Method for preparing platelets from pluripotent stem cells and its composition

Abstract

The present invention provides methods for preparing platelets from pluripotent stem cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These methods can be carried out without forming embryoid bodies or pluripotent stem cell clusters, and can be carried out without using stroma-induced cells. Additionally, the yield and / or purity are higher than previously reported for methods of preparing platelets from pluripotent stem cells. Compositions and pharmaceutical formulations comprising platelets, preferably platelets prepared from pluripotent stem cells, are also provided.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201380073429.7, with an application date of December 21, 2013 and an invention title of "Methods and Compositions for Producing Platelets from Pluripotent Stem Cells".

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 61 / 740,699, filed on December 21, 2012, and U.S. Provisional Application No. 61 / 787,476, filed on March 15, 2013, both with the invention title of "Methods and Compositions for Producing Platelets from Pluripotent Stem Cells", the contents of both applications are hereby incorporated by reference in their entirety. Background of the Invention

[0005] Platelets are small blood cells that perform important and highly specific blood coagulation functions. In the blood of an average person, there are almost 1 trillion platelets circulating, and platelets are completely replaced every 10 days. This means that platelets are produced continuously in large quantities. Platelets have a highly organized cytoskeleton and store more than 300 proteins intracellularly, which are secreted at the site of vascular injury. Platelets also play a role in inflammation, blood vessel growth, and tumor metastasis.

[0006] After vascular injury, platelets rapidly adhere to the damaged blood vessel and trigger a complex cascade of events that lead to thrombus formation. The demand for platelet transfusions has been increasing over the past few decades (51). Using conventional methods, platelets can only be stored for less than a week, thus continuously posing challenges to donation-dependent procedures. The shortage of platelet supply can have potentially life-threatening consequences, especially for patients who must undergo multiple transfusions. Repeated transfusions may also lead to tolerance reactions related to immune-mediated host responses and may require expensive patient matching (52, 53). The ability to generate platelets in vitro, especially patient-matched platelets, has significant advantages in these clinical scenarios.

[0007] For transfusion-dependent patients with unusual / rare blood types, especially those who are alloimmunized, and cancer or leukemia patients who frequently develop platelet alloimmunization, the limitations in platelet supply can have life-threatening consequences. Since the half-life of transfused human platelets is 4 - 5 days, these patients must be transfused with platelets frequently clinically. In addition, platelets from volunteer donation programs often carry the risk of various pathogen contaminations. Platelets cannot be cryopreserved using conventional techniques, so the ability to generate platelets in vitro will bring significant progress to platelet replacement therapy in clinical situations.

[0008] For more than a decade, megakaryocyte (MK) and platelet production have been studied from human hematopoietic stem cells (HSCs, CD34+) derived from bone marrow (BM), cord blood (CB), or peripheral blood (PB). Substantial success has been achieved in preparing functional platelets from HSCs by using certain combinations of cytokines, growth factors, and / or stromal feeder cells (1; 2). However, HSCs still need to be collected from donors, and their expansion ability is limited under current culture conditions, which hinders large-scale production and future clinical applications.

[0009] Human embryonic stem cells (hESCs) can be propagated and expanded infinitely in vitro, providing a potentially inexhaustible donor-free cell source for human therapy. In the past decade, extensive studies have been conducted on the in vitro differentiation of hESCs into hematopoietic cells. Directed hematopoietic differentiation of hESCs has been successfully achieved in vitro through two different culture systems. One of them involves co-culturing hESCs with stromal feeder cells in a serum-containing medium (3; 4). Another method uses suspension culture conditions with cytokines and with / without serum in an ultra-low cell-binding culture plate (5 - 7); the end point is the formation of cell aggregates or embryoid bodies ("EBs"). Hematopoietic precursors, as well as mature, functional progeny representing the erythroid, granulocyte, macrophage, megakaryocyte, and lymphoid lineages, have been found in both of the above two differentiated hESC culture systems (3 - 6: 8 - 14). Previous studies have also generated megakaryocytes / platelets from hESCs by co-culturing with stromal cells in the presence of serum (15; 16). However, the yield of megakaryocytes / platelets was low in the above studies (15; 16). Summary of the Invention

[0011] The present invention provides methods for preparing platelets from pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells ((iPSCs or iPS cells), e.g., human induced pluripotent stem cells (hiPSCs or hiPS cells). These methods may or may not generate embryoid bodies and may not require the use of stromal induction cells. Additionally, the yield and / or purity can be higher than previously reported methods for preparing platelets from pluripotent stem cells. Because platelets can be prepared more efficiently and on a larger scale, the methods and compositions of the present invention have great potential in medical transfusion applications. Moreover, because platelets do not have a nucleus and contain only a minimal amount of genetic material, the preparations of the present invention can be irradiated prior to infusion to effectively remove any contaminating nucleated cells, such as undifferentiated hESCs. Therefore, the possible presence of nucleated cells does not pose a safety problem.

[0012] Platelets collected from donors have a very limited shelf life, and the demand for prophylactic transfusions of platelets in patients is growing. Unlike umbilical cord blood or bone marrow CD34+ hematopoietic stem cells that rely on donors, human embryonic stem cells (hESCs) can be a very good alternative source for continuously preparing platelets in vitro under controlled conditions. As further described herein, the present invention provides a system and method for preparing megakaryocytes (MKs) from pluripotent stem cells under serum-free and matrix-free conditions. In a specific embodiment, pluripotent stem cells are directed to megakaryocytes through the differentiation of hematopoietic endothelial cells (PVE-HE, further described below). At the end of PVE-HE culture, a transient multipotent cell population expressing CD31, CD144, and CD105 markers is identified. In feeder-free, serum-free suspension culture, in the presence of TPO, SCF, and other cytokines, up to 100-fold expansion from hESCs or hiPS cells to MKs can be achieved within 18-20 days. This method can generate large amounts of MKs in vitro from pluripotent stem cells. When cultured under feeder-free conditions, the MKs produced by pluripotent stem cells can be used to produce platelet-like particles (platelets or platelet-like particles are produced from human-induced pluripotent stem cells (hiPSC-PLT) or human embryonic stem cells (ES PLT)). These hiPSC-PLT and ES-PLT respond to thrombin stimulation and can participate in microaggregate formation.

[0013] On the one hand, the present invention provides a pharmaceutical preparation suitable for human patients, which contains at least 10 8 platelets.

[0014] In addition, the present invention provides a pharmaceutical preparation containing platelets differentiated from human stem cells, for example, containing at least 10 8 platelets. Optionally, the preparation may be substantially free of white blood cells. Optionally, substantially all of the platelets may be functional.

[0015] The pharmaceutical preparation described above may contain 10 9 -10 14 platelets, optionally 10 9 、10 10 、10 11 、 10 12 、10 13 or 10 14 platelets.

[0016] Platelets may have one or more of the following characteristics: an average platelet volume in the range of 9.7-12.8 fL; a unimodal distribution of sizes in the preparation; and / or a logarithmic distribution of platelet volumes, where one standard deviation is less than 2 μm 3 (preferably less than 1.5 μm 3 、1 μm 3or even 0.5 μm 3 )。

[0017] Platelets may be positive for at least one of the following markers: CD41a and CD42b.

[0018] The platelets may be human platelets.

[0019] At least 50%, 60%, 70%, 80% or 90% of the platelets are functional and optionally are functional after storage at room temperature for at least 2, 3 or 4 days.

[0020] On the other hand, the present invention provides a bioreactor having weakly adherent or non - adherent megakaryocytes, which can produce functional platelets without feeder cells.

[0021] On the other hand, the present invention provides a composition comprising at least 10 9 megakaryocyte - lineage - specific precursors (MLPs).

[0022] On the other hand, the present invention provides a cryopreserved composition comprising MLPs.

[0023] On the other hand, the present invention provides a library comprising cryopreserved MLPs.

[0024] The MLPs may have a defined HLA type.

[0025] The cryopreserved composition may be HLA - matched to a patient.

[0026] On the other hand, the present invention provides a cryopreserved composition or library comprising 10 9 - 10 14 MLPs, optionally comprising 10 9 、10 10 、10 11 、10 12 、10 13 or 10 14 MLPs.

[0027] On the other hand, the present invention provides a method for preparing platelets from megakaryocytes or MPLs, comprising the steps of: (a) providing a non - adherent culture of megakaryocytes; (b) contacting the megakaryocytes or MPLs with TPO or a TPO agonist, thereby causing the formation of pro - platelets in the culture, wherein the pro - platelets release platelets; and (c) isolating the platelets.

[0028] On the other hand, the present invention provides a method for preparing platelets from megakaryocytes or MPL, comprising the steps of: (a) providing a non-adherent culture of megakaryocytes or MPL; (b) contacting the megakaryocytes or MPL with a hematopoietic expansion medium and optionally (1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or (2) TPO or a TPO agonist, SCF and IL-11, which can result in the formation of proplatelets in the culture, wherein the proplatelets release platelets. The method may further comprise (c) isolating the platelets.

[0029] The TPO agonist comprises one or more of the following: ADP, epinephrine, thrombin, collagen, a TPO-R agonist, a TPO mimetic, a second-generation thrombopoietic agent, romiplostim, eltrombopag (SB497115, Promacta), recombinant human thrombopoietin (TPO), pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, a TPO non-peptide mimetic, AKR-501, a monoclonal TPO agonist antibody, a polyclonal TPO agonist antibody, a TPO minibody, VB22B sc(Fv)2, a domain subclass-converted TPO agonist antibody, MA01G4G344, recombinant human thrombopoietin, a recombinant TPO fusion protein or a TPO non-peptide mimetic.

[0030] Optionally, substantially all of the isolated platelets may be functional.

[0031] The non-adherent culture of megakaryocytes or MPL may be a feeder cell-free culture.

[0032] The culture of step (b) may be in a medium containing one or more of the following: stem cell factor (SCF) at 0.5 - 100 ng / ml, thrombopoietin (TPO) at 10 - 100 ng / ml and interleukin-11 (IL-11) at 10 - 100 ng / ml, at least one ROCK inhibitor and / or heparin at 2.5 - 25 units / ml.

[0033] The culture of step (b) may be in a medium containing one or more of the following: TPO at 10 - 100 ng / ml, SCF at 0.5 - 100 ng / ml, IL-6 at 5 - 25 ng / ml, IL-9 at 5 - 25 ng / ml, at least one ROCK inhibitor and / or heparin at 2.5 - 25 units / ml.

[0034] The at least one ROCK inhibitor described may comprise Y27632, wherein the concentration of Y27632 may be 2 - 20 μM, about 3 - 10 μM, about 4 - 6 μM, or about 5 μM.

[0035] The method described may further comprise applying shear force to megakaryocytes.

[0036] Each megakaryocyte may produce at least 2, 3, 4, or 5 platelets.

[0037] Each megakaryocyte may produce at least 50 platelets.

[0038] Each megakaryocyte may produce at least 100, 500, 1000, 2000, 5000, or 10000 platelets.

[0039] At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the platelets described are CD41a+ and / or CD42b+, such as CD41a+ and CD42b+.

[0040] The platelets described may be prepared in the absence of feeder cells and / or in the absence of stromal feeder cells.

[0041] The platelets described may be prepared in the absence of any xenogeneic cells.

[0042] The platelets described may be human platelets.

[0043] Megakaryocytes or MPL may be cultured in the presence of an exogenous protease inhibitor. The megakaryocytes or MPL may be cultured in the presence of an exogenous MMP inhibitor. The megakaryocytes or MPL may be cultured in the presence of an exogenous MMP8 inhibitor. The megakaryocytes or MPL may be cultured in the presence of an exogenous MMP-specific inhibitor and a pan-MMP inhibitor.

[0044] Megakaryocytes or MPL may be cultured at a temperature of about 39°C.

[0045] Megakaryocytes or MPL may be prepared by the following steps, including: (a) culturing pluripotent stem cells to form pro-hematopoietic endothelial cells (PVE-HE); (b) culturing pro-hematopoietic endothelial cells to form MLP; and optionally (c) culturing MLP to form megakaryocytes. The pluripotent stem cells may be human pluripotent stem cells.

[0046] Pro-hematopoietic endothelial cells may be cultured in the presence of a BET inhibitor in step (b). The BET inhibitor may be IBET151.

[0047] Pro-hematopoietic endothelial cells may be generated without embryoid body formation.

[0048] The pluripotent stem cells can be induced pluripotent stem cells (iPSCs). The iPSCs can be human iPSCs.

[0049] Hematopoietic endothelial cells can be generated without embryoid body formation.

[0050] The hematopoietic endothelial cells can be differentiated from pluripotent stem cells under hypoxic conditions containing 1%-10% oxygen, 2%-8% oxygen, 3%-7% oxygen, 4%-6% oxygen, or approximately 5% oxygen.

[0051] Megakaryocytes can be formed by differentiating MLP at a temperature of 38-40 °C or approximately 39 °C.

[0052] On the one hand, the present invention provides a pharmaceutical preparation comprising platelets prepared by any of the methods described herein (e.g., any of the above methods).

[0053] The described preparation can be applicable to human patients. For example, the preparation can be applicable to human patients and is substantially free of white blood cells. The preparation can contain at least 10 8 platelets.

[0054] On the other hand, the present invention provides the use of a composition comprising platelets (e.g., the composition described herein, as described in the previous paragraph) or a composition comprising platelets prepared by the method described herein (e.g., the method described in the previous paragraph) in the preparation of a medicament for treating a patient in need thereof or a patient suffering from a disease or disorder affecting blood coagulation or a disease or disorder that can be treated thereby.

[0055] The described disease or disorder can include thrombocytopenia, trauma, blood-borne parasites, or malaria.

[0056] On the other hand, the present invention provides a method for treating a patient in need of platelet transfusion, comprising administering to the patient a composition comprising platelets (e.g., the composition described herein, as described in the previous paragraph) or a composition comprising platelets prepared by the method described herein (e.g., the method described in the previous paragraph).

[0057] The described method can effectively treat diseases or disorders including thrombocytopenia, trauma, blood-borne parasites, or malaria.

[0058] On the other hand, the present invention provides a composition comprising isolated PVE-HE cells, which are optionally derived from pluripotent stem cells and are optionally prepared by the method described herein.

[0059] On the one hand, the present invention provides a composition comprising isolated iPS-PVE-HE cells, which are optionally prepared according to the method described herein.

[0060] On the one hand, the present invention provides a composition comprising isolated hES-PVE-HE cells, optionally prepared according to the methods described herein.

[0061] On the one hand, the present invention provides a composition comprising isolated PVE-HE-MLP, optionally derived from pluripotent stem cells and optionally prepared according to the methods described herein.

[0062] On the one hand, the present invention provides a composition comprising isolated iPS-PVE-HE-MLP, optionally prepared according to the methods described herein.

[0063] On the one hand, the present invention provides a composition comprising isolated hES-PVE-HE-MLP, optionally prepared according to the methods described herein.

[0064] On the one hand, the present invention provides a composition comprising isolated PVE-HE-MLP-MK, optionally derived from pluripotent stem cells and optionally prepared according to the methods described herein.

[0065] On the one hand, the present invention provides a composition comprising isolated iPS-PVE-HE-MLP-MK, optionally prepared according to the methods described herein.

[0066] On the one hand, the present invention provides a composition comprising isolated hES-PVE-HE-MLP-MK, optionally prepared according to the methods described herein.

[0067] On the other hand, the present invention provides a pharmaceutical preparation suitable for human patients, containing at least 10 8 platelets, wherein the preparation is substantially free of white blood cells and substantially all platelets are functional.

[0068] In various embodiments, the pharmaceutical preparation is irradiated to remove or inactivate nucleated cells.

[0069] On the other hand, the present invention provides a pharmaceutical preparation suitable for human patients, which comprises at least 10 8 functional platelets, wherein the average plasma half-life of the functional platelets in the preparation is at least 4 days.

[0070] The pharmaceutical preparation described may contain 10 9 -10 14 platelets, optionally 10 9 、10 10 、10 11 、 10 12 、10 13 or 10 14 platelets.

[0071] The platelets contained in the pharmaceutical preparation may have one or more of the following characteristics: an average platelet volume in the range of 9.7 - 12.8 fL; a unimodal distribution of the size in the preparation; and / or a logarithmic platelet volume distribution with a standard deviation of less than 2 μm 3 (preferably less than 1.5 μm 3 、1 μm 3 or even 0.5 μm 3 ).

[0072] The platelets contained in the pharmaceutical preparation may be positive for at least one of the following markers: CD41a and CD41b.

[0073] At least half of the platelets are functional for at least 2, 3, 4 or 5 days after storage at room temperature (e.g., 22 - 25 °C). For example, at least 60%, 70%, 80% or 90% of the platelets are functional for at least two days. The platelets can be stored at room temperature for at least 5 days.

[0074] On the one hand, the present invention provides a cryopreserved library or preparation of MLP.

[0075] MLP can be obtained by collection when PVE-HE begins to be suspended in the suspension. Preferably, MLP is not plated, and preferably MLP is not allowed to adhere to the wall, thus avoiding differentiation into other cell types and promoting the generation of MK.

[0076] The said library or preparation may contain 10 9 to 10 14 MLP, optionally containing 10 9 、10 10 、10 11 、10 12 、10 13 or 10 14 MLP.

[0077] One MLP can produce 2, 3, 4, 5 or more platelets. In an exemplary embodiment, a composition providing 6x10 10 -1.2x10 11 MLP at a yield of 5 platelets per MLP is sufficient to produce a therapeutic dose of 300 - 600x10 9 platelets. In an exemplary embodiment, a composition providing 3 - 6x 10 9 MLP at a yield of at least 100 platelets per MLP is sufficient to produce a therapeutic dose.

[0078] On the other hand, the present invention provides a bioreactor having weakly adherent or non - adherent megakaryocytes, which produces functional platelets without feeder cells. Weakly adherent cells, including the megakaryocytes described, can be mechanically separated from each other or from a surface, for example, by gentle washing using a serum pipette with minimal force. Preferably, MKs are cultured under non - adherent conditions, which is thought to promote the maintenance of the MK phenotype. Shear forces can also be applied to the MK culture to increase the efficiency of platelet production. For example, microfluidic chambers or chips can be used to control shear stress, which can increase the platelet yield per MK. In one aspect, MKs can be seeded into a channel of a microfluidic chip and the culture medium can be flowed over the MKs at a near - physiological rate.

[0079] On the other hand, the present invention provides a composition containing at least 10 9 MLP.

[0080] On the other hand, the present invention provides a cryopreserved composition containing MLP.

[0081] On the other hand, the present invention provides a method for preparing platelets from megakaryocytes, comprising the steps of: a) providing a non - adherent culture of megakaryocytes; b) contacting the megakaryocytes with TPO or a TPO agonist, thereby causing the formation of pro - platelets in the culture, wherein the pro - platelets release platelets; and c) isolating the platelets.

[0082] Thrombopoietin (TPO) is considered a key cytokine involved in thrombosis and megakaryocytopoiesis and is the endogenous ligand for the thrombopoietin receptor expressed on the surface of platelets, megakaryocytes, and megakaryocyte precursors. TPO is a glycoprotein of 332 amino acids (95 kDa) and contains two domains: a receptor-binding domain (residues 1-153) and a highly glycosylated, carbohydrate-rich domain important for protein stability (residues 154-332). The TPO receptor c-Mpl (also known as CD110) is a typical hematopoietic cytokine receptor and contains two cytokine receptor homology modules. TPO binds only to the distal cytokine receptor homology module and thus initiates signal transduction. In the absence of the distal cytokine receptor homology module, c-Mpl becomes constitutively active, meaning that the distal cytokine receptor homology module functions as a c-Mpl inhibitor until it is bound by TPO. Binding of TPO activates the Janus kinase 2 (Jak2) - signal transducer and activator of transcription (STAT) signaling pathway, promoting cell proliferation and differentiation. Megakaryocyte growth and development factor (MGDF) is another platelet growth factor. Recombinant forms of TPO and MGDF, including human and pegylated forms, can be used to induce megakaryocyte and platelet differentiation and maturation. TPO receptor-activating peptides and fusion proteins (e.g., Fab59, the thrombopoietin agonist romiplostim / AMG 531, or pegylated (Peg-TPOmp)) can be used in place of TPO. Non-peptide mimetics (Eltrombopag (SB497115, Promacta) and AKR-501) bind and activate the TPO receptor by a mechanism different from that of TPO and may have an additive effect on TPO. TPO agonist antibodies (e.g., MA01G4G344) or minibodies (e.g., VB22B sc(Fv)2) that activate the TPO receptor can also be used to mimic the action of TPO. Exemplary TPO agonists are disclosed in Stasi et al., Blood Reviews 24(2010)179-190 and Kuter, Blood.2007;109:4607-4616, each of which is incorporated herein by reference in its entirety.Exemplary TPO agonists include: ADP, adrenaline, thrombin, and collagen, as well as other compounds identified in the literature as TPO-R agonists or TPO mimetics, second-generation thrombopoietic agents, thrombopoietin romiplostim, eltrombopag (SB497115, Promacta), first-generation platelet growth factors, recombinant human thrombopoietin (TPO), polyethylene glycolylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), TPO peptide mimetics, TPO receptor-activating peptides inserted into the Fab complementarity-determining regions (Fab 59), AMG 531 (a "peptibody" consisting of the human IgG1-HC constant region (Fc fragment) linked by two disulfide bonds, each covalently bound to residue 228 and having two identical peptide sequences linked by polyglycine), Peg-TPOmp (a polyethylene glycolylated TPO peptide agonist), orally available TPO agonists, TPO non-peptide mimetics, AKR-501, monoclonal TPO agonist antibodies, polyclonal TPO agonist antibodies, TPO minibodies such as VB22B sc(Fv)2, domain subclass-converted TPO agonist antibodies such as MA01G4G344, recombinant human thrombopoietin or recombinant TPO fusion proteins, TPO non-peptide mimetics. Wherever TPO is used in the embodiments of the present invention, the exemplary TPO agonists can replace TPO in further embodiments of the present invention.

[0083] On the other hand, the present invention provides a method for preparing platelets from megakaryocytes, comprising the steps of: a) providing a non-adherent culture of megakaryocytes or megakaryocyte progenitors, b) contacting the megakaryocytes or megakaryocyte progenitors with a composition containing a hematopoietic expansion medium, thereby causing the formation of proplatelets in the culture, wherein the proplatelets release platelets, and c) isolating the platelets.

[0084] In exemplary embodiments, substantially all of the isolated platelets are functional. In exemplary embodiments, the non-adherent culture of megakaryocytes or megakaryocyte progenitors is a culture free of feeder cells and / or free of xenogeneic cells. Accordingly, the present invention provides a method for preparing platelets without feeder cells.

[0085] The culture of step (b) can be carried out in a medium containing one or more of the following substances: stem cell factor (SCF), thrombopoietin (TPO), interleukin-11 (IL-11), ROCK inhibitors such as Y27632, and / or heparin. The culture of step (b) can be in a medium containing one or more of TPO, SCF, IL-6, IL-9, ROCK inhibitors such as Y27632, and / or heparin.

[0086] In one embodiment, the hematopoietic expansion medium comprises StemSpam TM ACF (ACF) (available from StemCell Technologies Inc.), and may further comprise TPO (thrombopoietin) or a TPO agonist, SCF (stem cell factor), IL-6 (interleukin 6), and IL-9 (interleukin 9), which may be provided in the form of the StemSpam TM CC220 cytokine combination (CC220) (available from StemCell Technologies Inc.). Optionally, it may comprise a ROCK inhibitor and / or heparin. TPO, SCF, IL-6, IL-9, and IL-11 are known to be megakaryocyte development and maturation factors (Stasi et al., Blood Reviews 24(2010) 179-190).

[0087] In one embodiment, the hematopoietic expansion medium comprises Stemline-II hematopoietic stem cell expansion medium (Stemline-II) (available from Sigma Aldrich), and may further comprise TPO or a TPO agonist, SCF, and IL-11. Optionally, it may comprise a ROCK inhibitor and / or heparin. The ROCK inhibitor can be, but is not limited to, Y27632.

[0088] In another embodiment, the hematopoietic expansion medium comprises Iscove’s Modified Dulbecco’s Medium (IMDM) as a basal medium, human serum albumin (recombinant or purified), iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), and cholesterol (which may be referred to herein as a defined-component medium), and may further comprise TPO or a TPO agonist, SCF, and IL-11. Optionally, it may comprise a ROCK inhibitor and / or heparin. The ROCK inhibitor can be, but is not limited to, Y27632.

[0089] In another embodiment, the hematopoietic expansion medium comprises Iscove’s Modified Dulbecco’s Medium (IMDM) as a basal medium, human serum albumin (recombinant or purified), iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), and cholesterol (which may be referred to herein as a defined component medium), and may further comprise TPO (thrombopoietin) or a TPO agonist, SCF (stem cell factor), IL-6 (interleukin 6), and IL-9 (interleukin 9). Optionally, it may comprise a ROCK inhibitor and / or heparin.

[0090] The culture of step (b) of determination can be carried out in a medium containing ACF, Stemline-II, or the defined component medium in the previous paragraph, and one or more of (1) SCF (e.g., 0.5 - 100 ng / ml), TPO (e.g., 10 - 100 ng / ml), IL-6 (e.g., 5 - 25 ng / ml), IL-9 (e.g., 5 - 25 ng / ml), and heparin (e.g., 2.5 - 25 units / ml); (2) TPO (e.g., 10 - 100 ng / ml), SCF (e.g., 0.5 - 100 ng / ml), IL-6 (e.g., 5 - 25 ng / ml), IL-9 (e.g., 5 - 25 ng / ml), Y27632 (e.g., 5 μM, or optionally 2 - 20 μM or another ROCK inhibitor at an effective concentration), and heparin (e.g., 2.5 - 25 units / ml); (3) TPO (e.g., 10 - 100 ng / ml), SCF (e.g., 0.5 - 100 ng / ml), IL-11 (e.g., 5 - 25 ng / ml), Y27632 (e.g., 5 μM, or optionally 2 - 20 μM or another ROCK inhibitor at an effective concentration), and heparin (e.g., 2.5 - 25 units / ml); or (4) one or more of TPO (e.g., 10 - 100 ng / ml), SCF (e.g., 0.5 - 100 ng / ml), IL-6 (e.g., 5 - 25 ng / ml), IL-9 (e.g., 5 - 25 ng / ml), Y27632 (e.g., 5 μM, or optionally 2 - 20 μM or another ROCK inhibitor at an effective concentration), and heparin (e.g., 2.5 - 25 units / ml).

