Method for preparing megakaryocytes

Abstract

Provided is a method for producing megakaryocytes having a platelet production ability, the method including the step of increasing the amount of at least one of the proteins constituting ubiquinol-cytochrome c reductase (UQCR) in the megakaryocytes.

Description

Technical Field

[0001] This invention relates to a method for preparing megakaryocytes, megakaryocytes produced by the method, and the uses of the megakaryocytes. Background Technology

[0002] Treatment or surgical procedures for blood-related diseases require a large number of blood cells. Among blood cells, platelets, essential for blood clotting or hemostasis, are particularly important. Platelets are in high demand in leukemia, bone marrow transplantation, and cancer treatment, making a stable supply crucial. To date, platelets have been collected not only through donor blood donations but also through methods such as administering TPO-like structural (mimicry) preparations and differentiating megakaryocytes from umbilical cord blood or bone marrow cells. Recently, techniques have been developed to induce the differentiation of pluripotent stem cells, such as embryonic stem cells (ES cells) and artificial pluripotent stem cells (iPS cells), in vitro and to modulate blood cells, including platelets.

[0003] To meet societal demand for platelets, the inventors have developed a technique for the stable in vitro production of platelet preparations using human iPS cells (Non-Patent Literature 1, 2). The inventors have established the following technique: introducing two factors (MYC / BMI1 or MYC / BCL-XL) or three factors (MYC / BMI1 / BCL-XL) into hematopoietic progenitor cells derived from human iPS cells to establish megakaryocyte lines, and maturing these megakaryocyte lines to produce large quantities of platelets (artificial platelet preparations derived from human iPS cells) (Patent Literature 1-4, Non-Patent Literature 1). Furthermore, culture conditions (fluid dynamic requirements, especially turbulent stimulation) that significantly increase platelet production during megakaryocyte maturation have been reported (Patent Literature 5, Non-Patent Literature 2).

[0004] Existing technical documents Patent documents Patent Document 1: International Publication No. 2011 / 034073; Patent Document 2: International Publication No. 2012 / 157586; Patent Document 3: International Publication No. 2014 / 123242; Patent document 4: International Publication No. 2021 / 075568; Patent Document 5: International Publication No. 2019 / 009364; Non-patent literature Non-patent literature 1: Cell Stem Cell. April 3, 2014; 14(4); 535-548; Non-patent literature 2: Cell. July 26, 2018; 174(3); 636-648. Summary of the Invention

[0005] The problem that the invention aims to solve However, the inventors have discovered the following problem: megakaryocytes (lines) induced by the above-mentioned existing differentiation induction methods show a decrease in platelet production capacity with passage, and the degree of decrease varies greatly among pluripotent stem cell lines derived from hematopoietic progenitor cells. Figure 1 Therefore, the objective of this invention is to provide a method for megakaryocytes induced by existing induction methods to proliferate while maintaining their platelet-producing capacity.

[0006] Methods for solving problems The inventors have discovered that megakaryocytes that are forced to express the core proteins (UQCRC1 and UQCRC2) of complex III (panthenol-cytochrome c reductase), which is responsible for the electron transport chain in mitochondria, still maintain platelet production capacity even after long-term passage culture.

[0007] Furthermore, it was found that adding adipocyte differentiation inducing factors (dexamethasone, IBMX, insulin) during the proliferation phase of megakaryocytes forcibly expressing UQCRC2 increased platelet production by approximately 1.5 times; further addition of JI051 increased platelet production by approximately 2 times. Additionally, it was found that adding HDAC5 (histone deacetylase 5) inhibitors or LSD1 (lysine-specific histone demethylase) inhibitors during the megakaryocyte proliferation phase maintained CD34 / CD41 co-positivity in megakaryocytes and preserved platelet production even after long-term culture.

[0008] The inventors verified the proliferative capacity of megakaryocytes derived from iPS cell lines. The results showed that, in megakaryocytes induced by the introduction of two megakaryocyte-inducing factors (i.e., the MYCdd gene and the BCL2L1 gene), the proliferative capacity of megakaryocytes differed depending on the iPS cell line origin. In investigating the cause of this difference, it was determined that a protein constituting the multicomb repressor complex 1.1 is related to megakaryocyte proliferative capacity; knocking down this protein successfully enhanced megakaryocyte proliferative capacity.

[0009] Furthermore, to further explore factors contributing to megakaryocyte proliferation, multiple transcription factors involved in hematopoietic lineage differentiation were analyzed. Unexpectedly, it was discovered that overexpression of BACH1, which contributes to erythropoiesis and lymphocyte production, could enhance megakaryocyte proliferation and platelet production. Based on these insights, the inventors conducted further and repeated studies, resulting in this invention.

[0010] That is, the present invention is as follows.

[0011] [1] A method for preparing megakaryocytes with platelet-producing capacity, comprising the step of increasing the amount of at least one of the proteins constituting ubiquitin-cytochrome c reductase (UQCR) in megakaryocytes.

[0012] [2] According to the method described in [1], at least one of the proteins constituting UQCR is a UQCR core protein (UQCRC).

[0013] [3] According to the method described in [2], at least one of the UQCRCs is UQCRC2.

[0014] [4-1] The method according to any one of [1] to [3], wherein the step of increasing the amount of the protein constituting the UQCR includes the step of introducing nucleic acid encoding the protein into megakaryocytes.

[0015] [4-2] The method according to any one of [1] to [4-1], wherein the nucleic acid is introduced via a viral vector.

[0016] [5-1] A method for preparing megakaryocytes that maintain platelet production capacity, comprising the step of inhibiting the function of polycomb inhibitory complex 1.1 in megakaryocytes.

[0017] [5-2] According to the method of [5-1], the step of inhibiting the function of the polycomb repressor complex 1.1 includes the step of reducing the amount of at least one of the proteins constituting the polycomb repressor complex 1.1.

[0018] [6] The method according to any one of [1] to [4-2] includes the step of inhibiting the function of the polycomb inhibitory complex 1.1 in megakaryocytes.

[0019] [7] According to the method of [6], the step of inhibiting the function of the polycomb repressor complex 1.1 includes the step of reducing the amount of at least one of the proteins constituting the polycomb repressor complex 1.1.

[0020] [8] According to the method of [5-2] or [7], wherein the protein constituting the polycomb inhibitory complex 1.1 is at least one selected from BCOR protein, BCORL1 protein and TRIM27 protein.

[0021] [9-1] The method according to any one of [1] to [8] includes the step of increasing the amount of BACH1 protein present in megakaryocytes.

[0022] [9-2] The method according to any one of [1] to [9-1] includes the steps of reducing the presence of (1) BCOR protein or BCORL1 protein and (2) TRIM27 protein in megakaryocytes and increasing the presence of (3) BACH1 protein.

[0023] [10-1] The method according to any one of [1] to [9-2] includes the step of culturing megakaryocytes in a culture medium containing an HDAC5 inhibitor.

[0024] [10-2] According to the method of [10-1], wherein at least one of the HDAC5 inhibitors is LMK235.

[0025] [11-1] The method according to any one of [1] to [10-2] includes the step of culturing megakaryocytes in a culture medium containing adipocyte differentiation inducing factor.

[0026] [11-2] The method according to any one of [1] to [11-1], wherein the adipogenic factor is a combination of dexamethasone, IBMX and insulin.

[0027]

[12] The method according to any one of [1] to [11-2], wherein the culture medium contains a compound (JI051) with the following structural formula.

[0028] [Chemical Formula 1]

[13] The method according to any one of [1] to

[12] includes the step of increasing the amount of MYC protein and apoptosis inhibitor protein in megakaryocytes.

[0029] [14-1] The method according to any one of [1] to

[13] , wherein the megakaryocytes are derived from pluripotent stem cells.

[0030] [14-2] The method according to any one of [1] to [14-1], wherein the megakaryocytes express the CD34 gene and the CD41 gene.

[0031] [14-3] The method according to any one of [1] to [14-2], wherein the megakaryocytes are derived from humans.

[0032]

[15] A megakaryocyte obtained by any one of [1] to [14-3].

[0033] [16-1] A megakaryocyte having all of the following characteristics (A) to (C).

