Method for inducing generation of megakaryocytes

Abstract

Provided is a use of a histone deacetylase (HDAC) inhibitor and / or JAK2 inhibitor in inducing hematopoietic stem progenitor cells, mononuclear cells or total nucleated cells to generate megakaryocytes and / or megakaryocyte progenitor cells. Also provided is a method for inducing hematopoietic stem progenitor cells, mononuclear cells or total nucleated cells to generate megakaryocytes and / or megakaryocyte progenitor cells, comprising exposing the cells to an HDAC inhibitor and / or exposing the cells to a JAK2 inhibitor.

Description

Methods for inducing megakaryocyte generation

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 2024101533064 filed on February 2, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of cell biology, and in particular, to a method for inducing the generation of megakaryocytes and / or megakaryocyte progenitor cells, and the use of a histone deacetylase inhibitor and / or a JAK2 inhibitor in inducing the generation of megakaryocytes and / or megakaryocyte progenitor cells. Background Art

[0004] Platelets play a vital role in hemostasis through their adhesion, aggregation, and granule release. Clinically, various etiologies, including hematopoietic malignancies, aplastic anemia, malignant tumors, hematopoietic stem cell transplantation, and chemotherapy and radiotherapy, can lead to thrombocytopenia or platelet dysfunction, resulting in varying degrees of bleeding, which can be life-threatening in severe cases. Platelet transfusions are commonly used clinically, but platelet sources are limited, platelets have a short lifespan, and are easily inactivated ex vivo. Repeated platelet transfusions can lead to ineffectiveness and increase the risk of transfusion-transmitted diseases. Statistics show that nearly 10 million patients in China require platelet transfusions annually, and the platelet market is estimated to be nearly 100 billion RMB. Currently, there is no effective alternative to platelets for clinical transfusion, requiring them to be collected from unpaid blood donors. In recent years, the shortage of blood and platelet products has become a significant challenge in clinical practice. Producing platelets ex vivo offers a potential solution. Currently, hematopoietic stem and progenitor cells from different sources, such as bone marrow, peripheral blood, umbilical cord blood, and human pluripotent stem cells, can be induced to differentiate into megakaryocytes and platelets. However, the preparation of platelets in vitro is difficult and costly. Studies have shown that CD34+ cells isolated from peripheral blood or umbilical cord blood can be induced to differentiate into megakaryocytes in vitro. The results showed that megakaryocytes induced in vitro can replace platelet transfusions or reduce the number of platelet transfusions. Literature reports that seed cells are mostly CD34+ cells, and mononuclear cells as seed cells for in vitro induced expansion have the advantages of low cost, simple operation, and low loss of hematopoietic stem cells.

[0005] Megakaryocytes are cells in the normal bone marrow that produce platelets. Platelets form when the edges of mature megakaryocytes rupture and detach. Each megakaryocyte produces an average of approximately 2,000 platelets in the body. Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells first differentiate into megakaryocyte progenitor cells, also known as colony-forming units (CFUs) of the megakaryotic lineage. The chromosomes within the nucleus of these progenitor cells are generally diploid. When these progenitor cells are diploid or tetraploid, they possess the ability to proliferate, marking the stage in which the megakaryocyte lineage increases in number. As the megakaryocyte progenitor cells further differentiate into megakaryocytes, which have a ploidy of 8-32, the cytoplasm begins to differentiate, and the endomembrane system gradually becomes complete. Finally, a membrane material divides the megakaryocyte cytoplasm into numerous small cells. Once each cell is completely separated, it becomes a platelet. Platelets shed from the megakaryocyte through the interstitial spaces between the endothelium of the venous sinusoidal wall and enter the bloodstream.

[0006] Most reported starting cells for megakaryocyte differentiation are CD34+ cells. However, the isolation of CD34+ cells is cumbersome, costly, and has limited differentiation efficiency. Studies have also shown that cord blood mononuclear cells can also serve as starting cells for induction of megakaryocyte differentiation. After a series of optimizations, the induction differentiation efficiency was approximately 50%. Therefore, there is still considerable room for improvement in the efficiency of mononuclear cell-to-megakaryocyte differentiation, which is crucial for both cost control and clinical effectiveness. Furthermore, there are relatively few studies on total nucleated cell-to-megakaryocyte differentiation systems. Using total nucleated cells as starting cells for megakaryocyte differentiation can further simplify the process and reduce costs. Furthermore, the purity of megakaryocytes is crucial for therapeutic applications. High purity not only ensures therapeutic efficacy but also reduces safety risks posed by non-target cells. Therefore, a method for preparing megakaryocytes in vitro that is simple to operate, has high differentiation efficiency, and produces high-purity products is needed.

[0007] According to other literature reports, many epigenetic regulatory small molecules used for somatic cell reprogramming also play an important role in the induced differentiation process of megakaryocytes. For example, the histone deacetylase (HDAC) inhibitor valproic acid (VPA), the G9a histone methyltransferase inhibitor Bix01294, the DNA methyltransferase inhibitor RG108, and the MAPK / ERK signaling pathway inhibitor PD0325901 can all improve the efficiency of induced differentiation. In addition, JAK kinase inhibitors have also been reported to promote the differentiation of pluripotent stem cells into megakaryocytes. Summary of the Invention

[0008] In one aspect, the present invention provides the use of histone deacetylase (HDAC) inhibitors and / or JAK2 inhibitors in inducing the differentiation of starting cells to generate megakaryocytes and / or megakaryocyte progenitor cells. In some embodiments, the starting cells are hematopoietic stem and progenitor cells, mononuclear cells or total nucleated cells. In some embodiments, the starting cells are total nucleated cells.

[0009] In some embodiments, the HDAC inhibitor and the JAK2 inhibitor are used in combination. In some embodiments, the HDAC inhibitor and the JAK2 inhibitor are used in a sequential order. In some embodiments, the HDAC inhibitor is used first, and then the HDAC inhibitor and the JAK2 inhibitor are used simultaneously.

[0010] In some embodiments, the HDAC inhibitor is one or more of Resminostat, Remetinostat, TMP 195, TMP269, Bufexamac, Tubastatin A, Pracinostat, Droxinostat, ACY-738, and Tubacin. In some embodiments, the HDAC inhibitor is Tubastatin A or ACY-738. In some embodiments, the HDAC inhibitor is Tubastatin A.

[0011] In some embodiments, the JAK2 inhibitor is one or more of AZD-1480, WP1066, Peficitinib, Fedratinib, Filgotinib, Momelotinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, Fedratinib, Momelotinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is Gandotinib.

[0012] In another aspect, the present invention provides a method for inducing the differentiation of starting cells to generate megakaryocytes and / or megakaryocyte progenitor cells, comprising exposing the starting cells to a histone deacetylase (HDAC) inhibitor, and / or exposing the starting cells to a JAK2 inhibitor. In some embodiments, the starting cells are hematopoietic stem and progenitor cells, mononuclear cells, or total nucleated cells. In some embodiments, the starting cells are total nucleated cells.

