, disorder, or condition is observed in a population suffering from the disease, disorder, or condition.

[0166] In the context of the invention, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk for a subject of acquiring or developing a given disease, disorder, or condition. The terms "prevent", "prevention" and "preventing" also comprise delaying the onset and / or reducing the frequency and / or intensity of clinical, histological and / or biochemical symptoms or parameters associated with this given disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis such that a therapy is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the risk of acquiring or developing the disease, disorder, or condition, and / or a statistically significant delay of the onset and / or a statistically significant decrease of the frequency and / or intensity of clinical, histological and / or biochemical symptoms or parameters associated with the disease, disorder or condition, is observed in a population susceptible to the disease, disorder, or condition.

[0167] In the context of the invention, the terms “therapeutically effective amount” refer to an amount effective of a therapeutic agent, at dosages and for periods of time necessary, to elicit the desired biological response in a subject. In other words, the therapeutic effective amount is the amount of the therapeutic agent which is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition and / or a symptom related thereto. The therapeutical effective amount will be adjusted to the individual requirements in each particular case. It can vary within wide limits depending upon numerous factors such as the nature and the severity of the disease, disorder or condition to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved. In the context of the invention, a “subject” may be any of mammalian animals including human, dog, cat, cattle, goat, pig, swine, sheep and monkey, preferably a human. A human subject can be known as a patient.

[0168] DETAILED DESCRIPTION

[0169] In a first object, the invention pertains to a method for the production of polyploid megakaryocytes comprising the steps of:

[0170] (a) providing a diploid megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, and

[0171] (b) inducing polyploidy of the diploid megakaryocyte by: i. suppressing the expression or activity of a protein chosen in the list consisting of TP53 or p21 , preferably suppressing the expression or activity of the combination of p21 and p19, and; ii. suppressing the expression or activity of a protein chosen in the list consisting of MPS1 and DYRK1 , preferably suppressing the expression or activity of MPSI and DYRK1 ; and

[0172] Hi. optionally, suppressing the expression or activity of ROCK, thereby producing a polyploid megakaryocyte.

[0173] Diploid megakaryocytes derived from a megakaryocyte progenitor, yet preferably derived from iPSC and means to obtain them are well known in the art and have already been disclosed in Bluteau et al. 2013.

[0174] Methods and means for suppressing the expression or activity of a protein are generally well known in biotechnology, and may encompass genetic modifications of the cells, for instance modification which silence the gene expressing the target protein, or the use of agents, such as inhibitors, which can inhibit the expression or activity of said protein.

[0175] Preferably, in the method of the invention,

[0176] (a) when present, suppressing the expression or activity of TP53 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of TP53;

[0177] (b) when present, suppressing the expression or activity of p21 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p21 ;

[0178] (c) when present, suppressing the expression or activity of p19 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p19, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p19;

[0179] (d) when present, suppressing the expression or activity of MPS1 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 , preferably reversine;

[0180] (e) when present, suppressing the expression or activity of DYRK1 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 , preferably harmine; and

[0181] (f) when present, suppressing the expression or activity of ROCK is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK, preferably Y27632.

[0182] In the context of the invention, the terms “when present”, used in reference to a step or feature of suppressing the expression or activity of a protein, should be understood as indicating that the phrase following these terms apply when said step or feature of suppressing the expression or activity of a protein is a mandatory feature of the embodiment ofthe method of the invention which is considered.

[0183] In the context of the invention, preferably, in those embodiments where the expression or activity of p21 and / or p19 is suppressed and where the expression or activity of MPS1 is suppressed, the expression or activity of MPS1 is suppressed by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 , preferably reversine.

[0184] Thus, preferably, in embodiments where suppressing the expression or activity of TP53 is a feature of the method of the invention, said feature is obtained by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of TP53.

[0185] Preferably, in embodiments where suppressing the expression or activity of p21 is a feature of the method of the invention, said feature is obtained by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p21 .

[0186] Preferably, in embodiments where suppressing the expression or activity of p19 is a feature of the method of the invention, said feature is obtained by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p19, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p19

[0187] Preferably, in embodiments where suppressing the expression or activity of MPS1 is a feature of the method of the invention, said feature is obtained by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 .

[0188] Preferably, in embodiments where suppressing the expression or activity of DYRK1 is a feature of the method of the invention, said feature is obtained by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 .

[0189] Preferably, in embodiments where suppressing the expression or activity of ROCK is a feature of the method of the invention, said feature is obtained by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK.

[0190] In the context of the invention, providing a diploid megakaryocyte genetically modified so as to silence a gene of interest should be construed as generally understood in the art. In a preferred embodiment, the diploid megakaryocyte is genetically modified by knocking out a gene of interest, chosen in the list consisting of TP53, p21 , p19, MPS1 , DYRK1 and ROCK. In a specific embodiment, the diploid megakaryocyte is genetically modified by introducing into the diploid megakaryocyte a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene of interest chosen in the list consisting of the genes encoding TP53, p21 , p19, MPS1 , DYRK1 and ROCK. Preferably, in this embodiment, the sequence encoding the shRNA is operably linked to a promoter allowing for stable expression of the shRNA molecule by the megakaryocyte, thereby inhibiting the expression of a functional protein by the gene of interest.

[0191] In the context of the invention, agents lowering the expression or inhibitors of the expression of a protein encompass RNA interference, including for transient inhibition of the translation of the mRNA encoding the protein, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against the mRNA sequence of said protein. These inhibitors of expression are for example anti-sense RNA molecules and anti-sense DNA molecules, and act to directly block the translation of the mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the protein, in the target cell. Antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the targeted protein can be used. They can be synthesized, e.g., by conventional phosphodiester techniques, and administered to the patients, by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131 ; 6,365,354; 6,410,323; 6,107,091 ; 6,046,321 ; and 5,981 ,732).

[0192] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53 is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the gene encoding TP53. Preferably, in the diploid megakaryocyte, the gene encoding TP53 is knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene encoding TP53.

[0193] Alternatively, inhibitors of the expression or activity of TP53 may be used. Such inhibitors of the expression or activity of TP53 comprise RNA interference against TP53, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against TP53 mRNA. In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising RNA interference against TP53, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against TP53 mRNA.

[0194] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the gene encoding p21 . Preferably, in the diploid megakaryocyte, the gene encoding p21 is knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene encoding p21 .

[0195] Alternatively, inhibitors of the expression or activity of p21 may be used. Such inhibitors of the expression or activity of p21 comprise RNA interference against p21 , for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against p21 mRNA, and the p21 inhibitor named UC2288.

[0196] In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising an inhibitor of the expression or activity of p21 chosen in the list consisting of RNA interference against p21 , for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against p21 mRNA, and UC2288.

[0197] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p19 is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the gene encoding p19. Preferably, in the diploid megakaryocyte, the gene encoding p19 is knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene encoding p19.