[0034] (A) Having at least one of the exogenous proteins that constitute ubiquitin-cytochrome c reductase (UQCR); (B) Possesses the ability to produce platelets; (C) Expression of CD34 and CD41 genes.

[0035] [16-2] The megakaryocyte according to [16-1] also has at least one of the following features (D) to (F).

[0036] (D) Derived from pluripotent stem cells; (E) Contains exogenous megakaryocyte inducing factors; (F) Has an inhibitor of expression of the TP53 gene and / or CDKN1A gene.

[0037] [17-1] The megakaryocyte according to [16-1] or [16-2], wherein at least one of the proteins constituting the UQCR is the UQCR core protein (UQCRC).

[0038] [17-2] The megakaryocyte according to [17-1], wherein at least one of the UQCRCs is UQCRC2.

[0039]

[18] A method for preparing platelets, comprising the step of maturing megakaryocytes as described in any one of

[15] to [17-2].

[0040] [19-1] The method according to

[18] includes an oscillating culture step.

[0041] [19-2] The method according to

[18] or [19-1], wherein the culture medium contains JI051.

[0042]

[20] A platelet obtained by any one of

[18] to [19-2].

[0043] [21-1] A blood preparation comprising the cells of any one of claims

[15] to [17-2] and

[20] .

[0044] [21-2] The preparations described in [21-1] are used to treat or prevent blood disorders or bleeding.

[0045] [22-1] A method for preparing megakaryocytes, comprising the step of culturing hematopoietic progenitor cells in a culture medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor.

[0046] [22-2] The method according to [22-1], wherein the culture medium contains an HDAC5 inhibitor and an LSD1 inhibitor.

[0047] [23-1] The method according to [22-1] or [22-2], wherein at least one of the HDAC5 inhibitors is LMK235.

[0048] [23-2] The method according to any one of [22-1] to [23-1], wherein at least one of the LSD1 inhibitors is tranylcypromine.

[0049]

[24] A method for treating or preventing blood disorders or bleeding, comprising administering or transplanting to a mammal an effective amount of any one of

[15] to [17-2] and

[20] cells.

[0050]

[25] The cells according to any one of

[15] to [17-2] and

[20] are used to treat or prevent blood diseases or bleeding.

[0051]

[26] The use of any one of the cells according to

[15] to [17-2] and

[20] in the manufacture of a treatment or preventive medicine for blood diseases or bleeding.

[0052] Invention Effects This invention enables the creation of megakaryocytes that maintain platelet production capacity over a long period, regardless of the type of pluripotent stem cell line. This, in turn, allows for a stable supply of high-quality platelets. Attached Figure Description

[0053] [ Figure 1 This graph shows the change in platelet production capacity of megakaryocytes from 15M41 human iPS cells introduced with MBX boxes over time. The horizontal axis represents the number of days since the start of culture in doxycycline-containing medium (megakaryocyte differentiation and proliferation medium), and the vertical axis represents the number of platelets released per megakaryocyte.

[0054] [ Figure 2 This graph represents the platelet production capacity of megakaryocytes from TkDNsev2 cells with introduced MBX boxes after knockdown of UQCRC1 and / or UQCRC2. The horizontal axis represents the type of shRNA introduced into the megakaryocytes, and the vertical axis represents the relative value of platelet production capacity compared to the control (megakaryocytes with introduced LacZ shRNA).

[0055] [ Figure 3The results represent the platelet-producing capacity of megakaryocytes induced by differentiation of 15M41 cells with MBX or MX boxes using lentiviral vectors, after which UQCRC1 or UQCRC2 was introduced and the cells matured. The horizontal axis represents the types of genes introduced using lentiviral vectors, and the vertical axis represents the number of platelets released per megakaryocyte. N=2, error bars: standard deviation.

[0056] [ Figure 4 This graph represents the results of introducing UQCRC2 into megakaryocytes induced by differentiation from 15M41 cells with an MBX box or 15M41 cells with an MX box using a lentiviral vector, followed by long-term amplification and maturation, and analysis of their platelet-producing capacity. The horizontal axis represents the number of days since the start of culture in the megakaryocyte differentiation and proliferation medium, and the vertical axis represents the number of platelets released by each megakaryocyte.

[0057] [ Figure 5 This graph represents the results of platelet production analysis of megakaryocytes expressing exogenous UQCRC2 from 15M41 cells introduced with an MBX cassette, cultured for more than 3 days in differentiation and proliferation medium supplemented with dextran, IBMX, and insulin, then transferred to maturation medium, and JI051 added after 3 days. The horizontal axis represents the added compounds, and the vertical axis represents the number of platelets released per megakaryocyte.

[0058] [ Figure 6 The results show the cell count (top) and CD34-positive cell percentage (bottom) measured by FACS after 3 days using differentiation and proliferation medium in which UM729 was replaced with LMK-235 to induce differentiation into megakaryocytes. The horizontal axis represents the compound used, and the number after LMK indicates the concentration of LMK-235 (unit: μM).

[0059] [ Figure 7 The results were obtained by inducing megakaryocyte differentiation into cells using differentiation and proliferation media in which 1 μM UM729 was replaced with any one of 200 nM LMK-235 (HDAC4, 5, 6 inhibitor), 100 nM TMP195 (HDAC4, 5, 7, 9 inhibitor), or 100 nM taquimod (HDAC4 inhibitor). The CD34 and CD41a positivity rates were measured by FACS on day 3 of proliferation culture. 200 nM LMK-235 and 100 nM TMP195 or 100 nM taquimod showed essentially the same HDAC inhibitory effect.

[0060] [ Figure 8The graph shows the platelet production capacity when differentiation induction and expansion culture were performed using differentiation and proliferation medium with 1 μM UM729 replaced with 200 nM LMK235, followed by maturation through static culture. The vertical axis represents the number of platelets released per megakaryocyte.

[0061] [ Figure 9 [A schematic diagram showing a method for inducing megakaryocytes from pluripotent stem cells using UM729.]

[0062] [ Figure 10 [This refers to the results of introducing the vectors outlined in the figure into iPS cell lines (15M41, MH09S01, MH15S01, MH15S01, MH23S01) to induce imMKCL, and measuring the CD34 positivity and CD41a positivity rates by FACS. A) FACS results on day 14 from the start of adding SCF, TA-316, UM729, and Dox. CD34 / CD41a co-positive fractions were sorted and then cultured in medium supplemented with SCF, TA-316, UM729, and Dox. B) After induction of imMKCL (day 1 of the start of Dox addition), cells from any iPS cell line maintained CD34 / CD41a co-positive expression for 42 days.

[0063] [ Figure 11 This figure shows the results of introducing the vector outlined in the diagram into the iPS cell line (QHJI14s04 / ABII-KO-03) to induce imMKCL, and measuring the CD34 positivity rate and CD41a positivity rate using FACS (the vertical axis represents the CD34 expression level, and the horizontal axis represents the CD41a expression level). On day 19 post-induction (day 1 of the first Dox addition), CD34 / CD41a co-expression fractions were sorted. FACS analysis was performed on days 28, 32, 36, and 42 post-imMKCL induction. The results showed the presence of CD34-negative and CD41a-positive cells, indicating difficulty in maintaining CD34 / CD41a expression.

[0064] [ Figure 12[A) shows the results of introducing the vector outlined in the figure into the iPS cell line (15M41) to induce imMKCL, and measuring the CD34 positivity and CD41a positivity rates by FACS. 15M41 is a cell line reported to have mutations in the BCOR and BRD3 genes. Left side of A): shows the results on day 14 from the start of adding SCF, TA-316, UM729, and Dox. CD34 / CD41a co-positive fractions were sorted and then cultured in medium supplemented with SCF, TA-316, UM729, and Dox. Right side of A): After induction of imMKCL (day 1 of the start of Dox addition), only cells from the 15M41 iPS cell line maintained CD34 / CD41a co-positive expression.

[0065] [ Figure 13 [This is in response to] Figure 11 Megakaryotic cell lines derived from QHJI14s04 / ABII-KO-03 (cell lines from day 28 of Dox addition) were used to knock down BCOR or BCORL1 with lentivirus. The results of CD34 and CD41a positivity rates were measured by FACS. By knocking down BCOR (BCL6 co-repressor) or BCORL1 (BCL6 co-repressor-like protein 1), megakaryotic cells maintaining co-positivity for CD34 / CD41a were successfully obtained.