[0091] The method may further comprise applying shear force to megakaryocytes.

[0092] The method can produce at least 2, 3, 4, or 5 platelets, at least 20, 30, 40, or 50 platelets, or at least 100, 500, 1000, 2000, 5000, or 10000 platelets per megakaryocyte.

[0093] At least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of said platelets may be CD41a+ and CD42b+.

[0094] Platelets can be prepared under feeder-free or stromal feeder cell conditions.

[0095] Platelets can be prepared in the absence of any xenogeneic cells.

[0096] Platelets can be human platelets.

[0097] Platelets can be CD41a+ and / or CD42b+.

[0098] On the other hand, the present invention provides a method for preparing megakaryocyte progenitor cells (also referred to herein as MLP) (such as those used in methods for preparing platelets or for other purposes), which may comprise the steps of: (a) culturing pluripotent stem cells to form hematopoietic endothelial cells (PVE-HE); and (b) culturing the hematopoietic endothelial cells to form megakaryocyte progenitor cells (MLP). Step (a) can be carried out in a medium free of animal components, the medium comprising Iscove’s Modified Dulbecco’s Medium (IMDM), human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, bone morphogenetic protein 4 (BMP4) (e.g., 50 ng / ml), basic fibroblast growth factor (bFGF) (e.g., 50 ng / ml) and vascular endothelial growth factor (VEGF) (e.g., 50 ng / ml). Step (b) can be carried out in Iscove’s modified Dulbecco’s medium (IMDM), Ham’s F-12 nutrient mixture, Albucult (recombinant human albumin), polyvinyl alcohol (PVA), linoleic acid, SyntheChol (synthetic cholesterol), monothioglycerol (a-MTG), recombinant human insulin-transferrin-selenium-ethanolamine solution, protein-free hybridoma medium II (PFHMII), ascorbic acid 2-phosphate, Glutamax I (L-alanyl-L-glutamine), penicillin / streptomycin, 25 ng / ml of stem cell factor (SCF), thrombopoietin (TPO) (e.g., 25 ng / ml), Fms-related tyrosine kinase 3 ligand (FL) (e.g., 25 ng / ml), interleukin-3 (IL-3) (e.g., 10 ng / ml), interleukin-6 (IL-6) (e.g., 10 ng / ml) and heparin (e.g., 5 units / ml).

[0099] On the other hand, the present invention provides a method for preparing megakaryocytes (such as those used in methods for preparing platelets or for other purposes), which can be prepared by the following steps: (a) culturing pluripotent stem cells to form hematopoietic endothelial cells (PVE-HE); (b) culturing the hematopoietic endothelial cells to form MLP; and (c) culturing MLP to form megakaryocytes, as shown in Examples 1 and 2. Steps (a) and (b) can be carried out as described in the previous paragraph. Step (c) can be carried out in Iscove’s Modified Dulbecco's Medium (IMDM) as a basal medium, human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, TPO (e.g., 30 ng / ml), SCF (e.g., 1 ng / ml), IL-6 (e.g., 7.5 ng / ml), IL-9 (e.g., 13.5 ng / ml) and optionally a ROCK inhibitor such as but not limited to Y27632 (e.g., 5 μM) and / or heparin (e.g., 5-25 units / ml).

[0100] On the other hand, the present invention provides a pharmaceutical preparation comprising platelets prepared by the above method. The preparation may be suitable for human patients and / or may contain at least 10 8 platelets, and / or may be substantially free of white blood cells.

[0101] On the other hand, the present invention provides the use of platelets of any composition prepared as described herein or by any method described herein in the preparation of a medicament for treating a patient in need thereof or suffering from a disease or disorder affecting blood coagulation.

[0102] On the other hand, the present invention provides a method for treating a patient in need of platelet transfusion, comprising administering to the patient platelets of any composition prepared as described herein or by any method described herein, which may be an effective dose for treating a disease or disorder affecting blood coagulation and / or other platelet functions and / or other diseases treatable thereby (such as thrombocytopenia or trauma). Disorders of platelet production, distribution or destruction lead to thrombocytopenia. It is common in a variety of medical conditions, including cirrhosis, HIV infection, autoimmune diseases, idiopathic thrombocytopenic purpura, chemotherapy-induced myelosuppression and bone marrow diseases. A low platelet count is associated with an increased risk of bleeding. Other typical diseases or disorders treatable thereby are malaria and other parasitic infections, and without being bound by theory, it is believed to be mediated by the ability of human platelet factor 4 to selectively lyse the digestive vacuoles of parasites and kill intraerythrocytic malaria parasites (see Love et.al., Cell Host Microbe 12(6):815-23, which is incorporated herein by reference in its entirety).

[0103] On the other hand, the present invention provides a method of administration, comprising administering to the patient any combination of platelets prepared as described herein or by any of the methods described herein, wherein the platelets deliver the drug. For example, due to the lifespan of platelets in vivo, the lack of engraftment and homing properties after administration, it is considered that they can be used as drug carriers. hESCs, hiPSCs and MPLs can be genetically modified to produce platelets that express the drugs required for treating diseases. In one aspect, hESCs, hiPSCs or MPLs can be genetically modified to express anti-tumor agents. Platelets prepared from the genetically modified hESCs, hiPSCs and MPLs can be used to deliver the anti-tumor agents to tumors for the treatment of tumor diseases.

[0104] The platelets of the present invention can be engineered to contain one or more therapeutic agents, which can be released by the platelets passively (diffusing out of the platelets over time) or actively (released due to platelet activation or degranulation). A wide range of drugs can be used. Engineered platelets can be prepared to contain one or more compounds selected from the group consisting of drugs acting at synapses and neuroeffector junctions; drugs acting on the central nervous system; drugs modulating the inflammatory response; drugs affecting renal and / or cardiovascular function; drugs affecting gastrointestinal function; antibiotics; anti-cancer drugs; immunomodulators; drugs acting on blood and / or blood-forming organs; hormones; hormone antagonists; drugs affecting calcification and bone turnover, vitamins, gene therapy agents; or other drugs such as targeting agents and the like.

[0105] In certain embodiments, the platelets have been engineered to contain one or more therapeutic agents, such as small molecule drugs, aptamers or other nucleic acid drugs or recombinant proteins, which can be stored in platelet granules (e.g., α-granules) and preferably released after platelet activation.

[0106] In certain embodiments, the platelets comprise one or more exogenous agents or combinations thereof that promote or accelerate normal wound healing, reduce scarring, and reduce fibrosis.

[0107] In certain embodiments, the platelets contain one or more exogenous anti-fibrotic drugs. Platelets engineered to deliver anti-thrombotic / anti-restenosis drugs can be used in angioplasty and thrombolytic therapy procedures. In certain embodiments, the engineered platelets can be used to prevent or reduce the severity of atherosclerosis. In certain embodiments, the engineered platelets can be used to prevent or reduce the severity of restenosis. In other embodiments, the engineered platelets can be part of the treatment of solid tumors. The engineered platelets can include one or more immune stimulants.

[0108] The present disclosure further provides methods for producing β2-microglobulin-deficient platelets, such as immunogenicity-reduced or "universal" platelets, including preparing platelets from cells engineered to lack β2-microglobulin expression, such as β2-microglobulin gene knockout pluripotent stem cells, using any of the methods disclosed herein. The present disclosure also provides β2-microglobulin-deficient platelets, megakaryocytes, or platelet progenitors that do not express β2-microglobulin. β2-microglobulin-deficient platelets typically have low or preferably undetectable class I MHC molecules on their cytoplasmic membranes, thereby reducing the immunogenicity of the platelets.

[0109] On the other hand, provided are methods for preparing platelets from megakaryocytes or MLP, including culturing a non-adherent population of megakaryocytes or MLP under shear force conditions in the presence of a protease inhibitor, and collecting the culture and optionally isolating platelets from the culture.

[0110] The protease inhibitor described above can be an MMP inhibitor.

[0111] The shear force conditions described above can be constant shear force conditions. The shear force conditions can include a shear force of 1-4.1 dynes / cm 2 of shear force.

[0112] The megakaryocytes or MLP can be cultured in a microfluidic device.

[0113] The megakaryocytes or MLP can be derived from iPS cells, ES cells, or naturally occurring CD34 + cells, optionally bone marrow or cord blood CD34 + cells.

[0114] The protease inhibitor described above can be an MMP inhibitor, such as GM6001.

[0115] The protease inhibitor described above can be an MMP-specific inhibitor, such as MMP8-I ((3R)-(+)-[2-(4-methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate]).

[0116] The protease inhibitor described above can be two or more protease inhibitors.

[0117] The two protease inhibitors described above can be an MMP general (pan) inhibitor and an MMP8-specific inhibitor.

[0118] The protease inhibitor can be added at the peak of platelet production in the culture.

[0119] The culturing of megakaryocytes or MPL in the presence of TPO or a TPO agonist results in the formation of proplatelets, from which platelets are released. The megakaryocytes or MPL are cultured in a hematopoietic expansion medium and optionally in (1) TPO or a TPO agonist, SCF, IL-6, and IL-9 or (2) TPO or a TPO agonist, SCF, and IL-11, thereby forming proplatelets in the culture, from which platelets are released.

[0120] The megakaryocytes or MPL can be cultured at a temperature higher than 37 °C and equal to or lower than 40 °C.

[0121] The megakaryocytes or MPL can be cultured at a temperature of about 39 °C.

[0122] On the other hand, a method for preparing platelets from megakaryocytes or MPL is provided, including culturing a population of non-adherent megakaryocytes or MPL derived from iPS cells or ES cells at a temperature higher than 37 °C and equal to or lower than 40 °C, and collecting and optionally isolating platelets from the culture.

[0123] The megakaryocytes or MPL can be cultured at a temperature of about 39 °C.

[0124] On the other hand, a method for preparing MPL from PVE-HE is provided, including culturing a population of PVE-HE cells derived from iPS cells or ES cells in the presence of a BET inhibitor, and collecting and optionally isolating MPL from the culture.

[0125] The inhibitor of BET can be I-BET151.

[0126] The inhibitor of BET can be added to the PVE-HE cells during the last 48 hours, last 36 hours, last 24 hours, last 18 hours, last 12 hours, or last 6 hours of the culture.

[0127] On the other hand, a method for preparing MPL from PVE-HE cells is provided, including culturing a population of PVE-HE cells derived from iPS cells or ES cells in the presence of a c-myc inhibitor or inhibitor, and collecting and optionally isolating MPL from the culture.

[0128] The inhibitor or inhibitor of c-myc can be added to the PVE-HE cells during the last 48 hours, last 36 hours, last 24 hours, last 18 hours, last 12 hours, or last 6 hours of the culture. Brief Description of the Drawings

[0130] Figure 1. Depict the step - by - step process of generating platelets from pluripotent stem cells. This figure shows the differentiation process through pluripotent hematopoietic endothelial cells (PVE - HE) generated from pluripotent stem cells, through megakaryocyte - lineage specific progenitors (MLP), and through megakaryocytes (MK).

[0131] Figure 2. Differentiation process of iPS cells through highly differentiated morphological cells (ADM). This figure shows the development of iPS cells into PVE - HE. Figure 2A . After 48 hours under feeder - free conditions, the adherent cells show the typical morphology of pluripotent stem cells. Figure 2B , showing that the transformation from pluripotent stem cells to dispersed small cell clusters is basically completed. Figure 2C , highly differentiated morphology is observed 96 - 146 hours after the initiation of PVE - HE differentiation, showing small, dense cell clusters growing on a single - cell layer.

[0132] Figures 3A-3B . Identification of the highly differentiated morphology of PVE - HE. 120 hours after the initiation of PVE - HE differentiation, the morphological changes ( Figure 3B ) of late - differentiated morphological (ADM) cells and successful PVE - HE differentiation were analyzed. Briefly, flow cytometry analysis of lineage - specific markers CD31 (PECAM), CD105 (endoglin), CD144 (VE - cadherin) was performed on a small cell sample. This figure shows the phenotype and morphology of ADM cells that have differentiated into PVE - HE. Figure 3A shows that at this differentiation stage, ADM cells show the PVE - HE phenotype CD31 + CD144 (VE - Cad) + CD105 + . Figure 3B shows the morphological changes of ADM cells.

[0133] Figure 4 . Identification of megakaryocyte - lineage specific progenitors (MLP) derived from iPS - PVE - HE by flow cytometry. This figure shows that the phenotype of human iPS - PVE - HE - MLP cells is CD34 + CD31 + CD41a + CD43 + CD13 + CD14 - CD42b - / + .

[0134] Figures 5A-5C.Morphological analysis of mature MK cells derived from iPS-PVE-HE-MLP. Figures 5A-5C show the nuclei within NK cells (as indicated by "N"), and proplatelet-forming cells with elongated pseudopodia that are easily observable (as indicated by arrows). Figure 5A and Figure 5B show that at 72 hours after the initiation of platelet differentiation, as the maturation process progresses, very large polyploid MKs (50 μM) become abundant, among which Figure 5A the scale bar in Figure 5B is 100 μM, and Figure 5A and Figure 5C show that between 72 - 96 hours, proplatelet-forming cells with elongated pseudopodia are easily visible under the microscope (as indicated by arrows).

[0135] Figure 6A -E. Comparison of the phenotype and purity of platelet preparations from different sources. This figure shows the flow cytometry analysis of the morphology and cell surface expression of CD41a and CD42b of human platelets from peripheral blood and platelets derived from iPS-PVE-HE-MLP-MK. Between 72 - 96 hours after initiation, the amount of CD41a+CD42b+ platelets increases sharply, with a level up to approximately 70% ( Figure 6D ). Figures 6A-6B shows the forward scatter ("FSC-A") and side scatter ("SSC-A") of human platelets from blood donors (6A) and hES-derived platelets (hES-PVE-HE-MLP) prepared as in Example 3 (6B). Figure 6C -E shows the expression of CD41a and CD42B of human platelets from blood donors (6C), iPS-derived platelets (iPS-PVE-HE-MLP) (6D), and hES-derived platelets (hES-PVE-HE-MLP) (6E), with the latter two samples prepared as described in Example 3.

[0136] Figure 7 .Ultrastructural comparison of human platelets from peripheral blood and platelets differentiated from human induced pluripotent stem cells (hiPSC-PLT) by transmission scanning microscopy. This figure shows the similarity in the cell characteristics of human platelets from peripheral blood and hiPSC-PLT disclosed in the present invention. hiPSC-PLT are discoid.

[0137] Figure 8. Comparison of human platelets (hPRP) derived from peripheral blood and hiPSC-PLT. This figure shows the similarity between the two cell preparations in platelet diameter (left panel) and the expression of the structural cell proteins β1-tubulin and F-actin (bound by FITC-Phalloidin) (right panel), and the said structural proteins are involved in activation-induced platelet morphological changes. Hoechst staining negative (right panel) confirmed the absence of nuclear DNA in iPSC-PLT and PLT from blood donors.

[0138] Figure 9 . Comparison of human platelets (hPRP) derived from peripheral blood and hiPSC-PLT. This figure shows the similarity in morphological characteristics between human platelets derived from peripheral blood and hiPSC-PLT of the present invention disclosed by differential interference contrast (DIC) live cell microscopy. When bound to a negatively charged glass surface, both human platelets derived from peripheral blood and hiPSC-PLT showed pseudopod protrusion indicating activation.

[0139] Figure 10. Comparison of human platelets (hPRP) derived from peripheral blood and hiPSC-PLT. This figure shows the similarity in α-granule expression demonstrated by thrombospondin 4 (TSP4) and platelet factor 4 (PF4) labeling. TSP4 and PF4 are chemokines released from the α-granules of activated platelets. TSP4 and PF4 labeling confirmed that, relative to normal human platelets ( Figure 10A ), hiPSC-PLTs ( Figure 10B ) have normal α-granule expression.

[0140] Figure 11 . Functional evaluation of hiPSC-PLT. This figure shows the in vitro activation of hiPSC-PLT by thrombin measured by the upregulation of two adhesion molecules CD62p and αIIbβIII (measured by PAC-1Ab).

[0141] Figure 12. Functional comparison of circulating human platelets and platelets derived from human iPSC and ESC. This figure shows the in vivo clot formation ability of circulating human platelets, hiPSC-PLT and hESC-PLT. Experiments were performed using native human platelets (“hPLT”), iPSC-PLT and hES cell-derived platelets (“hESC-PLT”). Figure 12A Representative images of thrombi formed in a mouse model of vascular wall injury are shown. Figure 12B The graph illustrates the average number of platelets bound to the thrombus in each experiment. The binding of platelets was inhibited by the action of an anti-αIIbβIII antibody fragment ReoPro, indicating that the binding depends on αIIbβIII as expected. ( Figure 12B)。

[0142] Figure 13 . Platelet kinetics study in macrophage-depleted NOD / SCID mice after infusion. The figure shows the detectable circulation of hiPSC-PLT and hESC-PLT for 8 hours.

[0143] Figure 14A -D. Typical process flow chart for preparing platelets from pluripotent stem cells.

[0144] Figure 15 . FACS analysis of CD41a, CD42b-MLP derived from hiPSC.

[0145] Figure 16 . Representative MLP derived from hiESC.

[0146] Figure 17 . FACS analysis of CD31+, CD43+ MLP derived from hiPSC.

[0147] Figure 18 . Representative MK derived from hESC.

[0148] Figure 19 . Formation of proplatelets from MK.

[0149] Figure 20 . FACS analysis of CD41a, CD42b.

[0150] Figure 21 . DIC microscopy of β1-tubulin staining, human donor PLT (top), hESC-PLT (bottom).

[0151] Figure 22 . Changes in platelet purity over time in the presence of DMSO (diamonds), MMP inhibitor GM6001 (squares), and GM6001 and 8% dextran (referred to as "viscosity") (triangles) under constant shear force culture conditions. The X-axis corresponds to the sample number, and samples were taken every 30 minutes during a 6+ hour culture.

[0152] Figure 23 . Changes in platelet number over time in the presence of DMSO (diamonds), MMP inhibitor GM6001 (squares), and GM6001 and 8% dextran (referred to as "viscosity") (triangles) under constant shear force culture conditions. GM6001 was added to the culture on day 0.

[0153] Figure 24. Platelet counts in the presence of DMSO (left bar), MMP inhibitor GM6001 (middle bar) under constant shear force culture conditions, and platelet counts under static culture conditions (in the absence of MMP inhibitor GM6001) (right bar).

[0154] Figure 25 . Platelet purity over time in a microfluidic device at a flow rate of 12 μl / min (squares) and 16 μl / min (triangles) in the presence of MMP inhibitor GM6001. The control (diamonds) is in the presence of DMSO since the MMP inhibitor is dissolved in DMSO.

[0155] Figure 26 . Platelet counts as a function of flow rate. Left bar: 12 μl / min, right bar: 16 μl / min.

[0156] Figure 27 . Platelet purity in static culture in the presence of MMP-specific inhibitor MMP8-I (second bar), pan-MMP inhibitor GM6001 (third bar), or a combination of MMP8-I and GM6001 (fourth bar). MMP8-I is (3R)-(+)-[2-(4-methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydroxamate] and is available from Millipore. The first bar is the control.

[0157] Figure 28 . Platelet counts in static culture in the presence of MMP-specific inhibitor MMP8-I (second bar), pan-MMP inhibitor GM6001 (third bar), or a combination of MMP8-I and GM6001 (fourth bar). The first bar is the control.

[0158] Figure 29 . Platelet purity as a function of time when cultured at 37 °C (left bar in each pair) and 39 °C (right bar in each pair). The temperature was set at the start and maintained throughout the culture.

[0159] Figure 30 . Platelet counts as a function of time when cultured at 37 °C (left bar in each pair) and 39 °C (right bar in each pair).

[0160] Figure 31 . Number of megakaryocyte precursors (MLP) collected on day 6+4 (i.e., day 10 of differentiation) as a function of increasing dose of iBET-151 (μM). iBET was added to the culture over a 6+3 day period and the MLP were exposed to iBET for approximately 24 hours before collection. iBET concentrations: 0 (left bar), 0.1 μM (middle bar), 0.25 μM (right bar).

[0161] Figure 32 . On day 6+4, relative quantification analysis of mRNA of c-myc and GATA-1 as a function of increasing dose of iBET-151. iBET concentration: for each triplicate group, 0 (left bar), 0.1 μM (middle bar), 0.25 μM (right bar).

[0162] Figure 33 . On day 6+4, purity of CD14+ cells in the collected cell population as a function of increasing dose of iBET-151. iBET concentration: for each triplicate group, 0 (left bar), 0.1 μM (middle bar), 0.25 μM (right bar). DETAILED DESCRIPTION OF THE INVENTION

[0164] Definitions and abbreviations used in this disclosure of the invention are provided at the end of the detailed description of the invention.

[0165] As described above, limitations in platelet supply have potentially life-threatening consequences for patients dependent on transfusions. Pluripotent stem cells can proliferate indefinitely in vitro and represent an inexhaustible, donor-free source of platelets for human therapy. The ability to establish a bank of hESCs with matched or reduced incompatibility may potentially reduce or eliminate the need for immunosuppressive drugs and / or immunomodulatory regimens. Exemplary embodiments provide a method comprising preparing patient-specific iPS cells (e.g., using the methods described herein or any other methods known in the art), and preparing patient-specific platelets from said patient-specific iPS cells, which platelets can be used to treat a patient, e.g., a patient who has developed platelet allogeneic immunity or is at risk of developing the same.

[0166] Exemplary embodiments provide an efficient method for generating megakaryocytes (MKs) from pluripotent stem cells under serum-free and feeder-free conditions. Preferably, the MKs are prepared from megakaryocyte lineage-specific progenitors (MLPs, further described herein). Preferably, the MKs are not prepared from angioblasts or angiogenic colony-forming cells, such as those disclosed in U.S. Patent No. 8,017,393. Using the methods disclosed herein, pluripotent stem cells (iPSCs and ESCs) are directed to differentiate into MKs. The efficiency of differentiation from MLPs to megakaryocytes has been very high (up to 90%). Without further purification, 85% of the viable cells in the MK suspension culture are CD41a+CD42b+, and the mature MKs are also CD29+ and CD61+. These in vitro-generated MKs can undergo endomitosis in the nucleus to become mature polyploid MKs. Importantly, proplatelet-forming cells with elongated pseudopods are observed at the end of MK culture, indicating that the MKs generated in this system are capable of terminal differentiation and producing functional platelets under feeder-free conditions.

[0167] Described herein is an efficient system suitable for large-scale in vitro megakaryocyte preparation using iPSCs or other pluripotent stem cells as the cell source under controlled conditions. Additionally, sufficient amounts of platelets can be prepared to supplement or replace the need for donor-derived platelets. Further, the disclosed methods can be used to prepare platelets and platelet progenitors in a predictable manner, and thus the cells can be prepared "on demand" or in amounts required to meet anticipated needs. The cells express CD41a and CD42b, undergo endomitosis, and form mature polyploid MKs. Upon further maturation, they produce functional or activated platelets that become positive for the cell adhesion molecules CD62p and αIIbβIII upon stimulation with thrombin (thought to occur due to conformational changes in αIIbβIII upon activation that expose its PAC-1 binding site, while CD62p is thought to be exposed on the outer membrane due to granule release). These two markers are known to be expressed on the surface of activated platelets and were detected on hiPSC-PLTs using PAC-1 and CD62p (p-selectin) binding assays. Since stroma-inducing cells are not required in megakaryocyte preparation, the methods described herein can provide feeder-free platelet preparation, such as without the use of any xenogeneic cells for platelet preparation.

[0168] In exemplary embodiments, other factors including estradiol, vitamin B3, matrix metalloproteinase inhibitors (MMPs), c-myc expression inhibitors, and extracellular matrix proteins can also be used to enhance platelet preparation, such as by stimulating megakaryocyte maturation and / or stimulating thrombopoiesis, which can be carried out under stroma-free conditions.

[0169] The generation of megakaryocytes under serum-free and stromal-cell-free conditions allows for the screening of factors that are important for regulating megakaryopoiesis and thrombus formation under very defined conditions. Factors so identified may be useful for clinical applications. Progress in this area may also provide an understanding of the cellular and molecular mechanisms that regulate different aspects of megakaryopoiesis, including lineage commitment, expansion, and maturation.

[0170] Exemplary embodiments incorporate a stepwise induction of megakaryocyte differentiation from pluripotent stem cells. Further optimization and establishment of process quality control can be performed to improve the consistency and efficiency of the system for clinical applications. It is not necessary to fully define the underlying cellular or extracellular mechanisms that regulate megakaryocyte maturation to practice these methods. Additional factors that promote polyploidization and cytoplasmic maturation can be identified and included to enhance the terminal differentiation of in vitro-generated megakaryocytes. For example, at least one ROCK kinase inhibitor can be used to induce endomitosis in megakaryocyte nuclei at an early stage. However, this effect is thought to be due to an artificial block of chromosome segregation and cytokinesis rather than the programmed cellular and nuclear maturation of differentiated megakaryocytes. This may be beneficial for achieving a balance among proliferation, endomitosis, and cytoplasmic maturation to obtain the highest in vitro megakaryocyte yield, terminal differentiation state, and downstream production of functional platelets under defined conditions.

[0171] These current results demonstrate that platelets derived from pluripotent stem cells have the same morphological and functional properties as normal blood platelets. These pluripotent stem cell-derived human platelets are also functional in vivo.