[0198] Alternatively, inhibitors of the expression or activity of p19 may be used. Such inhibitors of the expression or activity of p19 comprise RNA interference against p19, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against p19 mRNA. In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising RNA interference against p19, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against p19 mRNA.

[0199] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the gene encoding MPS1 . Preferably, in the diploid megakaryocyte, the gene encoding MPS1 is knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene encoding MPS1 .

[0200] Alternatively, inhibitors of the expression or activity of MPS1 may be used. Such inhibitors of the expression or activity of MPS1 comprise RNA interference against MPS1 , for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against MPS1 mRNA, reversine, BAY1217389, AZ3146, NMS-P715, Luvixasertib hydrochloride, MPI-0479605, Mps1-IN-8, BOS- 172722, Empesertib, Mps1-IN-1 , Mps1-IN-2, Mps1-IN-7, Mps1-IN-3 , CCT251455, Mps-BAY2a, RMS-07, TC-Mps1-12, Mps1-IN-5, Mps1-IN-1 dihydrochloride, Mps1-IN-4. In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising an inhibitor of the expression or activity of MPS1 chosen in the list consisting of RNA interference against MPS1 , for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against MPS1 mRNA, reversine, BAY1217389, AZ3146, NMS-P715, Luvixasertib hydrochloride, MPI-0479605, Mps1-IN-8, BOS-172722, Empesertib, Mps1-IN-1 , Mps1- IN-2, Mps1-IN-7, Mps1-IN-3 , CCT251455, Mps-BAY2a, RMS-07, TC-Mps1-12, Mps1-IN-5, Mps1- IN-1 dihydrochloride and Mps1-IN-4.

[0201] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the gene encoding DYRK1. Preferably, in the diploid megakaryocyte, the gene encoding DYRK1 is knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene encoding DYRK1 .

[0202] Alternatively, inhibitors of the expression or activity of DYRK1 may be used. Such inhibitors of the expression or activity of DYRK1 comprise RNA interference against DYRK1 , for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against DYRK1 mRNA, harmine or other known inhibitors of DYRK1 , such as AZ191 , GSK-626616, T025, Protein kinase inhibitor 1 hydrochloride, Dyrk1A-IN-8, GNF2133, DYRK1-IN-1 , ILK-IN-3, AZ-Dyrk1 B-33, ML167, Dyrk1A-IN-5, EHT 5372, GNF4877, Dyrk1A-IN-4, CLK1-IN-3 or ON 108600. In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising an inhibitor of the expression or activity of DYRK1 chosen in the list consisting of RNA interference against DYRK1 , for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against DYRK1 mRNA, harmine, AZ191 , GSK-626616, T025, Protein kinase inhibitor 1 hydrochloride, Dyrk1A-IN-8, GNF2133, DYRK1 -IN-1 , ILK-IN-3, AZ-Dyrk1 B-33, ML167, Dyrk1A-IN- 5, EHT 5372, GNF4877, Dyrk1A-IN-4, CLK1-IN-3 and ON 108600.

[0203] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the gene encoding ROCK; Preferably, in the diploid megakaryocyte, the gene encoding ROCK is knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene encoding ROCK.

[0204] Alternatively, inhibitors of the expression or activity of ROCK may be used. Such inhibitors of the expression or activity of ROCK comprise RNA interference against ROCK, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against ROCK mRNA, Y27632, Chroman 1 , Fasudil Hydrochloride, Belumosudin, Thiazovivin, Afuresertib, or GSK269962A. In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising an inhibitor of the expression or activity of ROCK chosen in the list consisting of RNA interference against ROCK, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against ROCK mRNA, Y27632, Chroman 1 , Fasudil Hydrochloride, Belumosudin, Thiazovivin, Afuresertib and GSK269962A

[0205] As is disclosed in the experimental part, the method of the invention provides very good results in particular when the suppression of the expression or activity of a combination of proteins, namely TP53, MPS1 , DYRK1 , and ROCK, is performed in the diploid megakaryocyte. This condition indeed leads to a very high number of polyploid megakaryocytes being produced, and further translates into a very high number of platelets or platelet-like particles per polyploid megakaryocyte, as shown in figure 7. Further, the method of the invention also provides exceptional results when the suppression of the expression or activity of a combination of proteins, namely p21 , p19, MPS1 , DYRK1 , and .ROCK, is performed in the diploid megakaryocyte. In addition to the above, the inventors have shown that, in embodiments comprising the suppression of the expression or activity of both p21 and p19, the yield of platelets or platelet-like particles per polyploid megakaryocyte may be increased by inhibiting CDK4, CDK6, or both CDK4 and CDK6.

[0206] In a preferred embodiment of the method of the invention, step b) of inducing polyploidy of the diploid megakaryocyte (thereby producing a polyploid megakaryocyte) is by suppressing the expression or activity of TP53 or of the combination of p21 and p19, and by suppressing the expression or activity of MPS1 , DYRK1 , and ROCK.

[0207] In such preferred embodiment when the expression or activity of the combination of p21 and p19 is suppressed, the method preferably further comprises suppressing the expression or activity of CDK4 and CDK6.

[0208] In the method of the invention, providing a diploid megakaryocyte genetically modified so as to silence the genes encoding CDK4 and CDK6 is preferably performed by providing a diploid megakaryocyte genetically modified so as to permanently silence the genes encoding CDK4 and CDK6. Preferably, in the diploid megakaryocyte, the genes encoding CDK4 and CDK6 are knocked out. In an embodiment, the diploid megakaryocyte comprises a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the genes encoding CDK4 and CDK6.

[0209] Alternatively, inhibitors of the expression or activity of CDK4 and CDK6 may be used. Such inhibitors of the expression or activity of CDK4 and CDK6 comprise RNA interference against CDK4 and CDK6, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against CDK4 and CDK6 mRNAs, and the CDK4 and CDK6 inhibitor named Palbociclib.

[0210] In this case, the method of the invention preferably comprises a step of culturing the diploid megakaryocyte in cell culture medium comprising an inhibitor of the expression or activity of CDK4 and CDK6 chosen in the list consisting of RNA interference against CDK4 and CDK6, for example small interfering RNAs (siRNAs) or other antisense nucleic acids directed against CDK4 and CDK6 mRNA, and Palbociclib. When Palbociclib is used, it may notably be used at a concentration between 10 nM and 2000 nM.

[0211] Preferably, in embodiments where suppressing the expression or activity of CDK4 and CDK6 is a feature of the method of the invention, said feature is obtained by: i) providing a diploid megakaryocyte genetically modified so as to silence the genes encoding CDK4 and CDK6, orii) culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of CDK4 and CDK6.

[0212] In a preferred embodiment of the method of the invention, step b) of inducing polyploidy of the diploid megakaryocyte (thereby producing a polyploid megakaryocyte) is by suppressing the expression or activity of TP53, MPS1 , DYRK1 , and ROCK.