[0013] In some embodiments, the method comprises exposing the starting cells to an HDAC inhibitor and exposing the starting cells to a JAK2 inhibitor. In some embodiments, the method comprises exposing the starting cells to the HDAC inhibitor and the JAK2 inhibitor in a sequential order. In some embodiments, the method comprises: exposing the starting cells to the HDAC inhibitor first, and then exposing the starting cells to the HDAC inhibitor and the JAK2 inhibitor simultaneously.

[0014] In some embodiments, the HDAC inhibitor is one or more of Resminostat, Remetinostat, TMP 195, TMP269, Bufexamac, Tubastatin A, Pracinostat, Droxinostat, ACY-738, and Tubacin. In some embodiments, the HDAC inhibitor is Tubastatin A or ACY-738. In some embodiments, the HDAC inhibitor is Tubastatin A.

[0015] In some embodiments, the HDAC inhibitor is present at a concentration of 0.1-1000 nM. In some embodiments, the HDAC inhibitor is present at a concentration of 100-1000 nM. In some embodiments, the HDAC inhibitor is present at a concentration of 200-800 nM.

[0016] In some embodiments, the JAK2 inhibitor is one or more of AZD-1480, WP1066, Peficitinib, Fedratinib, Filgotinib, Momelotinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, Fedratinib, Momelotinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is Gandotinib.

[0017] In some embodiments, the JAK2 inhibitor is present at a concentration of 0.1-1000 nM. In some embodiments, the JAK2 inhibitor is present at a concentration of 100-200 nM. In some embodiments, the JAK2 inhibitor is present at a concentration of about 200 nM.

[0018] In some embodiments, the method further comprises exposing the starting cells to stem cell factor, thrombopoietin, and / or an interleukin.

[0019] In yet another aspect, the present invention provides use of an HDAC inhibitor and / or a JAK2 inhibitor in the preparation of a medicament for treating a disease requiring megakaryocyte and / or platelet transfusion.

[0020] In yet another aspect, the present invention provides a method for treating a disease requiring megakaryocyte and / or platelet transfusion, comprising: administering to an individual suffering from the disease at least one selected from the following i) to ii): i) an HDAC inhibitor and / or a JAK2 inhibitor; ii) megakaryocytes prepared by the method.

[0021] In some embodiments, the disease is thrombocytopenia or a platelet dysfunction disorder.

[0022] In some embodiments, the HDAC inhibitor and the JAK2 inhibitor are used in combination. In some embodiments, the HDAC inhibitor and the JAK2 inhibitor are used in a sequential order. In some embodiments, the HDAC inhibitor is used first, and then the HDAC inhibitor and the JAK2 inhibitor are used simultaneously.

[0023] In some embodiments, the HDAC inhibitor is one or more of Resminostat, Remetinostat, TMP 195, TMP269, Bufexamac, Tubastatin A, Pracinostat, Droxinostat, ACY-738, and Tubacin. In some embodiments, the HDAC inhibitor is Tubastatin A or ACY-738. In some embodiments, the HDAC inhibitor is Tubastatin A.

[0024] In some embodiments, the JAK2 inhibitor is one or more of AZD-1480, WP1066, Peficitinib, Fedratinib, Filgotinib, Momelotinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, Fedratinib, Momelotinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, and Gandotinib. In some embodiments, the JAK2 inhibitor is Gandotinib.

[0025] Compared to existing technologies, the present invention offers the following advantages: It provides a method for preparing megakaryocytes and / or megakaryocyte progenitor cells in vitro that promotes efficient differentiation of starting cells. In some approaches, the starting cell acquisition process is simple, resulting in high-purity megakaryocyte progenitor cells and mature megakaryocytes. A limited number of added factors are required, meeting clinical needs while also reducing costs. Furthermore, the low proportion of T cells in the differentiation products reduces the risk of GVHD caused by cell transfusion. DETAILED DESCRIPTION

[0026] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] The term “comprise” or variations thereof such as “comprises”, “has” or “includes” means including stated steps or elements but not excluding any other steps or elements.

[0028] The term "and / or" should be understood to refer to any one element or any combination of several elements connected by this term.

[0029] The term "about," when used with a numerical variable, means that the value of the variable is within ±10% of the specified value.

[0030] The term "megakaryocyte progenitor cells" refers to precursor cells that are differentiated from pluripotent stem cells and are capable of proliferating and differentiating to produce megakaryocytes, including all megakaryocyte progenitor cells from the early high-proliferative potential mixed megakaryocyte colony-forming unit stage to the late pure megakaryocyte colony-forming unit stage with weaker proliferative ability.

[0031] The term "megakaryocyte" refers to a precursor cell that is generated by proliferation and differentiation of megakaryocyte progenitor cells and is capable of producing platelets after maturation, and includes all megakaryocytes from the early immature megakaryocyte stage to the mature megakaryocyte stage.

[0032] The term "hematopoietic stem and progenitor cells" refers to a general term for hematopoietic stem cells and / or hematopoietic progenitor cells, which are generally characterized by the presence of the antigenic marker CD34 (CD34+). They can be present in blood (e.g., umbilical cord blood, peripheral blood) and / or immune organs (e.g., bone marrow) and can be differentiated from pluripotent stem cells (ESCs) / iPSCs.

[0033] The term "mononuclear cells" refers to a general term for cells with a single nucleus present in blood (e.g., umbilical cord blood, peripheral blood) and / or immune organs (e.g., bone marrow), including but not limited to lymphocytes (including T cells, B cells, NK cells, NKT cells), monocytes, dendritic cells, etc.

[0034] The term "total nucleated cells" refers to the collective term for all nucleated cells present in blood (e.g., umbilical cord blood, peripheral blood) and / or immune organs (e.g., bone marrow). Total nucleated cells include mononuclear cells and hematopoietic stem and progenitor cells.

[0035] The terms "histone deacetylase inhibitor" and "HDAC inhibitor" are used interchangeably herein and refer to any substance that inhibits histone deacetylase, which can include, for example, a substance that inhibits HDAC expression, a substance that promotes HDAC activity reduction or inactivation, or a substance that promotes HDAC degradation. In non-limiting embodiments, an HDAC inhibitor can be a compound or a combination of compounds. "Small molecules" described herein can refer to such HDAC inhibitors. Exemplary HDAC inhibitors include Resminostat, Remetinostat, TMP 195, TMP269, Bufexamac, Tubastatin A, Pracinostat, Droxinostat, ACY-738, and Tubacin, which can promote the differentiation of starting cells (e.g., hematopoietic stem and progenitor cells, mononuclear cells, or total nucleated cells) into megakaryocytes / megakaryocyte progenitor cells.

[0036] Among them, Tubastatin A (CAS No.: 1252003-15-8) has the structure shown in Formula I:

[0037] ACY-738 (CAS No. 1375465-91-0) has the structure shown in Formula VII:

[0038] The term "JAK2 inhibitor" refers to any substance that inhibits JAK2, which may include, for example, a substance that inhibits JAK2 expression, a substance that promotes a decrease in JAK2 activity or inactivation, or a substance that promotes JAK2 degradation. In a non-limiting embodiment, the JAK2 inhibitor may be a compound or a combination of compounds. The "small molecule" described herein may refer to such a JAK2 inhibitor. Exemplary JAK2 inhibitors include AZD-1480, WP1066, Peficitinib, Fedratinib, Filgotinib, Momelotinib, and Gandotinib, which can promote the differentiation of starting cells (e.g., hematopoietic stem cells, mononuclear cells, or total nucleated cells) into megakaryocytes / megakaryocyte progenitor cells.