[0172] Additional hemodynamic events occur during platelet thrombus formation and proliferation in a living organism that cannot be fully mimicked by in vitro systems. The application of in vivo imaging techniques provides a means to directly detect and quantify platelet-dependent thrombus formation processes that occur in complex in vivo systems following vascular injury. Using in vivo high-speed wide-field microscopy, the inventors demonstrated that pluripotent stem cell-derived platelets, similar to normal human blood platelets, incorporate into developing mouse platelet thrombi at sites of laser-induced injury to the wall of small arteries in living mice. Pretreatment of pluripotent stem cell-derived and control platelets with ReoPro significantly reduced the number of donor-derived and pluripotent stem cell-derived platelets incorporated into thrombi, demonstrating that the described binding is mediated by αIIbβIII integrin. These results indicate that pluripotent stem cell-derived platelets function at sites of vascular injury in living animals.

[0173] Platelets are anucleate cells that adhere to tissues and to each other in response to vascular injury. This process is mainly mediated by platelet integrin αIIbβIII, which binds several adhesive substrates such as von Willebrand Factor (vWF) and fibrinogen, bridging and further activating platelets in the growing thrombus (36). The results herein show that platelets derived from pluripotent stem cells are functionally similar to normal blood platelets in vitro and in live animals. Platelets derived from pluripotent stem cells show important functions in participating in hemostasis, including the ability to aggregate when stimulated by physiological agonists. Additionally, results from immunofluorescence and transmission electron microscopy further demonstrate that platelets derived from pluripotent stem cells are similar to normal blood platelets.

[0174] As further described in the following examples, many similarities were found between platelets derived from pluripotent stem cells and purified normal human platelets. These similarities include the following.

[0175] hiPSC-PLTs are discoid (confirmed by transmission electron microscopy).

[0176] hiPSC-PLTs are mostly identical to circulating human platelets in ultrastructure (confirmed by transmission electron microscopy).

[0177] The size of hiPSC-PLTs was confirmed to be comparable to that of circulating human PLTs (2.38 μm ± 0.85 μm vs. 2.27 μm ± 0.49 μm) by DIC and β1-tubulin IF microscopy.

[0178] hiPSC-PLTs were shown by DIC live cell microscopy to be able to spread on glass and form filopodia and lamellipodia.

[0179] hiPSC-PLTs are anucleate - comparable to circulating human PLTs (confirmed by Hoeschst staining).

[0180] hiPSC-PLTs have a normal tubulin cytoskeleton relative to circulating human PLTs (confirmed by β1-tubulin staining).

[0181] hiPSC-PLTs have normal filamentous actin relative to circulating human PLTs (confirmed by phalloidin staining).

[0182] hiPSC-PLTs have normal α-granule expression relative to circulating human PLTs (confirmed by TSP4 and PF4 staining).

[0183] Another scientific and clinical question is whether pluripotent stem cell-derived platelets are functional in the complex in vivo environment. During the past decade, a large number of experimental models have been established to study thrombosis in mice, including the laser injury thrombosis model used by several recent groups (37:38:39). The laser-induced thrombosis model initiates platelet thrombosis as fast as 5 - 30 seconds after injury. Thus, this model can be used to monitor the real-time incorporation of rapidly cleared human platelets and hESC-PLT into developing murine platelet thrombi, which involves a large number of signaling pathways, enzymatic cascades, and interactions of numerous cellular and protein components. This model can also reflect the inflammatory response associated with thrombin-induced thrombosis.

[0184] Using the laser-induced vascular injury model, in vivo microscopy analysis confirmed that hiPSC-PLT and hESC-PLT, like blood platelets, incorporated into developing murine platelet thrombi via αIIbβIII integrin after vascular injury ( Figure 12A ). Pretreatment with ReoPro determined that the functionality of hiPSC-PLT and hESC-PLT was mediated by αIIbβIII, where ReoPro is the Fab fragment of a human-mouse chimeric monoclonal antibody that specifically binds αIIbβIII and inhibits platelet function ( Figure 12B ). These results provide evidence that hESC-PLT is functional at the site of in vivo vascular injury. Importantly, for the first time, it was shown that platelets derived from pluripotent stem cells in the absence of serum and feeder cells can promote in vivo blood coagulation and thrombosis.

[0185] Two previous studies have reported the preparation of MK from hESC. The yields of these systems were very low and, unlike the present system, they relied on co-culture with animal stromal cells supplemented with serum (15, 16). Additionally, functionality in vivo was not reported (15, 16). Eliminating these two variables during the differentiation of pluripotent stem cells allows platelet production without exposure to animal products. Furthermore, the present disclosure confirms that the feeder-free system described herein can produce MK with high efficiency and can effectively generate functional platelets under feeder-free conditions.

[0186] Thrombosis is a very complex process, accompanied by complex reorganization of membranes and microtubules and precise distribution of granules and organelles (40). Despite recent advances in the understanding of platelet formation mechanisms, the detailed mechanisms regarding membrane reorganization, proplatelet initiation, transport of platelet organelles and secretory granules, and control of platelet size remain to be elucidated. The ability to generate MK under serum-free and feeder-free conditions should facilitate the screening of factors crucial for regulating different aspects of megakaryocytopenia under defined conditions, including lineage commitment, expansion, and maturation.

[0187] The present invention discloses various methods for preparing PVE-HE cells, MLP, MK, proplatelets, and platelets in vitro (or ex vivo), wherein the cells are derived from iPS or ESC.

[0188] The present invention discloses methods for converting iPS cells or ES cells into PVE-HE cells, or MLP, or MK, or platelets. The present invention discloses methods for converting PVE-HE cells into MLP, or MK, or platelets. The present invention discloses methods for converting MLP into MK or platelets. The present invention discloses methods for converting MK into platelets. These different cultures are briefly described below.

[0189] PVE-HE cells can be prepared from iPS or ES by a method including culturing iPS or ES in a medium containing Iscove’s Modified Dulbecco’s Medium (IMDM) as a basal medium, human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low-density lipoprotein (LDL), cholesterol, and further containing bone morphogenetic protein 4 (BMP4) (e.g., 50 ng / ml), basic fibroblast growth factor (bFGF) (e.g., 50 ng / ml), and vascular endothelial growth factor (VEGF) (e.g., 50 ng / ml). This culture period can last for an average of 6 days.

[0190] MLP can be prepared from PVE-HE cells by a method including culturing the PVE-HE cells in a culture medium comprising Iscove’s modified Dulbecco’s medium (IMDM), Ham’s F-12 nutrient mixture, Albucult (recombinant human albumin), polyvinyl alcohol (PVA), linoleic acid, SyntheChol (synthetic cholesterol), monothioglycerol (α-MTG), recombinant human insulin-transferrin-selenium-ethanolamine solution, protein-free hybridoma medium II (PFHMII), ascorbic acid 2-phosphate, Glutamax I (L-alanyl-L-glutamine), penicillin / streptomycin, and further comprising stem cell factor (SCF) (e.g., 25 ng / ml), thrombopoietin (TPO) (e.g., 25 ng / ml), Fms-related tyrosine kinase 3 ligand (FL) (e.g., 25 ng / ml), interleukin-3 (IL-3) (e.g., 10 ng / ml), interleukin-6 (IL-6) (e.g., 10 ng / ml), and optionally heparin (e.g., 5 units / ml). The culture period can last for an average of 3-4 days. In the second half of the culture, including in the last 48 hours, in the last 36 hours, in the last 24 hours, in the last 18 hours, in the last 12 hours, or in the last 6 hours, a BET inhibitor can be added to the culture, preferably at a sub-cytotoxic level. The MLP harvested from these cultures can be stored at low temperature or immediately used for platelet preparation or other analyses.

[0191] Megakaryocytes can be prepared from MLP by a method including culturing the MLP in a culture medium comprising Iscove’s Modified Dulbecco's Medium (IMDM), human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, and further comprising TPO (e.g., 30 ng / ml), SCF (e.g., 1 ng / ml), IL-6 (e.g., 7.5 ng / ml), IL-9 (e.g., 13.5 ng / ml), and optionally a ROCK inhibitor such as Y27632 (e.g., 5 μM) and / or heparin (e.g., 5-25 units / ml).

[0192] Megakaryocytes can be prepared from MLP by a method including culturing MLP in a medium comprising Iscove’s Modified Dulbecco's Medium (IMDM), human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, and further comprising one or more of TPO (e.g., 10 - 100 ng / ml), SCF (e.g., 0.5 - 100 ng / ml), IL-11 (e.g., 5 - 25 ng / ml), and optionally a ROCK inhibitor such as Y27632 (e.g., 5 μM) and / or heparin (e.g., 2.5 - 25 units / ml).

[0193] The latter culture produces MK and platelets, depending on the length of the culture. Platelets are typically observed after about 3 - 4 days of culture. It should be understood that during the culture process, MLP will mature into MK, MK will mature into proplatelets, and proplatelets will mature into platelets.

[0194] Thus, platelets can be prepared from MLP or MK by a method including culturing MLP or MK in a medium comprising Iscove’s Modified Dulbecco's Medium (IMDM), human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, and further comprising TPO (e.g., 30 ng / ml), SCF (e.g., 1 ng / ml), IL-6 (e.g., 7.5 ng / ml), IL-9 (e.g., 13.5 ng / ml), and optionally a ROCK inhibitor such as Y27632 (e.g., 5 μM) and / or heparin (e.g., 5 - 25 units / ml). The culture period can last 4 - 8 days or longer.

[0195] Platelets can be prepared from MLP or MK by a method including culturing MLP or MK in a medium comprising Iscove’s Modified Dulbecco's Medium (IMDM), human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, and further comprising one or more of TPO (e.g., 10 - 100 ng / ml), SCF (e.g., 0.5 - 100 ng / ml), IL-11 (e.g., 5 - 25 ng / ml), and optionally a ROCK inhibitor such as Y27632 (e.g., 5 μM) and / or heparin (e.g., 2.5 - 25 units / ml).

[0196] These platelet preparation methods include culturing MLP or MK under static or shear force culture conditions. Static culture conditions are those in which the culture medium in contact with the cultured cells is relatively static. Shear force culture conditions are those that involve intentional and continuous movement of the culture medium in contact with the cultured cells. Shear force is measured in dynes / cm 2 and in some examples, the shear force approximates that which occurs in the bone marrow hematopoietic environment. The shear force in BM sinusoids has been reported to be about 1.3 - 4.1 dynes / cm 2 . The present disclosure relates to shear force culture that can be carried out at shear forces in the range of about 1 to about 4.5 dynes / cm 2 or about 1.3 to about 4.1 dynes / cm 2 including any force and any range of forces included between about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0 and about 4.1 dynes / cm 2 .

[0197] It should be understood that the culture can be carried out at a flow rate that generates such shear force. For a given culture, shear force is related to the flow rate (volume / time). For any given culture or culture device, using the existing knowledge in the art, the shear force can be determined based on the knowledge of the flow rate. Some experimental results provided herein compare the platelet yields at flow rates of 12 μl / min and 16 μl / min in a given microfluidic device. In some examples, the flow rate can be in the range of 5 - 25 μl / min, or within 10 - 20 μl / min. In some examples, the flow rate can be in the range of 10 - 15 μl / min, and in other examples, it can be in the range of 16 - 20 μl / min.

[0198] When using shear force culture, the platelet preparation method can include static culture for 3 - 4 days or until the first culture period in which platelets are observed, followed by a second culture in a shear force environment such as a microfluidic device or other device that can induce shear force.

[0199] It should be understood that small samples can be harvested from such cultures and the platelet content measured using, for example, FACS. This will identify the peak period of platelet production.

[0200] It should also be understood that platelet preparation methods often start with a mixture of MLP and MK as the starting material, or at certain points during the culture process, include a mixture of MLP and MK in their culture medium.

[0201] The present disclosure relates to culturing MLP or MK in a microfluidic device or other device where shear forces can be obtained. In some instances, the device is designed such that the MLP or MK is immobilized in the device but does not adhere to the device. In some instances, the device is made of a synthetic resin such as polydimethylsiloxane (PDMS) or dimethyl silicone oil. These are commonly used in microfluidic devices and chips.

[0202] The present disclosure further relates to obtaining better platelet yields and functionality when preparing platelets using shear force culture conditions if a particular protease inhibitor is added during the culture. It has been found according to the present disclosure that when platelets are subjected to shear forces, the cell surface CD42b can be lost from the platelet surface, reducing platelet activity. To avoid this and still obtain the benefits of shear force culture, the present disclosure relates to using protease inhibitors to prevent the shedding or loss of CD42b. Examples of such inhibitors include metalloprotease inhibitors, more specifically matrix metalloprotease (MMP) inhibitors. Another example of an inhibitor that can be used for shear force culture is plasminogen activator inhibitor. These inhibitors can be pan-inhibitors, meaning that a single inhibitor can inhibit more than one and possibly all such proteases. Alternatively, they can be specific inhibitors, meaning that a single inhibitor inhibits one protease either in total or predominantly.

[0203] In some cases, the culturing can be carried out in the presence of an MMP inhibitor. The inhibitor can be a small molecule such as a small organic molecule, an antibody or antibody fragment, an antisense or RNAi nucleic acid, and so on.

[0204] Examples of MMP inhibitors include, but are not limited to, GM6001 (a pan-inhibitor), N-dansyl-D-phenylalanine, 4-epi-chlortetracycline, Hydrochloride Pyridoxatin, ARP 100, ARP 101, Batimastat, Chlorhexidine, Dihydrochloride, cis-ACCP, CL 82198 Hydrochloride, Minocycline, Hydrochloride, Alendronate, Sodium Salt, GM 1489, TAPI-1, TAPI-2, GM 6001, Marimastat, MMP Inhibitor II, MMP Inhibitor III, EGTA, MMP Inhibitor V, MMP-13 Inhibitor, MMP-2 Inhibitor I, MMP-2 Inhibitor II, CP 471474, MMP-2 / MMP-3 Inhibitor I, MMP-2 / MMP-3 Inhibitor II, MMP-2 / MMP-9 Inhibitor I, MMP-2 / MMP-9 Inhibitor II, MMP-2 / MMP-9 Inhibitor V. Ecotin, Escherichia coli, MMP-3 Inhibitor, MMP-3 Inhibitor III, MMP-3 Inhibitor IV, Actinomycin D, MMP-3 Inhibitor V, MMP-3 Inhibitor VIII, MMP-7 Antisense Oligonucleotide, Sodium Salt, MMP-8 Inhibitor I, MMP-9 Inhibitor I, MMP-9 / MMP-13 Inhibitor I, MMP-9 / MMP-13 Inhibitor II, NNGH, NSC 23766, PD166793, Pro-Leu-Gly Hydroxamic Acid, Ro 32-3555, PF-356231, SB-3CT, Amiphenone Phosphate, WAY 170523, UK 370106, UK 356618, Barium Chloride Dihydrate, Luteolin, Isobavachalcone, Doxycycline Hyclate, Collagenase Inhibitor I, o-Phenanthroline, and TAPI-0 from Santa Cruz Biotechnology, Inc.They also include TIMP-1, TIMP-2, TIMP-3, TIMP-4, GM6001, methylprednisolone, batimastat, marimastat, prinomastat, BAY 12-9566, MMI270(B), BMS-275291, metastat and other inhibitors of MMP-1 to MMP-26. It should be understood that the MMP inhibitors may only inhibit one MMP family member or they may inhibit more than one or all of the MMP family members.

[0205] Certain synthetic MMP inhibitors generally contain chelating groups which can tightly bind to the catalytic zinc atom at the MMP activation site. Common chelating groups include hydroxamic acid (ester), carboxylic acid (ester), mercapto and phosphono groups.

[0206] Other MMP inhibitors include BB-94, Ro 32-3555, BB-1101, BB-2516, SE205, CT1746, CGS27023A, AG3340, BAY 12-9566, D2163, D1927, PNU-142372, CMT-1 and actinomycin.

[0207] Many MMP inhibitors are commercially available.

[0208] MMP inhibitors are well-known in the prior art, and more examples are provided in U.S. Patent Nos. 4,877,805; 5,837,224; 6,365,630; 6,630,516; 6,683,069; 6,919,072; 6,942,870; 7,094,752; 7,029,713; 6,942,870; 6,919,072; 6,906,036; 6,890,937; 6,884,425; 6,858,598; 6,759,432; 6,750,233; 6,750,228; 6,713,074; 6,699,486; 6,645,477; 6,630,516; 6,548,667; 6,541,489; 6,379,667; 6,365,630; 6,130,254; 6,093,398; 5,962,466; 5,837,224; 7,705,164; 7,786,316; 8,008,510; 7,579,486; 8,318,945 and 7,176,217; Published U.S. Patent Applications 20070037253; 20060293345; 20060173183; 20060084688; 20050058709; 20050020607; 20050004177; 20040235818; 20040185127; 20040176393; 20040067883; 20040048852; 20040034098; 20040034086; 20040034085; 20040023969; 20040019055; 20040019054; 20040019053; 20040006137; 20040006077; 20030212048; 20030004165; 20020198176; 20020177588; 20020169314; 20020164319; 20020106339; 20020061866; 20020054922; 20020049237; 20020010162; 20010039287 and 20010014688 and Published PCT Applications WO 02 / 064552, WO 05 / 1103399, WO 06 / 028523, WO2008 / 024784, WO 02 / 064552, WO 05 / 110399, WO 06 / 028523, WO01 / 62261 and WO2008 / 024784, each of which is hereby incorporated by reference.

[0209] MMP inhibitors have also been described in the scientific literature, see, e.g., Whittaker et al. Chem Rev. 99:2735-2776, 1999; Whittake et al. Cell transmissions 17(1):3-14 (Table AB) and Harrison, Nature Reviews Drug Discovery 6: 426-427, 2007 (Table AC), the specific teachings of which are hereby incorporated by reference.

[0210] Some MMP inhibitors may be

[0211]

[0212] In some instances, an MMP8 inhibitor such as a specific MMP8 inhibitor can be used in static or shear force cultures. In some instances, an MMP8 inhibitor can be used in the culture of naturally occurring MLP and MK, such as MLP (e.g., CD34 + progenitor cells) or MK derived from bone marrow or umbilical cord blood.

[0213] An example of an MMP-specific inhibitor is MMP8-I, the chemical name of which is (3R)-(+)-[2-(4-methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid oxime], which is commercially available from Millipore.

[0214] In some instances, the protease inhibitor can be a plasminogen activator inhibitor. Examples of plasminogen activator inhibitors include, but are not limited to, plasminogen activator inhibitor 1 (PAI-1), plasminogen activator inhibitor 2 (PAI-2), and tissue plasminogen activator (tPA) inhibitors. Other plasminogen activator inhibitors include those described in U.S. Patent 4,923,807; International PCT Application WO / 13063331; WO / 1316974; and the references Fortenberry YM. Plasminogen activator inhibitor-1 inhibitors: a patent review (2006-present). Expert Opin Ther Pat. 2013 Jul; 23(7):801-15; and Pannekoek et al., EMBO J. 1986; 5(10):2539-44. All are hereby incorporated by reference.

[0215] In some instances, two or more protease inhibitors can be used together in culture. As an example, the MMP inhibitor GM6001 can be used together with the MMP8-specific inhibitor MMP8-I.

[0216] In some instances, culturing can be performed at a temperature above 37°C. The culturing temperature can be in the range of 37°C - 45°C, or 37°C - 42°C, or 38°C - 41°C, or 39°C - 40°C or about 39°C or about 40°C. Culturing can be performed at a set temperature. Culturing at a temperature above 37°C is herein referred to as culturing at an elevated temperature.

[0217] In some instances, the method for preparing MLP from PVE-HE cells can be carried out in the presence of an inhibitor of the BET family of proteins containing a bromodomain. The BET inhibitor can be any molecule or compound that inhibits BET family members and can be nucleic acids such as DNA and RNA aptamers, antisense oligonucleotides, siRNA and shRNA, small peptides, antibodies or antibody fragments, and small molecules such as small chemical compounds. The BET inhibitor can prevent or reduce the binding of the bromodomain of at least one BET family member to the acetyl-lysine residue of a protein. It should be understood that the BET inhibitor can inhibit only one BET family member or it can inhibit more than one or all BET family members.

[0218] Examples of BET inhibitors are described in US 2011143651, WO2009 / 084693A1, WO 2011143669, WO2011143660, WO 2011054851, and JP 2008156311, which are hereby incorporated by reference.

[0219] Known BET examples in the prior art include, but are not limited to, RVX-208 (Resverlogix), PFI-1 (Structural Genomics Consortium), OTX015 (Mitsubishi Tanabe Pharma Corporation), BzT-7, GSK525762A (iBET, GlaxoSmithKline), JQ1 (Cell 2011 146(6):904-17), and the following compounds (WO 2011054851, GlaxoSmithKline):

[0220]

[0221] In some embodiments, the BET inhibitor is a small molecule compound (e.g., JQ1 or its derivatives) that binds to the binding pocket of the first bromodomain of BET family members (e.g., BRD1, BRD2, BRD3, BRD4, BRD7, BRDT; see WO2011143669). Other BET inhibitors include JQ1S, JQ1R, JQ20, JQ8, JQ6, JQ13, JQ19, JQ18, JQ11, JQ21, JQ24B, and KS1.

[0222] Another example of a BET inhibitor (referred to herein as iBET) is GSK1210151A (referred to herein as I-BET-151). Other BET inhibitors include IBET151 and IBET762.

[0223] Many BET inhibitors can be used as anti-leukemia drugs. When used in the methods disclosed in the present invention, they are typically used at low concentrations (i.e., below the level at which they exhibit cytotoxic effects).

[0224] The present disclosure relates to the use of BET inhibitors during the culture process of differentiating (or maturing) PVE-HE into MLP. This culture period typically lasts about 4 days. The BET inhibitor is typically added during the second half of the culture, including during the last 48 hours, last 36 hours, last 24 hours, last 12 hours, or last 6 hours of the culture.

[0225] Myc inhibitors include 10058-F4 and CX-3543.

[0226] Megakaryocyte lineage progenitor cells

[0227] Various embodiments of the present disclosure provide methods for generating megakaryocyte lineage progenitor cells (MLP) from pluripotent stem cells (including human iPS and human ES), as well as compositions of MLP.

[0228] Early lineage hematopoietic endothelial cells are CD41a negative and express CD41a at the late stage of hematopoietic differentiation of hematopoietic progenitor cells. CD42b is only expressed on mature megakaryocytes. MLP cultures may be heterogeneous, having a high percentage of CD41+ cells and a low percentage of CD42+ cells.

[0229] The percentage of CD41a and CD42b double-positive cells in MLP can be determined by FACS analysis. CD41a is a subunit of the fibrinogen receptor (αIIbβIII), and CD42b is a subunit of the von Willebrand factor receptor (GPIb-V-IX). The expression of both receptors is megakaryocyte lineage-specific and both are required for platelet function.

[0230] Before cryopreservation during harvesting, the approximate percentage of adherent cells in the MLP and the degree of differentiated large cells with a low nuclear-cytoplasmic ratio can be determined. The adherent cells may appear as discrete colonies without clear colony boundaries. There may be a large number of floating MLPs resting on top of the adherent cell population. Live floating MLPs may appear clear, with minimal birefringence, and are demarcated by a smooth cell membrane.

[0231] The MLP and its compositions may optionally be provided as cryopreserved compositions.

[0232] In another embodiment, the present disclosure provides a method for screening cell differentiation regulators, comprising: providing a quantity of PVE-HE cells or megakaryocyte progenitor cells (MLP); contacting the PVE-HE cells or MLP with a test compound; determining whether there is a functional effect in the contact of the PVE-HE cells or MLP with the test compound, wherein the presence of a functional effect indicates that the test compound is a megakaryocytopoietic factor, thrombopoietic factor, and / or hematopoietic factor that regulates cell differentiation, and the absence of a functional effect indicates that the test compound is not a megakaryocytopoietic factor, thrombopoietic factor, and / or hematopoietic factor that regulates cell differentiation. In other embodiments, the megakaryocytopoietic factor, thrombopoietic factor, and / or hematopoietic factor are related to proliferation, endomitosis, cytoplasmic maturation, and terminal differentiation of functional platelets.

[0233] Megakaryocyte

[0234] The various embodiments of the present disclosure provide methods for generating megakaryocytes from pluripotent stem cells (including human iPS and human ES) under stroma-free and / or serum-free conditions. These embodiments include generating megakaryocytes. Further embodiments provide methods for preparing megakaryocytes from pluripotent-derived hematopoietic endothelial cells. The megakaryocytes preferably are capable of producing platelets, such as when cultured under the conditions described herein.

[0235] In one embodiment, the method comprises: providing pluripotent stem cells; and differentiating the pluripotent stem cells into megakaryocytes. In one embodiment, the pluripotent stem cells are human cells. In another embodiment, the pluripotent stem cells are hESCs, which are optionally prepared without damaging the embryo, such as the NED7 cell line. In another embodiment, the pluripotent cells are human ES cells. In another embodiment, the megakaryocytes are derived from induced pluripotent stem cells. In another embodiment, the pluripotent stem cells are human iPS cells, which are derived from reprogramming somatic cells. In one embodiment, the somatic cells are from fetal tissue. In another embodiment, the somatic cells are from adult tissue.

[0236] In another embodiment, the present invention discloses a method for screening regulators of cell differentiation, comprising: providing a certain amount of megakaryocytes (MK); contacting the MK with a test compound; and determining whether there is a functional effect in the contact between the MK and the test compound, wherein the presence of a functional effect indicates that the test compound is a megakaryocyte-forming factor, a thrombogenic factor, and / or a hematopoietic factor that regulates cell differentiation, and the absence of a functional effect indicates that the test compound is not a megakaryocyte-forming factor, a thrombogenic factor, and / or a hematopoietic factor that regulates cell differentiation. In other embodiments, the megakaryocyte-forming factor, the thrombogenic factor, and / or the hematopoietic factor are related to proliferation, endomitosis, cytoplasmic maturation, and terminal differentiation of functional platelets.

[0237] platelet

[0238] Other embodiments of the present invention disclose methods for preparing platelets from human embryonic stem cells and pluripotent stem cells (including iPSCs and human iPSCs). In one embodiment, the method comprises: providing human embryonic stem cells (hESCs); forming PVE-HE cells; differentiating the PVE-HE cells into megakaryocytes; and differentiating the megakaryocytes into platelets.