[0213] In a preferred embodiment of the method of the invention, step b) of inducing polyploidy of the diploid megakaryocyte (thereby producing a polyploid megakaryocyte) is by suppressing the expression or activity of p21 , p19, MPS1 , DYRK1 , and ROCK.

[0214] In a yet preferred embodiment of the method of the invention, the diploid megakaryocyte is genetically modified so as to silence TP53, p21 or a combination of p21 and p19, and

[0215] (a) suppressing the expression or activity of MPS1 is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 ;

[0216] (b) suppressing the expression or activity of DYRK1 is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 ;

[0217] (c) suppressing the expression or activity of ROCK is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK.

[0218] Preferably, in the method of the invention, including in the above preferred embodiment:

[0219] (a) suppressing the expression or activity of MPS1 is obtained by culturing the diploid megakaryocyte in the presence of reversine, preferably reversine at a concentration of between 1 and 5 pM, yet preferably reversine at a concentration of about 2.5 pM; (b) suppressing the expression or activity of DYRK1A is obtained by culturing the diploid megakaryocyte in the presence of harmine, preferably harmine at a concentration of between 2 and 5 pM, yet preferably harmine at a concentration of about 2.5 pM;

[0220] (c) suppressing the expression or activity of ROCK is obtained by culturing the diploid megakaryocyte in the presence ofY27632, preferably Y27632 at a concentration of between 1 and 10 pM, yet preferably Y27632 at a concentration of about 10 pM; or

[0221] (d) any combination of (a) to (c).

[0222] Preferably, the diploid megakaryocyte is derived from a human iPSC, yet preferably derived from a human HLA-universal induced pluripotent stem cell (iPSC) line.

[0223] Human HLA-universal induced pluripotent stem cell (iPSC) lines and methods to obtain them, as well as megakaryocytes derived therefrom, are known in the art, and have been described for instance by Borger AK,et al., and reviewed by Figueiredo C et al. (2021), Figueiredo C, et al. (2014), Suzuki D, et al..

[0224] Methods to obtain diploid megakaryocytes derived from a megakaryocyte progenitor, yet preferably from iPSC are known, yet the inventors have set forth an optimized method to produce a large number of diploid megakaryocytes in a safe and efficient manner, and which can be implemented in the context of the invention.

[0225] In a preferred embodiment of the method of the invention, the diploid megakaryocyte derived from an iPSC is obtained by

[0226] (a) inducing differentiation of an iPSC into an hematopoietic progenitor cell by: i. Culturing an iPSC in the presence of molecules chosen from the list consisting of BMP4, CHIR, VEGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 12 and 36 hours, preferably for about 24 hours; ii. Culturing a cell obtained from step a) i) in the presence of molecules chosen from the list consisting of BMP4, VEGF, bFGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 60 and 84 hours, preferably for about 72 hours;

[0227] Hi. Culturing a cell obtained from step a) ii) in the presence of molecules chosen from the list consisting of VEGF, bFGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 36 and 60 hours, preferably for about 48 hours; iv. Culturing a cell obtained from step a) iii) in the presence of molecules chosen from the list consisting of VEGF, bFGF, SCF, Flt3L and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 12 and 36 hours, preferably for about 24 hours; v. Culturing a cell obtained from step a) iv) in the presence of molecules chosen from the list consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL6 and a combination thereof, preferably at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 132 and 156 hours, preferably for about 144 hours, thereby obtaining a hematopoietic progenitor cell, preferably a hematopoietic progenitor cell expressing CD34 and CD43; and

[0228] (b) inducing differentiation of the hematopoietic progenitor cell into a diploid megakaryocyte by culturing an hematopoietic progenitor cell in the presence of molecules chosen from the list consisting of SCF, TPO and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 324 and 348 hours, preferably for about 336 hours, thereby obtaining a diploid megakaryocyte.

[0229] The method of the invention, as indicated above, enables the reliable production of a large number of polyploid megakaryocytes, that is to say of population of cells comprising a high ratio of polynucleated cells, otherwise called polyploid megakaryocytes. Those cells may advantageously be stored in the form of a composition comprising the cells and an appropriate cell culture medium, for instance in the presence of relevant inhibitors.

[0230] Another object of the invention is a megakaryocyte produced by the method of the invention.

[0231] Another object of the invention is a population of megakaryocytes produced by the method of the invention.

[0232] Preferably, the population of megakaryocytes comprises at least 30%, 40% 50%, 60% preferably 65% of polyploid megakaryocytes. Yet preferably, the population of megakaryocytes comprises at least 20, advantageously at least 23%, polyploid megakaryocytes having 4N chromosomes. Yet preferably, the population of megakaryocytes comprises at least 30%, advantageously at least 33%, polyploid megakaryocytes having at least 8N chromosomes. Yet preferably, the population of megakaryocytes comprises at least 20%, advantageously at least 24%, polyploid megakaryocytes having at least 16N chromosomes. Yet preferably, the population of megakaryocytes comprises at least 5%, preferably at least 8%, polyploid megakaryocytes having at least 32N chromosomes. Yet preferably, the population of megakaryocytes comprises at least 1 % polyploid megakaryocytes having at least 64N chromosomes.

[0233] Another object of the invention is a composition comprising the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, and a cell culture medium, wherein the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of TP53, p21 , p19, MPS1 , DYRK1 , and ROCK.

[0234] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0235] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53, and

[0236] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 , DYRK1 , and ROCK.

[0237] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK.

[0238] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0239] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding TP53 and MPS1 , and

[0240] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and ROCK.

[0241] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of ROCK.

[0242] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein: - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding TP53 and DYRK1 , and

[0243] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and ROCK.

[0244] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of ROCK.

[0245] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0246] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding TP53 and ROCK, and

[0247] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and DYRK1 .

[0248] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of DYRK1 .

[0249] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0250] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 , and

[0251] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , DYRK1 , and ROCK.

[0252] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p19.

[0253] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0254] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and p19, and

[0255] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 , DYRK1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0256] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of DYRK1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0257] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0258] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and MPS1 , and

[0259] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, DYRK1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0260] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of DYRK1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0261] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0262] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and DYRK1 , and - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0263] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of MPS1. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0264] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0265] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and ROCK, and

[0266] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , and DYRK1 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0267] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of MPS1. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of CDK4 and CDK6.

[0268] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0269] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding p19, and

[0270] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , MPS1 , DYRK1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0271] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of p21 and an inhibitor of the expression or activity of CDK4 and CDK6.

[0272] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0273] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19 and MPS1 , and

[0274] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , DYRK1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0275] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of DYRK1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0276] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0277] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19 and DYRK1 , and

[0278] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , MPS1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of MPS1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0279] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0280] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19 and ROCK, and

[0281] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , and DYRK1 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0282] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of MPS1. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of CDK4 and CDK6. In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0283] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 , and

[0284] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, DYRK1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0285] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of CDK4 and CDK6. In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0286] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding MPS1 and DYRK1 , and

[0287] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0288] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. , Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0289] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0290] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding MPS1 and ROCK, and

[0291] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, and DYRK1 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0292] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1 . Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of CDK4 and CDK6.