[0039] Among them, Filgotinib (CAS No.: 1206161-97-8), Peficitinib (CAS No.: 944118-01-8), Gandotinib (CAS No.: 1229236-86-5), Fedratinib (CAS No.: 936091-26-8) and Momelotinib (CAS No.: 1056634-68-4) have the structures shown in Formula II, Formula III, Formula IV, Formula V, and Formula VI, respectively:

[0040] Those skilled in the art are aware that the HDAC inhibitors and / or JAK2 inhibitors described herein may exist in the form of salts or esters or other types of derivatives without substantially affecting their function in inducing the differentiation of initial cells into megakaryocytes and / or megakaryocyte progenitor cells. Such salts or esters or other types of derivatives are included within the scope of the HDAC inhibitors and / or JAK2 inhibitors described herein. Salts thereof include, but are not limited to, salts formed with inorganic bases, organic bases, inorganic acids, organic acids, or basic or acidic amino acids.

[0041] This application provides the use of an HDAC inhibitor and / or a JAK2 inhibitor to induce differentiation of starting cells into megakaryocytes and / or megakaryocyte progenitor cells, wherein the starting cells are hematopoietic stem cells, mononuclear cells, or total nucleated cells. The starting cells can be derived from bone marrow, peripheral blood, or umbilical cord blood. The hematopoietic stem cells can be hematopoietic stem cells differentiated from pluripotent stem cells (ESCs) / iPSCs. In some embodiments, the HDAC inhibitor and the JAK2 inhibitor can be used in combination. Those skilled in the art will appreciate that "combined use" does not necessarily mean that the two must be used simultaneously. For example, in some embodiments, the HDAC inhibitor and the JAK2 inhibitor can be used sequentially. In some embodiments, the HDAC inhibitor is used first, followed by the simultaneous use of the HDAC inhibitor and the JAK2 inhibitor. In some embodiments, the HDAC inhibitor Tubastatin A or ACY-738 is used in combination with the JAK2 inhibitors Filgotinib, Peficitinib, or Gandotinib. In some embodiments, the HDAC inhibitor Tubastatin A or ACY-738 and the JAK2 inhibitors Filgotinib, Peficinib, or Gandotinib are used sequentially. In some embodiments, the HDAC inhibitor Tubastatin A or ACY-738 is used first, followed by the simultaneous use of the HDAC inhibitor Tubastatin A or ACY-738 and the JAK2 inhibitors Filgotinib, Peficinib, or Gandotinib. In some preferred embodiments, the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib are used in combination. In some preferred embodiments, the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib are used sequentially. In some preferred embodiments, the HDAC inhibitor Tubastatin A is used first, followed by the simultaneous use of the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib.

[0042] The present application also provides a method for inducing differentiation of starting cells into megakaryocytes and / or megakaryocyte progenitor cells, comprising exposing the starting cells to an HDAC inhibitor and / or exposing the starting cells to a JAK2 inhibitor. "Exposure" to a specified substance refers to a state in which the cells have the opportunity to come into contact with the specified substance, including but not limited to introducing the specified substance into the cell's environment (e.g., culture medium).

[0043] It should be understood that when referring to "exposing the starting cells to" a specified substance, the starting cells may undergo a certain degree of change (for example, but not limited to, proliferation and / or differentiation) during the exposure process, and such starting cells that undergo a certain degree of change during the exposure process are also included in the scope of the "starting cells" described in this application. For example, when describing "exposing the starting cells to an HDAC inhibitor and a JAK2 inhibitor in sequence," it means that the starting cells are first exposed to the HDAC inhibitor and then exposed to the JAK2 inhibitor. For another example, when describing "exposing the starting cells to a stem cell factor," such exposure to the stem cell factor can occur before, at the same time as, or after the starting cells are exposed to the HDAC inhibitor.

[0044] In some embodiments, the method comprises exposing the starting cells to an HDAC inhibitor, and exposing the starting cells to a JAK2 inhibitor. In some embodiments, the method comprises exposing the starting cells to an HDAC inhibitor and a JAK2 inhibitor in sequence. In some embodiments, the method comprises exposing the starting cells to the HDAC inhibitor Tubastatin A, and exposing the starting cells to the JAK2 inhibitors Filgotinib or Gandotinib. In some embodiments, the method comprises exposing the starting cells to the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Filgotinib or Gandotinib in sequence. In some embodiments, the method comprises: exposing the starting cells first to an HDAC inhibitor, and then simultaneously exposing the HDAC inhibitor and the JAK2 inhibitor. In some embodiments, the method comprises exposing the starting cells to the HDAC inhibitor Tubastatin A or ACY-738, and exposing the starting cells to the JAK2 inhibitors Filgotinib, Peficinib, or Gandotinib. In some preferred embodiments, the method comprises sequentially exposing the starting cells to the HDAC inhibitor Tubastatin A or ACY-738 and the JAK2 inhibitor Filgotinib, Peficinib, or Gandotinib. In some embodiments, the method comprises: first exposing the starting cells to the HDAC inhibitor Tubastatin A or ACY-738, and then simultaneously exposing the starting cells to the HDAC inhibitor Tubastatin A or ACY-738 and the JAK2 inhibitor Filgotinib, Peficinib, or Gandotinib. In some preferred embodiments, the method comprises exposing the starting cells to the HDAC inhibitor Tubastatin A, and exposing the starting cells to the JAK2 inhibitor Gandotinib. In some preferred embodiments, the method comprises sequentially exposing the starting cells to the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib. In some embodiments, the method comprises: exposing the starting cells to the HDAC inhibitor Tubastatin A first, and then exposing the starting cells to the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib simultaneously.

[0045] In some embodiments, the HDAC inhibitor to which the starting cells are exposed can be present at a concentration of 0.1-1000 nM, such as, but not limited to, 0.1 nM, 1 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM; preferably, at a concentration of 100-1000 nM; more preferably, at a concentration of 200-800 nM. In some embodiments, the JAK2 inhibitor to which the starting cells are exposed can be present at a concentration of 0.1-1000 nM, such as, but not limited to, 0.1 nM, 1 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM; preferably, at a concentration of 100-200 nM; more preferably, at a concentration of about 200 nM.

[0046] In some embodiments, the method may further include exposing the starting cells to stem cell factor, thrombopoietin and / or an interleukin. In some embodiments, the stem cell factor may be present at a concentration of 20-50 ng / mL. In some embodiments, the thrombopoietin may be present at a concentration of 50-100 ng / mL. In some embodiments, the interleukin may be interleukin-3 and / or interleukin-11, which may be present at a concentration of 20-50 ng / mL.