[0239] In another embodiment, the method for preparing platelets comprises: providing PVE-HE cells; differentiating the PVE-HE cells into MLP or megakaryocytes; and optionally, differentiating the MLP into megakaryocytes; then differentiating (or maturing) the megakaryocytes into platelets, typically through the proplatelet step. The process of differentiating PVE-HE cells into megakaryocytes can be carried out as described above. In one embodiment, the PVE-HE cells are derived from human ES cells. In another embodiment, the PVE-HE cells are derived from induced pluripotent stem cells (iPSCs). In one embodiment, the iPS cells are human iPS cells reprogrammed from somatic cells. In one embodiment, the somatic cells are from fetal tissue. In another embodiment, the somatic cells are from adult tissue. In each embodiment, the process of differentiating megakaryocytes into platelets comprises continuously culturing the megakaryocytes to differentiate the megakaryocytes into platelets. In each embodiment, the process of differentiating megakaryocytes into platelets is carried out under feeder-free conditions and comprises collecting megakaryocytes differentiated from megakaryocyte lineage-specific progenitors.

[0240] In a further embodiment, platelets are collected from megakaryocyte cultures from day 4 to day 10 in MK-M medium or other medium, said other medium comprising Iscove’s Modified Dulbecco's Medium (IMDM) as a basal medium, human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, TPO (e.g., 30 ng / ml), SCF (e.g., 1 ng / ml), IL-6 (e.g., 7.5 ng / ml), IL-9 (e.g., 13.5 ng / ml) and optionally a ROCK inhibitor such as Y27632 (e.g., 5 μM), and / or heparin (e.g., 5 - 25 units / ml). In a preferred embodiment, platelets are collected 3 - 5 days after the first appearance of proplatelet-forming cells in megakaryocyte cultures in MK-M medium. In certain embodiments, platelets are purified using density gradient centrifugation. In a further embodiment, density gradient centrifugation uses Percoll medium. In a still further embodiment, density gradient centrifugation uses BSA / HAS medium. In another embodiment, the platelet purification method separates CD41a-negative particles. In another embodiment, the platelet purification method separates CD42b-negative particles. In another embodiment, the platelet purification method preserves cell viability and morphological integrity. In other embodiments, platelets express CD41a and CD42b. In other embodiments, platelets are responsive to thrombin stimulation. In another embodiment, platelets can spread on fibrinogen and von Willebrand factor (vWF) surfaces. In yet another embodiment, platelets have the ability to bind PAC-1 and integrin activation. In another embodiment, platelets form microaggregates and promote clot formation and contraction. In another embodiment, platelets are not activated in the presence of adenosine triphosphate diphosphatase and / or EDTA.

[0241] Methods of using pluripotent stem cell-derived platelets are provided in various embodiments disclosed herein. In certain embodiments, pluripotent stem cell-derived platelets are used for platelet transfusion. The method can include providing an amount of pluripotent stem cell-derived platelets; administering an amount of pluripotent stem cell-derived platelets to a subject in need thereof. In various embodiments, the pluripotent stem cell-derived platelets can be patient-matched platelets. In another embodiment, the platelets are derived from iPSC cells. In one embodiment, the platelets are derived from human iPS cells. In other embodiments, the platelets are stored in a solution that does not cause HLA allogeneic immune responses in the subject after administration of the platelets to the subject. In further specific embodiments, the pluripotent stem cell-derived platelets can be substantially free of white blood cells, such as containing less than 5%, less than 4%, less than 3%, less than 2% or less than 1% white blood cells, preferably less than 0.1%, 0.001% or even 0.0001%. Another specific embodiment provides a preparation of pluripotent stem cell-derived platelets, which contains less than 10 6 white blood cells, more preferably less than 10 5 , 10 4 or even 10 3 white blood cells.

[0242] Another specific embodiment provides a composition containing at least 10 8 platelets, more preferably containing at least 10 9 , at least 10 10 or at least 10 11 platelets.

[0243] Using current blood bank storage conditions of 22-24 °C, the survival and function of human platelets (from blood collection) can only be maintained for 5 days - the limited storage time is considered due to platelet aging and the increasing risk of bacterial proliferation. It is expected that the platelets prepared by the methods of the present invention will have a longer survival period than blood bank platelets, such as being able to maintain for at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or even 15 days at 22-24 °C, and maintaining appropriate viability for use in human patients.

[0244] In certain embodiments, after isolation or in one or more culturing steps for generating platelets, the platelets can be treated with one or more agents that extend platelet storage at 22-24 °C, refrigerated (e.g., 4 °C) or frozen.

[0245] For example, the present invention relates to treating platelets with an agent or derivatizing platelets under certain conditions, which agent or conditions can reduce sialidase activity and (optionally) inhibit the proliferation of one or more bacteria in a platelet product formulation. The method can include the step of contacting a platelet product formulation with an amount of a sialidase inhibitor to obtain a sialidase-treated platelet product formulation that has reduced sialidase activity and inhibited proliferation of one or more bacteria compared to a platelet product formulation that has not been treated with a sialidase inhibitor.

[0246] The types of bacteria to be inhibited include those commonly found in platelet product preparations. Examples of such bacteria include: Aspergillus, Bacillus sp, Bacteroides eggerthii, Candida albicans, Citrobacter sp, Clostridium perfringens, Corynebacterium sp, Diphtheroid, Enterobacter aerogenes, Enterobacter amnigenus, Enterobacter cloacae, Enterococcus avium, Enterococcus faecalis, Escherichia coli, Fusobacterium spp., Granulicatella adiacens, Heliobacter pylori, Klebsiella sp (Klebsiella pneumonia, Klebsiella oxytoca), Lactobacillus sp, Listeria sp, Micrococcus sp, Peptostreptococcus, Proteus vulgaris, Pseudomonas sp, Pseudomys oxalis, Propionibacterium sp, Salmonella sp, Serratia sp, Serratia marcescens Staphylococcus sp (Coagulase-negative Staphylococcus, Staphylococcus epidermidis, Staphylococcus aureus), Streptococcus sp (S. gallolyticus, S. bovis, S. pyogenes, S.viridans)) and Yersinia enterocolitica.

[0247] Sialidase inhibitors that can be used in conjunction with the present invention include, for example, fetuin, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) or a pharmaceutically acceptable salt thereof; ethyl (3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-1-cyclohex-1-ene-1-carboxylate; (2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylic acid; (4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylic acid; and (1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0248] One or more polysaccharide modifiers can be added to platelets. These polysaccharide modifiers include, for example, CMP-sialic acid, CMP-sialic acid precursors, UDP-galactose, or combinations thereof. In one aspect, an enzyme that converts CMP-sialic acid precursors to CMP-sialic acid can also be added to platelets.

[0249] In certain embodiments, platelets can be treated with at least one polysaccharide modifier in an amount effective to reduce the clearance rate of the platelet population. In some embodiments, the polysaccharide modifier is selected from UDP-galactose and UDP-galactose precursors. In some preferred embodiments, the polysaccharide modifier is UDP-galactose.

[0250] In certain embodiments, platelets can be treated with certain sugar molecules that glycosylate GlcNAc residues exposed on GP1b, thereby reducing platelet clearance, blocking platelet phagocytosis, increasing platelet circulation time, and / or increasing platelet storage time.

[0251] In certain embodiments, platelets can be treated with trehalose or other low molecular weight polysaccharides.

[0252] In certain embodiments, platelets can be treated with protease inhibitors, such as matrix metalloproteinase inhibitors.

[0253] In certain embodiments, the in vivo circulation time of the platelets can be increased by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 150%, 200% or more.

[0254] The platelets of the present invention can be cryopreserved for at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days or at least about 28 days.

[0255] Furthermore, the generation of platelets from stem cells in culture provides the opportunity to pre-treat platelets and megakaryocytes, and even the opportunity to genetically modify stem cells or progenitor cells such as megakaryocytes, thereby extending the shelf life of platelets as well as increasing the yield and survival after refrigeration and / or cryopreservation. For example, MK can be engineered to participate in changes in the expression levels of genes involved in membrane lipid ratios, protein glycosylation patterns, stress-induced proteins, 14-3-3ζ translocation, etc.

[0256] In further embodiments, the platelets are functional platelets. In further embodiments, the percentage of functional platelets is at least about 60%, at least about 70%, at least about 80% or at least about 90%. In still further embodiments, the functional platelets are viable for at least two days when stored at 22 - 37°C.

[0257] The present disclosure further relates to platelets produced according to the methods provided herein that can be engineered to contain one or more drugs, which can be released from the platelets passively (e.g., they can diffuse out of the platelets over time) or actively (e.g., released upon platelet activation and degranulation). A variety of drugs can be used, including antibiotics, antivirals, anesthetics, steroids, anti-inflammatory drugs, anti-tumor drugs, antigens, vaccines, antibodies, decongestants, antihypertensive drugs, sedatives, contraceptives, progestogens, anticholinergics, analgesics, antidepressants, antipsychotics, β-adrenergic blockers, diuretics, cardiovascular active agents, vasoactive agents, non-steroidal anti-inflammatory agents, nutritional agents, etc.

[0258] For example, engineered platelets can be prepared to include one or more compounds selected from the group consisting of the following drugs: drugs acting on synapses and neuroeffector junctions (e.g., acetylcholine, methacholine, pilocarpine, atropine, scopolamine, physostigmine, succinylcholine, epinephrine, norepinephrine, dopamine, dobutamine, isoproterenol, albuterol, propranolol, serotonin); drugs acting on the central nervous system (e.g., clonazepam, diazepam, lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, ropivacaine, amitriptyline, fluoxetine, paroxetine, valproic acid, carbamazepine, bromocriptine, morphine, fentanyl, naltrexone, naloxone); drugs modulating the inflammatory response (e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, cromolyn sodium, theophylline); drugs affecting renal and / or cardiovascular function (e.g., furosemide, thiazide, amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem, nifedipine, verapamil, digoxin, isordil, dobutamine, lidocaine, quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin, mevalonate);Drugs affecting gastrointestinal function (e.g., omeprazole, sucralfate); antibiotics (e.g., tetracycline, clindamycin, amphotericin B, quinine, methicillin, vancomycin, penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline, acyclovir, zidovudine (AZT), ddC, ddI, ribavirin, cefaclor, cephalexin, streptomycin, gentamicin, tobramycin, chloramphenicol, isoniazid, fluconazole, amantadine, interferon); anti-tumor agents (e.g., cyclophosphamide, methotrexate, fluorouracil, cytarabine, mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, decarbazine); immunomodulators (e.g., interleukin, interferon, GM-CSF, TNFα, TNFβ, cyclosporine, FK506, azathioprine, steroid); drugs acting on blood and / or blood-forming organs (e.g., interleukin, G-CSF, GM-CSF, erythropoietin, vitamins, iron, copper, vitamin B12, folic acid, heparin, warfarin, coumarin).Hormones (e.g., growth hormone (GH), prolactin, luteinizing hormone, TSH, ACTH, insulin, FSH, CG, somatostatin, estrogen, androgen, progesterone, gonadotropin-releasing hormone (GnRH), thyroxine, triiodothyronine); hormone antagonists; drugs affecting calcification and bone turnover (e.g., calcium, phosphoric acid, parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin, fluoride), vitamins (e.g., riboflavin, niacin, pyridoxine, pantothenic acid, biotin, choline, inositol, camitine, vitamin C, vitamin A, vitamin E, vitamin K), gene therapy agents (e.g., viral vectors, nucleic-acid-bearing liposomes, DNA-protein conjugates, antisense preparations); or other drugs, such as targeted drugs, etc.;

[0259] In certain embodiments, platelets are engineered to contain one or more drugs, such as small molecule drugs, aptamers or other nucleic acid drugs, or recombinant proteins, i.e., they can be stored in the granules of platelets (e.g., α-granules) and are preferably released due to platelet activation, such as at sites of vascular injury or other wounds, atherosclerotic plaques or endothelial erosion, infection or a prothrombotic environment capable of activating platelets, such as the vasculature of solid tumors. In other embodiments, engineered platelets can be used to reduce the severity or prevent fibrosis, such as in the treatment of pulmonary fibrosis, i.e., for example, they can be selected from pulmonary fibrosis, pulmonary arterial hypertension, chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis.

[0260] In certain embodiments, the platelets comprise one or more exogenous agents that promote or accelerate normal wound healing, reduce scar formation, reduce fibrosis, or combinations thereof. An exemplary recombinant protein that can be expressed in megakaryocytes and packaged in the granules of platelets produced therefrom is erythropoietin. Topical administration of erythropoietin accelerates fibrin-induced wound healing responses, and platelets loaded with recombinant EPO can be used for the treatment of open wounds and ulcers, including diabetic ulcers, burns, etc., and closed (internal) wounds, including surgical procedures (surgical injuries such as those resulting from laminectomy, discectomy, joint surgery, abdominal surgery, or thoracotomy). Other wound healing proteins, particularly non-fibrotic growth factors, that can be expressed in MK cells and stored in platelet granules according to the present invention include insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), transforming growth factor (TGFββ-3), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), keratinocyte growth factor (KGF), fibronectin, vitronectin, thrombospondin, laminin, tenasin.

[0261] In certain embodiments, the platelets comprise one or more exogenous anti-fibrotic agents, such as, but not limited to, antibodies (particularly single-chain antibodies) including anti-TGFβ-1, TGFβ-2, and / or PDGF; binding proteins (e.g., peptides comprising receptor-binding site sequences) or soluble forms of growth factor receptors or growth factor-binding domains of these receptors that can prevent TGFβ-1, TGF β-2, and / or PDGF from binding to their receptors by binding to the growth factor itself or to its receptor; or aptamers that inhibit receptor-ligand interactions.

[0262] In certain embodiments, the platelets comprise one or more exogenous agents that modulate wound healing, such as proteases; vasoactive substances such as serotonin and / or histamine; fibronectin; collagenase; plasminogen activator; neutrophil protease; elastin; collagen; proteoglycan; epidermal growth factor (EGF); hormones such as estrogen, testosterone, or progesterone; macrophage-derived growth factor (MDGF); adrenomedullin; angiogenin; angiopoietin-1; angiopoietin-related growth factors; brain-derived neurotrophic factor; corticotropin-releasing hormone; Cyr16; follistatin; hepatocyte growth factor; interleukin; midkine; neurokinin A; neuropeptide Y (NPY); pleiotrophin; progranulin, prolifern; secretoneurin; substance P; VG5Q; factors that recruit pericytes, and becaplermin.

[0263] In certain embodiments, platelets comprise one or more nuclear aptamers that can promote wound healing and / or reduce fibrosis and scar formation at the wound site. Scar formation is thought to be due to persistent inflammation and hyperactive fibroblast activation. Osteopontin (OPN) is a cytokine that promotes cell activation. Lack of OPN in vivo reduces scar formation in the skin. RNA aptamers are short RNA molecules that bind to target proteins with high affinity. The OPN-R3 (R3) blocks OPN signaling. In certain embodiments, platelets can be loaded with OPN-R3 released either actively or passively, preferably actively released to the platelet activation site. Exemplary OPN inhibitory aptamers are described in US20110190386.

[0264] In certain embodiments, platelets include one or more small (organic) agents that can promote wound healing and / or reduce fibrosis and scar formation at the wound site. For example, A2A receptor agonists, such as CGS-21680, can significantly accelerate excisional wound closure (Montesinos et al., JEM 1997, 186(9), p1615-1620). Thus, by way of illustration only, platelets can be loaded with A2A receptor agonists released either actively or passively, preferably actively released to the platelet activation site. Other small molecule agents include steroids, non-steroidal anti-inflammatory compounds (NSAIDs), 5-lipoxygenase (5-LO) inhibitors, leukotriene B4 (LTB4) receptor antagonists, leukotriene A4 (LTA4) hydrolase inhibitors, 5-HT agonists, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) inhibitors, H2 antagonists, anti-tumor agents, and cyclooxygenase-2 inhibitors.

[0265] In certain embodiments, the platelets comprise one or more exogenous antibiotics. Exemplary antibiotics include chloramphenicol, chlortetracycline, clyndamycin, clioquinol, erythromycin, framycetin, gramicidin, fusidic acid, gentamicin, mafenide, mupiroicin, neomycin, polymyxin B, bacitracin, silver sulfadiazine, tetracycline, chlortetracycline, tobramycin, amikacin, vancomycin, ramoplanin, levofloxacin, ofloxacin, moxifloxacin, clindamycin, or combinations thereof.

[0266] In certain embodiments, the platelets comprise one or more exogenous analgesic or anesthetic and / or anti-inflammatory agents. Exemplary anti-inflammatory drugs may be selected from acetaminophen, aspirin, ibuprofen, diclofenac, indometacin, piroxicam, fenoprofen, flubiprofen, ketoprofen, naproxen, suprofen, loxoprofen, cinnoxicam, tenoxicam, and combinations thereof.

[0267] Platelets processed to deliver antithrombotic / anti-restenosis drugs can be employed during angioplasty and thrombolytic therapy.

[0268] In certain embodiments, engineered platelets can be used to prevent or reduce the severity of atherosclerosis and may contain one or more exogenous anti-atherosclerotic agents (i.e., drugs that reduce atherosclerotic lesions or prevent their formation), and may include: anti-proliferative / anti-mitotic drugs, including natural products such as vinca alkaloids (i.e., vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e., etoposide, teniposide), antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), and mitomycin; enzymes (the systemic metabolism of L-asparagine and the deprivation of L-asparaginase of cells that do not have the ability to synthesize their own asparagine); anti-proliferative / anti-mitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide, and analogs, melphalan, chlorambucil), ethylenimines, and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); anti-proliferative / anti-mitotic antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs (fluorouracil, floxuridine, and cytarabine), purine analogs, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones (such as estrogen); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migration agents;Secretory inhibitor (breveldin); anti-inflammatory agents: such as corticosteroids (Cortisol, cortisone, fludrocortisone, prednisone, prednisolone, 6α-methylprednisolone, triamcinolone, betamethasone, and dexamethasone), non-steroidal drugs (salicylic acid derivatives such as aspirin); p-aminophenol derivatives such as acetaminophen; indole and indene acetic acids (indomethacin, sulindac, and etodalac), heteroaryl acetic acids (tolmetin, diclofenac, and ketorolac), aryl propionic acids (ibuprofen and derivatives), anthranilic acids (mefenamic acid and meclofenamic acid), enolic acids (piroxicam, tenoxicam, phenylbutazone, and Oxyphenthatrazone), nabumetone, gold compounds (auranofin, aurothioglucose, gold sodium thiomalate); immunosuppressive agents (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); angiogenesis drugs: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides and combinations thereof; cell cycle inhibitors, mTOR inhibitors, growth factor receptor signal transduction kinase inhibitors; retinoids; cyclin / CDK inhibitors; HMG-CoA reductase inhibitors (statins); and protease inhibitors.

[0269] In certain embodiments, engineered platelets can be used to prevent or reduce the severity of restenosis and may include one or more exogenous anti-proliferative substances, anti-inflammatory and anti-thrombotic compounds as active agents. Exemplary active agents for restenosis include sirolimus, everolimus, somatostatin, tacrolimus, roxithromycin, dunaimycin, ascomycin, bafilomycin, erythromycin, midecamycin, josamycin, concanamycin, clarithromycin, troleandomycin, folimycin, cerivastatin, simvastatin, lovastatin, fluvastatin, rosuvastatin, atorvastatin, pravastatin, pitavastatin, vinblastine, vincristine, vindesine, vinorelbine, etoboside, teniposide, nimustine, carmustine, lomustine, cyclophosphamide, 4-hydroxyoxycyclophosphamide, estramustine, melphalan, ifosfamide, tropfosfamide, chlorambucil, bendamustine, dacarbazine, busulfan, procarbazine, treosulfan, temozolomide, thiotepa, daunorubicin, doxorubicin, aclarithrubicin, epirubicin(epirubicin), mitoxantrone, idarubicin, bleomycin, mitomycin, dactinomycin, methotrexate, fludarabine, fludarabine-5'-dihydrogenphosphate, cladribine, mercaptopurine, thioguanine, cytarabine, fluorouracil, gemcitabine, capecitabine, docetaxel, carboplatin, cisplatin, oxaliplatin, amsacrine, irinotecan, topotecan, hydroxycarbamide, miltefosine, pentostatin, aldesleukin, tretinoin, asparaginase, pegasparase, anastrozole, exemestane, letrozole, formestane, aminoglutethemide, adriamycin, azithromycin, spiramycin, cepharantin, smc proliferation inhibitor-2w, epothilone A and B, mitoxantrone, azathioprine, mycophenolatmofetil, c-myc-antisense, b-myc-antisense, betulinic acid, camptothecin, lapachol, β-lapachone, podophyllotoxin, betulin, podophyllicacid2-ethylhydrazide), molgramostim, peginterferon α-2b, lenograstim; filgrastim, macrogol, dacarbazine, basiliximab, daclizumab, selectin, cytokine antagonist, CETP inhibitor, cadherin, cytokinin inhibitor, COX-2 inhibitor, NF. B, angiotensin, ciprofloxacin, camptothecin, fluroblastin, monoclonal antibody that inhibits muscle cell proliferation, bFGF antagonist, probucol, prostaglandin, 1,11-dimethoxycanthin-6-one, 1-hydroxy-11-methoxycanthin-6-one, scopolectin, colchicine, NO donors, pentaerythritol tetranitrate, syndnoeimines, S nitrosoderivatives model positive active agent, tamoxifen, staurosporine, β-estradiol, α-estradiol, estriol, estrone, ethinylestradiol, fosfestrol, medroxyprogesterone, estradiol cypionates, estradiol benzoates, tranilast, kamebakaurin and other terpenoid compounds for cancer treatment, verapamil, tyrosine kinase inhibitor, tyrphostines, cyclosporine AA), paclitaxel and its derivatives, baccatin, taxotere, and other macrocyclic oligomers of low-carbon monoxide (MCS) and their derivatives from synthetic or natural sources, mofebutazone, acemetacin, diclofenac, lonazolac, dapsone, o-carbamoylphenoxyacetic acid, lidocaine, ketoprofen, mefenamic acid, piroxicam, meloxicam, chloroquine phosphate, penicillamine, hydroxychloroquine, auranofin, sodium aurothiomalate, oxaceprol, celecoxib, β-sitosterin, adenosylmethionine, myrtecaine, polidocanol, nonivamide, levomenthol, benzocaine, aescin, ellipticine, Calbiochem D 24851, colcemid, cytochalasins A-E, indanocine, nocadazole, s-100 protein, bacitracin, vitronectin receptor antagonist, azelastine, guanocyclase-stimulating tissue inhibitors of metalloproteinases-1 and -2, free nucleic acids, nucleic acids bound to viral vectors, DNA and RNA fragments, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, antisense oligonucleotides, VEGF inhibitors, IGF-1, antibiotics, antithrombotic agents, argatroban, aspirin, abciximab, synthetic antithrombin, bivalirudin, coumadin, enoxoparin, N-Desulfated and reacetylated heparin, tissue plasminogen activator, GpIIb / IIIa platelet membrane receptor, factor X inhibitor antibody, hirudin, r-hirudin, PPACK, protamin, pro-urokinase, streptokinase, warfarin, urokinase, vasodilator, dipyramidole, trapidil, nitroprussides, PDGF antagonist, triazolopyrimidine and seramin, ACE inhibitor, captopril, cilazapril, lisinopril, enalapril, losartan, thiol protease inhibitor, prostacyclin, vapiprost, interferon α, β and, histamine antagonist, 5-hydroxytryptamine blocker, apoptosis inhibitor, apoptosis regulator, p65 NF-κB and Bcl xL antisense oligonucleotide, halofuginone, nifedipine, tocopherol, tranirast, molsidomine, tea polyphenol, epicatechin gallate, epigallocatechin gallate, Boswellic acids and its derivatives, leflunomide, anakinra, etanercept, sulfasalazine, etoposide, dicloxacillin, tetracycline, triamcinolone, mutamycin, procainimid, retinoic acid, quinidine, disopyrimide, flecainide, propafenone, sotolol, amidorone, natural and synthetic steroids, bryophyllin A, inotodiol, maquirosideA, ghalakinoside, mansonine, strebloside, hydrocortisone, betamethasone, dexamethasone, fenoporfen, ibuprofen, indomethacin, naproxen, phenylbutazone, antiviral drugs, antifungals, antiprozoal agents, natural terpenoids, hippocaesculin, barringtogenol-C21 angelate, 14-dehydroagrostistachin, agroskerin, agrostistachin, 17-hydroxyagrostistachin, ovatodiolids, 4,7-oxycycloanisomelic acid, baccharinoids B1, B2, B3 and B7, tubeimoside, bruceanol A, B and C, bruceantinoside C, yadanziosides N and P, isodeoxyelephantopin, tomenphantopin A and B, dihydroxypropyltheophylline A, B, C and D, ursolic acid, hyptatic acid A, zeorin, iso-iridogermanal, maytenfoliol, effusantin A, excisanin A and B, longikaurin B, sculponeatin C, kamebaunin, leukamenin A and B, 13,18-dehydro-6-α-senecioyloxychaparrin, taxamairin A and B,regenilol, triptolide, cymarin, apocymarin, aristolochic acid, anopterin, hydroxyanopterin, anemonin, protoanemonin, berberine, cheliburin chloride, cictoxin, sinococuline, bombrestatin A and B, cudraisoflavone A, curcumin, dihydronitidine, nitidine chloride, 12-β-hydroxypregnadien-3,20-dione, bilobol, ginkgol, ginkgolic acid, helenalin, indicine, indicine-N-oxide, lasiocarpine, inotodiol, glycoside la, podophyllotoxin, justicidin A and B, larreatin, malloterin, mallotochromanol, isobutyrylmallotochromanol, maquiroside A, marchantin A, maytansine, lycoridicin, margetine, pancratistatin, liriodenine, bisparthenolidine, oxoushinsunine, aristolactam-AII, bisparthenolidine, periplocosideA), ghalakinoside, ursolic acid, deoxypsorospermin, psycorubin, ricin A, sanguinarine, manwu wheat acid, methylsorbifolin, sphatheliachromen, stizophyllin, mansonine, strebloside, akagerine, dihydrousambaraensine, hydroxyusambarine, strychnopentamine, strychnophylline, usambarine, usambarensine, berberine, liriodenine, oxoushinsunine, daphnoretin, lariciresinol, methoxylariciresinol, syringaresinol, umbelliferon, afromoson, acetylvismione B, desacetylvismione A or vismione A and B.