[0293] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0294] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 , and

[0295] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, MPS1 , and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0296] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of p21 . and an inhibitor of the expression or activity of CDK4 and CDK6.

[0297] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0298] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding DYK1 and ROCK, and

[0299] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, and MPS1 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0300] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of CDK4 and CDK6.

[0301] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0302] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK, and - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, DYRK1 , and MPS1 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0303] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of CDK4 and CDK6.

[0304] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein: - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , p19 and MPS1 , and

[0305] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0306] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of ROCK. Yet more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0307] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0308] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , p19 and DYRK1 , and

[0309] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0310] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of ROCK. Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK., and an inhibitor of the expression or activity of CDK4 and CDK6.

[0311] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0312] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , MPS1 , and DYRK1 and - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19 and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0313] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19 and an inhibitor of the expression or activity of ROCK. Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0314] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0315] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19, MPS1 , and DYRK1 , and

[0316] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 and ROCK, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0317] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 and an inhibitor of the expression or activity of ROCK. Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0318] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0319] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p21 , MPS1 and ROCK, and

[0320] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and p19, and optionally an inhibitor of the expression or activity of CDK4 and CDK6. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity o p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of p19. Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0321] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0322] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p19, MPS1 and ROCK, and

[0323] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and p21 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0324] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of p21 . Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of CDK4 and CDK6.

[0325] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0326] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p21 , DYRK1 and ROCK, and

[0327] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and p19, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0328] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity o p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of p19. Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0329] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0330] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p19, DYRK1 and ROCK, and

[0331] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and p21 , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0332] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of p21 . Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of p21 and an inhibitor of the expression or activity of CDK4 and CDK6.

[0333] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0334] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding MPS1 , DYRK1 and ROCK, and

[0335] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 and p19, and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0336] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity o p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 and an inhibitor of the expression or activity of p19. Yet more preferably in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of CDK4 and CDK6.

[0337] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein: - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , p19, MPS1 , DYRK1 and

[0338] - the cell culture medium comprises an inhibitor of the expression or activity of ROCK, , and optionally an inhibitor of the expression or activity of CDK4 and CDK6.

[0339] Another object of the invention is a megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, wherein said megakaryocyte is genetically modified so as to silence:

[0340] (a) a gene selected in the list consisting of the genes encoding p21 , p19, MPS1 , DYRK1 , and ROCK, and

[0341] (b) optionally, the gene encoding TP53.

[0342] The megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC may be genetically modified for instance by knocking out the gene of interest, or by introducing into the megakaryocyte a polynucleotide having a sequence encoding a shRNA, as already detailed herein.

[0343] In a preferred embodiment, the megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC is genetically modified by knocking out a gene of interest, chosen in the list consisting of p21 , p19, MPS1 , DYRK1 and ROCK and optionally TP53. In a specific embodiment, the megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC is genetically modified by introducing into the diploid megakaryocyte a polynucleotide having a sequence encoding a shRNA, where the shRNA targets the mRNA which is the expression product of the gene of interest chosen in the list consisting of the genes encoding of p21 , p19, MPS1 , DYRK1 and ROCK and optionally TP53. Preferably, in this embodiment, the sequence encoding the shRNA is operably linked to a promoter allowing for stable expression of the shRNA molecule by the megakaryocyte, thereby inhibiting the expression of a functional protein by the gene of interest.

[0344] Such megakaryocytes are indeed particularly useful for producing high levels of platelets. They may be diploid or megakaryocytes.

[0345] The polyploid megakaryocytes as produced by the method of the invention or of the invention can provide very high yields of platelets or platelet-like particles and are therefore of particular for the production of such particles.

[0346] Another object of the invention is a method for the production of iPSC-derived platelet-like particles comprising culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention in conditions appropriate for the production of plateletlike particles, wherein said conditions comprise culturing the polyploid megakaryocyte or the population of megakaryocytes in the presence of an inhibitor of the expression or activity of a protein chosen from the list consisting of TP53, p21 , p19, MPS1 , DYRK1 , and ROCK.

[0347] Methods and conditions appropriate for the production of platelet and platelet-like particles using megakaryocytes are well known in the art and have for instance been described by Heazlewood et al. (2014), Nakamura et al. (2014), Moreau et al. (2016) and Chang et al. (2007).

[0348] Such conditions typically include culturing the polyploid megakaryocyte in a cell culture medium appropriate for producing platelets or platelet-like particles. In a preferred embodiment, the method comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention in a cell culture medium comprising at least Iscove’s Modified Dulbecco’s Mediu (IMDM), Bovin Serum Albumine (BSA), Liposomes and Insulin Transferrin Selenium (ITS).

[0349] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0350] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53,and

[0351] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 , DYRK1 , and ROCK.

[0352] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0353] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding TP53 and MPS1 , and

[0354] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and ROCK.

[0355] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of ROCK.

[0356] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0357] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding TP53 and DYRK1 , and

[0358] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and ROCK.

[0359] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of ROCK.

[0360] In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0361] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding TP53 and ROCK, and

[0362] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and DYRK1 .

[0363] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of DYRK1 .

[0364] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0365] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 , and

[0366] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , DYRK1 , and ROCK.

[0367] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p19.

[0368] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0369] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and p19, and

[0370] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 , DYRK1 , and ROCK.

[0371] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of DYRK1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK.

[0372] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0373] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and MPS1 , and

[0374] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, DYRK1 , and ROCK.

[0375] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of DYRK1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK.

[0376] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0377] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and DYRK1 , and

[0378] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , and ROCK.

[0379] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of MPS1. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK.

[0380] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0381] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 and ROCK, and

[0382] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , and DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of MPS1. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 .

[0383] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0384] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding p19, and

[0385] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , MPS1 , DYRK1 , and ROCK.

[0386] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 .

[0387] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0388] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19 and MPS1 , and

[0389] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , DYRK1 , and ROCK.

[0390] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of DYRK1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK.

[0391] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0392] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19 and DYRK1 , and - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , MPS1 , and ROCK.

[0393] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of MPS1 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK.

[0394] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0395] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19 and ROCK, and

[0396] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19, MPS1 , and DYRK1 .

[0397] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of MPS1. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0398] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 , and

[0399] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, DYRK1 , and ROCK.

[0400] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 . Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 .

[0401] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein: - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding MPS1 and DYRK1 , and

[0402] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, and ROCK.

[0403] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK.

[0404] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0405] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding MPS1 and ROCK, and

[0406] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, and DYRK1 .

[0407] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1 . In a particular embodiment, the composition of the invention comprises the megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0408] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 , and

[0409] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, MPS1 , and ROCK.