[0047] The present application also provides the use of HDAC inhibitors and / or JAK2 inhibitors in the preparation of drugs for treating diseases that require megakaryocyte and / or platelet transfusions. In some embodiments, the disease is thrombocytopenia. Thrombocytopenia is generally manifested as a decrease in platelet count, which may be accompanied by bleeding symptoms or may not be accompanied by bleeding symptoms but by other symptoms or bleeding risks. Thrombocytopenia may be thrombocytopenia caused by platelet production disorders, such as but not limited to bone marrow suppression or bone marrow failure caused by radiotherapy and chemotherapy; it may also be dilutional thrombocytopenia, such as but not limited to dilutional platelet reduction caused by large-scale transfusion of blood products. In some embodiments, the disease is a platelet dysfunction disease. Platelet dysfunction diseases may be platelet dysfunction, such as but not limited to thrombasthenia, uremia, giant platelet syndrome, severe liver disease, etc.

[0048] The present invention will be described in detail below with reference to the following examples. Unless otherwise specified, the test reagents used in the following examples are all conventional biochemical reagents; and the experimental methods described are all conventional methods unless otherwise specified.

[0049] Example 1: Isolation of starting cells

[0050] 1) Isolation of total nucleated cells from umbilical cord blood:

[0051] A. Gently shake 6% hydroxyethyl starch (HES) (w / v) and add 10 ml to a 50 ml EP tube. Add 30 ml of anticoagulated cord blood (purchased from Shandong Umbilical Cord Blood Bank) to each tube (blood:HES = 3:1), gently shake to mix, let stand at room temperature for 30 minutes, and then centrifuge at 50g for 10 minutes at 20°C to sediment red blood cells.

[0052] B. Use a 50ml EP tube to collect as much supernatant as possible after centrifugation in step A, avoiding aspirating red blood cells;

[0053] C. Centrifuge at 400g for 10 min at 4℃ to collect total nucleated cells for later use.

[0054] 2) Isolation of mononuclear cells: Based on 1), mononuclear cells were separated from total nucleated cells using conventional Ficoll density gradient centrifugation method.

[0055] 3) Isolation of hematopoietic stem and progenitor cells (CD34+ cells): Based on 2), use EasySep TM Human CD34 Positive Selection Kit II (Stem cell) was used to isolate and purify CD34+ cells. The obtained target cells were stained and analyzed by flow cytometry to confirm the purity of CD34+ cells.

[0056] Example 2: Study on the effects of different small molecules on the differentiation of total nucleated cells into megakaryocytes (CD41 / CD61+)

[0057] A. Dissolve Tubastatin A, Peficitinib, Filgotinib, Fedratinib, Momelotinib, and Gandotinib small molecules in DMSO to prepare a 200 mM stock solution. Add a specific volume of small molecule to achieve a final concentration of 200 nM. For the control group, the volume of DMSO added should be consistent with the volume of small molecule added. For example, if 1 mL of 200 μM small molecule stock solution is added to 1 mL of culture medium, add 1 μL of DMSO to 1 mL of culture medium for the control group.

[0058] B. The total nucleated cells obtained in Example 1 were resuspended in SFEMII medium supplemented with stem cell factor (SCF, 20 ng / mL), thrombopoietin (TPO, 50 ng / mL), and interleukin-3 (IL-3, 20 ng / mL), and DMSO, HDAC inhibitor Tubastatin A (concentration of 200 nM), or JAK2 inhibitor (one of peficitinib, filgotinib, fedratinib, momelotinib, or gandotinib, concentration of 200 nM) were added at a concentration of 1×10 6 The cells were placed in a 48-well plate at a density of 3 × 10 5 Cells, volume 300mL. The starting cell number is determined by counting the viable cells using a Countstar cell counter. The specific counting method is to add 20μL of cell suspension to the counting chamber. After inserting the counting chamber, select the cell counting mode to obtain the concentration of the cell suspension.

[0059] C. On day 4 of differentiation induction, the culture medium was replaced with fresh culture medium containing DMSO, HDAC inhibitor Tubastatin A (200 nM), or JAK2 inhibitor (Peficitinib, Filgotinib, Fedratinib, Momelotinib, or Gandotinib, 200 nM).

[0060] D. On the 7th day of differentiation induction, the medium was replaced with SFEMII medium supplemented with thrombopoietin (TPO, 50 ng / mL) and interleukin-11 (IL-11, 20 ng / mL), and DMSO, HDAC inhibitor Tubastatin A (concentration of 200 nM), or JAK2 inhibitor (one of peficitinib, filgotinib, fedratinib, momelotinib, or gandotinib, concentration of 200 nM) were added, and differentiation induction was continued for 7 days.

[0061] E. Harvest cells on day 14 of differentiation induction. Mix the cell suspension in the well plate by pipetting, then pipette 20 μL of the cell suspension into a counting plate. Use a Countstar cell counter to obtain the concentration of the cell suspension, which is then multiplied by the volume to obtain the number of harvested cells. 500,000 cells are collected (if the number is less than 500,000, all cells are used for staining). Resuspend the cells in a PBS buffer containing 0.5% BSA with an antibody cocktail (PE-Cy7-conjugated anti-human CD41 / CD61, BV650-conjugated anti-human CD42b, APC-conjugated anti-human CD45, and Pacific Blue-conjugated anti-human CD3). Mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 min. After incubation, 1 mL of PBS buffer containing 0.5% BSA was added to each tube to wash the cells. The cells were centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS buffer and stained with DAPI before loading. DAPI-negative cells were living cells, while DAPI-positive cells were dead cells.

[0062] As shown in Table 1, compared to the DMSO control group, the addition of either the HDAC inhibitor Tubastatin A or a JAK2 inhibitor promoted the differentiation of total nucleated cells into megakaryocytes, as evidenced by increases in the ratios of CD41 / CD61+ cells and CD41 / CD61+CD42b+ cells. The proportion of T cells was also significantly reduced in the group supplemented with the HDAC inhibitor Tubastatin A, improving the safety of cell transplantation.

[0063] Table 1: Statistics of differentiation of total nucleated cells under the action of different additives:

[0064] The first column represents different experimental groups, including the DMSO control group added throughout the differentiation process (DMSO was selected as a negative control because the other small molecules were dissolved in DMSO), the HDAC inhibitor Tubastatin A, the JAK2 inhibitor Peficitinib, the JAK2 inhibitor Filgotinib, the JAK2 inhibitor Fedratinib, the JAK2 inhibitor Momelotinib, and the JAK2 inhibitor Gandotinib. The second and third columns are the number of total nucleated cells at the initial inoculation and the number of cells harvested on the 14th day of differentiation, respectively. The more cells harvested, the better. The fourth, fifth, and sixth columns represent the proportion of different cell subtypes in the harvested cells. CD41 / CD61+ represents megakaryocytes and megakaryocyte progenitors, CD41 / CD61+CD42b+ represents mature megakaryocytes, and CD3+ represents T cells. The proportions of different cell subpopulations were obtained by staining and flow cytometry analysis.