[0270] In still some other embodiments, the engineered platelets can be part of the treatment of solid tumors. Solid tumors generate a prothrombotic environment that can activate platelets. Recent findings have shown that activated platelets are key regulators of tumor vascular homeostasis as they can prevent tumor bleeding. Surprisingly, this effect does not depend on the ability of platelets to form thrombi, but rather on the secretion of their granular content. Thus, harnessing platelet secretory activity to direct the release of anti-tumor and / or anti-angiogenic drugs represents a way to specifically kill tumor cells and / or disrupt tumor blood vessels. In certain preferred embodiments, the engineered platelets can be loaded with anti-angiogenic agents and / or tumor blood vessel disrupting agents.

[0271] For further illustration, engineered platelets can be loaded with anti-cancer agents such as anti-tumor agents or chemotherapeutic agents, which can include (a) alkylating agents such as mechlorethamine, cyclophosphamide, ifosfamide, melphaan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, etc.; (b) antimetabolites such as methotrexate, 5-fluorouracil (5-FU), FudR, cytarabine, 6MP, thioguanine, pentostatin, etc.; (c) natural products such as taxol, vinblastine, vincristine, etoposide, teniposide, etc.; (d) antibiotics such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin c, etc.; (e) enzymes such as L-asparaginase, heparinase, chondroitinase, etc.; (f) interferons and interleukins such as interferon-α, interferon-γ, tumor necrosis factor, etc.; (g) platinum complexes such as cisplatin, carboplatin or their derivatives; and (h) various other reagents such as mitoxantrone, nitrosourea nitrogen mustard, hydroxyurea, chloroethyl cyclohexyl nitrosourea, prednisone, diethylstilbestrol, medroxyprogesterone, tamoxifen, mitotane, procarbazine, aminoglutethimide, progestogens, androgens, anti-androgens, leuprolide, etc.

[0272] In an exemplary embodiment, the engineered platelets comprise a recombinant protein that acts as a VEGF inhibitor (i.e., a VEGF antagonist). Such proteins include antibodies and antibody mimetics (such as single-chain antibodies, monobodies, antigen-binding sites, etc.), such as ranibizumab; VEGF traps, such as aflibercept, which is a soluble protein comprising the ligand-binding domains of VEGF receptors, which bind VEGF or VEGF receptors and block receptor activation. In a preferred embodiment, the polypeptide VEGF antagonist is expressed in MK cells in a manner incorporated into the granules of platelets, particularly α-granules, and is released upon platelet activation.

[0273] Although the preferred use of the engineered platelet composition is for the treatment of tumors, including solid tumors and myelomas, it can also be used in the treatment of other pathological conditions based on abnormal angiogenesis. Other pathological conditions may include arthritis, retinopathy, psoriasis, solid tumors, benign tumors, Kaposi's sarcoma, and malignant blood diseases. This may include the drugs described previously; or, for example, in the case of arthritis, it may include disease-modifying antirheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), colchicine, methotrexate, etc.

[0274] In an exemplary embodiment, the engineered platelets can be loaded with an anti-cancer drug selected from the group consisting of: acivicin, aclarubicin, acodazole, acronycine, adozelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgens, anguidine, aphidicolin glycinate, asaley, asparaginase, 5-azacytidine, azathioprine, Bacillus calmette-guerin (BCG), Baker's Antifol (soluble), β-2'-thioguanosine, bisantrene HCl, bleomycin sulfate, busulfan, buthionine sulfoximine, BWA 773U82, BW 502U83.HCl, methanesulfonic acid, ceracemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, glucocorticoids, Corynebacterium parvum, CPT-11, crisnatol, cytarabine, cytembena, dabis maleate, dacarbazine, dactinomycin, daunorubicinHCl), deazauridine, dexrazoxane, dianhydrogalactitol, diaziquone, dibromodulcitol, didemnin B, diethyldithiocarbamate, diglycoaldehyde, dihydro-5-azacytidine, doxorubicin, echinomycin, edatrexate, edelfosine, eflornithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin, estramustine phosphate, estrogen, etanidazole, ethiofos, etoposide, fadrazole, fazarabine, fenretinide, filgrastim, finasteride, flavone acetic acid, floxuridine, fludarabine phosphate, 5-fluorouracil, Fluosol, flutamide, gallium nitrate, gemcitabine, goserelin acetate, hepsulfam, hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate, hydroxyurea, 4-hydroxyandrostenedione, hydrozyurea, idarubicin HCl, ifosfamide, interferon α, interferon β, interferon γ, interleukin-1α and β, interleukin-3, interleukin-4, interleukin-6, 4-ipomeanol, iroplatin, isotretinoin, leucovorincalcium), leuprolide acetate, levamisole, liposomal doxorubicin, liposome-coated daunorubicin, lomustine, lonidamine, maytansine, mechlorethamine hydrochloride, melphalan, menogaril, merbarone, 6-mercaptopurine, mesna, methanol-extracted residue of Bacillus Calmette-Guérin, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin C, mitotane, mitoxantrone hydrochloride, monocyte / macrophage colony-stimulating factor, nabilone, nafoxidine, neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, pala, pentostatin, piperazinedione, pipobroman, pirarubicin, piritrexim, piroxantrone hydrochloride, PIXY-321, plicamycin, porfimer sodium, prednimustine, procarbazine, progestin, pyrazofurin, razoxane, sargramostim, semustine, spirogermanium, spiromustine, streptonigrin, streptozocin, sulofenur, suraminsodium), tamoxifen, taxotere, tegafur, teniposide, terephthalamidine, teroxirone, thioguanine, thiotepa, thymidine injection, tiazofurin, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluridine, trimetrexate, tumor necrosis factor, uracil mustard, vinblastine sulfate, vincristine sulfate, vinorelbine, vindesine, vinzolidine, Yoshi 864, zorubicin, and combinations thereof.

[0275] The engineered platelets may include one or more immunostimulatory agents to promote immune activation against tumor cells and may thus include, for example, Toll-like receptor (TLR) agonists, TLR4, TLR7, TLR9, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), lipopolysaccharide (LPS), genetically modified and / or degraded LPS, alum, dextran, colony-stimulating factors, EPO, GM-CSF, G-CSF, M-CSF, pegylated G-CSF, SCF, IL-3, IL-6, PIXY 321, interferons, gamma-interferon, alpha-interferon, interleukins, IL-2, IL-7, IL-12, IL-15, IL-18, MHC class II-binding peptides, saponins, QS21, unmethylated CpG sequences, I-methyltryptophan, arginase inhibitors, cyclophosphamide, or antibodies that block immunosuppressive functions, anti-CTLA4 antibodies, or mixtures of two or more thereof.

[0276] In some cases, particularly for creating engineered platelets that include small molecule drugs and / or nucleic acids, the active agent can be introduced into the platelets by adding the active agent to the medium of megakaryocytes, proplatelets, or other cells in the differentiation pathway, or by providing the active agent in the medium / solution in which the platelets are incubated.

[0277] In other cases, particularly for creating engineered platelets that incorporate proteinaceous drugs, the active agent can be recombinantly expressed by megakaryocytes, proplatelets, or other cells in the differentiation pathway such that it is present in the platelets produced. In a preferred embodiment, the recombinant protein is packaged within platelet granules (particularly α-granules). Certain recombinant proteins, such as factor VIII, are automatically incorporated into the granules, while others may require the use of fusion proteins that include a granule targeting moiety that transports the fusion protein to the platelet granules. A typical granule targeting moiety is platelet factor 4 (PF4), or a portion thereof sufficient to transport the resulting fusion protein to the platelet granules. See, e.g., Briguet-Laugier et al., J Thromb Haemost. 2004 2(12):2231-40; El Goli et al., J Biol Chem. 2005 280(34):30329-35.

[0278] An exemplary embodiment of a recombinant protein that does not require addition of a granule transport moiety is factor VIII. The present invention relates to platelets engineered to store factor VIII in their α-granules, which release the recombinant protein, for example, upon activation at a wound site. Hemophilia A is an X-linked bleeding disorder caused by a deficiency in the factor VIII (FVIII) gene, affecting approximately 1 in 5000 male individuals. Current treatment methods include factor replacement therapy using pooled FVIII concentrates or recombinant products. Limitations of these products include their cost and their limited ability to prevent long-term sequelae unless used in a strict prophylaxis regimen. Approximately 10% of hemophilia A patients develop inhibitors to the infused products, requiring alternative, even more expensive and less effective forms of treatment. Even with new manufacturing techniques, concerns about infectious complications from blood-derived replacement products remain a problem. The high cost of treatment, infectious and immune complications of treatment, and limitations in preventing long-term complications of hemophilia A make platelet delivery methods for the treatment of hemophilia A an attractive alternative form of therapy.

[0279] For further illustration, an expression construct useful for generating the factor VIII-engineered platelets of the present invention is described in Yarovoi et al., Blood, 2003 102(12):4007. In particular, the (human) factor VIII coding sequence can be placed under the control of the megakaryocyte-specific glycoprotein Ib (GPIbα) proximal promoter region. See Fujita et al., Blood. 1998;92:488-495. Factor VIII expressed exogenously in developing megakaryocytes is stored in the α-granules contained within the platelets produced by the MKs and is then released by circulating platelets upon activation.

[0280] Pharmaceutical preparation

[0281] Exemplary compositions disclosed by the present invention may be formulations suitable for treating human patients, such as pyrogen-free or substantially pyrogen-free and pathogen-free. When administered, the pharmaceutical preparations for use in the present invention may be in a pyrogen-free, pathogen-free, and physiologically acceptable form.

[0282] Additional exemplary compositions disclosed by the present invention may be irradiated, for example, before administration. For example, cells may be irradiated with γ-rays, such as a dose of about 25 Gy. For example, the composition may be irradiated with a dose sufficient to inactivate any nucleated eukaryotic cells and / or pathogens in the composition mitotically, such as a dose sufficient to inactivate any pluripotent stem cells, MK, white blood cells, and / or PVE-HE that may be contained therein mitotically.

[0283] Delivery of platelets

[0284] A variety of membranes, devices, and methods for their manufacture have been proposed and evaluated as means for transplanting cells and their secretory products into the human body, which are collectively referred to as bioartificial implants in the patent literature. Generally, they have a common operating principle, that is, cells are encapsulated in a chamber defined by a semipermeable membrane. Long-term cell viability is thought to depend on the continuous diffusional exchange of nutrients and wastes with adjacent vascular tissues. (U.S. Patent Nos 6,372,244, 6,113,938, 6,322,804, 4,911,717, 5,855,613, 6,083,523, 5,916,554, 6,511,473, 6,485,723). There are three main types of devices described in the scientific and patent literature for cell implantation into various tissue compartments, including: planar disk designs, hollow fiber-based designs, and geometric entity-based designs. These devices are typically designed to be placed in a body cavity.

[0285] Definitions

[0286] "Embryoid body" refers to a mass or aggregate of pluripotent stem cells (e.g., iPSC or ESC) that can be formed by culturing pluripotent stem cells under non-adherent conditions, such as on a low-adhesion substrate or in a "hanging drop". In these cultures, pluripotent stem cells can form cell aggregates or masses, which are named embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb;6(2):88-95, which is incorporated herein by reference in its entirety. Typically, embryoid bodies begin to form as solid masses of pluripotent stem cells, and over time, some embryoid bodies begin to include fluid-filled cavities, with the former referred to as "simple" EBs and the latter as "cystic" embryoid bodies in the literature.

[0287] As used herein, the term “embryonic stem cell” (ES cell) is consistent with that in the prior art. This term includes cells from the inner cell mass of a human blastocyst or morula, including those that have been serially passaged as a cell line. ES cells may be derived from the fertilization of an egg by a sperm, and the production of ES cells using DNA, nuclear transfer, parthenogenesis, or by homozygosity in the HLA region. ES cells may also be from a fertilized egg, blastomere, or mammalian embryo at the blastocyst stage produced by the fusion of a sperm and an egg, nuclear transfer, parthenogenesis, androgenesis, or chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane to produce cells. Embryonic stem cells, regardless of their source or the specific method used to produce them, can be determined based on: (i) the ability to differentiate into cells of all three germ layers, (ii) the expression of at least Oct 4 and alkaline phosphatase, and (iii) the ability to form teratomas when transplanted into immunodeficient animals. Embryonic stem cells that can be used in the embodiments of the present invention include, but are not limited to, human ES cells (“ESC” or “hES cells”), such as MA01, MA09, ACT-4, No.3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Additional exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also the NIH Human Embryonic Stem Cell Registry. One available exemplary human embryonic stem cell line is the MA09 cell. The isolation and preparation of the MA09 cell have been previously described in Klimanskaya, et al. (2006) “Human Embryonic Stem Cell lines Derived from Single Blastomeres.” Nature 444:481-485. The isolation and preparation of other ES cells that can be used in accordance with the disclosure of the present invention have also been described in Chung et al. (2008) “Human Embryonic Stem Cell Line Generated Without Embryo Destruction”, Cell Stem Cell, 2:113-117. The human ES cells used in the exemplary embodiments of the present invention can be produced and maintained according to GMP standards.

[0288] As used herein, the term "pluripotent stem cell" includes embryonic stem cells, stem cells from an embryo, induced pluripotent stem cells, regardless of the method of preparing the pluripotent stem cell. A pluripotent stem cell is functionally defined as a stem cell that: (a) can cause a teratoma when transplanted into an immunodeficient (SCID) mouse; (b) can differentiate into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal cell types); and (c) expresses one or more embryonic stem cell markers (e.g., expresses Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). In certain embodiments, the pluripotent stem cell expresses one or more markers selected from the following: Oct4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated, for example, using methods known in the prior art. Exemplary pluripotent stem cells include embryonic stem cells from the ICM of a blastocyst stage embryo, and embryonic stem cells from one or more blastomeres of a cleavage stage or a morula stage embryo (optionally without destroying other parts of the embryo). Such embryonic stem cells can be generated from embryonic material prepared by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPSCs), which are generated by reprogramming somatic cells by expressing a combination of multiple factors (herein referred to as reprogramming factors). iPSCs can be generated using fetal, neonatal, infant, adolescent, or adult somatic cells.

[0289] In certain embodiments, factors that can reprogram somatic cells into pluripotent stem cells include, for example, a combination of OCT4 (sometimes referred to as OCT3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of OCT4, SOX2, Nanog, and Lin28. In certain embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. Induced pluripotent stem cells are functionally defined to include cells reprogrammed using various methods (integrating vectors, non-integrating vectors, chemical means, etc.). Pluripotent stem cells can be genetically modified or otherwise modified to increase lifespan, potency, homing, to prevent or reduce allogeneic immune responses, or to provide desired factors in cells differentiated from such pluripotent cells (e.g., platelets).

[0290] “Induced pluripotent stem cells” (iPS cells or iPSCs) can be generated by protein transduction of reprogramming factors into somatic cells. In certain embodiments, at least two reprogramming proteins are transduced into somatic cells to successfully reprogram the somatic cells. In other embodiments, at least three reprogramming proteins are transduced into somatic cells to successfully reprogram the somatic cells. In other embodiments, at least four recombinant proteins are transduced into somatic cells to successfully reprogram the somatic cells.

[0291] Pluripotent stem cells can be from any species. Embryonic stem cells have been successfully obtained, for example, in mice, various species of non-human primates, and humans, and embryonic stem cell-like cells have been generated from many additional species. Thus, one of ordinary skill in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), felines (domestic and wild, such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including the giant panda), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0292] Similarly, iPS cells can be derived from any species. iPS cells have been successfully generated using mouse and human cells. In addition, iPS cells have been successfully generated using embryonic, fetal, neonatal, and adult tissues. Thus, one can readily generate iPS cells using donor cells from any species. Therefore, one can generate iPS cells from any species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), felines (domestic and wild felines such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including the giant panda), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0293] Induced pluripotent stem cells can be prepared using any somatic cell at almost any developmental stage as a starting point. For example, the cells can be from embryonic, fetal, neonatal, juvenile, or adult donors. Exemplary somatic cells that can be used include fibroblasts, such as skin fibroblasts obtained from a skin sample or biopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. Although skin and cheek provide readily available and easily accessible suitable cell sources, almost any cell can be used. In certain embodiments, the somatic cell is not a fibroblast.

[0294] Induced pluripotent stem cells can be prepared by expressing or inducing the expression of one or more reprogramming factors in somatic cells. The somatic cells can be fibroblasts, such as skin fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblast somatic cells. The somatic cells can be reprogrammed by causing the expression (such as by viral transduction, integration or non-integration vectors, etc.) and / or contact (e.g., using protein transduction domains, electroporation, microinjection, cationic amphiphiles, fusion with lipid bilayers, detergent permeabilization, etc.) of at least 1, 2, 3, 4, 5 reprogramming factors. The reprogramming factors can be selected from Oct3 / 4, SOX2, NANOG, Lin28, c-myc, and Klf4. The expression of the reprogramming factors can be induced by contacting the somatic cells with at least one reagent capable of inducing the expression of the reprogramming factors, such as an organic small molecule reagent.

[0295] Further exemplary pluripotent stem cells include induced pluripotent stem cells, which are generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors ("reprogramming factors"). iPS cells can be obtained from a cell bank. The preparation of iPS cells can be the first step in preparing differentiated cells. iPS cells can be specifically generated using materials from a particular patient or a matched donor, with the aim of generating tissue-matched megakaryocytes and platelets. iPS cells can be prepared from cells that are not significantly immunogenic in the intended recipient, for example, from autologous cells or cells that are compatible with the tissues of the intended recipient.

[0296] Somatic cells can also be reprogrammed using a combination of methods that express reprogramming factors (e.g., using viral vectors, plasmids, etc.) and induce the expression of reprogramming factors (e.g., using small organic molecules). For example, infection with a viral vector, such as a retroviral vector or a lentiviral vector, can be utilized to express reprogramming factors in somatic cells. At the same time, non-integrating vectors, such as episomal plasmids, can be used to express reprogramming factors in somatic cells. See, for example, Yu et al., Science. 2009 May 8; 324(5928): 797 - 801, which is incorporated herein by reference in its entirety. When using a non-integrating vector to express reprogramming factors, somatic cells can be transfected, electroporated, or transformed with the vector to express the factors in the cells. For example, in mouse cells, expressing four factors (Oct3 / 4, SOX2, c-myc, KLF4) using an integrating viral vector is sufficient to reprogram somatic cells. In human cells, expressing four factors (Oct3 / 4, SOX2, NANOG, and Lin28) using an integrating viral vector is sufficient to reprogram somatic cells.

[0297] Once the reprogramming factors are expressed in the cell, the cell can be cultured. Over time, cells with ES characteristics appear in the culture dish. Cells can be selected and passaged based on, for example, ES morphology or the expression of selectable or detectable markers. The cells can be cultured to produce a cell culture similar to ES cells - these are putative iPS cells.

[0298] To confirm the pluripotency of iPS cells, the cells can be tested using one or more methods for testing pluripotency. For example, the expression of ES cell markers in the cells can be tested; the ability of the cells to form teratomas when transplanted into SCID mice can be evaluated; the ability of the cells to differentiate into cell types of all three germ layers can be evaluated. Once pluripotent iPSCs are obtained, they can be used to generate megakaryocytes and platelets.

[0299] As used herein, the term "prohematopoietic endothelial cells (PVE-HE)" refers to cells that can differentiate into hematopoietic cells or endothelial cell types, which can express PECAM-1, VE-cadherin, and / or Endoglin (e.g., PECAM1+VE-Cad+Endoglin+ hematopoietic PVE-HE), and optionally are derived from pluripotent stem cells. These cells can be described based on a number of structural and functional characteristics, including but not limited to the expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. PVE-HE cells are characterized by the expression of the marker CD31 (PECAM1). For example, at least about 90%, at least about 95%, or at least about 99%. Additionally, immunofluorescence and transmission electron microscopy results further demonstrate that 9% of the PVE-HE cells in the population may be CD31+. PVE-HE cells can also express the markers CD105 (endoglin) and CD144 (VE-cadherin). For example, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the PVE-HE cells in the population may be CD105+, CD144+, and CD31+. In certain embodiments, PVE-HE cells adhere loosely to each other. CD31, platelet endothelial cell adhesion molecule-1 (PECAM-1), has been used as a marker for the development of endothelial progenitor cells, angiogenesis, and vasculogenesis. CD31 is constitutively expressed on the surface of adult and embryonic endothelial cells, is a major component of endothelial cell junctions (where up to 10 6 PECAM-1 molecules are concentrated) and is weakly expressed on many peripheral leukocytes and platelets.

[0300] In an exemplary embodiment, PVE-HE may have one or more, preferably all, of the following characteristics: (1) A significant amount of CD31+ PVE-HE cells can be detected as early as 72 hours after the start of PVE-HE differentiation. (2) It will reach a peak at approximately 120 to 146 hours after the start of PVE-HE differentiation. (3) The CD31+ PVE-HE cell population can be isolated and cryopreserved. (3) They express almost all endothelial progenitor cell surface markers such as CD31, CD105 (Endoglin), CD144 (VE-cadherin). They can also express CD34, CD309 (KDR), and CD146. (4) The CD31+ PVE-HE cell subset also expresses CXCR4 (CD184). (5) Endothelial lineage capacity can be demonstrated by culturing CD31+ PVE-HE cells in fibronectin of an endothelial cell (EC)-specific medium (such as EGM-2 or EndoGro) to obtain a monolayer of cells with a typical endothelial cell morphology. (6) Endothelial cells derived from PVE-HE (PVE-HE-EC) not only express CD31 (localized at cell-cell junctions), but also express von Willebrand factor (vWF) and are capable of LDL uptake. (7) When cultured on Matrigel, PVE-HE-EC is capable of forming a three-dimensional network structure. (8) When plated at very low density on fibronectin of an EC-specific medium, CD31+ PVE-HE cells can form colonies with a typical endothelial cell morphology, confirming their colony-forming ability. (9) When grown as blast-colonies on methylcellulose medium, blast-colonies can only be generated from the CD31+ portion. Different from CD31- cells, CD34- and CD105- are capable of generating blast-colonies, suggesting that hematopoietic capacity is only maintained in the CD31 portion. (10) Newly generated PVE-HE-EC maintains hematopoietic potential and will generate hematopoietic cells if cultured under conditions favorable for the hematopoietic lineage.

[0301] As used herein, the term "megakaryocyte lineage specific progenitor cell" ("MLP") refers to a mononuclear hematopoietic stem cell that is at least committed to the megakaryocyte lineage and includes, but is not limited to, cells in cord blood, bone marrow, and peripheral blood, as well as hematopoietic stem cells, cells derived from human embryonic stem cells, and cells derived from induced pluripotent stem cells. These cells can be characterized based on a number of structural and functional properties, including, but not limited to, the expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. The MLP disclosed in the present invention can be a mixture of immature and mature cells. The percentage of mature to immature MLP may vary based on the length of time in culture in the MLP derivation and expansion medium (MLP-DEM, also referred to herein as APEL) (as described in Example 2). Immature MLP is characterized by the expression of the markers CD41a, CD31, CD34, CD13 and the lack of expression of the markers CD14 and CD42b. The exemplary methods disclosed in the present invention provide methods for detecting and / or purifying MLP, including detecting the expression of CD13 and / or enriching or purifying CD13 positive cells. Optionally, in these methods, the expression of CD13 can be detected and / or combined with one or more other markers of immature or mature MLP as described herein and used as a basis for cell purification. Mature MLP is characterized by the expression of the markers CD41a, CD31, CD34, CD13, CD42b and the lack of expression of the marker CD14. In certain embodiments, MLP generated in feeder-free culture may be semi-attached or fully detached and may float in the culture medium. For example, MLP can be collected from PVE-HE when they begin to float in suspension. Preferably, MLP is not allowed to plate and adhere, which may cause it to differentiate into lineages other than MK or non-MK lineages (such as endothelial cells). MLP is preferably cultured in suspension to produce MK. Optionally, MLP can be cryopreserved.

[0302] As used herein, the term "megakaryocyte" (MK) refers to large polyploid hematopoietic cells that produce platelets, as well as smaller MKs (which can be produced by the methods herein), which may be diploid but are fully capable of producing platelets. A major morphological feature of mature MKs is the production of a large polyploid nucleus. Mature MKs cease proliferation but continue to increase their DNA content by endomitosis; concomitantly, the cell size increases. The large polyploid nuclei, large cell volume, and abundant cytoplasm of MKs enable each cell to produce thousands of platelets. MKs can be described based on these and many other structural and functional properties, including, but not limited to, the expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. Mature MKs express the markers CD41a and CD42b. Mature MKs may also express CD61 and CD29. For example, the MKs disclosed herein preferably have the function of being able to produce platelets, such as when cultured under the conditions described herein. Exemplary embodiments provide methods for detecting mature MKs, including detecting CD29 expression and identifying CD29-positive cells as mature MKs. Additional exemplary embodiments provide methods for purifying MKs, including purifying CD29-positive cells from a cell population, such as using magnetic bead depletion, FACS, other immunoaffinity-based methods, etc. Optionally, in these methods, the expression of CD29 can be detected and / or combined with the expression of one or more other markers of mature MKs as a basis for cell purification, such as those shown in Table 1 (which provides the expression of more exemplary cell surface markers of mature MKs).

[0303] In vivo, MKs arise from precursor cells of hematopoietic stem cells in the bone marrow. These multipotent stem cells are located in the bone marrow sinusoids and are capable of producing all types of blood cells depending on the signals they receive. The major signal for MK generation is TPO. TPO induces progenitors in the bone marrow to differentiate into the final MK phenotype. The development of MKs follows the following lineage: CFU-ME (multipotent hematopoietic stem cell or hemocytoblast), megakaryoblast, promegakaryocyte, megakaryocyte. The cell ultimately reaches the megakaryoblast stage and loses the ability to divide. However, it is still able to replicate its own DNA and continue to develop into a polyploid. The cytoplasm continues to expand, and its DNA complement can increase to more than 64N.