[0410] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of ROCK, and an inhibitor of the expression or activity of p21 .

[0411] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein: - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding DYK1 and ROCK, and

[0412] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, and MPS1 .

[0413] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of MPS1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p19. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1.

[0414] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0415] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK, and

[0416] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 , p19, DYRK1 , and MPS1 .

[0417] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 , an inhibitor of the expression or activity of MPS1 , and an inhibitor of the expression or activity of p21. Even more preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19, an inhibitor of the expression or activity of MPS1 , an inhibitor of the expression or activity of DYRK1 , and an inhibitor of the expression or activity of p21 .

[0418] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0419] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , p19 and MPS1 , and

[0420] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and ROCK.

[0421] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of ROCK.

[0422] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0423] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , p19 and DYRK1 , and

[0424] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of ROCK.

[0425] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0426] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , MPS1 , and DYRK1 and

[0427] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p19 and ROCK.

[0428] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19. Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p19 and an inhibitor of the expression or activity of ROCK.

[0429] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0430] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p19, MPS1 , and DYRK1 , and

[0431] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 and ROCK.

[0432] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 and an inhibitor of the expression or activity of ROCK.

[0433] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein: - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p21 , MPS1 and ROCK, and

[0434] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and p19.

[0435] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity o p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of p19.

[0436] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0437] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p19, MPS1 and ROCK, and

[0438] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of DYRK1 and p21 .

[0439] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of DYRK1 and an inhibitor of the expression or activity of p21 .

[0440] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0441] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p21 , DYRK1 and ROCK, and

[0442] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and p19.

[0443] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity o p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of p19.

[0444] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0445] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding, p19, DYRK1 and ROCK, and

[0446] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of MPS1 and p21 .

[0447] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of MPS1 and an inhibitor of the expression or activity of p21 .

[0448] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0449] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding MPS1 , DYRK1 and ROCK, and

[0450] - the cell culture medium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of p21 and p19.

[0451] Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 . Preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity o p19. Yet preferably, in this embodiment, the cell culture medium comprises an inhibitor of the expression or activity of p21 and an inhibitor of the expression or activity of p19.

[0452] In a particular embodiment, the method of the invention comprises culturing the polyploid megakaryocyte produced by the method of the invention or the population of megakaryocytes of the invention, wherein:

[0453] - the megakaryocyte or the population of megakaryocytes of the invention derives from a diploid megakaryocyte genetically modified so as to silence the genes encoding p21 , p19, MPS1 , DYRK1 and the cell culture medium comprises an inhibitor of the expression or activity of ROCK. Another object of the invention are iPSC-derived platelet-like particles produced by the method of the invention above.

[0454] Another object of the invention is the use of iPSC-derived platelet-like particles produced by the method of the invention above to manufacture a medicament, in particular for the treatment or prevention of thrombocytopenia, preferably neonatal thrombocytopenia or thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL. An other object of the invention is the iPSC-derived platelet-like particles produced by the method of the invention for use as a medicament, in particular for the treatment or prevention of thrombocytopenia, preferably neonatal thrombocytopenia or thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL.

[0455] In other terms, the invention also provides a method for the treatment or prevention of thrombocytopenia in a subject in need thereof wherein it comprises a step of administering iPSC- derived platelet-like particles produced by the method of the invention, preferably administering a therapeutically effective amount of iPSC-derived platelet-like particles produced by the method of the invention. Preferably the “subject” or “subject in need” refers to human that is suffering from neonatal thrombocytopenia or is suspected of suffering from neonatal thrombocytopenia or has been diagnosed with neonatal thrombocytopenia. Preferably the “subject” or “subject in need” refers to human that is suffering from thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL or is suspected of suffering from thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL or has been diagnosed with thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL.

[0456] The invention is illustrated in the examples below, which should not be construed as limiting the scope of the invention.

[0457] EXAMPLES

[0458] Materials and methods

[0459] Flow cytometry and cell sorting

[0460] The cells were resuspended in a serum-free medium containing 3% (v / v) Bovine Serum Albumin (Sigma). All antibodies were from BD Biosciences and are listed in Table 1. The cells were assayed on BD Fortessa and data were analyzed with FlowJo software. Cell sorting was performed on BD Influx or BD FACSAria Fusion. A sorting strategy was used to obtain a pure iPSC-derived MK population where CD41 +CD33+ were excluded from the sorting; keeping only the CD41 +CD42+CD33- cells. Tablel . List of antibodies

[0461] Hematopoietic differentiation from iPSC.

[0462] Clumps from iPSC colonies were seeded on Geltrex-coated plates in iPS Brew medium at day -1. The starting cell concentration was at a 20% confluency range that corresponded to approximately 500 to 1000 single cells from 1 iPSC clone per experiment. At day 0, StemPro-34 SFM (Gibco; Thermo Fisher Scientific) xeno-free medium was used, supplemented with 1 % penicillin / streptomycin (v / v; Gibco; Thermo Fisher Scientific), 1 % l-glutamine (v / v; Gibco), 0.04 mg / mL of 1 -thioglycerol (MilliporeSigma), and 50 mg / mL of ascorbic acid (MilliporeSigma). This medium was retained until day 13 and supplemented with different small molecules, cytokines, and growth factors in a time-dependent manner, as described in Table 2.

[0463] Table 2. Growth factors used for the hematopoietic differentiation from iPSCs

[0464] 3’ single cell RNA sequencing

[0465] CD34-positive hematopoietic progenitors from iPSC, cord blood or apheresis were cultured under megakaryocytic condition using a serum free medium containing SCF (10 ng / ml) and TPO (25 ng / ml). Mature MK CD41 CD42-positive cells were sorted at day 5 (iPSC), day 14 (cord blood) or day 10 (apheresis) of culture according to their ploidy level (2N and 8N MKs). Single-cell suspensions were loaded onto a Chromium Single Cell Chip (10x Genomics) according to the manufacturer’s instructions for co-encapsulation with barcoded Gel Beads at a target capture rate of ~10,000 individual cells per sample. Captured mRNAs were barcoded during cDNA synthesis using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (1 OX Genomics). All samples were processed with the Chromium Controller (10X Genomics) and the resulting libraries were prepared in parallel in a single batch. We pooled all of the libraries for sequencing in a single SP Illumina flow cell. All of the libraries were sequenced with an 8-base index read, a 28-base Readl containing cell-identifying barcodes and unique molecular identifiers (UMIs), and a 91 -base Read2 containing transcript sequences on an Illumina NovaSeq 6000. iPSC genome editing.

[0466] In order to invalidate TP53 (p53), CDKN2D (p19) and CDKN1A (p21) a CRISPR ribonuclueoprotein strategy was used. A total of 250 000 iPS cells were electroporated with 2.5 pg of gRNA targeting a coding exon of the gene together with 10 pg of Cas9 protein (all from Synthego) by using the Neon electroporater. TP53 mutant clones were selected at day 12 after electroporation.