[0065] Example 3: Optimization of small molecule action mode

[0066] From Example 2, it can be found that the HDAC inhibitor Tubastatin A and some JAK2 inhibitors (Peficitinib, Filgotinib, and Gandotinib) play an important regulatory role in the differentiation of total nucleated cells into megakaryocytes. Next, we want to compare the effects of using small molecules alone and in combination. The specific steps are as follows:

[0067] A. Dissolve Tubastatin A, Peficitinib, Filgotinib, and Gandotinib in DMSO to prepare a 200 μM stock solution. Add a specific volume of the small molecule to achieve a final concentration of 200 nM. For the control group, use the same volume of DMSO as the small molecule. For example, if 1 μL of 200 μM small molecule stock solution is added to 1 mL of culture medium, add 1 μL of DMSO to 1 mL of culture medium.

[0068] B. The total nucleated cells obtained in Example 1 were resuspended in SFEMII medium supplemented with stem cell factor (SCF, 20 ng / mL), thrombopoietin (TPO, 50 ng / mL), and interleukin-3 (IL-3, 20 ng / mL), and DMSO, HDAC inhibitor Tubastatin A (concentration of 200 nM), or JAK2 inhibitor (Filgotinib or Gandotinib, concentration of 200 nM) were added at a concentration of 1×10 6 The differentiation was started at a cell density of 10 / mL and 2.5×10 5 Cells, volume 250 μL. The starting cell number is determined by counting the viable cells using a Countstar cell counter. The specific counting method is to add 20 μL of cell suspension to the counting chamber. After inserting the counting chamber, select the cell counting mode to obtain the concentration of the cell suspension.

[0069] C. On the 4th day of differentiation induction, the culture medium was replaced with fresh culture medium containing different small molecules;

[0070] D. On the 7th day of differentiation induction, the medium was replaced with SFEMII medium supplemented with thrombopoietin (TPO, 50 ng / mL) and interleukin-11 (IL-11, 20 ng / mL), and DMSO, HDAC inhibitor Tubastatin A (concentration of 200 nM), or JAK2 inhibitor (Filgotinib or Gandotinib, concentration of 200 nM) were added, and differentiation was continued for 7 days.

[0071] E. Harvest cells on day 14 of differentiation induction. Mix the cell suspension in the well plate by pipetting, then pipette 20 μL of the cell suspension into a counting plate. Use a Countstar cell counter to obtain the concentration of the cell suspension, which is then multiplied by the volume to obtain the number of harvested cells. 500,000 cells are collected (if the number is less than 500,000, all cells are used for staining). Resuspend the cells in a PBS buffer containing 0.5% BSA with an antibody cocktail (PE-Cy7-conjugated anti-human CD41 / CD61, BV650-conjugated anti-human CD42b, APC-conjugated anti-human CD45, and Pacific Blue-conjugated anti-human CD3). Mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 min. After incubation, 1 mL of PBS buffer containing 0.5% BSA was added to each tube to wash the cells. The cells were centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS buffer and stained with DAPI before loading. DAPI-negative cells were living cells, while DAPI-positive cells were dead cells.

[0072] As shown in Table 2, the HDAC inhibitor Tubastatin A and the JAK2 inhibitors (Filgotinib and Gandotinib) alone can promote the differentiation of total nucleated cells into megakaryocytes, as reflected in the increase in the ratios of CD41 / CD61+ cells and CD41 / CD61+CD42b+ cells compared with the control group. The combination of the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib has advantages over other groups in terms of differentiation efficiency, harvested cell number, and control of T cell proportion.

[0073] Table 2: Statistics of differentiation of total nucleated cells under the action of different small molecule additives:

[0074] The first column represents the different experimental groups, including the DMSO control group, the HDAC inhibitor Tubastatin A, the JAK2 inhibitor Filgotinib, or the JAK2 inhibitor Gandotinib, which were added throughout the differentiation process. Tubastatin A+Filgotinib indicates that the HDAC inhibitor Tubastatin A was added during the first 7 days of differentiation and the JAK2 inhibitor Filgotinib was added during the last 7 days of differentiation. Tubastatin A+Gandotinib indicates that the HDAC inhibitor Tubastatin A was added during the first 7 days of differentiation and the JAK2 inhibitor Gandotinib was added during the last 7 days of differentiation. The small molecule concentration was 200 nM in both groups. The second and third columns, respectively, represent the number of total nucleated cells initially seeded and the number of cells harvested on day 14 of differentiation. The greater the number of cells harvested, the greater the advantage. The fourth, fifth, and sixth columns represent the proportions of different cell subtypes in the harvested cells. CD41 / CD61+ represents megakaryocytes and megakaryocyte progenitors, CD41 / CD61+CD42b+ represents mature megakaryocytes, and CD3+ represents T cells. The proportions of different cell subpopulations were determined by staining and flow cytometry analysis.

[0075] Example 4: Optimization of small molecule concentration

[0076] From Example 3, we found that the combination of the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib can efficiently induce the differentiation of total nucleated cells into megakaryocytes, and has good effects on the control of harvested cell number and T cell ratio. Next, we want to further improve the effect by optimizing the concentration of small molecules. The specific steps are as follows:

[0077] A. Dissolve Tubastatin A and Gandotinib in DMSO to prepare a 200 μM stock solution. Add the small molecules to the final concentrations shown in Table 3. For the control group, the volume of DMSO added should be consistent with the volume of small molecules added. For example, if 1 μL of 200 μM small molecule stock solution is added to 1 mL of culture medium, add 1 μL of DMSO to the control group.

[0078] B. The total nucleated cells obtained in Example 1 were resuspended in SFEMII medium supplemented with stem cell factor (SCF, 20 ng / mL), thrombopoietin (TPO, 50 ng / mL), and interleukin-3 (IL-3, 20 ng / mL), and DMSO or different concentrations of HDAC inhibitor Tubastatin A were added to the suspension at a concentration of 1×10 6 The differentiation was started at a cell density of 5 × 10 / mL in 24 wells.5 Cells, volume 500 μL. The starting cell number is determined by counting the viable cells using a Countstar cell counter. The specific counting method is to add 20 μL of cell suspension to the counting chamber. After inserting the counting chamber, select the cell counting mode to obtain the concentration of the cell suspension.

[0079] C. On the fourth day of differentiation induction, the culture medium was replaced with fresh culture medium containing DMSO or different concentrations of the HDAC inhibitor Tubastatin A;

[0080] D. On the 7th day of differentiation induction, the medium was replaced with SFEMII medium supplemented with thrombopoietin (TPO, 50 ng / mL) and interleukin-11 (IL-11, 20 ng / mL), and DMSO or different concentrations of the JAK2 inhibitor Gandotinib were added, and differentiation induction was continued for another 7 days.

[0081] E. Harvest cells on day 14 of differentiation induction. Mix the cell suspension in the well plate by pipetting, then pipette 20 μL of the cell suspension into a counting plate. Use a Countstar cell counter to obtain the concentration of the cell suspension, which is then multiplied by the volume to obtain the number of harvested cells. 500,000 cells are collected (if the number is less than 500,000, all cells are used for staining). Resuspend the cells in a PBS buffer containing 0.5% BSA with an antibody cocktail (PE-Cy7-conjugated anti-human CD41 / CD61, BV650-conjugated anti-human CD42b, APC-conjugated anti-human CD45, and Pacific Blue-conjugated anti-human CD3). Mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 min. After incubation, 1 mL of PBS buffer containing 0.5% BSA was added to each tube to wash the cells. The cells were centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS buffer and stained with DAPI before loading. DAPI-negative cells were living cells, while DAPI-positive cells were dead cells.