[0304] Once the cells have completed differentiation and become mature megakaryocytes, the process of platelet production begins. TPO plays a role in inducing MKs to form small proplatelets. Platelets are retained within these internal membranes in the MK cytoplasm. Two mechanisms for platelet release have been proposed. In one scenario, these proplatelet processes burst and fragment into platelets. Alternatively, the cell may form platelet strands that enter the blood vessels. The described platelet strands are formed by pseudopodia and they are capable of continuously releasing platelets into the circulation. In either case, each of these proplatelet process ruptures can produce 2,000 - 5,000 new platelets. Overall, more than 75% of these newly produced platelets will remain in the circulation, while the remainder will be retained in the spleen.

[0305] As used herein, the term "platelet" refers to an anucleate cytoplasmic body that derives from cells involved in the major hemostatic cell mechanism leading to blood clot formation. Platelets (thrombocytes) are very small, irregularly shaped, transparent cell fragments with a diameter of 2 - 3 μm that are produced in the body by fragmentation of precursor megakaryocytes (MKs). Platelets can be identified based on a number of structural and functional characteristics, including, but not limited to, the expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. Platelets express the markers CD41a and CD42b. Platelets adhere to tissues and to each other in response to vascular injury.

[0306] As used herein, the term "functional platelet" or "platelet having functionality" refers to a platelet that can be activated by thrombin and is involved in blood clot retraction. In an exemplary embodiment, whether a platelet is a functional platelet can be determined using an animal model, for example, as described in Example 4 (Figure 12), for example by comparison with naturally occurring platelets that can be of the same species. Additionally, activated platelets can be identified by the expression of CD62p and αIIbβIII, and functional platelets can be identified (optionally quantified) by the expression of these markers upon activation, such as upon activation by thrombin. The term "substantially all platelets are functional" refers to a composition or preparation containing platelets, wherein, for example, at least 60% of the platelets are functional platelets, or at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the platelets are functional platelets. Functional platelets may be active for at least 5 days when stored at 22 - 37°C.

[0307] PECAM-1 (CD31) is a member of the immunoglobulin (Ig) superfamily and is expressed on the surface of circulating platelets, monocytes, neutrophils, and particularly subsets of T cells. It is also a major component of endothelial cell junctions, where up to approximately one million molecules are concentrated. Because of this cellular expression pattern, PECAM-1 is involved in several functions, including leukocyte transendothelial migration, angiogenesis, and integrin activation. The immunoglobulin superfamily mediates cell adhesion (e.g., NCAM1, ICAM1, and VCAM1) or antigen recognition (e.g., immunoglobulins, T cell receptors, and MHC molecules). In addition, a subgroup of 30 members has been identified, characterized by the presence of one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within their cytoplasmic domains. PECAM-1, which has six ITIMs within its cytoplasmic domain, is a member of this subfamily.

[0308] Endoglin (ENG), also known as CD105, is a homodimeric membrane glycoprotein that is mainly associated with human vascular endothelium. It has also been found on bone marrow proerythroblasts in childhood leukemia, activated monocytes, fibroblasts, smooth muscle cells, and lymphoblasts. Endoglin is a component of the transforming growth factor β (TGFB) receptor complex and binds TGFB1 with high affinity. Endoglin is involved in cytoskeletal organization, which affects cell shape and migration, and processes such as cardiovascular system development and blood vessel remodeling. Its expression is regulated during heart development. Experimental mice without the Endoglin gene die of cardiovascular abnormalities.

[0309] VE-cadherin (CD144) is a classical cadherin from the cadherin superfamily. VE-cadherin plays an important role in endothelial cell biology by controlling co-adhesion and the organization of cell-cell junctions, thereby maintaining endothelial integrity. VE-cadherin is essential for normal blood vessel development. Studies in transgenic mouse models have confirmed that the lack of VE-cadherin is embryonically lethal due to vascular defects. VE-cadherin serves to maintain newly formed blood vessels.

[0310] As used herein, the term "ROCK inhibitor" refers to any substance that inhibits or reduces the function of Rho-associated kinase or its signaling pathway within a cell, such as small molecules, siRNA, miRNA, antisense RNA, and the like. As used herein, "ROCK signaling pathway" may include any signal processor involved in a ROCK-related signaling pathway, such as the intracellular Rho-ROCK-Myosin II signaling pathway, its upstream signaling pathway, or its downstream signaling pathway. An exemplary ROCK inhibitor that may be used is Stemgent's Stemolecule Y27632, a rho-associated protein kinase (ROCK) inhibitor (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):681-6). Other ROCK inhibitors include, for example, H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, and SB-772077-B. Doe et al., J. Pharmacol. Exp. Ther., 32:89-98, 2007; Ishizaki, et al., Mol. Pharmacol., 57:976-983, 2000; Nakajima et al., Cancer Chemother. Pharmacol., 52:319-324, 2003; and Sasaki et al., Pharmacol. Ther., 93:225-232, 2002, each of which is incorporated herein by reference in its entirety. ROCK inhibitors may be used at concentrations and / or culture conditions known in the prior art, such as, for example, as described in US PGPub No. 2012 / 0276063, which is incorporated herein by reference in its entirety. For example, the concentration of a ROCK inhibitor may be from about 0.05 to about 50 μM, such as, for example, at least or about 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μM, including any range derivable therein, or any concentration that effectively promotes cell growth or survival.

[0311] For example, the survival of pluripotent stem cells can be improved by adding a ROCK inhibitor. In one specific embodiment, pluripotent stem cells can be maintained under feeder-free conditions. In another embodiment, the survival of megakaryocyte lineage-specific progenitor cells can be improved by adding a ROCK inhibitor. In another embodiment, the survival of megakaryocytes can be improved by adding a ROCK inhibitor. In another specific embodiment, megakaryocyte lineage-specific progenitor cells can be maintained under feeder-free conditions.

[0312] Abbreviations

[0313] iPS: Induced pluripotent

[0314] iPSC: Induced pluripotent stem cell

[0315] hiPSC: Induced human pluripotent stem cell

[0316] hES: Human embryonic stem

[0317] hESC: Human embryonic stem cell

[0318] MK: Megakaryocyte

[0319] MLP: Megakaryocyte lineage-specific progenitor cell, also known as megakaryocyte progenitor cell (MKP)

[0320] PVE-HE: Hemogenic endothelial cell, which is optionally PECAM1+VE-cadherin+Endoglin+ and optionally derived from pluripotent stem cells

[0321] iPS-PVE-HE: iPS cell-derived hemogenic endothelial cell (such as PECAM1+VE-cadherin+Endoglin+ cell)

[0322] hES-PVE-HE: hES cell-derived hemogenic endothelial cell (such as PECAM1+VE-cadherin+Endoglin+ cell)

[0323] PVE-HE-MLP: Megakaryocyte lineage-specific progenitor cell prepared from hemogenic endothelial cell

[0324] iPS-PVE-HE-MLP: PVE-HE-MLP prepared from iPS cells

[0325] hES-PVE-HE-MLP: PVE-HE-MLP prepared from hES cells

[0326] PVE-HE-MLP-MK: Megakaryocyte prepared from megakaryocyte lineage-specific progenitor cell, said progenitor cell being prepared from hemogenic endothelial cell

[0327] iPS-PVE-HE-MLP-MK: PVE-HE-MLP-MK prepared from iPS cells

[0328] hES-PVE-HE-MLP-MK: PVE-HE-MLP-MK prepared from hES cells

[0329] PLT: Platelet

[0330] hiPSC-PLT: Platelets or platelet-like particles prepared from induced human pluripotent stem cells

[0331] hESC-PLT: Platelets or platelet-like particles prepared from human embryonic stem cells

[0332] ADM: Highly differentiated morphology.

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[0394] All publications are hereby incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not admitted that any of the information provided herein is prior art with respect to the present invention, or that any publication incorporated by reference herein is prior art, whether explicitly or implicitly. Examples

[0395] The following examples are provided to better illustrate the present invention and should not be construed as limiting the scope of the present invention. The ranges of specific substances mentioned are for illustrative purposes only and are not intended to limit the present invention. Equivalent methods or reactants can be developed by those skilled in the art without creative effort and without departing from the scope of the present invention.

[0396] Example 1 - Generation of pluripotent-derived hematopoietic endothelial cells (PVE-HE).

[0397] Generation of pluripotent hematopoietic endothelial cells (PVE-HE) from induced pluripotent stem cells (iPS cells).

[0398] First, expand and culture iPS cells on Matrigel (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) in feeder-free pluripotent stem cell medium mTeSR1. Briefly, harvest human iPS cells by dissociating them using a chemically defined cell dissociation buffer (CDB) with EDTA as the sole active ingredient. No enzymes or any other animal products are used in the medium or CDB. It should be understandable to researchers with ordinary skills in the art that other chemically defined matrices, such as recombinant vitronectin or SyntheMax II, or media, such as mTeSR2, or other compatible cell dissociation reagents may be used.

[0399] Second, prepare to differentiate the harvested human iPS cells into pluripotent PVE-HE that are PECAM1+VE-cadherin+Endoglin+. No embryoid bodies (EBs) need to be formed. Briefly, resuspend the harvested cells in mTeSR1 and plate the cells onto the extracellular matrix human collagen IV (Advanced BioMatrix, cat#5022). Add 10 μM of the small molecule ROCK inhibitor Y27632 to the culture, which is thought to help the harvested iPS cells adhere to the collagen IV-coated surface. Incubate the iPS cells at 37 °C and 5% CO2 for 12 - 48 hours for adhesion. As Figure 2A shown, under feeder-free conditions, the adhered cells showed a typical pluripotent stem cell morphology after 48 hours.

[0400] Third, differentiate the prepared human iPS cells into PVE-HE cells. Briefly, remove the mTeSR1 medium containing Y27632 and add the differentiation initiation medium (DIM). DIM is an animal component-free medium (ACF) composed of Iscove’s Modified Dulbecco’s Medium (IMDM) as the basal medium, human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, 50 ng / ml bone morphogenetic protein 4 (BMP4), 50 ng / ml basic fibroblast growth factor (bFGF), and 50 ng / ml vascular endothelial growth factor (VEGF). After incubating at 37 °C and 5% CO2 for 48 hours, the early morphology of the desired PVE-HE differentiation was observed. In Figure 2B it can be seen that the transformation from pluripotent stem cells to dispersed small cell clusters is basically completed. After 96 - 146 hours of the start of PVE-HE differentiation, the late differentiation morphology showing small, tightly packed cell clusters growing on top of a single cell layer was observed ( Figure 2C ).

[0401] Fourth, 120 hours after the initiation of PVE-HE differentiation, morphological changes ( Figure 3B ) of late differentiated morphology (ADM) cells and successful PVE-HE differentiation were analyzed. Briefly, flow cytometry analysis of lineage-specific markers CD31 (PECAM), CD105 (endoglin), and CD144 (VE-cadherin) was performed on a small cell sample. At this differentiation stage, ADM cells showed the PVE-HE phenotype CD31 + CD144 + CD105 + ( Figure 3A ).

[0402] Example 2 - Generation of non-adherent megakaryocyte lineage-specific progenitors (MLP) from human iPS-derived PVE-HE cells

[0403] First, MLP differentiation was initiated 120 hours after the initiation of PVE-HE differentiation. Briefly, the medium containing 50 ng / ml BMP4, 50 ng / ml bFGF, and 50 ng / ml VEGF was removed and replaced with MLP-derived and expanded medium (MLP-DEM, also known as APEL or Stemline II, as Figure 1 shown), which consists of Iscove’s Modified Dulbecco’s Medium (IMDM), Ham’s F-12 nutrient mixture, Albucult (recombinant human albumin), polyvinyl alcohol (PVA), linoleic acid, SyntheChol (synthetic cholesterol), alpha-mercaptoethanol (α-MTG), recombinant human insulin-transferrin-selenium-ethanolamine solution, protein-free hybridoma medium II (PFHMII), ascorbic acid 2-phosphate, Glutamax I (L-alanyl-L-glutamine), penicillin / streptomycin, 25 ng / ml stem cell factor (SCF), 25 ng / ml thrombopoietin (TPO), 25 ng / ml Fms-related tyrosine kinase 3 ligand (FL), 10 ng / ml interleukin-3 (IL-3), 10 ng / ml interleukin-6 (IL-6), and 5 units / ml heparin. The cells were then incubated at 37 °C and 5% CO2 for up to 8 days. Maintain MLP differentiation for 8 days.

[0404]

[0405] Table 1 shows the flow cytometry comparative analysis of marker expression of mature MLP and mature MK cells. This analysis characterized the phenotype of PVE-HE-derived MLP cells before cryopreservation and their subsequent differentiation into MK cells with a defined phenotype.

[0406] Second, use a serum pipette to gently rinse and collect the floating and semi-detached MLP above the adherent cell population. Transfer the medium containing MLP to a conical tube, centrifuge at 300 x g for 5 minutes to collect the MLP. Discard the medium, resuspend the MLP pellet in phosphate-buffered saline for morphological analysis (Figure 5) and flow cytometry analysis using the markers shown in Table 1. Selected results are shown in Figure 4 . The results confirmed that the MLP collected at this stage mainly (more than 90%) consisted of two populations: one characterized by a relatively immature MLP population, showing CD41a + CD31 + CD34 + CD14 - CD13 + CD42b - , and a relatively mature MLP population, showing CD41a + CD31 + CD34 + CD14 - CD13 + CD42b + .

[0407] Third, cryopreserve the MLP. Briefly, cryopreservation of the MLP was performed using cell freezing medium CS10 (Sigma) containing 10% DMSO.

[0408] Example 3 - Preparation of mature platelets from megakaryocytes (MK) derived from human iPS-PVE-HE-MLP

[0409] First, as described above, initiate platelet differentiation using human iPS-PVE-HE-derived MLP cells. Seed the MLP onto the non-adherent surface of MK medium (MK-M), which consists of Iscove’s Modified Dulbecco’s Medium (IMDM) as the basal medium, human serum albumin, iron-saturated transferrin, insulin, β-mercaptoethanol, soluble low density lipoprotein (LDL), cholesterol, 30 ng / ml of TPO, 1 ng / ml of SCF, 7.5 ng / ml of IL-6, 13.5 ng / ml of IL-9, 5 μM of Y27632, and 5 - 25 units / ml of heparin. Then incubate the cells at 37 °C and 5% CO2 for 3 days.

[0410] In some cases, MLP was seeded onto the non-adherent surface of the culture medium, which consisted of Iscove’s Modified Dulbecco’s Medium (IMDM), Ham’s F-12 nutrient mixture, Albucult (recombinant human albumin), polyvinyl alcohol (PVA), linoleic acid, linolenic acid, SyntheChol (synthetic cholesterol), alpha-mercaptoethanol (α-MTG), recombinant human insulin-transferrin-selenium-ethanolamine solution, protein-free hybridoma medium II (PFHMII), ascorbic acid 2-phosphate, Glutamax I (L-alanyl-L-glutamine), penicillin / streptomycin, 30 ng / ml of TPO, 1 ng / ml of SCF, 7.5 ng / ml of IL-6, 13.5 ng / ml of IL-9, 5 μM of Y27632, and 5-25 units / ml of heparin. The cells were then incubated at 37 °C and 5% CO2 for 3 days.

[0411] Second, the mature MK cells derived from iPS-PVE-HE-MLP were analyzed. At 72 hours after the initiation of platelet differentiation, as maturation progressed, very large polyploid MKs (50 μm) became very abundant ( Figure 5A & The scale bar in 5A is 100 μm, and N in 5B indicates the nucleus within the MK). Between 72-96 hours, proplatelet-forming cells with slender pseudopodia were readily visible under the microscope. ( Figure 5A & 5C, as indicated by the arrow), and the appearance of CD41a+CD42b+ platelet granules was detected by flow cytometry analysis (Figure 6). In Figure 6, the cells in "A" are circulating human platelets, the cells in "B" are hES-PVE-HE-MLP, the cells in "C" are human platelets derived from peripheral blood, the cells in "D" are iPS-PVE-HE-MLP, and the cells in "E" are hES-PVE-HE-MLP). At 84 hours after initiation, the amount of CD41a+CD42b+ platelets increased significantly, reaching a level as high as 70% ( Figure 6D ).

[0412] Third, without damaging the mature MKs in the culture, collect high-quality platelets for at least 5 consecutive days or possibly longer to increase the platelet yield from MLP by at least 3 - 5 times. To isolate large MKs from the platelet-containing medium, centrifuge the cell suspension at 50 x g for 10 minutes, remove the supernatant, and resuspend the MK cell pellet in fresh MK-M to enable them to produce more platelets later. To separate platelets from proplatelets, large cell debris, and small MKs, use the BSA / HAS gradient precipitation method to obtain a more pure platelet population from the 50 x g centrifugation supernatant. The purified platelets are suspended in MK-M and kept at room temperature. Identify the quality of platelets regarding granularity, transparency, size, and cell surface marker expression by FACS analysis and comparison with human platelets from peripheral blood.( Figure 6A -E). Store the purified platelets at room temperature in MK-M medium within a non-adhesive surface to ensure minimal loss of functional platelets.

[0413] Example 4 - Analysis of mature platelets from megakaryocytes (MK) derived from iPS-PVE-HE-MLP

[0414] First, analyze the morphology of iPS-PVE-HE-MLP-derived platelets (hiPSC-PLT). Perform immunofluorescence and transmission electron microscopy analyses according to the previously described method (Cell Research 2011 21:530-45). It was found that hiPSC-PLTs are discoid and the ultrastructure is basically the same as that of circulating human PLTs (as confirmed by transmission electron microscopy, Figure 7 ). As confirmed by DIC and β1-tubulin IF microscopy, the average size of hiPSC-PLTs is similar to that of circulating human PLTs (2.38 μm ± 0.85 μm vs. 2.27 μm ± 0.49 μm) ( Figure 8 ). hiPSC-PLTs spread on glass - forming filopodia and lamellipodia (as confirmed by DIC live cell microscopy imaging shown in Figure 9 ). hiPSC-PLTs have no nuclei, similar to circulating human PLTs (as confirmed by Hoechst staining, Figure 8 ). It was found that hiPSC-PLTs have a normal tubulin cytoskeleton relative to circulating human PLTs (as confirmed by β1-tubulin labeling, Figure 8 ). It was found that hiPSC-PLTs have normal filamentous actin relative to circulating human PLTs (as confirmed by phalloidin labeling, Figure 8 ). It was found that hiPSC-PLTs have normal α-granule expression relative to circulating human PLTs (as confirmed by TSP4 and PF4 labeling in Figure 10A &10B).

[0415] Second, the functionality of hiPSC-PLTs was analyzed by measuring the expression of cell adhesion molecules on activated platelets through in vitro activation assays. Briefly, two adhesion molecules were analyzed using anti-CD62p and PAC-1 antibodies. PAC-1 recognizes αIIbβIII integrin. Both CD62p and αIIbβIII are expressed on the surface of activated platelets. The experiments were conducted according to the method described previously (Cell Research 2011 21:530-45). Upon thrombin stimulation, the binding of both PAC-1 and P-selectin increased( Figure 11 ).

[0416] Third, the functionality of hiPSC-PLTs was analyzed by measuring thrombus formation in macrophage-depleted NOD / SCID mice using an in vivo assay system. Briefly, cremaster arteriole intravital microscopy was performed as described previously (Cell Research 2011 21:530-45). Macrophage-depleted NOD / SCID mice were anesthetized by intraperitoneal injection of ketamine (125 mg / kg) and xylazine (25 mg / kg). An endotracheal tube was inserted and the mice were placed on a temperature-controlled blanket. After scrotal incision, the cremaster muscle was removed from the abdomen and placed on an intravital microscopy stage. Throughout the experiment, the muscle preparation was perfused with temperature-controlled (37 °C) and gassed (95% N2, 5% CO2) bicarbonate-buffered saline. The wall of the cremaster arteriole was damaged by microspot laser ablation using a microspot laser system (Photonics Instruments). The formation of developing murine platelet thrombi was visualized by injecting Dylight 649-conjugated anti-mouse CD42c antibody (Emfret Analytics, 0.05 μg / g body weight) through the jugular vein annulus. 3x10 6 calcein AM-labeled hPLTs, iPSC-PLTs, and ESC-PLTs with or without ReoPro were infused into the mice at 100 x g. Two to four thrombi were formed in one mouse. Fluorescent and bright-field images were recorded using an Olympus BX61W microscope with a 60x / 1.0 NA water immersion objective and a high-speed camera (Hamamatsu C9300) through an intensifier (Video Scope International). Data were collected 5 minutes after vessel wall injury and analyzed using Slidebook v5.0 (Intelligent Imaging Innovations). Figure 12AThe results shown indicate that hiPSC-PLTs contribute to clot formation in the in vivo environment. It was also determined that the functionality of hiPSC-PLTs is mediated by αIIbβIII. Specifically, hiPSC-PLTs were pretreated with ReoPro, which is the Fab fragment of a human-mouse chimeric monoclonal antibody that specifically binds to αIIbβIII and inhibits platelet function( Figure 12B ). Asterisks indicate statistically significant differences relative to controls not treated with ReoPRO (p < 0.01 relative to controls, Student's t-test).

[0417] Fourth, the kinetics of hiPSC-PLT infusion in macrophage-depleted NOD / SCID mice were determined. To determine whether the kinetics of hiPSC-PLTs and ESC-PLTs in vivo are similar to those of hPLTs, iPSC- or ESC-PLTs were infused into macrophage-depleted NOD / SCID mice, and blood collected at various time points was analyzed by flow cytometry. Briefly, macrophages were depleted by intravenous injection of liposome-encapsulated clodronate as previously described. Clodronate liposomes were injected into mice via the tail vein on day 0 (100 μl) and day 2 (50 μl). On day 3, platelet-rich human plasma was obtained by centrifuging citrate-treated blood at 200 x g for 25 minutes, and then at 800 x g for 10 minutes in the presence of 0.5 μM PGE1 and 10% citrate buffer. The pellet was resuspended in HEPES-Tyrode buffer containing 0.5 μM PGE1 and 10% citrate buffer and centrifuged at 800 x g for 5 minutes. Platelets were resuspended in HEPES-Tyrode buffer containing 0.1% fatty acid-free BSA. Isolated human platelets (hPLT, 1.5 x 10 7 ), induced pluripotent stem cell-derived platelets of the present disclosure (hiPSC-PLT, 1.5 x 10 7 ), and embryonic stem cell-derived platelets of the present disclosure (ESC-PLT, 1.0 x 10 7 ) were intravenously infused into macrophage-depleted mice. At different time points (10, 30, 60, 120, 240, 360, and 480 minutes), 30 μl of mouse blood was collected via the jugular vein and analyzed by flow cytometry using APC-conjugated anti-human CD41 and Dylight 488-conjugated anti-mouse CD42c antibodies. The results confirmed that hiPSC-PLTs circulated for several hours in macrophage-depleted NOD / SCID mice( Figure 13 ).

[0418] Example 5. Preparation of platelets from pluripotent cells

[0419] This example provides a further exemplary method for preparing platelets from pluripotent stem cells.

[0420] As described above, hESC-PLT and hiPSC-PLT are human platelets prepared from human embryonic and induced pluripotent stem cells. The initial in vitro and in vivo characterizations described above have confirmed that hESC-PLT and hiPSC-PLT are morphologically and functionally similar to human donor platelets. For example, hiPSC-PLT can spread on a glass substrate.

[0421] The preparation of a large amount of intermediate product (MK progenitor cells) starts from thawing a sample vial from an approved hiPSC or hESC master cell bank (MCB). The process flow diagram for preparing a large amount of intermediate product is shown in Figure 14A -E. A cryotube containing the master or working cell bank of 1-2 million hiPSC or hESC is removed from the cGMP liquid nitrogen storage vapor phase and transferred to a clean room (ISO class 7). All operations are performed in a certified biosafety cabinet (ISO class 5 BSC). After thawing, DMSO is removed by washing; the cells are seeded into the defined medium of mTeSR1 in a Matrigel-coated vessel. Care is taken to keep the cells as aggregates. The culture plates are labeled with the date, batch number, and passage number, and the lid of each well is labeled with a unique number (e.g., 1-6). The seeded culture plates are placed in an incubator at 37 °C and 5% CO2. The cultures are observed daily using a stereomicroscope and an inverted microscope. The observations on colony size, cell morphology, including the degree of differentiation and the color of the medium, are recorded in a working table. Usually, the mTeSR1 medium is changed every 1-2 days until the cultures reach 60-90% confluence. When the morphological and confluence evaluations indicate that the cultures require cell passage, the cell colonies are harvested using a cell dissociation buffer. Again, care is taken to keep the cells as aggregates. The cultures are re-seeded into the medium of mTeSR1 in a Matrigel-coated container. Based on the number of harvested and 2 seeded cells per cm 2 and per cm

[0422] Three days after seeding for hematopoietic cell differentiation, the cultures are examined to determine cell attachment and outgrowth. The cultures at this stage usually have a low density of attached cells, covering less than 10% of the total surface area. Well-attached cells should show outgrowth with a significant transition from the pluripotent stem cell morphology to that of differentiated cells: larger cells, significantly more cytoplasm relative to the nuclear size, and a more diffuse growth without distinct colony boundaries.

[0423] When it is estimated that the stem cell expansion phase has met the cell yield requirement, initiate hematopoietic differentiation. Discard one representative container, harvest and prepare a single cell suspension. Quantify the cell concentration to establish culture seeding parameters and discard the remaining cells. Carefully harvest the colonies using a cell dissociation buffer and re-seed them at a density of 5,000 cells / cm 2 into mTeSR1 medium supplemented with 10 μM Y27632 (ROCK inhibitor) in a container coated with type IV collagen. Place the seeded container in an incubator at 37 °C and 5% CO2.