[0467] Polyploid megakaryocyte differentiation from Hematopoietic Progenitor Cell-like cells (HPC- LC)

[0468] At day 13, CD34+CD43+Hematopoietic Progenitor Cell-like cells (HPC-LCs) of the supernatant were sorted and seeded to undergo megakaryocyte differentiation in a serum-free medium containing 10 ng / ml hSCF (Peprotech) and 25 ng / ml hTPO (Kirin Brewery). To increase megakaryocyte ploidy level CD34+CD43+HPC-LCs are first incubated with 2.5 pM of DYRK1 A inhibitor (Harmine, Selleckchem, #S3868) for 48h, then with 2.5 pM of MPS1 inhibitor (Reversine, Selleckchem, #S7588) for additional 72h.

[0469] Assessment of ploidy level

[0470] DNA staining was performed by adding Hoechst (Thermo Fisher) staining solution to culture at a concentration of 10 pg / ml for 1 hour at 37 °C, 5% CO2. Cells were then washed and resuspended in the same staining medium (PBS 1X with Hoechst 10 pg / ml) and suspended in staining medium. CD41 a-APC and CD42a-PE antibodies were used for BD Fortessa analyses.

[0471] Platelet assay

[0472] At day 11 , CD34+CD43+HPC-LCs of the supernatant were sorted and seeded to undergo megakaryocyte differentiation in a serum-free medium containing 10 ng / ml hSCF (Peprotech) and 25 ng / ml hTPO (Kirin Brewery). To increase megakaryocyte ploidy level CD34+CD43+HPC-LCs are first incubated with 2.5 pM of DYRK1A inhibitor (Harmine, Selleckchem, #S3868) for 48h, then with 2.5 pM of MPS1 inhibitor (Reversine, Selleckchem, #S7588) for additional 48h. To optimize platelet release from polyploid megakaryocytes under static culture conditions, the cells were incubated with 10 pM ROCK inhibitor (Bio-techne, Y27632 #1254) for additional 6 days. Platelet counts were determined by resuspending the cells for each condition before they were labeled with CD41 and CD42 and analyzed by flow cytometry. Since not all MKs will give rise to proplatelets, the platelet yields per MK were determined by dividing the total number of platelets by the number of MK bearing proplatelets for each condition. This latter was deduced by determining the percentage of MK bearing proplatelets. Results

[0473] Changes in megakaryocyte molecular signatures revealed by scRNA seq. scRNA-seq data were carried out on 2N and 8N MKs derived from iPSC (embryo), cord blood (fetal) and apheresis (adult) CD34-positive hematopoietic progenitor cells (HPCs) (Figure 1A). The cell sorting purity of 2N and 8N MK was verified by May Grunwald Giemsa coloration (Figure 1 B). Analysis of cell cycle signatures (Seurat) revealed that almost all embryonic MK clusters were in G1 phase, whereas fetal and adult MK clusters were either in G1 , S, or G2 / M (Figure 2). This embryonic G1 signature was associated with an enrichment in the TP53 signature compared to other ontogenetic stages (Figure 3A). In parallel, our data showed an increase in the expression of spindle assembly checkpoints such as TTK (MPS1) within polyploid (8N) MKs during ontogenetic progression (Figure 3B).

[0474] TP53KO together with MPS1 and DYRK1A inhibition cooperate synergistically to increase MK ploidy.

[0475] It is established that alteration of the mitotic spindle leads to entry into G1 without separation of sister chromatids and without cytokinesis. These tetrapioid cells can stop in G1 or undergo apoptosis through activation of the TP53 checkpoint. In a pilot experiment, CRISPR Cas9 TP53 invalidation in embryonic MKs did not increase their ploidy level (Figure 4) even though it increased their proliferation (Figure 5A, 5B). Interestingly, selective inhibition of MPS1 with reversine in a pilot experiment had a minor effect on the ploidy of TP53mMKs while it significantly increased the percentage of >4N MKs in TP53KO Iikecontext (18% vs. 1 .5% for the control) (Figure 4). Furthermore, it was already demonstrated that DYRK1 A phosphorylates TP53 to negatively regulate the G1 phase of the cell cycle (Atas-Ozcan et al. 2021). Recently, Elagib et al. showed that DYRK1A inhibition dramatically enhanced MK ploidy in vitro in human and in vivo in mice (Elagib et al. 2022). The authors showed that Dyrkl inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal MKs and those derived from induced pluripotent stem cells responded in a similar manner with 15-20% of MK beyond 4N ploidy level. In our experiments, DYRK1A inhibition alone had a minor effect on the percentage of >4N MKs derived from iPSC either in TP53m(5% vs. 1 .8%) or TP53KO Iike(7% vs. 1 .8%) context (Figure 4). Importantly, the simultaneous inhibition of DYRK1A and MPS1 had a synergistic effect on ploidization particularly in the TP53KO Iikecontext with 8N modal ploidy level versus. 2.2N in control and 40% of MK were beyond 4N ploidy level (Figure 4). Therefore, we selected this condition by inhibiting MS1 and DYRK1A simultaneously either in TP53KO Iikeor TP53 Knock-down (TP53KD) contexts forthe next experiments. Similar results were obtained with the two inhibitors in both contexts (Figure 6). Indeed, MPS1 i together with DYRKlAi increased the size (mean FSC) and granularity (mean SSC) of iPSC-derived MKs in the control (shSCR) and even more in TP53KD(Figure 6A). This was associated with high ploidy levels that were significantly increased in both controls (wild-type and shSCR) with the two inhibitors but even more in TP53KO Iikeor TP53KDreaching up to 40% of >4N iPSC-derived MKs compared with 20% in their respective controls (Figure 6B). Subsequently, MPS1 i / DYRK1Ai significantly increased the modal ploidy from 2N to 4N in both controls and nearly up to 8N in TP53KO-like or TP53KD context (Figure 6B). Finally, the ploidy level was correlated to the cytoplamic volume of MK as revealed by our MGG coloration (Figure 6C). Interestingly, while small cytoplasmic volume of MK was observed in DMSO condition for shSCR or shTP53, MPS1 i / DYRK1Ai increased the cytoplasmic volume in the polyploid MK for shSCR but even much more for shTP53 when compared to their respective controls (Figure 6C). Note that all the image acquisition parameters for all conditions were the same. Altogether, MPS1 i / DYRK1 Ai strongly increase the ploidy level of TP53KD MKs that subsequently increase their size and cytoplasmic volume.

[0476] The increased MK ploidy level with MPS1 I / DYRK1 i / ROCKi in TP53KO Iikeor TP53KDis correlated with platelet production.