[0066] [ Figure 14 This study compares platelet production capacity in megakaryocytes derived from QHJI14s04 / ABII-KO-03 (No.3 cells; control), megakaryocytes obtained by knocking down BCOR in No.3 cells (sh BCOR), and megakaryocytes obtained by knocking down BCORL1 in No.3 cells (shBCORL1). Data for No.3 cells were obtained on day 29 from the start of Dox administration, while data for sh BCOR and sh BCORL1 were obtained on day 36 from the start of Dox administration. The results show that sh BCOR and No.3 sh BCORL1 produced 2–3 times more platelets compared to No.3 cells.

[0067] [ Figure 15Plasmids integrating BACH1 were prepared (numbered 1-5 in the figure for summary). Various combinations of these plasmids (combinations 1, 4, and 5; combinations 2, 4, and 5; combinations 3, 4, and 5) were introduced into the iPS cell line (QHJI14s04 / ABII-KO-03) via lipid transfection to induce megakaryocytes. Combinations 1, 4, and 5 resulted in overexpression of c-MYCdd, overexpression of BACH1 and BCL-XL, and knockdown of BCOR in the presence of Dox. Combinations 2, 4, and 5 resulted in overexpression of c-MYCdd, overexpression of BACH1 and BCL-XL, and knockdown of BCOR and BCORL1 in the presence of Dox. Combinations 3, 4, and 5 resulted in overexpression of c-MYCdd, overexpression of BACH1 and BCL-XL, and knockdown of BCOR and TRIM27 (containing the triple motif 27) in the presence of Dox.

[0068] [ Figure 16-1 A) will introduce Figure 15 iPS cell lines containing plasmids 1, 4, and 5 (QHJI14s04 / AB II-KO-03) were induced into megakaryocytes. After sorting CD34 / CD41a co-positive cells, the FACS results on day 58 post-induction (day 1 of the initial Dox addition) are shown. B) [The text abruptly ends here, likely due to an incomplete translation or missing information.] Figure 15 iPS cell lines containing plasmids 2, 4, and 5 (QHJI14s04 / AB II-KO-03) were induced into megakaryocytes. After sorting CD34 / CD41a co-positive cells, the FACS results on day 58 post-induction (day 1 of the initial Dox addition) are shown. C) [The text abruptly ends here, likely due to an incomplete translation or missing information.] Figure 15 iPS cell lines containing plasmids 3, 4, and 5 (QHJI14s04 / AB II-KO-03) were induced into megakaryocytes. After sorting CD34 / CD41a co-positive cells, the FACS results on day 58 post-induction (day 1 of the first Dox addition) were shown. Megakaryocytes maintaining CD34 / CD41a co-positivity were successfully obtained by expressing BACH1.

[0069] [ Figure 16-2 [D] Comparison of platelet-producing capacity of different cell types. Megakaryocytes (BCOR / TRIM27) induced by the introduction of plasmids 3, 4, and 5 produced the most platelets. The leftmost figure shows the number of platelets produced by megakaryocytes introduced with plasmids 4 and 5 (c-MYC / BCL-XL / UQCRC2).

[0070] [ Figure 17-1[A) The results of introducing plasmids 3, 4, and 5 (shown in the figure) into iPS cells (QHJI14s04 / ABII-KO-11) via lipid transfection to induce megakaryocytes, and measuring CD34 and CD41a positivity rates by FACS. A) The iPS cell line (QHJI14s04 / ABII-KO-11) with introduced plasmids 3, 4, and 5 was induced into megakaryocytes. After sorting CD34 / CD41a co-positive cells, the FACS results on day 64 after induction (day 1 of the start of Dox addition) are shown.

[0071] [ Figure 17-2 This image shows a comparison of platelet-producing capacity between megakaryocytes (No.3 cells) from QHJI14s04 / ABII-KO-3 and megakaryocytes (No.11 cells) from QHJI14s04 / ABII-KO-11. From left to right in the figure, the platelet production counts are as follows: No.3 cells (c-MYC / BCL-XL / sh p21 (p21 knocked down with shRNA) / shp53 (p53 knocked down with shRNA)) on day 40 from the start of Dox administration; No.3 UQ2 cells (c-MYC / BCL-XL / UQCRC2 / sh p21 / shp53) on day 36 from the start of Dox administration; No.3 UQ2 BACH1 cells (c-MYC / BCL-XL / UQCRC2 / BACH1 / sh BCOR / sh TRIM27 (TRIM27 knocked down with shRNA)) on days 26, 27, 33, 42, 48, and 50 from the start of Dox administration (showing the mean ± standard deviation of platelet count at each time point); No.11 cells (c-MYC / BCL-XL / sh p21 / shp53) on day 40 from the start of Dox administration. Platelet production of p21 / shp53 cells on day 40 from the start of Dox addition; platelet production of No.11 UQ2 cells (c-MYC / BCL-XL / UQCRC2 / sh p21 / shp53) on day 36 from the start of Dox addition; and platelet production of No.11 UQ2 BACH1 cells (c-MYC / BCL-XL / UQCRC2 / BACH1 / sh BCOR / shTRIM27) on days 26 and 29 from the start of Dox addition (showing mean ± standard deviation of platelet count at each time point). No.11 UQ2 BACH1 cells produced the most platelets.

[0072] [ Figure 18-1[A] The results of introducing the plasmids outlined in the figure into iPS cells (QHJI14s04 / ABII-KO-3 and QHJI14s04 / ABII-KO-11) via lipid transfection to induce megakaryocytes, and measuring the CD34 positivity and CD41a positivity rates by FACS. A) Shows the FACS results on day 48 (for megakaryocytes from QHJI14s04 / ABII-KO-3) or day 42 (for megakaryocytes from QHJI14s04 / ABII-KO-11) after inducing each iPS cell line with introduced plasmids into megakaryocytes and sorting CD34 / CD41a co-positive cells following induction (day 1 of the start of Dox addition).

[0073] [ Figure 18-2 B) Comparison of platelet-producing capacity of various megakaryocytes. The graphs on the left, from left to right, represent: platelet production of No. 3 cells (c-MYC / BCL-XL) on day 40 from the start of Dox addition; platelet production of No. 3 UQ2 cells (c-MYC / BCL-XL / UQCRC2) on day 36 from the start of Dox addition; platelet production of No. 3 UQ2 tetBACH1 cells (c-MYC / BCL-XL / UQCRC2 / tetBACH1) on days 33, 35, 39, 43, and 46 from the start of Dox addition (showing the mean ± standard deviation of platelet count at each time point); platelet production of No. 11 cells (c-MYC / BCL-XL) on day 40 from the start of Dox addition; platelet production of No. 11 UQ2 cells (c-MYC / BCL-XL / UQCRC2) on day 36 from the start of Dox addition; and platelet production of No. 11 UQ2 tetBACH1 cells (c-MYC / BCL-XL / UQCRC2 / tetBACH1). Platelet production in BACH1 cells (c-MYC / BCL-XL / UQCRC2 / tetBACH1) at days 26, 28, 32, 36, 39, and 46 from the start of Dox administration (showing mean ± standard deviation of platelet count at each time point). The figure shows that No.11 UQ2 tet BACH1 cells produced the most platelets. The right panel shows the results of platelet production in No.11 UQ2 tet BACH1 cells measured over time.