[0424] On the next day, remove the medium, carefully removing as few floating cell clusters as possible, and replace it with StemSpan ACF (StemCell Technologies Inc.) supplemented with 50 ng / ml recombinant human (rh) BMP-4, 50 ng / ml rh VEGF, and 50 ng / ml rh bFGF. Transfer the culture to an environment with low O2 (∼5%), 37 °C, and 5% CO2 and leave it undisturbed for 2 days. After 2 days, perform a morphological evaluation of cell attachment and protrusions in the culture. Remove the medium, carefully removing as few floating cell clusters as possible, and replace it with fresh medium. Return the culture to the low O2 (∼5%), 37 °C, and 5% CO2 environment for another 2 days. After this period, replace the medium again and place the culture under normoxic culture conditions. After 2 days of normoxic culture, remove the medium, carefully removing as few floating cell clusters as possible, and replace it with MLP medium containing STEMdiff APEL (StemCell Technologies Inc.) and supplemented with 5 units / ml heparin, 25 ng / ml rh TPO, 25 ng / ml rhSCF, 25 ng / ml rh FL, rh IL-6, and rh IL-3. Maintain these conditions for the next 2 - 6 days. Perform a morphological evaluation of the culture daily to determine the quality of floating MK progenitors. Do not replace the medium, but if the culture starts to show depleted medium (the medium appears yellow), add additional medium. Sample the culture regularly and measure the expression of CD41a and CD42b by FACS. When the culture morphology and the expression of CD41a and CD42b are appropriate (∼≥15% double positive), harvest the culture and then cryopreserve the cells at 3 - 5 million viable MLP / mL / vial in cryopreservation medium containing 10% dimethyl sulfoxide and 90% fetal bovine serum (Hyclone). Perform cryopreservation as follows: Place the cryovials in a freezer (Nalgene) and then store them in an -80 °C refrigerator for 1 - 3 days, and then transfer them to the vapor phase of a cGMP liquid nitrogen storage system.

[0425] Thaw the approved bulk intermediate to initiate the process for the preparation of the final product hESC-PLT or hiPSC-PLT. The cryotube containing 3 - 5 million MLP that has passed the bulk intermediate quality test is removed from the cGMP liquid nitrogen storage vapor phase and transferred to the clean room. At approximately 2.3x10 5 / cm 2 The cells are seeded into an ultra-low attachment (ULA) vessel in StemSpan ACF medium supplemented with 30 ng / ml rh TPO, 1 ng / ml rh SCF, 7.5 ng / ml rh IL-6, 13.5 ng / ml rh IL-9, and 5 μM Y27632 (ROCK inhibitor). The culture vessels are labeled with the date and batch number, and a unique number (e.g., 1 - 6) is marked on the lid of each vessel. The cultures are maintained in a humidified 10% CO2 atmosphere at 39°C. Usually, there is no obvious cell expansion during this culture stage. After culturing for approximately 3 to 4 days, larger mature megakaryocytes (MK) will start to appear. Samples are taken regularly to monitor the cultures and FACS analysis for CD41a and CD42b expression is performed. Proplatelet formation is usually first observed after 5 days of culture. As proplatelet formation progresses and CD41a and CD42b expression increases to approximately 30% - 70%, the cultures can be harvested (days 6 - 7).

[0426] The culture medium is harvested by centrifugation at 50 x g to remove the megakaryocyte slurry. The supernatant is removed and the platelets are concentrated by centrifugation at 1000 x g. The platelets are resuspended and then loaded onto a discontinuous albumin (human) (HSA) gradient (12%, 10%, 7%, 5%, and 2%) for further platelet separation. The platelet HSA gradient is centrifuged at 80 x g for 15 minutes. The platelets are harvested from the gradient and PGE1 is added to the suspension to prevent activation. The platelets are concentrated by centrifugation at 1000 g for 10 minutes. Currently, hESC and hiPSC platelets are stored in the medium. For the planned research purposes, the stability of the target product will be studied for 48 - 72 hours. Preliminary studies have shown that PAC1 binding results before and after storage determine a minimum stability of 24 hours.

[0427] FACS analysis (CD31 and CD43) of the MLP cell population in the bulk intermediate has shown that approximately 98% of the cells have formed the hematopoietic endothelial cell line or the hematopoietic lineage and have thus differentiated beyond pluripotency.

[0428] At a more downstream stage of the process, the differentiation of the prepared MK and the cells present during the PLT harvest stage have been tested by the expression of vWF (von Willebrand factor). Approximately 100% of the cells at this preparation stage are vWF+. The maintenance of a highly differentiated cell population indicates that the culture has not undergone clonal expansion of undifferentiated progenitor cells.

[0429] Preliminary studies have shown that hiPS-derived MK cell populations are completely devoid of pluripotent cells as analyzed by IFA staining for OCT4, NANOG, TRA-1-60, TRA-1-81, SSE3, SSE4, and alkaline phosphatase. Further studies using IFA staining for pluripotency markers will examine hES-derived and hiPS-derived MLP and MK, as well as purified PLT populations derived from MK, to screen for the presence of pluripotent cells. Spiking studies will be performed to determine the LOD of this assay for detecting stem cells in various cell populations.

[0430] Preliminary studies have been performed to characterize the MK population by FACS analysis of pluripotency markers (SSEA4 and TRA-1-60). Spiking studies have been performed in which hES cells at 1% and 0.1% concentrations were mixed with MK. The results showed that the limit of detection was 0.1%. Initial studies of SSEA4 and TRA-1-60 to characterize MK showed <0.1% SSEA4 / TRA-1-60 positive cells in the MK population (below the level of detection of this assay).

[0431] Referring to step 2-3 of FIG. 14, accurate cell counting cannot be performed because stem cells harvested with dissociation buffer form cell clumps. For this reason, trypsin-EDTA is used to harvest representative culture vessels to ensure formation of a single cell suspension. This suspension is counted in a hemocytometer, and the number of harvested cells is normalized per cm 2 Then the cells used for counting are discarded. Other culture products are harvested with dissociation buffer and the cell yield is calculated based on and multiplied by the normalized cell number per cm 2 The calculated cell yield is used to set the cell density per cm 2 required to seed vessels when inducing hematopoietic cell differentiation. 2 .

[0432] Referring to Figure 14, step 10, starting from day 2 to day 6 in MLP medium, cell sampling was performed from representative culture vessels, combined, and the percentage of CD41a and CD42b double-positive cells was determined by FACS analysis. CD41a is a subunit of the fibrinogen receptor (αIIbβIII), and CD42b is a subunit of the von Willebrand factor receptor (GPIb-V-IX). The expression of both receptors is MK lineage-specific and both are essential for platelet function. Early lineage hematopoietic endothelial cells are CD41a-negative and express CD41a during the late hematopoietic differentiation of hematopoietic progenitor cells. CD42b is only expressed in mature MK. At this point, the culture is heterogeneous, with a high percentage of CD41+ cells and a low percentage of CD42+ cells (Pineault, et. Al., Megakaryocyte and platelet production from human cord blood stem cells. Methods Mol Biol. 2012;788:219-47).

[0433] Sample preparation and FACS analysis were performed as follows. Briefly, floating cells were collected and combined. A gentle stream of growth medium was used on the surface of the culture using a serological pipette to dislodge any loosely adherent MLP and combine it with the free-floating cells. A well-suspended cell sample (100 - 200 μL) of the combined cells was collected and centrifuged (160 x g, 5 minutes). The cell pellet was resuspended in DPBS, recentrifuged, and resuspended in DPBS supplemented with 3% FBS (FACS buffer) containing CD41a-APC-conjugated (allophycocyanin) and CD42b-PE-conjugated (phycoerythrin) (BD Bioscience, San Jose, CA). Fluorescent-conjugated antibodies and appropriate isotype controls (mouse IgG1k-APC and mouse IgG1k-PE) were present for 15 minutes at room temperature. The labeled cells were then diluted into FACS buffer, centrifuged, and resuspended in FACS buffer. FACS analysis was performed, monitoring 10,000 events. Cultures with an acceptable percentage of double-positive (CD41a+ CD42b+) MLP cells were harvested and cryopreserved. The tentative minimum specification is 10% double-positive. Figure 15 Shown is a representative two-dimensional dot plot of MLP derived from hiPSC. In this plot, the cell population is 86.4% CD41a+; 31.2% CD42b+, and 29.9% of the population stains double-positive.

[0434] Prior to cryopreservation at harvest, the approximate percentage of adherent cells and the degree of differentiated large cells with a low nuclear to cytoplasmic ratio in the MLP culture are determined. The adherent cells should present as dispersed colonies without clear colony boundaries. There should be a large number of floating MLP resting on top of the adherent cell population. The live floating MLP should appear clear, with minimal birefringence, and be demarcated by a smooth cell membrane. Typically, a surface area of 1 cm 2 can generate 5,000 to 15,000 MLP per day. Figure 16 Shows a micrograph (Hoffman Modulation Contrast, x 400) of a representative MLP population derived from the hiPSC cell line MA-iPS-01.

[0435] Prior to cryopreserving the MLP, viable cell counts are performed using a hemocytometer by the trypan blue exclusion method. Cells are evaluated based on the viable cell number and percentage viability. Representative sample vials are tested for mycoplasma and sterility.

[0436] In addition, the CD31 and CD43 expression of cryopreserved MLP in representative cryovials was determined as described below. CD31 is a hematopoietic endothelial cell marker and is expressed on both the endothelial cell lineage and the hematopoietic cell lineage. Expression of CD43 confirms hematopoietic function. The cryopreserved MLP sample vials are rinsed and thawed, resuspended in DPBS supplemented with 3% FBS (FACS buffer), and centrifuged (160 x g for 5 minutes). The cell pellet is resuspended in DPBS supplemented with 3% FBS (FACS buffer) containing CD31-APC-conjugated (allophycocyanin) and CD43-FITC-conjugated (phycoerythrin) (BD Bioscience, San Jose, CA). The thawed MLP samples are incubated with the fluorescent-conjugated antibodies and appropriate isotype controls (mouse IgG-APC and mouse IgG-FITC) for 15 minutes at room temperature. The labeled cells are then diluted into FACS buffer, centrifuged, and resuspended in FACS buffer. FACS analysis is performed, monitoring 10,000 events. An acceptable MLP bank has >= 50% CD31 positive cells and >= 50% CD43 positive cells. Figure 17 Shown is a representative two-dimensional dot plot of MLP derived from the hiPSC line MA-iPS-01. In the shown embodiment, the cell population is 99.8% CD31+; 98.5% CD43+, and 98.4% of the population is stained double positive.

[0437] In addition, FACS analysis of cryopreserved MLP in representative cryovials was performed using pluripotent markers such as SSEA4, TRA-1-60, confirming the absence of pluripotent cells.

[0438] Thaw the cryotube of the cryopreserved MLP sample, and measure its post-thaw viability and recovery. The MLP is further processed to the pre-platelet formation point, and MK morphology determination and FACS analysis of CD41a+ and CD42b+ are performed during MK maturation. Monitor pre-platelet formation and platelet formation in the culture, and FACS analysis to identify double-stained (CD41a+ and CD42b+) cells. Acceptable criteria include: sterility (<USP 21> immersion test negative), mycoplasma negative (tested by direct (agar & broth) test or indirect test (cell culture)), at least 90% CD31 and CD43 positive in FACS, at least 70% viability by trypan blue staining, and negative expression of pluripotent cell markers.

[0439] Referring to Figure 14, Steps 13 - 14, 2 - 3 days after thawing, determine the appearance of MK lineage free-floating cells in the culture inoculated with MLP. At this time, the cells are heterogeneous and range in size from 10 - 50 microns in diameter. Determine the approximate proportion of live cells by visual observation using an inverted optical microscope for healthy MK precursor cells and MKs showing bright cytoplasm and smooth cell membranes. Figure 18 Micrographs (Hoffman Modulation Contrast, x 400) showing a representative MK population derived from hESC are presented.

[0440] Referring to Figure 14, Steps 13 - 14, 2 - 3 days after MLP thawing, take cell samples from representative culture vessels, pool them, process them, label them with fluorescently conjugated antibodies against CD41a and CD41b, and perform FACS analysis. MLP cells expressing CD41a+ and CD42b+ with an acceptable percentage are further processed (preferably at least 10% double-positive cells).

[0441] Referring to Figure 14, Step 15, from 3 days to 5 days after MLP thawing, determine the appearance of pre-platelets in the MK culture. Figure 19 The arrows in [Figure 14] indicate pre-platelet morphology. MKs with pre-platelets exhibit long protrusions extending from the cell, showing some beading and branching, and then fragmentation.

[0442] Referring to Figure 14, Steps 15-16, once the appearance of preplatelets is confirmed, starting 3 days after MLP thawing and ending at 8 days, preplatelet and platelet samples are collected from representative cultures. Briefly, using a 10 mL serum pipette, the samples are transferred to 50 mL conical tubes and pipetted up and down at least 5 times to generate shear forces sufficient to break the preplatelets. The samples are returned to a 39 °C incubator filled with 10% CO2 and allowed to stand for 30 minutes. Approximately 250 μL of the supernatant containing suspended platelets is stained and subjected to FACS analysis for CD41a and CD42b. When the platelet peak is detected, typically when 30-70% of the platelets are double positive stained (CD41a+CD42b+), harvesting and subsequent processing of platelets are initiated.

[0443] The pH, platelet count, and identity (determined by the expression of CD61, CD41a, CD42b), the expression of platelet activation markers (CD62P, PAC1, platelet factor 4), physiological responses (aggregation (microplate method), TEG (thromboelastography), and spreading) of hiPSC-PLT and hESC-PLT were identified and their morphology was evaluated by electron microscopy and DIC microscopy with β1-tubulin staining.

[0444] The sterility of the cells (<USP 21> immersion test result negative), endotoxin (gel method USP <85> less than 5 EU / ml), mycoplasma (negative for European Pharmacopoeia test and US Pharmacopoeia test), identity (by FACS, at least 70% CD41a+, CD42b+), PLT count / mpPLT (FACS analysis of CD61), morphology (DIC microscopy with β1-tubulin staining), and PAC1 binding potency (activated) were further examined.

[0445] Purified platelets were stained with fluorescently conjugated antibodies against CD41a and CD41b and then subjected to FACS analysis as described above. Shown below is a representative two-dimensional dot plot of PLT derived from iPS-01. In Figure 20 the example shown, the population is 81.8% CD41a+; 69.2% CD42b+ and 68.2% of the population is double positive stained.

[0446] Binding Figure 20CD41a+ and CD42b+ FACS was performed in [the medium]. During CD staining, propidium iodide (PI) was added to the sample to determine viability. As previously described, FACS analysis was performed, counting 10,000 events, and additional events were collected in the fluidic path 3 to detect PI-positive events (inactive platelets). The analysis data gave the percentage of CD41a+ / CD41b+ platelets and the percentage of viability (total platelets detected - PI+ platelets / total platelets detected).

[0447] For each batch of the final platelet product, CD61-positive events were detected by flow cytometry analysis to determine the number of platelets and the number of platelet microparticles. This determination used a known number of fluorescent beads to standardize the data to obtain the absolute numbers of PLTs / μL and mpPLT / μL. Briefly, 5 - 10 μL of the platelet sample was diluted into 0.9% saline and aliquoted into each of two tubes:

[0448] 1) One TruCount (BD Cat#340334) tube containing a lyophilized pellet with a known number of fluorescent beads and phycoerythrin-conjugated anti-CD61 IgG, with a final reaction volume of 60 μL / tube, and

[0449] 2) One isotype control tube containing PE-conjugated mouse IgG for non-specific first antibody binding, with a final reaction volume of 60 μL / tube.

[0450] The tubes were gently mixed and incubated at 20 - 24 °C for 20 minutes, then 400 μL of cold (2 - 8 °C) 1% formaldehyde was added and stored in the cold and dark for two hours. Before analyzing the fixed platelet sample, the settings of the appropriate peak path, threshold, axis, quadrant position, and region marker boundaries for data acquisition of the FACS instrument were determined by collecting at least 10,000 events from a sample of freshly sonicated latex beads (CML Cat#C37483) with a uniform diameter of 1 μM diluted in 0.9% saline. After applying these settings, at least 100,000 events were required for both the isotype control and the CD61-stained samples. The counts obtained from the CD61 sample would record the events as CD-61 positive fluorescently stained PLT, CD-61 positive mpPLT, and the number of Trucount fluorescent beads detected. The events counted in the predetermined region were analyzed within the platelet size range of 2 - 4 microns and within the mpPLT quadrant corrected for non-specific binding detected in the isotype control. The data was standardized using the following formula based on the known number of Trucount beads per tube provided by the manufacturer relative to the validity of the detected Trucount beads.

[0451] Events Counted # / Beads Counted #X TruCount Beads per Tube # / Sample Volume = mpPLT / μL or PLT / μL.

[0452] In addition, the morphology of hESC-PLTs and hiPSC-PLTs was determined by microscopic observation. Morphological determination was performed by differential interference contrast microscopy (DIC) and β1-tubulin staining to confirm the unique characteristic microtubule circumferential band of platelets. Briefly, platelets were fixed in 4% formaldehyde and centrifuged onto 1 μg / ml poly-L-lysine-coated coverslips, permeabilized with 0.5% Triton X-100, blocked in immunofluorescence blocking buffer (0.5 g BSA, 0.25 ml 10% sodium azide, and 5 ml FCS in 50 ml PBS) for at least two hours, and then antibody-labeled. To distinguish permeabilized cells, samples were incubated with a rabbit polyclonal primary antibody against human β1-tubulin, which was generated against the C-terminal peptide sequence CKAVLEEDEEVTEEAEMEPEDKGH (Genemed Synthesis, Inc.) (SEQ ID NO:1). Samples were then treated with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 568 nm (Invitrogen; Molecular Probes) and washed thoroughly with PBS between and after incubation. Coverslips were mounted (Aqua Polymount; Polysciences) on microscope slides. As a background control, slides were incubated with the secondary antibody alone, and images were adjusted for non-specific binding of the antibody. Samples were examined using a microscope equipped with an oil immersion objective or a differential interference contrast objective. Images were acquired using a charge-coupled device camera. Images were analyzed using MetaMorph image analysis software (Molecular Devices) and ImageJ (National Institutes of Health). The characteristics of resting platelets were determined as follows: Differential interference contrast (DIC) microscopy: discoid, approximately 2-3 μM in diameter; β1-tubulin staining: prominent microtubule circumferential band, similar diameter to resting platelets. See Figure 21.

[0453] Platelets (hESC-PLTs and hiPSC-PLTs) were further tested to confirm the absence of pluripotent cells (e.g., by PCR, immunofluorescence, and / or FACS to detect pluripotency markers). To increase sensitivity, the detection method can be coupled with a process that can concentrate potential cell contaminants, which is captured by filtration, matrix, or gradient combined with low-speed centrifugation of precipitated cells while leaving the platelets in the supernatant.

[0454] The presence of microorganisms in platelets (hESC-PLT and hiPSC-PLT) was further tested according to the immersion method USP <21>, CFR 610.12.

[0455] The endotoxin of platelets (hESC-PLT and hiPSC-PLT) was further tested. The Gel-Clot Endotoxin System (Associates of Cape Cod, Inc.) was used to quantify Gram-negative bacterial endotoxin. Appropriate negative, positive, and positive product controls were prepared. The positive product control is an inhibitory control and consists of a specimen or specimen diluent to which standard endotoxin has been added. The sample was added directly to the reagent and mixed well. The reaction tubes were incubated at 37 °C ± 1 °C for 60 ± 2 minutes. The formation of a gel that does not disappear even when the test tube is inverted indicates a positive test. Endotoxin was quantified by determining the endpoint of serial dilution of the specimen. In the series without endotoxin, known concentrations of positive controls can be included in the test. The use of PLT samples will be verified using endotoxin assays and conducted in a manner consistent with the 1987 FDA guidance on endotoxin validation.

[0456] The mycoplasma of platelets (hESC-PLT and hiPSC-PLT) was further tested. After centrifuging the platelets before the HSA gradient, the supernatants (e.g., consisting of conditioned StemSpan ACF medium) were collected and pooled. Platelet samples purified from the final product batch and conditioned medium were sent for mycoplasma testing. Mycoplasma detection was performed according to European Pharmacopoeia and US Pharmacopoeia guidelines by indirect culture on indicator cell cultures and direct inoculation on agar plates and in broth.

[0457] Example 6. Determination of platelet potency and PAC-1 binding

[0458] The methods described in this example can be used to evaluate the potency and / or PAC-1 binding of the platelets disclosed in the present invention.

[0459] In vivo, platelet activation induces a conformational change in αIIbβ3 integrin, activates the fibrinogen receptor function of the GPIIb / IIIa complex, and causes enhanced ligand binding. Activated platelets bind substrates including fibrinogen and von Willebrand factor, stimulating thrombus formation at the site of vascular injury. To determine the extent of functional αIIbβ3 integrin expression upon platelet activation, hESC-PLT, iPSC-PLT, and purified normal human platelets were activated and compared with resting control PLT, and the degree of PAC-1 binding was evaluated. PAC-1 is a fibrinogen mimic that specifically binds to the activated conformation of αIIbβ3 integrin.

[0460] Count PLTs. At least 100,000 PLTs are taken out and diluted in another culture medium to a density of approximately 20 PLTs / μL. PAC1-FITC (BD, diluted 1:100) and antibodies (CD41a-APC-conjugated (allophycocyanin) 1:100 and CD42b-PE-conjugated (phycoerythrin) 1:100 (BD Bioscience, San Jose, CA)) are added to the PLT sample. Half of the sample (250 μL) is added to each of two 5-mL FACS tubes. Thrombin (Sigma) is added to one tube to a final concentration of 1 U / mL. The thrombin-treated activated PLTs and the control sample are incubated at room temperature for 15 - 20 minutes. The samples are subjected to FACS analysis, and forward and side scatter gating is determined using human blood platelets as a control. The binding (activation) of PAC-1 is quantified by comparing the number of CD41a and PAC-1 positive events in the activated and non-activated controls.

[0461] Example 7. Use of proteases such as MMP inhibitors to increase the yield or purity of platelets generated under shear stress

[0462] The present disclosure also relates to a method for preparing platelets using shear force conditions (e.g., the culture medium is flowing during the culture). The described culture is carried out using a microfluidic device, where the flow rate is in the range of several tens of microliters per minute, approaching the shear force in the bone marrow cavity during hematopoiesis. In some cases, the flow rate is in the range of 5 - 25 microliters per minute. When culturing megakaryocytes generated from the iPS cells or ES cells disclosed in the present invention under shear force, it is found that they release platelets in a more efficient manner.

[0463] The device for culturing MLP must be able to immobilize the MLP without it adhering to the wall. In some cases, this means that the MLP is located in an area or chamber of the device where it can come into contact with the flowing culture medium but cannot move significantly by itself.

[0464] The present disclosure believes that the platelet yield and purity can be improved in these culture systems. On the one hand, the improvement can be obtained by using one or more defined protease inhibitors in shear force culture. This example demonstrates the benefits of adding a matrix metalloproteinase (MMP) inhibitor under these culture conditions. It should be understood that the MMP inhibitor is representative of other protease inhibitors that can be similarly used in these methods, such as but not limited to plasminogen activator inhibitor.

[0465] On the other hand, an increase in platelet yield and purity can be obtained by culturing under increased shear force. The shear force can be 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 dynes / cm 2 。

[0466] Use mature MKs derived from HA-iPS and MA09 hES lines. The same number of MKs suspended in MK-specific medium were loaded into a microfluidic device with or without 20 μM MMP inhibitor GM6001. A constant shear force was applied to the MKs throughout the culture period. MK medium downstream of the MKs was collected every 30 minutes over a 6-hour period. The number and purity of platelets (i.e., the percentage of CD41a + CD42b + in total cells) in the collected medium were measured by flow cytometry.

[0467] Figures 22-24 Results of platelet production from MKs derived from HA-iPS are provided. Platelets were generated under a constant shear force with a flow rate set at [Please provide] μl / min. Adding an MMP inhibitor at the start of the culture significantly increased the purity ( Figure 22 ) and yield ( Figure 24 ) of newly generated platelets. Purity was expressed as the percentage of CD41a + CD42b + cells in the total harvested cells. Adding 8% dextran increased the viscosity of the medium, which had a negative impact on platelet formation.

[0468] Figure 23 Illustrate that significantly more platelets are generated from MKs in the presence of the MMP inhibitor GM6001 during shear force culture.

[0469] Figure 24 Illustrate the differences in platelet yield in the presence of the MMP inhibitor GM6001 or DMSO during shear force culture, or in static culture.

[0470] Figure 25 Illustrate that similar results were obtained when MA09 (ES)-derived MKs were used for platelet preparation. Figure 25 and 26 Also illustrate that a moderate change in shear force (reflected as a change in flow rate from 12 μl / min - 16 μl / min) can also increase the purity and yield of platelets.

[0471] Example 8. MMP-specific inhibitors as protective inhibitors contribute to platelet formation in static culture

[0472] Proteases (such as MMPs,) are involved in the shedding of CD42b (also known as GPIbα,). Platelet CD42b is a receptor for vWF, which mediates the initial platelet participation in wound healing. Therefore, a reduction in CD42b results in poorer platelet quality. The broad-spectrum MMP inhibitor GM6001 has been reported to inhibit the shedding of platelet CD42b.

[0473] In a comparative study using several specific MMP inhibitors, it was determined that the MMP8 inhibitor has a greater ability to protect platelet function than GM6001. As Figure 26 shown, when the MMP8 specific inhibitor was added at the peak of MK platelet production, the purity of iPS-derived platelets was significantly increased from 43.7% to 65.5%, which is approximately 10% higher than the purity obtained from GM6001-treated cultures. Compared with GM6001, significantly more CD41a + CD42b + platelets were produced in the presence of the MMP8 specific inhibitor. When the MMP specific inhibitor and GM6001 were used in combination, the purity level was not affected ( Figure 27 ), but the platelet production was increased ( Figure 28 ). The peak of platelet production was determined by measuring the platelet content in the culture, for example, over several days on a daily basis. It usually occurs 4 - 7 days after the start of the MLP differentiation culture phase, but it may be extended or may vary.