[0477] In order to assess whether the high ploidy level correlates with platelet production, shSCR and shTP53 hematopoietic progenitors (CD34+ / CD43+) were seeded under megakaryocytic culture conditions in the presence of DYRK1 Ai / MPS1 i for 5 days to allow polyploid MK formation. Due to the risk of shrinkages of MKs during their sorting particularly those with high ploidy, platelet yields experiments were performed without MK sorting. This allowed a better platelet yield compared to that obtained when MKs were sorted (data not shown). Since Rho-ROCK pathway, a negative regulator of proplatelet formation (Chang et al. 2007), was enriched in 8N versus. 2N iPSC-derived MKs from our scRNAseq (Figure 7A), ROCK1 / 2 inhibitor was added to the obtained polyploid MK to enhance platelet release (Figure 7B). Indeed, ROCKi alone or in combination with DYRK1Ai / MPS1 i significantly increased the mean percentage of MK forming proplatelets either in shSCR or shTP53 context compared to their respective controls (Figure 7B). ROCKi alone slightly increased the platelet yields, reaching around 16 platelets per MK, in shTP53 context compared to the non-treated condition (Figure 7B). Importantly, when ROCKi was added to the TP53KD MKs treated with MPS1 i / DYRK1Ai, the highest platelet yields were obtained reaching around 60 platelets per MK instead of 5 platelets per MK in shSCR; representing a 12-fold increase relative to control (Figure 7C).

[0478] Due to the risk of shrinkages of MKs during their sorting particularly those with high ploidy, another set of experiments were performed without MK sorting (Figure 8A). This allowed a better platelet yield with MPS1 i / DYR1 Ai / ROCKi in TP53KDcontext with 14 platelet per MK, corresponding to a 28- fold increase relative to control (Figure 8B).

[0479] TP53KD MKs together with MPS1 i / DYRK1 Ai / ROCKi do not alter platelet function

[0480] Thereafter, we verified the functionality of platelets derived from TP53KD MK precursors in presence of MPS1 i / DYRK1 Ai. To be functional, the platelet must be able to undergo adhesion and activation before aggregation in contact of an agonist such as: thrombin or ADP. Once activated, the platelet should undergo surface membrane activation of glycoproteins complex called GPIIbllla and actin remodeling to lamellipodia and stress fibers. In order to verify their activation, GPIIbllla complex formation was assessed using PAC1 antibody from CD42-positive platelets by flow cytometry in absence or presence of a thrombin agonist (Figure 12A). Thrombin significantly increased the percentage of PAC1 + platelets compared to the non-stimulated condition (from 4.5% to 50%) (Figure 12B). To assess actin remodeling, platelets were adhered on fibrinogen in absence or presence of the thrombin agonist before they were analyzed by confocal microscopy. Stress fibers formation was significantly increased by thrombin reaching up to 80% of adhered platelets instead of 8% only in control (Figure 12C). These data strongly suggest that the combination of the inhibitors of MPS1 , DYRK1A and ROCK in the context of TP53KD does not alter the platelet function.

[0481] Invalidation of p21, the major target of TP53, recapitulates the high ploidy phenotype in presence of MPS1i / DYRK1Ai.

[0482] In order to minimize genetic instability and cytogenetic abnormalities that could occur in vitro when TP53 gene is invalidated, we hypothesized that a knock-out of p21 could mimic the high ploidy phenotype observed with MPS1 i / DYRK1Ai in the context of TP53KO. Preliminary results showed p21K0, similarly to TP53KO, increased MKs proliferation but not their polyploidization (Figure 9A, 9B). While MPS1 i / DYRK1Ai increased the percentage of >4N from 1.34% to 10.2% in control, these inhibitors allowed 37% of >4N MK in p21K0context, similar to what was already observed in theTP53Ko-iikeor TP53KDcontext (See Figure 6).

[0483] The invalidation of the CDK inhibitor p19lNK4D, known to negatively regulate endomitosis of adult MKs (Gilles et al. 2008), in iPSC-derived MKs, allowed an increase in proliferation, but not in their ploidization. MPS1 i / DYRK1Ai increased the percentage of >4N until 30% versus. 1.9% in control. Interestingly, p19K0combined with p21K0increased not only the proliferation but also the percentage of >4N MK (10% versus. 1.7%). Importantly, MSP1 i / DYRK1Ai dramatically increased the ploidy level until 70% of >4N MK with a modal ploidy of 10N in p19KO / p21KOversus. 3N in control (Figure 9A, 9B). Platelet generation experiments are ongoing to confirm that this condition allows the highest platelet yields.

[0484] MK morphology analyses by MGG coloration showed that the increase in the ploidy level in presence of both MPS1 i / DYRK1 Ai was accompanied by a slight gain in the cytoplasmic volume in the control which was much more important in p19KO / p21 KO MKs after MGG coloration (Figure 9C). Note that the image acquisition parameters were the same for all conditions.

[0485] Invalidation of both p21 and p19 slightly increase platelet yields in presence of MPS1 i / DYRK1Ai / ROCKi which is further enhanced by Palbociclib

[0486] In order to assess terminal MK differentiation and their ability to generate platelet, p19KO, p21 KO, p19KO / p21 KO and control hematopoietic progenitors (CD34+ / CD43+) were seeded under megakaryocytic culture conditions in the absence or presence of DYRK1Ai / MPS1 i for 5 days to allow polyploid MK formation. Thereafter, ROCKi was added alone or in combination to the above inhibitors. ROCKi alone or in presence of MPS1 i / DYRK1Ai increased the percentage of MK bearing proplatelets compared to their respective controls (Figure 10A). The mean number of platelets per MK was not increased by ROCKi alone while its combination with MPS1 i / DYRK1Ai slightly increased the platelet yields in control, p19KO, p21 KO and p19KO / p21 KO contexts (Figure 10A). Unexpectedly, this platelet yield corresponded to 2-fold increase only from the highly polyploid p19KO / p21 KO MK compared to untreated wild-type (Figure 10A). Morphology analyses showed highly basophilic p19KO / p21 KO MK resembling immature cells that did not finish their cycling activity. Thus, we hypothesized that if the cell cycle is blocked then these MK could undergo better maturation. Indeed, the addition of Palbociclib, an inhibitor of CDK4 / CDK6, at different concentrations significantly enhanced their ability to produce platelets in vitro increasing by nearly a 3-fold the platelet yields compared to untreated p19KO / p21 KO MKs (Figure 10B). Further experiments are ongoing to compare the platelet yields per MK in p19KO / p21 KO TP53KD in presence of MPS1 i / DYRK1Ai / ROCKi with or without Palbociclib. To further enhance cell cycle arrest to stop ploidization and trigger MK maturation, we plan to add a CDK2 inhibitor, such as Tagtociclib, alone or in combination to Palbociclib.

[0487] MPS1i alone in the context of p19KO / p21 KO recapitulates the ploidy level of TP53KD MKs with MPS1i / DYRK1Ai.