[0074] [ Figure 19The results of cell number and FACS analysis after 10 days of culture in imMKC cells induced by differentiation of 15M41 cells introduced with MX boxes showed that the addition of SCF, TA316, and Dox with UM729 (1 μM), LMK235 (200 nM), 2PCPA (trans-2-phenylcyclopropylamine) (1 μM) or a combination of LMK235 (200 nM) and 2PCPA (1 μM) resulted in low cell numbers and FACS analysis. Although the effects of LMK235 and 2PCPA alone in maintaining CD34 / CD41a expression were not high, the combined use of LMK235 and 2PCPA maintained CD34 / CD41a expression. Detailed Implementation

[0075] 1. Methods for preparing megakaryocytes This invention provides a method for preparing megakaryocytes with maintained platelet-producing capacity. Specifically, it provides a method for preparing megakaryocytes with maintained platelet-producing capacity (hereinafter sometimes referred to as "the preparation method of this invention"), which includes the step of increasing the amount of at least one of the proteins constituting ubiquitin-cytochrome c reductase (UQCR) in megakaryocytes. In another embodiment, this invention provides a method for preparing megakaryocytes with maintained platelet-producing capacity, which includes the step of inhibiting the function of polycomb repressive complex 1.1 (PRC1.1) (also known as non-classical PRC1.1) in megakaryocytes (unless otherwise stated, this method is also included in "the preparation method of this invention"). Further, these methods can be combined; specifically, it also provides a method for preparing megakaryocytes with maintained platelet-producing capacity, which includes the steps of increasing the amount of at least one of the proteins constituting UQCR in megakaryocytes and inhibiting the function of PRC1.1 in megakaryocytes. When these steps are combined, they can be performed sequentially or simultaneously. In addition, in this specification, "method for preparing megakaryocytes" can also be appropriately interpreted as "method for the proliferation of megakaryocytes" or "method for the large-scale culture of megakaryocytes".

[0076] In this specification, unless otherwise stated, "cells" such as megakaryocytes include "cell populations". Additionally, unless otherwise stated, "cell" refers to cells obtained through cell culture. A cell population may consist of one type of cell or two or more types of cells.

[0077] Megakaryocytes can be, for example, cells characterized as CD41a-positive / CD42a-positive / CD42b-positive. In addition to these markers, megakaryocytes may further express one or more markers selected from CD9, CD34, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. Megakaryocytes may also express GATA1, FOG1, NF-E2, and β1-tubulin. Megakaryocytes can be multinucleated, mononuclear, or binucleated. Furthermore, megakaryocytes can be immortalized as megakaryocyte lines or as clonal cell populations.

[0078] Megakaryocytes that have not undergone multinucleation can proliferate through expanded culture and are sometimes referred to as immortalized megakaryocyte progenitor cell lines (imMKCL). Immortalized megakaryocytes mature to produce functional platelets. Megakaryocyte maturation refers to the differentiation of megakaryocytes into the desired multinucleated state, enabling them to produce functional platelets. Functional platelets can be, for example, CD42b-positive platelets. Megakaryocyte maturation can also be confirmed by increased expression of megakaryocyte maturation-related gene groups such as GATA1, FOG1, FLI1, NF-E2, and β1-tubulin.

[0079] In one embodiment, the megakaryocytes used in this invention are CD34-positive and CD41-positive cells. In another embodiment, the megakaryocytes used in this invention are CD38-negative, CD90-positive, and / or CD49f-positive. In this specification, CD41-positive cells refer to CD41a-positive cells.

[0080] In one embodiment of the invention, the megakaryocyte possesses an exogenous gene encoding a MYC protein and an exogenous gene encoding an apoptosis-inhibiting protein, and preferably also possesses an exogenous gene encoding a polycomb family protein. In this specification, these MYC proteins, apoptosis-inhibiting proteins, and polycomb family proteins are collectively referred to as "megakaryocyte-inducing factors."

[0081] Genes encoding MYC proteins (hereinafter also referred to as "MYC genes") include, for example, the c-MYC gene, the N-MYC gene, and the L-MYC gene. The c-MYC gene is more preferred. Examples of c-MYC genes include genes with the nucleic acid sequence shown in NCBI accession number NM_002467.

[0082] The c-MYC gene can be a c-MYC gene encoding a protein fused with a destabilizing domain. A "destabilizing domain" is a domain that destabilizes a operatively linked protein. The destabilizing domain can be operatively linked to the N-terminus or C-terminus of the protein. Examples of destabilizing domains include: ubiquitin, PEST sequences (sequences rich in proline, glutamate, serine, and threonine), cyclin destruction boxes, hydrophobic segments of amino acids, *E. coli* dihydrofolate reductase (ecDHFR), human estrogen receptor ligand-binding domain (ERLBD), FK506-binding protein (FKBP12), and their variants. Examples of destabilizing domains include FKBP12 and its variants. Variants of FKBP12 used as destabilization domains include, for example, F15S, V24A, H25R, E60G, L106P, M66T, R71G, D100G, D100N, E102G, and K105I variants (Banaszynski et al., Cell 126:995 (2006)). Commercially available products are also available for the destabilization domain, such as those sold by TAKARA Bio (Clontech ProteoTuner™ Shield System C, #631072).

[0083] As a "poptosis-inhibiting gene," any gene that inhibits apoptosis is acceptable; there are no particular limitations. Examples include: BCL2 gene, BCL2L1 gene (protein name: Bcl-xL), Survivin, MCL1, etc. The BCL2L1 gene is preferred. Examples of BCL2L1 genes include those with nucleic acid sequences represented by NCBI accession numbers NM_001191 or NM_138578.

[0084] In this specification, the term "gene encoding polycomb family proteins" (hereinafter also referred to as "polycomb gene") means a gene known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and function to prevent cellular senescence. Specifically, examples of multicomb genes include: BMI1 (multicomb complex protein BMI-1, multicomb family ring finger protein 4 (PCGF4), ring finger protein 51 (RNF51)), Mel18 (multicomb family ring finger protein 2), Ring (ring finger protein) 1a / b, Phc (multi-homological homologs) 1 / 2 / 3, Cbx (pigment boxes) 2 / 4 / 6 / 7 / 8, Ezh2 (enhancer of the second subunit of the Zeste 2 multicomb repression complex), Eed (embryonic ectoderm development), Suz12 (the second subunit of the SUZ12 multicomb repression complex), HADC (histone deacetylase), Dnmt (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b, etc., with BMI1 being the preferred gene. Examples of BMI1 genes include those with the nucleic acid sequence shown in NCBI accession number NM_005180.

[0085] The source of the genes encoding megakaryocyte-inducing factors (hereinafter also referred to as "megakaryocyte-inducing factor genes") is not particularly limited, but mammals (e.g., humans, mice, rats, monkeys, cattle, horses, pigs, dogs, etc.) are preferred, with humans being the most preferred. Alternatively, homologs of human genes from mammalian species other than humans are also preferred as megakaryocyte-inducing factor genes. In addition, genes with high base sequence identity to wild-type genes (e.g., having identity of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) are also preferred.

[0086] Each megakaryocyte-inducing factor gene is preferably linked under the regulation of a drug-responsive promoter. A drug-responsive promoter is a promoter that expresses or inhibits gene expression in the presence of a corresponding drug. Examples of drug-responsive promoters include: a fusion protein of reverse tetR (rtetR) and VP16AD (rtTA) or a fusion protein of tetR and VP16AD (rTA), and a TRE promoter that can bind in the presence or absence of the corresponding drug. Examples of such drugs include: doxycycline (Dox), tetracycline, or their derivatives (hereinafter referred to as "Dox et al."). Additionally, examples of drug-responsive promoters include metallothionein promoters (corresponding drugs: heavy metal ions) and steroid-responsive promoters (corresponding drugs: steroid hormones or their derivatives). Vectors containing these drug-responsive promoters are also called drug-responsive vectors. In addition to drug-responsive promoters, light-responsive promoters (induced by light) and heat shock protein promoters (induced by heat shock) can also be used. These promoters induced by drugs or stimuli are also called inducible promoters.

[0087] In this specification, "maintained platelet-producing capacity" means not only that the platelet-producing capacity of megakaryocytes does not decrease with increasing proliferation days, but also that the platelet-producing capacity is higher compared to the control (specifically, megakaryocytes cultured under the same conditions without increasing the amount of proteins constituting the corresponding UQCR, or megakaryocytes cultured under the same conditions without inhibiting PRC1.1 function (in one protocol, without reducing the amount of proteins constituting the corresponding PRC1.1)). Furthermore, in this specification, "platelet-producing capacity" refers to the ability of megakaryocytes to produce platelets by forming cell body processes after entering the maturation phase. The maturation of megakaryocytes can be achieved through the "steps for maturing megakaryocytes" described below. Additionally, as shown in the examples below, if the proportion of platelets (platelet count / megakaryocyte count) recovered after 6 days of culture following the maturation of megakaryocytes into the maturation phase on day 28 after the onset of proliferation is higher than the platelet count of the control, then the platelet-producing capacity of the megakaryocytes can be considered maintained.