[0474] Despite the data using iPS-derived megakaryocytes to produce Figure 27 and 28 , the present disclosure provides the use of the MMP8 specific inhibitor in culturing natural sources of platelets such as bone marrow and umbilical cord blood CD34 + progenitor cells.

[0475] Example 9. iPS platelet production at elevated temperature

[0476] Figure 29 and 30 show that culturing megakaryocytes under warm conditions significantly improves the purity and yield of platelets. The percentage of CD41a + CD42b + platelets was significantly higher when cultured at 39 °C than when cultured at 37 °C. The most obvious effect was before reaching the peak of platelet production. In addition to increasing purity, culturing megakaryocytes at a higher temperature also helps to increase the yield of platelets produced from the same starting number of megakaryocytes, indicating that the elevated temperature has no adverse effect on megakaryocytes or platelets in the culture system provided herein.

[0477] Example 10. iBET promotes the ability of megakaryocytes and increases the overall platelet yield by downregulating the c-myc gene

[0478] In our new method of megakaryocyte lineage-specific differentiation, the appearance of megakaryocyte progenitors most suitable for platelet production can only be harvested in a short period of 3 - 4 days starting from the culture with PVE-HE. Myeloid CD14 +The gradual increase in cells seems to be associated with a decrease in megakaryocyte mass and platelet production. In the effort to obtain higher and better megakaryocyte progenitor yields, in order to identify new megakaryocyte promoting factors, the c-myc gene was found to be an important regulatory gene for early megakaryopoiesis.

[0479] GSK1210151A (I-BET151) is an orally active, imidazolonoquinoline-based inhibitor of BET family bromodomains. By inducing early cell cycle arrest and apoptosis, I-BET151 has great potency against human and murine MLL fusion leukemia cell lines. Part of this mode of action is through the inhibition of the transcription of key genes (BCL2, C-MYC, and CDK6) by the translocation of BRD3 / 4, PAFc, and SEC components between chromatin.

[0480] Although high doses of i-BET-151 trigger severe apoptosis, treatment of cells undergoing in vitro megakaryopoiesis at low doses in the micromolar range (or submicromolar range) results in an increase in the number of MK progenitors. Figure 31 Quantitative mRNA analysis demonstrates the dose-dependent inhibition of c-myc gene expression by i-BET-151. In contrast, i-BET-151 upregulates the pro-MK (pro-megakaryocyte) gene GATA1 ( Figure 32 ). Treatment with i-BET-151 also leads to a dose-dependent decrease in the number of CD14 + myeloid cells.

[0481] Therefore, using the method for in vitro preparation of iPS-derived (or ES-derived) megakaryocytes and platelets provided herein, it was further discovered that inhibiting the gene expression of endogenous c-myc in cells undergoing megakaryopoiesis can alter the balance of cell differentiation contributing to the MK lineage.

[0482] The present invention relates to the following embodiments:

[0483] 1. A pharmaceutical preparation suitable for human patients, comprising at least 10 8 platelets, wherein the preparation is substantially free of white blood cells, and wherein substantially all platelets are functional.

[0484] 2. The pharmaceutical preparation of embodiment 1, comprising 10 9 -10 14 platelets, optionally 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 1014 Platelets.

[0485] 3. The pharmaceutical preparation of any one of embodiments 1-2, wherein the platelets have one or more of the following characteristics: a mean platelet volume in the range of 9.7-12.8 fL; a unimodal size distribution in the preparation; and / or a logarithmic platelet volume distribution with a standard deviation of less than 2 μm. 3 (Preferably less than 1.5 μm 3 , 1μm 3 or even 0.5 μm 3 ).

[0486] 4. The preparation of any one of embodiments 1-3, wherein the platelets are positive for at least one of the following markers: CD41a and CD42b.

[0487] 5. The preparation of any one of embodiments 1-4, wherein the platelets are human platelets.

[0488] 6. The preparation of any one of embodiments 1-5, wherein at least 50%, 60%, 70%, 80% or 90% of the platelets are functional after storage at room temperature for at least 2, 3 or 4 days.

[0489] 7. A bioreactor with weakly adherent or non-adherent megakaryocytes that produces functional platelets without the need for feeder cells.

[0490] 8. A composition comprising at least 10 9 MLPs.

[0491] 9. A cryopreservable composition comprising MLPs.

[0492] 10. A library comprising cryopreserved MLPs.

[0493] 11. The cryopreserved composition or library of embodiment 9 or 10, wherein said MLPs have a defined HLA type.

[0494] 12. The cryopreserved composition of embodiment 9, which is HLA-matched to the patient.

[0495] 13. The cryopreserved composition or library of any one of embodiments 9 to 12, comprising 10 9 -10 14 MLPs, choose 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 MLPs.

[0496] 14. A method for preparing platelets from megakaryocytes, comprising the following steps:

[0497] a) Providing a non-adherent culture of megakaryocytes;

[0498] b) Contacting the megakaryocytes with TPO or a TPO agonist, thereby causing the formation of proplatelets in the culture, wherein the proplatelets release platelets; and

[0499] c) Isolating the platelets.

[0500] 15. A method for preparing platelets from megakaryocytes, comprising the following steps:

[0501] a) Providing a non-adherent culture of megakaryocytes;

[0502] b) Contacting the megakaryocytes with a hematopoietic expansion medium and optionally with (1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or (2) TPO or a TPO agonist, SCF and IL-11, thereby causing the formation of proplatelets in the culture, wherein the proplatelets release platelets; and

[0503] c) Isolating the platelets.

[0504] 16. The method of embodiment 14 or 15, wherein the TPO agonist comprises one or more of the following: ADP, epinephrine, thrombin, collagen, a TPO-R agonist, a TPO mimetic, a second-generation thrombopoietic agent, romiplostim, eltrombopag (SB497115, Promacta), recombinant human thrombopoietin (TPO), polyethylene glycolated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, a TPO non-peptide mimetic, AKR-501, a monoclonal TPO agonist antibody, a polyclonal TPO agonist antibody, a TPO minibody, VB22B sc(Fv)2, a domain subclass-converted TPO agonist antibody, MA01G4G344, recombinant human thrombopoietin, a recombinant TPO fusion protein or a TPO non-peptide mimetic.

[0505] 17. The method of any one of embodiments 14-16, wherein substantially all of the isolated platelets are functional.

[0506] 18. The method of any one of embodiments 14-17, wherein the non-adherent culture of megakaryocytes is a feeder cell-free culture.

[0507] 19. The method according to any one of Embodiments 14 - 18, wherein the culture in step (b) is in a culture medium containing one or more of the following substances: stem cell factor (SCF) at 0.5 - 100 ng / ml, thrombopoietin (TPO) at 10 - 100 ng / ml, and interleukin - 11 (IL - 11) at 10 - 100 ng / ml, at least one ROCK inhibitor, and / or heparin at 2.5 - 25 units / ml.

[0508] 20. The method according to any one of Embodiments 14 - 18, wherein the culture in step (b) is in a culture medium containing one or more of the following substances: TPO at 10 - 100 ng / ml, SCF at 0.5 - 100 ng / ml, IL - 6 at 5 - 25 ng / ml, IL - 9 at 5 - 25 ng / ml, at least one ROCK inhibitor, and / or heparin at 2.5 - 25 units / ml.

[0509] 21. The method according to Embodiment 19 or 20, wherein the at least one ROCK inhibitor comprises Y27632.

[0510] 22. The method according to Embodiment 21, wherein the concentration of the Y27632 is 2 - 20 μM, about 3 - 10 μM, about 4 - 6 μM, or about 5 μM.

[0511] 23. The method according to any one of Embodiments 14 - 22, further comprising applying shear force to the megakaryocytes.

[0512] 24. The method according to any one of Embodiments 14 - 23, wherein each megakaryocyte produces at least 2, 3, 4, or 5 platelets.

[0513] 25. The method according to Embodiment 24, wherein each megakaryocyte produces at least 50 platelets.

[0514] 26. The method according to Embodiment 25, wherein each megakaryocyte produces at least 100, 500, 1000, 2000, 5000, or 10000 platelets.

[0515] 27. The method according to any one of Embodiments 14 - 26, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the platelets are CD41a+ and CD42b+.

[0516] 28. The method according to any one of Embodiments 14 - 27, wherein the platelets are prepared in the absence of feeder cells or stromal feeder cells.

[0517] 29. The method according to any one of Embodiments 14 - 28, wherein the platelets are prepared under conditions in which no xenogeneic cells are present.

[0518] 30. The method according to any one of Embodiments 14 - 29, wherein the platelets are human platelets.

[0519] 31. The method according to any one of Embodiments 14 - 30, wherein the megakaryocytes are cultured in the presence of an externally added protease inhibitor.

[0520] 32. The method according to any one of Embodiments 14 - 30, wherein the megakaryocytes are cultured in the presence of an externally added MMP inhibitor.

[0521] 33. The method according to any one of Embodiments 14 - 30, wherein the megakaryocytes are cultured in the presence of an externally added MMP8 inhibitor.

[0522] 34. The method according to any one of Embodiments 14 - 30, wherein the megakaryocytes are cultured in the presence of an externally added MMP - specific inhibitor and a pan - MMP inhibitor.

[0523] 35. The method according to any one of Embodiments 14 - 34, wherein the megakaryocytes are cultured at a temperature of about 39°C.

[0524] 36. The method according to any one of Embodiments 14 - 35, wherein the megakaryocytes are prepared by steps comprising:

[0525] (a) culturing pluripotent stem cells to form hematopoietic endothelial cells (PVE - HE);

[0526] (b) culturing the hematopoietic endothelial cells to form MLP; and

[0527] (c) culturing the MLP to form megakaryocytes.

[0528] 37. The method of Embodiment 36, wherein a BET inhibitor is added to the culture in step (b).

[0529] 38. The method of Embodiment 37, wherein the BET inhibitor is IBET151.

[0530] 39. The method according to any one of Embodiments 36 - 38, wherein the pluripotent stem cells are human pluripotent stem cells.

[0531] 40. The method according to any one of Embodiments 36 - 39, wherein the hematopoietic endothelial cells are derived without embryoid body formation.

[0532] 41. The method according to any one of embodiments 36 - 40, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0533] 42. The method of embodiment 41, wherein the iPSCs are human iPSCs.

[0534] 43. The method of embodiment 41 or 42, wherein the hematopoietic endothelial cells are derived without embryoid body formation.

[0535] 44. The method according to any one of embodiments 36 - 43, wherein the hematopoietic endothelial cells are differentiated from the pluripotent stem cells under hypoxic conditions comprising 1% - 10% oxygen, 2% - 8% oxygen, 3% - 7% oxygen, 4% - 6% oxygen, or approximately 5% oxygen.

[0536] 45. The method according to any one of embodiments 36 - 44, wherein the MLP is cultured at a temperature of 38 - 40 °C or approximately 39 °C to form megakaryocytes.

[0537] 46. A pharmaceutical preparation comprising platelets prepared by the method according to any one of embodiments 14 - 45.

[0538] 47. The preparation of embodiment 46, which is suitable for human patients and comprises at least 10 8 platelets.

[0539] 48. The preparation of embodiment 46, which is suitable for human patients and is substantially free of white blood cells.

[0540] 49. Use of the composition of any one of the foregoing embodiments or the composition prepared by the method of any one of the foregoing embodiments in the manufacture of a medicament for treating a patient in need thereof or a patient suffering from a disease or disorder affecting blood clotting or a disease or disorder treatable thereby.

[0541] 50. The use of embodiment 49, wherein the disease or disorder comprises thrombocytopenia, trauma, blood - borne parasites, or malaria.

[0542] 51. A method of treating a patient in need of platelet transfusion, comprising administering to the patient the composition of any one of the foregoing embodiments or the composition prepared by the method of any one of the foregoing embodiments.

[0543] 52. The method of embodiment 51, wherein the method is effective in treating a disease or disorder comprising thrombocytopenia, trauma, blood - borne parasites, or malaria.

[0544] 53. A method of preparing platelets from megakaryocytes or MLP, which comprises

[0545] Culturing a non - adherent population of megakaryocytes or MLP under shear force conditions in the presence of a protease inhibitor, and

[0546] Harvesting and optionally isolating platelets from the culture.

[0547] 54. The method of embodiment 53, wherein the protease inhibitor is an MMP inhibitor.

[0548] 55. The method of embodiment 53 or 54, wherein the shear force conditions are constant shear force conditions.

[0549] 56. The method according to any one of embodiments 53 - 55, wherein the shear force conditions comprise a shear force of 1 - 4.1 dynes / cm 2 of shear force.

[0550] 57. The method according to any one of embodiments 53 - 56, wherein the megakaryocytes or MLP are cultured in a microfluidic device.

[0551] 58. The method according to any one of embodiments 53 - 57, wherein the megakaryocytes or MLP are derived from iPS cells, ES cells or naturally occurring CD34 + cells, optionally bone marrow or cord blood CD34 + cells.

[0552] 59. The method according to any one of embodiments 53 - 58, wherein the protease inhibitor is GM6001.

[0553] 60. The method according to any one of embodiments 53 - 58, wherein the protease inhibitor is an MMP8 - specific inhibitor.

[0554] 61. The method of embodiment 60, wherein the MMP8 - specific inhibitor is MMP8 - I ((3R)-(+)-[2-(4 - methoxybenzenesulfonyl)-1,2,3,4 - tetrahydroisoquinoline - 3 - oximic acid salt]).

[0555] 62. The method according to any one of embodiments 53 - 61, wherein two or more protease inhibitors are used.

[0556] 63. The method of embodiment 62, wherein the two protease inhibitors are an MMP general inhibitor and an MMP8 - specific inhibitor.

[0557] 64. The method according to any one of embodiments 53 - 63, wherein the protease inhibitor is added at the platelet production peak in the culture.

[0558] 65. The method according to any one of embodiments 53 - 64, wherein the megakaryocytes or MPL are cultured in the presence of TPO or a TPO agonist to result in the formation of proplatelets, and wherein the proplatelets release platelets.

[0559] 66. The method according to any one of embodiments 53 - 65, wherein the megakaryocytes or MPL are cultured in a hematopoietic expansion medium and optionally in

[0560] (1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or

[0561] (2) TPO or a TPO agonist, SCF and IL-11 to form proplatelets in the culture, and wherein the proplatelets release platelets.

[0562] 67. The method according to any one of embodiments 53 - 66, wherein the megakaryocytes or MPL are cultured at a temperature higher than 37 °C and equal to or lower than 40 °C.

[0563] 68. The method of embodiment 67, wherein the megakaryocytes or MPL are cultured at a temperature of about 39 °C.

[0564] 69. A method for preparing platelets from megakaryocytes or MPL, which comprises

[0565] culturing a non-adherent population of megakaryocytes or MPL derived from iPS cells or ES cells at a temperature higher than 37 °C and equal to or lower than 40 °C, and

[0566] harvesting and optionally isolating platelets from the culture.

[0567] 70. The method of embodiment 69, wherein the megakaryocytes or MPL are cultured at a temperature of about 39 °C.

[0568] 71. A method for preparing MPL from PVE-HE cells, which comprises

[0569] culturing a population of PVE-HE cells derived from iPS cells or ES cells in the presence of an inhibitor of BET, and

[0570] harvesting and optionally isolating MPL from the culture.

[0571] 72. The method of embodiment 71, wherein the BET inhibitor is I-BET151.

[0572] 73. A method for preparing MPL from PVE-HE cells, which comprises

[0573] Culturing a PVE-HE cell population derived from iPS cells or ES cells in the presence of a c-myc repressor protein, and

[0574] Harvesting and optionally isolating MPL from the culture. Sequence Listing <110>Astellas Regenerative Medicine, Inc. <120>Method for Preparing Platelets from Pluripotent Stem Cells and Compositions Thereof <130>A1025.70046WO00 <140>TBD <141>2013-12-21 <150>61 / 787,476 <151>2013-03-15 <150>61 / 740,699 <151>2012-12-21 <160>1 <170>PatentIn version 3.5 <210>1 <211>24 <212>PRT <213>Homo Sapiens <400>1 Cys Lys Ala Val Leu Glu Glu Asp Glu Glu Val Thr Glu Glu Ala Glu 1 5 10 15 Met Glu Pro Glu Asp Lys Gly His 20

Claims

1. A method for producing megakaryocytes, comprising: (a) Culturing pluripotent stem cells under adherent conditions in a culture medium containing bone morphogenetic protein 4, basic fibroblast growth factor, and vascular endothelial growth factor to form a population of hematopoietic endothelial cells, wherein at least 70% of the cells in the population of hematopoietic endothelial cells express CD31 / PECAM1, CD105 / endoglin, and CD144 / VE-Cad; (b) Culturing the population of hematopoietic endothelial cells in a culture medium containing: (i) thrombopoietin (TPO), interleukin-3, Fms-related tyrosine kinase 3 ligand, stem cell factor, and interleukin-6 or (ii) a TPO agonist, interleukin-3, Fms-related tyrosine kinase 3 ligand, stem cell factor, and interleukin-6 to form megakaryocyte progenitors; and (c) Culturing the megakaryocyte progenitors under non-adherent culture conditions to form megakaryocytes, wherein the non-adherent culture conditions include: (i) a culture medium containing TPO, stem cell factor, interleukin-6, and interleukin-9, or (ii) a culture medium containing a TPO agonist, stem cell factor, interleukin-6, and interleukin-9, or (iii) a culture medium containing TPO, stem cell factor, and interleukin-11, or (iv) a culture medium containing a TPO agonist, stem cell factor, and interleukin-11.

2. The method according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells.

3. The method according to claim 1, wherein the population of hematopoietic endothelial cells is cultured in the presence of a BET inhibitor.

4. The method according to claim 3, wherein the BET inhibitor is IBET151.

5. The method according to claim 4, wherein the concentration of the IBET151 is in the micromolar or submicromolar concentration range.

6. The method according to claim 4, wherein the concentration of IBET151 is 0.25 μM.

7. The method according to any one of claims 1-6, wherein the culture medium in step (a) contains 50 ng / ml bone morphogenetic protein 4, 50 ng / ml basic fibroblast growth factor, and 50 ng / ml vascular endothelial growth factor.

8. The method according to any one of claims 1-6, wherein the culture medium in step (b) contains thrombopoietin, interleukin-3, Fms-related tyrosine kinase 3 ligand, stem cell factor, and interleukin-6.

9. The method according to any one of claims 1-6, wherein the culture medium in step (c) contains stem cell factor, thrombopoietin, interleukin-6, interleukin-9, and a ROCK inhibitor.

10. The method according to claim 9, wherein the ROCK inhibitor is Y27632.

11. The method according to any one of claims 1-6, wherein the population of hematopoietic endothelial cells differentiates from the pluripotent stem cells under hypoxic conditions containing 1% to 10% oxygen.

12. The method according to claim 11, wherein the hypoxic condition comprises 4% to 6% oxygen.

13. The method according to any one of claims 1-6, wherein the megakaryocyte progenitor cells are cultured at a temperature between 38-40 °C to form megakaryocytes.

14. The method according to any one of claims 1-6, wherein at least 90% of the cells in the population of hematopoietic endothelial cells express CD31 / PECAM1, CD105 / endoglin, and CD144 / VE-Cad.

15. The method according to any one of claims 1-6, wherein the step of culturing the pluripotent stem cells under adherent conditions comprises culturing the pluripotent stem cells on an extracellular matrix.

16. The method according to any one of claims 1-6, wherein the pluripotent stem cells in step (a) are cultured under feeder-free culture conditions.

17. The method according to any one of claims 1-6, wherein: The culture medium for step (a) comprises bone morphogenetic protein 4, basic fibroblast growth factor, and vascular endothelial growth factor; the culture medium for step (b) comprises stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-3, and interleukin-6; and the culture medium for step (c) comprises stem cell factor, thrombopoietin, interleukin-6, and interleukin-9.

18. A method for producing human megakaryocytes, comprising: (a) culturing a population of human hematopoietic endothelial cells in a culture medium comprising: (i) thrombopoietin (TPO), interleukin-3, Fms-related tyrosine kinase 3 ligand, stem cell factor, and interleukin-6 or (ii) a TPO agonist, interleukin-3, Fms-related tyrosine kinase 3 ligand, stem cell factor, and interleukin-6 to form human megakaryocyte progenitor cells, wherein at least 70% of the cells in the population of hematopoietic endothelial cells express CD31 / PECAM1, CD105 / endoglin, and CD144 / VE-Cad; and (b) culturing the human megakaryocyte progenitor cells under non-adherent culture conditions to form human megakaryocytes, wherein the non-adherent culture conditions comprise: (i) a culture medium comprising TPO, stem cell factor, interleukin-6, and interleukin-9, or (ii) a culture medium comprising a TPO agonist, stem cell factor, interleukin-6, and interleukin-9, or (iii) a culture medium comprising TPO, stem cell factor, and interleukin-11, or (iv) a culture medium comprising a TPO agonist, stem cell factor, and interleukin-11.

19. The method according to claim 18, further comprising culturing human pluripotent stem cells under adherent culture conditions and / or feeder-free culture conditions to form a population of human hematopoietic endothelial cells.

20. The method according to claim 18, wherein the population of human hematopoietic endothelial cells is cultured in the presence of a BET inhibitor.

21. The method according to claim 20, wherein the BET inhibitor is IBET151.

22. The method according to claim 18, wherein the culture medium for step (a) comprises thrombopoietin, interleukin-3, and interleukin-6.

23. The method according to claim 18, wherein the culture medium in step (b) comprises stem cell factor, thrombopoietin, interleukin-6, interleukin-9, and a ROCK inhibitor.

24. The method according to claim 23, wherein the ROCK inhibitor is Y27632.

25. The method according to claim 18, wherein the human megakaryocyte progenitor cells are cultured at a temperature between 38 - 40 °C to form human megakaryocytes.

26. The method according to claim 18, wherein the human megakaryocyte progenitor cells express CD41a.

27. The method according to claim 18, wherein the culture medium in step (a) comprises stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-3, and interleukin-6, and the culture medium in step (b) comprises stem cell factor, thrombopoietin, interleukin-6, and interleukin-9.

28. A method for generating human megakaryocytes, comprising: (a) culturing a population of human hematopoietic endothelial cells in a culture medium comprising (i) thrombopoietin, (ii) interleukin-3, (iii) interleukin-6, (iv) Fms-related tyrosine kinase 3, and (v) stem cell factor to form human megakaryocyte progenitor cells, wherein at least 70% of the cells in the population of hematopoietic endothelial cells express CD31 / PECAM1, CD105 / endoglin, and CD144 / VE-Cad; and (b) culturing the human megakaryocyte progenitor cells in a culture medium comprising thrombopoietin, stem cell factor, interleukin-6, interleukin-9, and a ROCK inhibitor under non-adherent culture conditions to form human megakaryocytes.

29. A method for producing hematopoietic endothelial (PVE-HE) cells, comprising in vitro culturing pluripotent stem cells in a culture medium comprising bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) for at least 4 days, wherein the at least 4 days includes a first hypoxic condition stage comprising 1% to 10% oxygen and a second normoxic condition stage, thereby forming a cell population comprising hematopoietic endothelial cells, wherein the pluripotent stem cells differentiate into the hematopoietic endothelial cells without forming embryoid bodies, and wherein at least 70% of the cells in the cell population express CD31 / PECAM1, CD105 / endoglin, and CD144 / VE-Cad.

30. The method according to claim 29, wherein the pluripotent stem cells differentiate in the presence of one or more extracellular matrix components.

31. The method according to claim 30, wherein the one or more extracellular matrix components are collagen.

32. The method according to claim 31, wherein the collagen is collagen IV.

33. The method according to claim 29, wherein the pluripotent stem cells are differentiated into hematopoietic endothelial cells by culturing the pluripotent stem cells in a feeder-free condition in a differentiation induction medium containing BMP4, bFGF, and VEGF until CD31 / PECAM1+ hematopoietic endothelial cells are formed.

34. The method according to claim 33, wherein the pluripotent stem cells are first cultured on a collagen-coated surface in a medium containing a ROCK inhibitor and then cultured in the differentiation induction medium.

35. The method according to claim 29, wherein the pluripotent stem cells are differentiated into hematopoietic endothelial cells by: (i) culturing the pluripotent stem cells on a surface coated with collagen IV in a feeder-free condition in the presence of a medium containing 10 μM ROCK inhibitor Y27632, (ii) replacing the medium in (i) with a differentiation induction medium containing BMP4, bFGF, and VEGF and culturing until CD31 / PECAM1+ hematopoietic endothelial cells are formed.

36. The method according to claim 29, wherein the hypoxic condition comprises 2% to 8% oxygen, 3% to 7% oxygen, 4% to 6% oxygen, or 5% oxygen.

37. The method according to claim 29, wherein the hypoxic condition comprises 5% oxygen.

38. The method according to claim 29, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

39. The method according to claim 29, wherein the pluripotent stem cells are embryonic stem cells (ES cells).

40. The method according to claim 29, wherein at least 90% of the cells in the cell population express CD31 / PECAM1, CD144 / VE-Cad, and CD105 / endoglin.

41. The method according to claim 29, wherein the hematopoietic endothelial cells express CD34, CD309 / KDR, CD146, and / or CD184 / CXCR4.

42. The method according to claim 29, further comprising cryopreserving the cell population containing the hematopoietic endothelial cells.

43. A composition comprising: a cell population, wherein at least 70% of the cells in the cell population are CD31 / PECAM1+, CD144 / VE-Cad+, and CD105 / endoglin+ hematopoietic endothelial cells, and wherein the hematopoietic endothelial cells are generated by in vitro differentiation of pluripotent stem cells.

44. The composition according to claim 43, wherein at least 80% of the cells in the cell population are CD31 / PECAM1+, CD144 / VE-Cad+, and CD105 / endoglin+ hematopoietic endothelial cells.

45. The composition according to claim 43, wherein the CD31 / PECAM1+, CD144 / VE-Cad+, and CD105 / endoglin+ hematopoietic endothelial cells express CD34, CD309 / KDR, CD146, and / or CD184 / CXCR4.

46. The composition according to claim 43, wherein the composition is cryopreserved.

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