[0488] To assess at which level MPS1 i alone can increase ploidy, p19KO / p21 KO and TP53KD hematopoietic progenitors were treated with MPS1 i in absence or presence of DYRKlAi (Figure 11A). The mean percentage of >4N MK or modal ploidy were slightly decreased in MPS1 i alone compared to MPS1 i / DYRK1Ai in both p19KO / p21 KO and TP53KD contexts (Figure 11A and B). Interestingly, MPS1 i alone in the context of p19KO / p21 KO allowed a MK modal ploidy that was at the same level when MPS1 i is combined with DYRKlAi in p53KD context (Figure 11 B). Ongoing experiments will determine the platelet yields that could be obtained with MPS1 i alone in both genetic contexts.

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Claims

1. CLAIMS1 . A method for the production of polyploid megakaryocytes comprising the steps of:(a) providing a diploid megakaryocyte, preferably derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, and(b) inducing polyploidy of the diploid megakaryocyte by: i. suppressing the expression or activity of a protein chosen in the list consisting of TP53 or p21 , preferably suppressing the expression or activity of the combination of p21 and p19, and; ii. suppressing the expression or activity of a protein chosen in the list consisting of MPS1 and DYRK1 , preferably suppressing the expression or activity of MPSI and DYRK1 ; andHi. optionally, suppressing the expression or activity of ROCK, thereby producing a polyploid megakaryocyte.

2. The method of claim 1 , wherein:(a) when present, suppressing the expression or activity of TP53 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding TP53, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of TP53;(b) when present, suppressing the expression or activity of p21 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p21 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p21 ;(c) when present, suppressing the expression or activity of p19 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding p19, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of p19;(d) when present, suppressing the expression or activity of MPS1 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding MPS1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 , preferably reversine;(e) when present, suppressing the expression or activity of DYRK1 is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding DYRK1 , or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 , preferably harmine; and(f) when present, suppressing the expression or activity of ROCK is performed by: i. providing a diploid megakaryocyte genetically modified so as to silence the gene encoding ROCK, or ii. culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK, preferably Y27632.

3. The method of claim 1 or claim 2, wherein step b) of inducing polyploidy of the diploid megakaryocyte is by suppressing the expression or activity of TP53 or of the combination of p21 and p19, and by suppressing the expression or activity of MPS1 , DYRK1 , and ROCK.

4. The method of claim 3, wherein the diploid megakaryocyte is genetically modified so as to silence TP53, p21 or a combination of p21 and p19, and(a) suppressing the expression or activity of MPS1 is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of MPS1 ;(b) suppressing the expression or activity of DYRK1 is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of DYRK1 ;(c) suppressing the expression or activity of ROCK is obtained by culturing the diploid megakaryocyte in the presence of an inhibitor of the expression or activity of ROCK.

5. The method of claim 4 wherein:(a) suppressing the expression or activity of MPS1 is obtained by culturing the diploid megakaryocyte in the presence of reversine, preferably reversine at a concentration of between 1 and 5 pM, yet preferably reversine at a concentration of about 2.5 pM;(b) suppressing the expression or activity of DYRK1 is obtained by culturing the diploid megakaryocyte in the presence of harmine, preferably harmine at a concentration of between 2 and 5 pM, yet preferably harmine at a concentration of about 2.5 pM;(c) suppressing the expression or activity of ROCK is obtained by culturing the diploid megakaryocyte in the presence of Y27632, preferably Y27632 at a concentration of between 1 and 10 pM, yet preferably Y27632 at a concentration of about 10 pM; or(d) any combination of (a) to (c).

6. The method of any one of claims 1 to 5, wherein the diploid megakaryocyte is derived from a human iPSC, yet preferably derived from a human HLA-universal induced pluripotent stem cell (iPSC) line.

7. The method of any one of claim 1 to 6, wherein the diploid megakaryocyte derived from an iPSC is obtained by(a) inducing differentiation of an iPSC into an hematopoietic progenitor cell by: i. Culturing an iPSC in the presence of molecules chosen from the list consisting of BMP4, CHIR, VEGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 12 and 36 hours, preferably for about 24 hours; ii. Culturing a cell obtained from step a) i) in the presence of molecules chosen from the list consisting of BMP4, VEGF, bFGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 60 and 84 hours, preferably for about 72 hours;Hi. Culturing a cell obtained from step a) ii) in the presence of molecules chosen from the list consisting of VEGF, bFGF and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno- free cell culture medium, for between 36 and 60 hours, preferably for about 48 hours; iv. Culturing a cell obtained from step a) iii) in the presence of molecules chosen from the list consisting of VEGF, bFGF, SCF, Flt3L and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 12 and 36 hours, preferably for about 24 hours; v. Culturing a cell obtained from step a) iv) in the presence of molecules chosen from the list consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL6 and a combination thereof, preferably at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 132 and 156 hours, preferably for about 144 hours, thereby obtaining a hematopoietic progenitor cell; and(b) inducing differentiation of the hematopoietic progenitor cell into a diploid megakaryocyte by culturing an hematopoietic progenitor cell in the presence of molecules chosen from the list consisting of SCF, TPO and a combination thereof, preferably the at least one cell is cultured in serum-free and / or xeno-free cell culture medium, for between 324 and 348 hours, preferably for about 336 hours, thereby obtaining a diploid megakaryocyte.

8. A polyploid megakaryocyte produced by the method of any one of claims 1 to 7.

9. A population of megakaryocytes produced by the method of any one of claims 1 to 8.

10. The population of megakaryocytes of claim 9, wherein the population of megakaryocytes comprises at least 30%, 40% 50%, 60% preferably 65% of polyploid megakaryocytes.

11. A composition comprising the polyploid megakaryocyte of claim 8 or the population of megakaryocytes of claim 9 or claim 10, and a cell culture medium, wherein the cell culturemedium comprises an inhibitor of the expression or activity of a protein chosen from the list consisting of TP53, p21 , p19, MPS1 , DYRK1 , and ROCK.

12. A megakaryocyte derived from a megakaryocyte progenitor, yet preferably derived from an iPSC, wherein said megakaryocyte is genetically modified so as to silence:- a gene selected in the list consisting of the genes encoding p21 , p19, MPS1 , DYRK1 , and ROCK, and- optionally, the gene encoding TP53.

13. A method for the production of iPSC-derived platelet-like particles comprising culturing the polyploid megakaryocyte of claim 8 or the population of megakaryocytes of claim 9 or claim 10 in conditions appropriate for the production of platelet-like particles, wherein said conditions comprise culturing the megakaryocyte or the population of megakaryocytes in the presence of an inhibitor of the expression or activity of a protein chosen from the list consisting of TP53, p21 , p19, MPS1 , DYRK1 , and ROCK.

14. iPSC-derived platelet-like particles produced by the method of claim 13.

15. iPSC-derived platelet-like particles produced by the method of claim 13, for use as a medicament, in particular for the treatment or prevention of thrombocytopenia, preferably neonatal thrombocytopenia or thrombocytopenia associated with Acute Megakaryoblast Leukemia (AMKL), advantageously associated with pediatric AMKL.

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