[0082] As can be seen from the results in Table 3, when the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib are used in combination, the differentiation effect is enhanced with increasing concentration. The best effect is achieved under the treatment conditions of 200nM HDAC inhibitor Tubastatin A and 200nM Gandotinib (high megakaryocyte ratio and low T cell ratio), indicating that the effects of these two small molecules are concentration-dependent.

[0083] Table 3: Statistics of differentiation of total nucleated cells under the action of small molecule additives at different concentrations:

[0084] The first column represents the different experimental groups: the control group, which received DMSO throughout the differentiation process; the remaining experimental groups, which received the HDAC inhibitor Tubastatin A for the first 7 days of differentiation and the JAK2 inhibitor Gandotinib for the last 7 days of differentiation. The number 20+20 in the table indicates that both the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib were added at a concentration of 20 nM. The same applies to the remaining groups. Units are all nM. The second and third columns represent the total number of nucleated cells seeded at the start and the number of cells harvested on day 14 of differentiation, respectively. A higher number of cells harvested is preferred. The fourth, fifth, and sixth columns represent the proportions of different cell subtypes in the harvested cells. CD41 / CD61+ represents megakaryocytes and megakaryocyte progenitors, CD41 / CD61+CD42b+ represents mature megakaryocytes, and CD3+ represents T cells. The proportions of different cell subpopulations were determined by staining and flow cytometry analysis.

[0085] Example 5: Optimization of the concentration of the HDAC inhibitor Tubastatin A small molecule

[0086] From Example 4, it can be found that the combination of HDAC inhibitor Tubastatin A and JAK2 inhibitor Gandotinib can achieve high-efficiency differentiation of total nucleated cells into megakaryocytes at a treatment concentration of 200 nM. In order to further improve the differentiation effect, the concentration of HDAC inhibitor Tubastatin A was optimized. The specific steps are as follows:

[0087] A. Dissolve Tubastatin A and Gandotinib in DMSO to prepare a 200 μM stock solution. Add the small molecules to the final concentrations shown in Table 4. For the control group, the volume of DMSO added should be consistent with the volume of small molecules added. For example, if 1 μL of 200 μM small molecule stock solution is added to 1 mL of culture medium, add 1 μL of DMSO to the control group.

[0088] B. The total nucleated cells obtained in Example 1 were resuspended in SFEMII medium supplemented with stem cell factor (SCF, 20 ng / mL), thrombopoietin (TPO, 50 ng / mL), and interleukin-3 (IL-3, 20 ng / mL), and DMSO or different concentrations of HDAC inhibitor Tubastatin A were added to the suspension at a concentration of 1×10 6 The cells were placed in a 48-well plate at a density of 3 × 10 5Cells, volume 300 μL. The starting cell number is determined by counting the viable cells using a Countstar cell counter. The specific counting method is to add 20 μL of cell suspension to the counting chamber. After inserting the counting chamber, select the cell counting mode to obtain the concentration of the cell suspension.

[0089] C. On the fourth day of differentiation induction, the culture medium was replaced with fresh culture medium containing DMSO or different concentrations of the HDAC inhibitor Tubastatin A;

[0090] D. On the 7th day of differentiation induction, the medium was replaced with SFEMII medium supplemented with thrombopoietin (TPO, 50 ng / mL) and interleukin-11 (IL-11, 20 ng / mL), and DMSO or JAK2 inhibitor Gandotinib (concentration of 200 nM) was added, and differentiation induction was continued for another 7 days.

[0091] E. Harvest cells on day 14 of differentiation induction. Mix the cell suspension in the well plate by pipetting, then pipette 20 μL of the cell suspension into a counting plate. Use a Countstar cell counter to obtain the concentration of the cell suspension, which is then multiplied by the volume to obtain the number of harvested cells. 500,000 cells are collected (if the number is less than 500,000, all cells are used for staining). Resuspend the cells in a PBS buffer containing 0.5% BSA with an antibody cocktail (PE-Cy7-conjugated anti-human CD41 / CD61, BV650-conjugated anti-human CD42b, APC-conjugated anti-human CD45, and Pacific Blue-conjugated anti-human CD3). Mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 min. After incubation, 1 mL of PBS buffer containing 0.5% BSA was added to each tube to wash the cells. The cells were centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS buffer and stained with DAPI before loading. DAPI-negative cells were living cells, while DAPI-positive cells were dead cells.

[0092] As can be seen from the results in Table 4, when the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib are used in combination, the ratio of megakaryocytes (CD41 / CD61+) gradually increases with the increase in the concentration of the HDAC inhibitor Tubastatin A, reaching saturation at 800 nM. When increased to 1000 nM, the effect is equivalent to that of 800 nM. From the perspective of cell harvest number and T cell ratio control, 800 nM has a better effect.

[0093] Table 4: Statistics of differentiation of total nucleated cells under the action of different concentrations of HDAC inhibitor Tubastatin A:

[0094] The first column represents the different experimental groups: the control group, which received DMSO throughout the differentiation process; the remaining experimental groups, which received the HDAC inhibitor Tubastatin A for the first 7 days of differentiation and the JAK2 inhibitor Gandotinib for the last 7 days of differentiation. The numbers 200+200 in the table indicate that both the HDAC inhibitor Tubastatin A and the JAK2 inhibitor Gandotinib were added at 200 nM concentrations, and the same applies to the remaining groups. Units are all nM. The second and third columns represent the total number of nucleated cells seeded at the start and the number of cells harvested on day 14 of differentiation, respectively. A higher number of cells harvested is preferred. The fourth, fifth, and sixth columns represent the proportions of different cell subtypes in the harvested cells. CD41 / CD61+ represents megakaryocytes and megakaryocyte progenitors, CD41 / CD61+CD42b+ represents mature megakaryocytes, and CD3+ represents T cells. The proportions of different cell subpopulations were determined by staining and flow cytometry analysis.

[0095] Example 6: Effects of different HDAC inhibitor small molecules on the differentiation of total nucleated cells into megakaryocytes (CD41 / CD61+)

[0096] In order to explore the regulatory effects of different HDAC inhibitors on the differentiation of total nucleated cells into megakaryocytes, the following experiments were conducted:

[0097] A. Dissolve the HDAC inhibitor small molecule in DMSO to prepare a stock solution at a concentration of 800 μM. When using, add a certain volume of the small molecule to achieve a final concentration of 800 nM. For the control group, the volume of DMSO added should be the same as the volume of the small molecule added. For example, if 1 μL of 800 μM small molecule stock solution is added to 1 mL of culture medium, the control group should add 1 μL of DMSO to 1 mL of culture medium.