[0088] Umbilol-cytochrome c reductase (UQCR) is one of four complexes present in the inner mitochondrial membrane, also known as "complex III". Within the mitochondria, UQCR catalyzes the electron transport reaction from umbilol to cytochrome c, while simultaneously actively transporting protons from the matrix side to the cytoplasmic side. While not bound by any theoretical constraints, it is speculated that the enhanced platelet production effect through forced expression of the UQCR core protein is due to increased megakaryocyte proliferation by improving the energy production capacity of mitochondria. Therefore, any protein capable of exerting this effect is acceptable; any protein constituting the UQCR other than the UQCR core protein can be used in this invention. Examples of proteins constituting the UQCR include UQCRC1, UQCRC2, UQCRB, BCS1L, UQCRQ, CYC1, TTC19, LYRM7, UQCC2, and UQCC3, with the UQCR core protein (i.e., UQCRC1 and UQCRC2) being preferred, and UQCRC2 being more preferred. These proteins can be used in single or multiple ways (e.g., combining UQCRC1 and UQCRC2).

[0089] An example of the base and amino acid sequence of human UQCRC1 is shown as SEQ ID NO: 1 (the coding sequence (CDS) of NCBI accession number NM_003365.3) and SEQ ID NO: 2 (NCBI accession number NP_003356.2). Another example of the base and amino acid sequence of human UQCRC2 is shown as SEQ ID NO: 3 (the CDS of NCBI accession number NM_003366.4) and SEQ ID NO: 4 (NCBI accession number NP_003357.2). An example of the base and amino acid sequence of human BACH1 is shown as SEQ ID NO: 5 (the CDS of NCBI accession number NM_001186.4) and SEQ ID NO: 6 (NCBI accession number NP_001177.1).

[0090] PRC1.1 is one of the complexes responsible for transcriptional repression based on polycomb family proteins, suppressing gene expression via Lys119 ubiquitination of histone H2A (H2AK119). PRC1.1 participates in various biological processes, such as cell differentiation or proliferation and tumorigenesis suppression, through epigenetic regulation of gene expression (e.g., Nakajima-Takagi Y. et al., Elife. 12:e83004 (2023)). PRC1.1 is mainly composed of PCGF1 (polycomb family ring finger protein 1), RING1A (ring finger protein 1A) or RING1B (ring finger protein 1B), KDM2B (lysine demethylase 2B), SKP1 (S-phase kinase-associated protein 1), and BCOR or BCORL1. In addition, TRIM27 and USP7 (ubiquitin-specific processing protease) can also be listed as proteins constituting PRC1.1 (Maat H. et al., iScience. 24(5): 102435 (2021)). In the preparation method of the present invention, there is no particular limitation on the method of inhibiting the function of PRC1.1, as long as the ubiquitination of Lys119 of the above-mentioned H2AK119 can be inhibited. For example, methods such as contacting the PRC1.1 inhibitor with megakaryocytes (typically culturing megakaryocytes in a culture medium containing the inhibitor) and reducing the amount of at least one of the proteins constituting PRC1.1 can be listed.

[0091] Examples of PRC1.1 inhibitors used in this invention include, but are not limited to, RING1B inhibitors such as PRT4165; KDM2B inhibitors such as GSK-J4, SD70, and CPI-455; BCOR inhibitors such as lenalidomide and pomalidomide; and PCGF1 inhibitors such as AUX-001 and Pevonedistat. These inhibitors may be used individually or in combination.

[0092] BCOR can be prevented from enriching into the target sequence due to partial deletion of its gene, frameshifting, or mutations introduced at its PCGF1 binding site. Other proteins constituting PRC1.1 can also be knocked down to reduce their protein content and thus inhibit PRC1.1 function. Therefore, the target proteins can be any of the PRC1.1 constituent proteins, such as PCGF1, RING1A, RING1B, KDM2B, SKP1, BCOR, BCORL1, TRIM27, or USP7, with BCOR, BCORL1, and TRIM27 being preferred. The target protein can be a single protein or multiple proteins.

[0093] As shown in the following examples, in the preparation method of the present invention, platelet production capacity can be improved by further overexpressing BACH1. Therefore, in one embodiment of the present invention, the preparation method of the present invention includes a step of increasing the amount of BACH1 protein in megakaryocytes. Therefore, the step of increasing the amount of BACH1 protein in megakaryocytes can be combined with the steps of increasing the amount of at least one of the proteins constituting UQCR and / or inhibiting the function of PRC1.1. When these steps are combined, the steps can be performed sequentially or simultaneously. BACH1 is a transcription factor that has been reported to promote erythropoiesis, etc., together with Bach2, by regulating heme metabolism in erythrocytes (e.g., Kato H. et al., Nat Immunol. 19(10):1059-1070 (2018)).

[0094] The proteins constituting the UQCR and the BACH1 protein used in this invention are not particularly limited in origin, but are preferably from mammals (e.g., humans, mice, rats, monkeys, cattle, horses, pigs, dogs, etc.), with humans being the most preferred. Furthermore, the proteins constituting the UQCR and the BACH1 protein used in this invention are preferably homologous proteins of human proteins from mammalian species other than humans. Alternatively, paralogous proteins may also be used. In addition, the proteins constituting the UQCR and the BACH1 protein include not only wild-type proteins but also variants with the same function. Examples of variants include proteins with high identity (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) relative to the amino acid sequence of a specific wild-type protein (e.g., the sequence shown in SEQ ID NO: 2, 4, or 6). In addition, it is preferred to have a variant thereof consisting of an amino acid sequence obtained by deleting, substituting, inserting and / or adding one or more (2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the amino acid sequence of a specific wild-type protein (e.g., the sequence shown in SEQ ID NO: 2, 4 or 6) and having the same function as the wild-type.

[0095] In this specification, "increasing the amount of a protein present" means that the amount of that protein present in megakaryocytes is at a higher level than the amount present in megakaryocytes before the step of increasing the amount of a specific protein. In megakaryocytes, a higher protein level can be maintained consistently or temporarily. There are no particular limitations on the method for increasing the amount of the protein constituting the UQCR in megakaryocytes. Examples include: introducing the nucleic acid (DNA or RNA) encoding the protein constituting the UQCR (hereinafter also referred to as "exogenous nucleic acid") from outside into megakaryocytes or cells capable of differentiating into megakaryocytes (e.g., hematopoietic progenitor cells, pluripotent stem cells, etc.) to force the expression of the protein; activating the expression of the endogenous gene encoding the protein constituting the UQCR by altering the promoter using methods such as genome editing; and adding the protein constituting the UQCR to the culture medium to deliver the protein into the cells. The protein constituting the UQCR or the nucleic acid encoding the protein can be introduced into the cells only once or multiple times. The method for increasing the amount of BACH1 protein in megakaryocytes is the same.

[0096] On the other hand, "reducing the amount of a protein" means that the amount of that protein present in megakaryocytes is low compared to the amount present in megakaryocytes before the step of reducing the amount of a specific protein. This amount of protein is typically the amount of wild-type (in other words, functionally normal) protein. In megakaryocytes, the low protein level can be maintained consistently or temporarily. There are no particular limitations on the methods for reducing the amount of proteins that constitute PRC1.1. Examples include introducing antisense nucleic acids (e.g., antisense oligonucleotides (ASOs)) (including the nucleic acid encoding the antisense) targeting the mRNA encoding the proteins that constitute PRC1.1, siRNA (including the nucleic acid encoding the antisense), heteroduplex oligonucleotides (HDOs), shRNA (including the nucleic acid encoding the antisense), and miRNAs (including the nucleic acid encoding the miRNA) into megakaryocytes or cells capable of differentiating into megakaryocytes to knock down the expression of each protein. When these nucleic acids are introduced into megakaryocytes, they can be introduced into the cells only once or multiple times. For example, if these nucleic acids are integrated into the cell's genome, even if they are introduced only once, their continuous expression within the cell can be expected. Alternatively, mutations that result in loss of function of the protein constituting PRC1.1, or mutations accompanied by reduced protein function and / or reduced protein expression, can be introduced into the gene using methods such as genome editing. Examples of such mutations include nonsense mutations, frameshift mutations, missense mutations, mutations with splicing abnormalities, mutations in gene regulatory regions (e.g., promoters, enhancers), and dominant / negative mutations.