[0098] B. The total nucleated cells obtained in Example 1 were resuspended in SFEMII medium supplemented with stem cell factor (SCF, 20 ng / mL), thrombopoietin (TPO, 50 ng / mL), and interleukin-3 (IL-3, 20 ng / mL), and DMSO or different concentrations of HDAC inhibitors were added to the suspension at a concentration of 1×10 6 The cells were placed in a 48-well plate at a density of 3 × 10 5 Cells, volume 300 μL. The starting cell number is determined by counting the viable cells using a Countstar cell counter. The specific counting method is to add 20 μL of cell suspension to the counting chamber. After inserting the counting chamber, select the cell counting mode to obtain the concentration of the cell suspension.

[0099] C. On the 4th day of differentiation induction, the culture medium was replaced with fresh culture medium containing DMSO or HDAC inhibitors;

[0100] D. On the 7th day of differentiation induction, the medium was replaced with SFEMII medium supplemented with thrombopoietin (TPO, 50 ng / mL) and interleukin-11 (IL-11, 20 ng / mL), and DMSO, HDAC inhibitor (concentration of 800 nM), or JAK2 inhibitor Gandotinib (concentration of 200 nM) were added, and differentiation induction was continued for 7 days.

[0101] E. Harvest cells on day 14 of differentiation induction. Mix the cell suspension in the well plate by pipetting, then pipette 20 μL of the cell suspension into a counting plate. Use a Countstar cell counter to obtain the concentration of the cell suspension, which is then multiplied by the volume to obtain the number of harvested cells. 500,000 cells are collected (if the number is less than 500,000, all cells are used for staining). Resuspend the cells in a PBS buffer containing 0.5% BSA with an antibody cocktail (PE-Cy7-conjugated anti-human CD41 / CD61, BV650-conjugated anti-human CD42b, APC-conjugated anti-human CD45, and Pacific Blue-conjugated anti-human CD3). Mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 min. After incubation, 1 mL of PBS buffer containing 0.5% BSA was added to each tube to wash the cells. The cells were centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS buffer and stained with DAPI before loading. DAPI-negative cells were living cells, while DAPI-positive cells were dead cells.

[0102] As shown in Table 5, different HDAC inhibitors can improve the differentiation of total nucleated cells into megakaryocytes. In addition, some HDAC inhibitors are more effective when used in combination with the JAK2 inhibitor Gandotinib. For example, the total number of harvested cells when Droxinostat and Gandotinib are used in combination is greater than when Droxinostat is used alone.

[0103] Table 5: Statistics of differentiation of total nucleated cells under the action of different HDAC inhibitor additives:

[0104] The first column represents the different experimental groups: a control group in which DMSO was added throughout the differentiation process; subsequent experimental groups in which different HDAC inhibitors, TMP195, Remetinostat, TMP269, Bufexamac, Droxinostat, or Tubacin, were added throughout the differentiation process; and experimental groups with plus signs indicate that different HDAC inhibitors were added during the first 7 days of differentiation and the JAK2 inhibitor Gandotinib was added during the last 7 days. The second and third columns, respectively, represent the total number of nucleated cells seeded at the start and the number of cells harvested on day 14 of differentiation, with a higher number of cells harvested being more advantageous. The fourth, fifth, and sixth columns represent the proportions of different cell subtypes in the harvested cells: CD41 / CD61+ represents megakaryocytes and megakaryocyte progenitors, CD41 / CD61+CD42b+ represents mature megakaryocytes, and CD3+ represents T cells. The proportions of different cell subpopulations were determined by staining and flow cytometry analysis.

[0105] Example 7: Effects of different HDAC inhibitor small molecules combined with different JAK2 inhibitors on the differentiation of total nucleated cells into megakaryocytes (CD41 / CD61+)

[0106] To explore the regulatory effects of different combinations of HDAC inhibitors and JAK2 inhibitors on the differentiation of total nucleated cells into megakaryocytes, the following experiments were conducted:

[0107] A. Dissolve the HDAC inhibitor and JAK2 inhibitor small molecules in DMSO to prepare stock solutions at 800 μM or 200 μM. Add a certain volume of small molecules according to the final concentration.

[0108] B. The total nucleated cells obtained in Example 1 were resuspended in SFEMII medium supplemented with stem cell factor (SCF, 20 ng / mL), thrombopoietin (TPO, 50 ng / mL), and interleukin-3 (IL-3, 20 ng / mL), and DMSO or different concentrations of HDAC inhibitors were added to the suspension at a concentration of 1×10 6 The cells were placed in a 48-well plate at a density of 3 × 10 5 Cells, volume 300 μL. The starting cell number is determined by counting the viable cells using a Countstar cell counter. The specific counting method is to add 20 μL of cell suspension to the counting chamber. After inserting the counting chamber, select the cell counting mode to obtain the concentration of the cell suspension.

[0109] C. On the 4th day of differentiation induction, the culture medium was replaced with fresh culture medium containing HDAC inhibitors.

[0110] D. On the 7th day of differentiation induction, the culture medium was changed to SFEMII medium supplemented with thrombopoietin (TPO, 50 ng / mL) and interleukin-11 (IL-11, 20 ng / mL), and a JAK2 inhibitor was added alone or simultaneously. The differentiation was continued for 7 days.

[0111] E. Harvest cells on day 14 of differentiation induction. Mix the cell suspension in the well plate by pipetting, then pipette 20 μL of the cell suspension into a counting plate. Use a Countstar cell counter to obtain the concentration of the cell suspension, which is then multiplied by the volume to obtain the number of harvested cells. 500,000 cells are collected (if the number is less than 500,000, all cells are used for staining). Resuspend the cells in a PBS buffer containing 0.5% BSA with an antibody cocktail (PE-Cy7-conjugated anti-human CD41 / CD61, BV650-conjugated anti-human CD42b, APC-conjugated anti-human CD45, and Pacific Blue-conjugated anti-human CD3). Mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 min. After incubation, 1 mL of PBS buffer containing 0.5% BSA was added to each tube to wash the cells. The cells were centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS buffer and stained with DAPI before loading. DAPI-negative cells were living cells, while DAPI-positive cells were dead cells.

[0112] The results in Table 6 show that, except for the combination of Tubastatin A-800 and Peficinib-200, the other small molecule combinations can all enhance the differentiation of total nucleated cells into megakaryocytes. In addition, the number of harvested cells shows that the simultaneous addition of HDAC inhibitors and JAK2 inhibitors on the last 7 days of differentiation is more advantageous than the experimental group in which only JAK2 inhibitors were added. For example, the number of harvested cells in experimental groups 5, 6, 7, and 8 was greater than that in experimental groups 1, 2, 3, and 4, and the number of harvested cells in experimental groups 13 and 15 was greater than that in experimental groups 9 and 11.