[0097] Nucleic acids such as antisense nucleic acids, used to suppress the expression of proteins constituting PRC1.1, can be appropriately designed with reference to known base sequence information. An example of the base and amino acid sequences of human BCOR is shown in SEQ ID NO: 7 (CDS of NCBI accession number NM_001123385.2) and SEQ ID NO: 8 (NCBI accession number NP_001116857.1). An example of the base and amino acid sequences of human BCORL1 is shown in SEQ ID NO: 9 (CDS of NCBI accession number NM_001184772.3) and SEQ ID NO: 10 (NCBI accession number NP_001171701.1). An example of the base and amino acid sequences of human TRIM27 is shown in SEQ ID NO: 11 (CDS of NCBI accession number NM_006510.5) and SEQ ID NO: 12 (NCBI accession number NP_006501.1).

[0098] Furthermore, in this specification, "increasing or decreasing the amount of protein in megakaryocytes" does not necessarily mean increasing or decreasing the amount of protein only at the megakaryocyte stage. It is also possible to first introduce the protein or the nucleic acid encoding the protein into cells capable of differentiating into megakaryocytes (e.g., pluripotent stem cells, hematopoietic progenitor cells), so that the amount of protein is already increased or decreased in cells before differentiation into megakaryocytes. Therefore, for example, the step of first increasing the expression level of a specific protein (i.e., BACH1 protein, any protein constituting UQCR) in pluripotent stem cells, maintaining this state, and then inducing the pluripotent stem cells to differentiate into megakaryocytes is also included in the step of "increasing (or decreasing) the amount of the target protein in megakaryocytes." The same applies to the step of "inhibiting the function of PRC1.1 or decreasing the amount of any protein constituting PRC1.1 in megakaryocytes."

[0099] Exogenous nucleic acids are typically introduced into cells (e.g., megakaryocytes, hematopoietic progenitor cells, pluripotent stem cells, etc.) in the form of expression vectors carrying the nucleic acid. Vectors for expressing exogenous nucleic acids can include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, as well as animal cell expression plasmids (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo). Retroviral or lentiviral vectors are preferred for implementation with a single introduction.

[0100] Examples of promoters used in expression vectors include the EF-α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rouse sarcoma virus) promoter, MoMuLV (Moloni mouse leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter. In addition to promoters, expression vectors may also contain enhancers, poly-A tailing signals, selection marker genes, and SV40 origin of replication, as needed. Useful selection marker genes include, for example, dihydrofolate reductase genes, neomycin resistance genes, and puromycin resistance genes.

[0101] Furthermore, the aforementioned inducible promoters can be used as promoters in the expression vector. This allows for the expression of exogenous nucleic acids at the desired time and period. Additionally, to excise exogenous nucleic acids from the vector using the Cre-loxP system, expression vectors with the loxP sequence configured by inserting a gene or promoter region, or both, into the loxP sequence can be used. This allows expression to be stopped at stages where it is no longer necessary.

[0102] Exogenous nucleic acids can be introduced into cells via transposon systems. Transposons are a collective term for short gene sequences that have been preserved during evolution and cause gene translocation. Transposon systems induce gene translocation through the pairing of a gene enzyme (transposase) with its specific recognition sequence. For example, PiggyBac can be used as a transposon system. TM Transposon subsystem. PiggyBac TM The transposon system utilizes transposons isolated from insects (Fraser MJ et al., Insect Mol Biol. 1996 May;5(2):141-51.; Wilson MH et al., Mol Ther. 2007 Jan; 15(1):139-45.) and can be efficiently integrated into mammalian chromosomes. PiggyBac TM Transposon systems are actually being used for gene introduction (see, for example, Nakazawa Y et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013, etc.). The transposon systems applicable to this invention are not limited to those utilizing PiggyBac. TMSystems that utilize SleepingBeauty (Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Cell 91: 501-510.), Frog Prince (Miskey C, Izsvak Z, Plasterk RH, Ivics Z (2003) Nucleic AcidsRes31: 6873-6881.), Tol1 (Koga A, Inagaki H, Bessho Y, Hori H. Mol Gen Genet.1995 Dec 10;249(4):400-5.; Koga A, Shimada A, Kuroki T, Hori H, Kusumi J, Kyono-Hamaguchi Y, Hamaguchi S. J Hum Genet. 2007;52(7):628-35. Epub 2007 Jun7.), Tol2 (Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11.; JohnsonHamlet MR, Yergeau DA, Kuliyev E, Takeda M, Taira M, Kawakami K, MeadPE (2006) Genesis 44: 438-445.; Choo BG, Kondrichin I, Parinov S, Emelyanov A, Go W, TohWC, Korzh V (2006) BMC Dev Biol 6: 5.) and other transposon systems.

[0103] The introduction operation using the transposon subsystem can be performed using conventional methods, as described in known literature (e.g., on PiggyBac). TM Transposon subsystems, refer to Nakazawa Y et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013; or Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5;(69):e4235), etc. In one embodiment, the above-mentioned gene is transmitted via PiggyBac TMThe transposon system is introduced into the cell. Typically, in PiggyBac... TM In the transposon subsystem, the code PiggyBac is prepared to be retained. TM The vector containing the transposase gene (transposase plasmid) and the gene encoding the target protein were created by PiggyBac. TM Vectors containing inverted repeat sequence-clamped structures (transposon plasmids; PiggyBac) TM These two vectors (transposons) are introduced into target cells. Genes encoding megakaryocyte-inducing factors can also be introduced into pluripotent stem cells and hematopoietic stem cells using transposon systems.

[0104] In addition, to introduce multiple genes simultaneously, genes can be vertically linked to obtain polycistronic vectors. To achieve polycistronic expression, foot-and-mouth disease virus 2A self-cleaving peptides (see Science, 322, 949-953, 2008, etc.) and IRES sequences can be linked between the genes to be forcibly expressed.

[0105] Various methods can be used to introduce exogenous nucleic acids into cells, including lipid transfection, liposome transfection, electroporation, nuclear transfection, calcium phosphate coprecipitation, DEAE-dextran transfection, microinjection, and gene gun transfection. When the exogenous nucleic acid is contained in a viral vector, a plasmid containing that nucleic acid is introduced into appropriate packaging cells (e.g., Plat-E cells) or supplementary cell lines (e.g., 293 cells). The virus generated in the culture supernatant is then recovered and brought into contact with the cells to infect them, thus introducing the virus into the cells.

[0106] When delivering proteins into cells, known methods for introducing proteins into cells can be employed. Examples of such methods include: methods using protein-introducing reagents, methods using protein-introducing domains (PTDs) or cell-penetrating peptides (CPPs) to fusion proteins, and microinjection. Commercially available protein-introducing reagents include BioPOTER protein delivery kits (Gene Therapy Systems), Pro-Ject™ protein transfection kits (PIERCE), and ProVectin (IMGENEX), all based on cationic lipids; Profect-1 (Targeting Systems), based on lipids; Penetrain Peptide (Q biogene) and ChariotKit (Active Motif), based on permeabilizing peptides; and GenomONE (Ishihara Sangyo), which utilizes the HVJ envelope (inactivated Sendai virus). Introduction can be performed according to the operating procedures accompanying these reagents.

[0107] In one embodiment, megakaryocytes can begin to proliferate by increasing the levels of MYC protein (especially c-MYC) and apoptosis-inhibiting proteins (especially Bcl-xL) (preferably further increasing the levels of polycomb family proteins (especially BMI1)). Therefore, in a preferred embodiment of the invention, the preparation method includes the step of increasing the levels of MYC protein and apoptosis-inhibiting proteins (preferably further increasing the levels of polycomb family proteins) in megakaryocytes. There are no particular limitations on the methods for increasing the levels of these proteins in megakaryocytes; examples include methods for increasing the expression level of nucleic acids encoding the proteins, or methods for adding proteins to the culture medium to deliver them into the cells. Specifically, methods for increasing the expression level of genes encoding proteins include, for example, introducing exogenous nucleic acids (DNA or RNA) encoding the proteins into megakaryocytes, or culturing megakaryocytes in the presence of corresponding drugs or stimuli using the aforementioned inducible promoters.

[0108] The culture method of this invention can be a suspension culture or an adhesion culture, typically a suspension culture. Alternatively, megakaryocytes isolated from an organism can be cultured using an adhesion culture. According to the preparation method of this invention, since megakaryocytes capable of maintaining platelet production can proliferate for a long period, there are no particular limitations on the culture period; typically it is 6 days or more, but it can also be 12 days or more, 18 days or more, 24 days or more, 30 days or more, 36 days or more, 42 days or more, 48 days or more, 54 days or more, or 60 days or more.

[0109] Furthermore, the period during which the levels of at least one of the proteins constituting the UQCR are maintained elevated in megakaryocytes, the period during which the levels of BACH1 protein are maintained elevated, and the period during which PRC1.1 function is inhibited are not particularly limited; they can be limited to the period of megakaryocyte proliferation or can be maintained during the period of megakaryocyte maturation. The aforementioned periods are typically 6 days or more, but can also be 12 days or more, 18 days or more, 24 days or more, 30 days or more, 36 days or more, 42 days or more, 48 days or more, 54 days or more, or 60 days or more. On the other hand, from the viewpoint of effective maturation, the period during which the levels of megakaryocyte-inducing factors (especially MYC protein) are maintained elevated in megakaryocytes is preferably limited to the period of megakaryocyte proliferation.

[0110] In this specification, "suspension culture" refers to culture conducted under conditions in which cells or cell aggregates are kept in suspension in the culture medium, that is, culture under conditions in which a strong cell-substratum junction is not formed between the cells or cell aggregates and the culture vessel. Suspension culture can be static culture or shaking culture. In the case of shaking culture, as long as the cells are not fixed in one location, it can be called suspension culture.

[0111] There are no particular limitations on the culture containers used for suspension culture. Examples include: flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multipurpose culture dishes, microplates, microwell plates, microwells, multi-slice plates, multi-well plates, chambered slides, culture dishes, tubes, trays, culture bags, roller flasks, etc. For the shaking culture (also known as "stirred culture") described below, a sealed culture container is preferred. Examples of such culture containers include: tissue culture flasks, culture bags, roller flasks, etc. In addition, in order to enable culture under non-adhesive conditions, the culture container is preferably cell-non-adhesive. As a cell-non-adhesive culture container, containers whose surfaces have not been artificially treated to improve cell adhesion (e.g., coating treatment with extracellular matrix, etc.) or containers that have undergone artificial adhesion inhibition treatment (e.g., coating treatment with poly-HEMA, etc.) can be used.

[0112] The megakaryocytes used in this invention can be obtained by known methods. Examples include methods for isolating megakaryocytes from biological tissues (e.g., bone marrow, umbilical cord blood, peripheral blood, etc.), methods for inducing differentiation of pluripotent stem cells or hematopoietic progenitor cells, and methods obtained from companies such as ATCC. For example, megakaryocytes can be isolated from biological tissues using flow cytometry or mass spectrometry, magnetic cell separation, or affinity columns immobilized with the desired antigen, using antigen expression as an indicator. The megakaryocytes used in this invention are preferably obtained by inducing differentiation of hematopoietic progenitor cells. Therefore, the preparation method of this invention may include a step of preparing (or “inducing differentiation”) megakaryocytes from pluripotent stem cells or hematopoietic progenitor cells (hereinafter also referred to as the “megakaryocyte preparation step”) before preparing megakaryocytes capable of maintaining platelet production. Hematopoietic progenitor cells can be obtained, for example, by methods for isolating megakaryocytes from biological tissues (e.g., bone marrow, umbilical cord blood, peripheral blood, etc.) or by methods for inducing differentiation from pluripotent stem cells.

[0113] There are no particular limitations on the megakaryocyte preparation steps, which can be carried out by known induction methods. As specific examples, megakaryocyte preparation can be performed using methods described in International Publication No. 2011 / 034073, International Publication No. 2012 / 157586, etc. In one embodiment, the megakaryocyte preparation steps include step (A) culturing pluripotent stem cells to prepare (or “differentiation-inducing”) hematopoietic progenitor cells, and / or step (B) culturing hematopoietic progenitor cells to prepare megakaryocytes.

[0114] The method for differentiating pluripotent stem cells into hematopoietic progenitor cells in step (A) is not particularly limited as long as the cells can differentiate into hematopoietic progenitor cells. For example, the method described in Takayama N. et al., J Exp Med. 2817-2830 (2010), in which pluripotent stem cells are cultured on C3H10T1 / 2 in the presence of VEGF and modulated by the resulting reticular structures (also called ES-sacs or iPS-sacs), can be cited. Here, "reticular structures" refers to a three-dimensional sac-like (with internal cavities) structure derived from pluripotent stem cells, formed by endothelial cell groups, etc., and containing hematopoietic progenitor cells. In addition, examples can be given of methods based on the formation of embryoids and the addition of cytokines (Chadwick et al., Blood 2003, 102: 906-15; Vijayaragavan et al., Cell Stem Cell 2009, 4: 248-62; Saeki et al., Stem Cells 2009, 27: 59-67) or methods co-culturing with stromal cells from xenogeneic sources (Niwa A et al., J Cell Physiol. 2009 Nov;221(2):367-77.). For example, further examples can be given of methods described in International Publication No. 2013 / 075222, International Publication No. 2016 / 076415, International Publication No. 2017 / 221975, Liu S. et al., Cytotherapy, 17(2015); 344-358, etc.

[0115] "Pluripotent stem cell" refers to a stem cell that possesses the ability to differentiate into various tissues or cells with different morphologies or functions in an organism, and can also differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in this invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells) obtained from cloned embryos through nuclear transfer, multipotent germline stem cells (mGS cells), and embryonic germline stem cells (EG cells), with iPS cells (more preferably human iPS cells) being preferred. When the aforementioned pluripotent stem cell is an ES cell or any cell derived from a human embryo, the cell can be prepared by destroying the embryo or by preparing a cell without destroying the embryo; from an ethical perspective, cells prepared without destroying the embryo are preferred.

[0116] ES cells are stem cells derived from the internal cell blocks of early embryos (e.g., blastocysts) in mammals such as humans or mice. They possess pluripotency and the ability to proliferate based on self-replication. ES cells were first discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and ES cell lines have since been established in primates such as humans and monkeys (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting an internal cell block from the blastocyst of a fertilized egg of the target animal and culturing the internal cell block on a feeder layer of fibroblasts. Alternatively, ES cells can be established using only a single blastomer from a pre-blastocyst cleavage embryo (Chung Y. et al. (2008), Cell Stem Cell 2:113-117) or using an arrested embryo (Zhang X. et al. (2006), Stem Cells 24:2669-2676).

[0117] As for the ES cell lines used in this invention, if they are mouse ES cells, various mouse ES cell lines established by companies such as inGenious Targeting Laboratory and RIKEN can be used; if they are human ES cell lines, various human ES cell lines established by companies such as the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Medical Research, and Cellartis can be used. Specifically, examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 sold by ESIBio, H1 and H9 sold by WiCellResearch, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 sold by RIKEN.

[0118] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are many types of iPS cells. Besides the iPSCs established by Yamanaka et al. by introducing four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), there are also iPSCs derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al., Cell, (2007) 131:861-872). Nanog-iPSCs, established by sorting and establishing Nanog expression after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.). There are also iPSCs prepared using a method that does not contain c-Myc (Nakagawa M, Yamanaka). S. et al., Nature Biotechnology, (2008) 26, 101-106; and iPSC established by introducing 6 factors through a virus-free method (Okita K et al., Nat. Methods 2011 May; 8(5):409-12, Okita K et al., Stem Cells. 31(3):458-66.). In addition, artificial pluripotent stem cells can be used, such as those created by Thomson et al. by introducing four factors: OCT3 / 4, SOX2, NANOG, and LIN28 (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), artificial pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and artificial pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Application Publication No. 2008-307007).