[0113] Table 6: Statistics of differentiation of total nucleated cells under the action of different inhibitor additives:

[0114] The second column represents the different experimental groups. HDAC inhibitors Tubastatin A or ACY-738 were added during the first 7 days of differentiation, and JAK2 inhibitors Gandotinib or Peficinib were added alone or simultaneously during the last 7 days of differentiation (experimental groups 5-8 and 13-16 had both inhibitors added during the last 7 days). The numbers following the different small molecules in the table indicate the concentration of the small molecule, all in nM. The third and fourth columns represent the total number of nucleated cells at the initial inoculation and the number of cells harvested on day 14 of differentiation, respectively. A higher number of cells harvested is more advantageous. The fifth, sixth, and seventh columns represent the proportion of different cell subtypes in the harvested cells. CD41 / CD61+ represents megakaryocytes and megakaryocyte progenitors, CD41 / CD61+CD42b+ represents mature megakaryocytes, and CD3+ represents T cells. The proportions of different cell subpopulations were obtained by staining and flow cytometry analysis.

[0115] In addition, it can be seen from the above experimental data that HADC inhibitors, when used alone or in combination with JAK2 inhibitors, can improve the differentiation of total nucleated cells into megakaryocytes to a certain extent. For example, Tubastatin A, Bufexamac, Droxinostat, Tubacin, and ACY-738 are HDAC6 inhibitors, TMP195 and TMP269 are inhibitors of HDAC4, HDAC5, HDAC7, and HDAC9, and Remetinostat is a hydroxamic acid-based HDAC inhibitor. Such small molecule inhibitors targeting different HDAC targets can all improve the differentiation efficiency of megakaryocytes to a certain extent. Based on the disclosure of the present invention, those skilled in the art can foresee that small molecule inhibitors with the same or similar targets as the above-mentioned HADC inhibitors can also improve the differentiation efficiency of megakaryocytes to a certain extent. For example, small molecule inhibitors that also target HDAC6 include Tubastatin A Hydrochloride, Resminostat Hydrochloride, ACY-775, WT-161, HPOB, BRD73954, Romidepsin, Ricolinostat, CAY10603, Nexturastat A, TH34, Citarinostat, ACY-1083, TYA-018, SW-100, KA2507, J22352, SS-208, T-518, and CG347B. Another example is small molecule inhibitors that share the same target as TMP195, such as YF479, YX968, KD 5170, Leuxinostat, Vorinostat, Psammaplin A, JPS016, DLC-50, and TNG260. Using small molecule inhibitors with different targets as HDAC inhibitors for the purposes defined herein falls within the scope of protection of the present invention.

[0116] Similarly, the experimental data of the present invention also show that JAK2 inhibitors, when used alone or in combination with HADC inhibitors, can significantly enhance the differentiation of total nucleated cells into megakaryocytes. Therefore, it is foreseeable that small molecule inhibitors targeting JAK2, such as Upadacitinib, CHZ868, Delgocitinib, NSC 42834, TG101209, Brepocitinib, Ilginatinib, and Flonoltinib, can also improve the differentiation efficiency of megakaryocytes to a certain extent. The use of small molecule inhibitors targeting JAK2 as JAK2 inhibitors for the applications defined in the present invention falls within the scope of protection of the present invention.

[0117] Although the above content refers to a particular preferred embodiment, it should be understood that the present invention is not limited thereto. Those skilled in the art will recognize that various modifications can be made to the disclosed embodiments, and these modifications are also intended to fall within the scope of the present invention.

Claims

1. Use of a histone deacetylase (HDAC) inhibitor and / or a JAK2 inhibitor in inducing the differentiation of starting cells into megakaryocytes and / or megakaryocyte progenitor cells, wherein the starting cells are hematopoietic stem and progenitor cells, mononuclear cells or total nucleated cells.

2. The use according to claim 1, wherein the starting cells are total nucleated cells.

3. Use of an HDAC inhibitor and / or a JAK2 inhibitor in the preparation of a medicament for treating a disease requiring megakaryocyte and / or platelet transfusion. The use according to claim 3 , wherein the disease is thrombocytopenia or platelet dysfunction disease.

5. The use according to any one of claims 1 to 4, wherein the HDAC inhibitor and the JAK2 inhibitor are used in combination.

6. The use according to any one of claims 1 to 5, wherein the HDAC inhibitor and the JAK2 inhibitor are used sequentially; or, the HDAC inhibitor is used first, and then the HDAC inhibitor and the JAK2 inhibitor are used simultaneously.

7. The use according to any one of claims 1 to 6, wherein the HDAC inhibitor is one or more of Resminostat, Remetinostat, TMP 195, TMP269, Bufexamac, Tubastatin A, Pracinostat, Droxinostat, ACY-738 and Tubacin.

8. The use according to claim 7, wherein the HDAC inhibitor is Tubastatin A or ACY-738.

9. The use according to any one of claims 1 to 8, wherein the JAK2 inhibitor is one or more of AZD-1480, WP1066, Peficitinib, Fedratinib, Filgotinib, Momelotinib and Gandotinib.

10. The use according to claim 9, wherein the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, Fedratinib, Momelotinib and Gandotinib.

11. The use according to claim 10, wherein the JAK2 inhibitor is one or more of Filgotinib, Peficitinib and Gandotinib.

12. The use according to claim 11, wherein the JAK2 inhibitor is Gandotinib.

13. A method for inducing initial cells to differentiate into megakaryocytes and / or megakaryocyte progenitor cells, comprising exposing the initial cells to an HDAC inhibitor, and / or exposing the initial cells to a JAK2 inhibitor, wherein the initial cells are hematopoietic stem and progenitor cells, mononuclear cells, or total nucleated cells.

14. The method of claim 13, comprising exposing the starting cell to an HDAC inhibitor, and exposing the starting cell to a JAK2 inhibitor.

15. The method according to claim 13 or 14, comprising: The starting cells are exposed to the HDAC inhibitor and the JAK2 inhibitor in sequence; alternatively, the starting cells are exposed to the HDAC inhibitor first and then exposed to the HDAC inhibitor and the JAK2 inhibitor simultaneously.

16. The method according to any one of claims 13-15, wherein the HDAC inhibitor is one or more of Resminostat, Remetinostat, TMP 195, TMP269, Bufexamac, Tubastatin A, Pracinostat, Droxinostat, ACY-738 and Tubacin.

17. The method of claim 16, wherein the HDAC inhibitor is Tubastatin A or ACY-738.

18. The method according to any one of claims 13 to 17, wherein the HDAC inhibitor is present at a concentration of 0.1-1000 nM, preferably at a concentration of 100-1000 nM, more preferably at a concentration of 200-800 nM.

19. The method of claim 18, wherein the JAK2 inhibitor is one or more of AZD-1480, WP1066, Peficitinib, Fedratinib, Filgotinib, Momelotinib, and Gandotinib.

20. The use according to claim 19, wherein the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, Fedratinib, Momelotinib and Gandotinib.

21. The method of claim 20, wherein the JAK2 inhibitor is one or more of Filgotinib, Peficitinib, and Gandotinib.

22. The method of claim 21, wherein the JAK2 inhibitor is Gandotinib.

23. The method according to any one of claims 13-22, wherein the JAK2 inhibitor is present at a concentration of 0.1-1000 nM, preferably at a concentration of 100-200 nM, more preferably at a concentration of about 200 nM.

24. The method of any one of claims 13-23, wherein the starting cells are total nucleated cells.

25. The method of any one of claims 13-24, further comprising exposing the starting cells to stem cell factor, thrombopoietin, and / or interleukins.

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