[0119] In addition, all publicly published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol. 3, No. 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7) Any artificial pluripotent stem cell known in the art as described in patent publications (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) may be used.

[0120] As an artificial pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, Kyoto University iPS Cell Research Foundation, etc. can be used. For example, if it is a human iPS cell line, examples include Riken's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains; Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and TkDN-sev2 strains; and the Kyoto University iPS Cell Research Foundation's FfI-01s04, Ff-MH23s01, 15M41 (Ff-I 01s01), and QHJI14s04 strains. Human iPS cell lines established other than those described above can also be used; for example, the TC-1133 strain from RONZA can be cited. In addition, iPS cell lines with mutations in the proteins constituting PRC1.1 (especially BCOR) and with reduced PRC1.1 function are preferred.

[0121] Cell lines obtained by further genetic modification of iPS cell lines, such as those that have undergone gene knockout or genetic modification to reduce antigenicity, are also preferred. Examples of such iPS cell lines include QHJI 14s04-AB II-KO-03, QHJI14s04-AB II-KO-11, and QHJI14s04-AB II-KO-12 established by the Kyoto University iPS Cell Research Foundation, as well as universal donor cells established by Healios Corporation.

[0122] The artificial pluripotent stem cells used in this invention can be cells derived from patients with hereditary diseases (such as hereditary blood disorders). Cells induced to differentiate from pluripotent stem cells from patients with hereditary blood disorders can serve as disease models reflecting the pathological state of the disease, and are therefore suitable for screening therapeutic or preventative drugs for the disease. Alternatively, pluripotent stem cells from patients with hereditary blood disorders can be genetically repaired using genome editing methods such as CRISPR-Cas systems, causing them to differentiate into target cells, thereby enabling these cells to be used as therapeutic agents for the disease.

[0123] There are no specific restrictions on the species from which pluripotent stem cells are derived. For example, they can be cells from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. Humans are the preferred source species.

[0124] In this specification, "hematopoietic progenitor cell" refers to a cell capable of differentiating into hematopoietic cell lines such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. In this specification, hematopoietic progenitor cells and hematopoietic stem cells are not distinguished and are considered the same cell type unless otherwise stated. Hematopoietic progenitor cells are typically cells that express the CD34 and / or CD43 genes.

[0125] Regarding the culture period in step (A), those skilled in the art can appropriately determine it while monitoring the number of hematopoietic progenitor cells. As long as hematopoietic progenitor cells can be obtained, there is no particular limitation on the number of days; typically, it is 6 days or more, preferably 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or 14 days or more (especially 14 days). A longer culture period does not pose a problem for the preparation of hematopoietic progenitor cells; typically, it is less than 30 days, but it can also be less than 20 days.

[0126] Step (B) can also be performed by known methods (e.g., the methods described in Patent Documents 1-4). Specifically, methods such as simultaneously forcibly expressing an apoptosis-inhibiting gene (e.g., the BCL2L1 gene) and a MYC gene (e.g., the c-MYC gene) in hematopoietic progenitor cells to culture the cells can be cited.

[0127] Regarding the culture period in step (B), those skilled in the art can appropriately determine it while monitoring the number of megakaryocytes. As long as megakaryocytes can be obtained, there is no particular limitation on the number of days; typically 5 days or more, preferably 6 days or more, and more preferably 7 days or more. There is also no particular limitation on the upper limit of the culture period; typically 20 days, preferably 17 days or less, and more preferably 14 days or less.

[0128] In one embodiment of the present invention, any step of the preparation method (e.g., the megakaryocyte preparation step) is carried out in the presence of a pyrimidoindole derivative. Examples of pyrimidoindole derivatives include UM171 ((1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indole-4-yl)cyclohexane-1,4-diamine) and UM729 (4-(3-(1-piperidinyl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylic acid methyl ester). Pyrimidoindole derivatives are preferred, and UM171 or UM729 are more preferred. Only one or more pyrimidoindole derivatives may be used.

[0129] The concentration of the pyrimidoindole derivative can be appropriately selected by those skilled in the art based on the pyrimidoindole derivative used. In the case of the pyrimidoindole derivative UM729, its concentration in the culture medium is typically 1 nM to 100 μM, preferably 10 nM to 100 μM, more preferably 100 nM to 10 μM (1 μM in one scheme).

[0130] Pyrimidine indole derivatives can be added to the culture medium when culturing hematopoietic progenitor cells, or when culturing pluripotent stem cells or cells at any stage of differentiation from pluripotent stem cells to hematopoietic progenitor cells. For example, pluripotent stem cells or cells at any stage of differentiation from pluripotent stem cells to hematopoietic progenitor cells can be cultured in a medium containing pyrimidine indole derivatives.

[0131] Any step of the preparation method of the present invention (e.g., the megakaryocyte preparation step, the megakaryocyte proliferation step) can be performed in the presence of an HDAC5 (histone deacetylase) 5 inhibitor and / or an LSD1 inhibitor. Therefore, in one embodiment, the preparation method of the present invention includes culturing megakaryocytes in a culture medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor (preferably a culture medium containing both an HDAC5 inhibitor (e.g., LMK235) and an LSD1 inhibitor (e.g., transphenylcyclopropane)). In another embodiment, the present invention also provides a method for preparing megakaryocytes, which includes culturing hematopoietic progenitor cells in a culture medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor.

[0132] The HDAC5 inhibitors used in this invention are not limited to HDAC5-specific inhibitors, but may also have inhibitory activity against other molecules (typically other HDACs). Examples of HDAC5 inhibitors include LMK235, TMP195, quinosstat (JNJ-26481585) 2HCl, CUDC-101, Pracinostat (SB939), TMP269, and Domatinostat (4SC-202), with LMK235 and TMP195 being preferred, and LMK235 being more preferred. Furthermore, antibodies, peptides, or aptamers targeting HDAC5 are also preferred. Alternatively, the HDAC5 inhibitor may also be an antisense nucleic acid, siRNA, shRNA, heteroduplex nucleic acid, or other HDAC5 expression inhibitor that inhibits HDAC5 expression. Only one HDAC5 inhibitor may be used, or multiple HDAC5 inhibitors may be used. Furthermore, it is preferred to combine the HDAC5 inhibitor with a pyrimidine indole derivative.

[0133] The concentration of the HDAC5 inhibitor can be appropriately selected by those skilled in the art based on the HDAC5 inhibitor used. When the HDAC5 inhibitor is LMK235, its concentration in the culture medium is typically 1 nM to 100 μM, preferably 10 nM to 100 μM, more preferably 20 nM to 1 μM (50 nM to 200 nM in one formulation).

[0134] LSD1 inhibitors are not particularly limited to any specific inhibitor that can inhibit the enzymatic activity of LSD1 (i.e., the demethylation activity of histones H3K4me1 / 2 and H3K9me1 / 2). Examples include transphenylcyclopropylamine (TCP), ORY-1001 (Iadademstat), GSK2879552, IMG-7289 (Bomedemstat), SP-2509, HCI-2509, RN-1, MC3324, SP-2577 (Seclidemstat), and Corin, with transphenylcyclopropylamine being preferred. Additionally, antibodies, peptides, or aptamers targeting LSD1 are also preferred. Alternatively, LSD1 inhibitors can be LSD1 expression inhibitors such as antisense nucleic acids, siRNA, shRNA, and heteroduplex nucleic acids that inhibit LSD1 expression. Only one LSD1 inhibitor or multiple inhibitors may be used. Furthermore, it is preferred to combine an LSD1 inhibitor with a pyrimidindole derivative.

[0135] The concentration of the LSD1 inhibitor can be appropriately selected by those skilled in the art based on the LSD1 inhibitor used. When the LSD1 inhibitor is transphenylcyclopropane, its concentration in the culture medium is typically 5 nM to 500 μM, preferably 50 nM to 500 μM, and more preferably 500 nM to 5 μM (1 μM in one formulation).

[0136] As shown in the following examples, inspired by phenomena within the bone marrow, megakaryocytes were cultured in the presence of adipocyte differentiation inducing factors, resulting in a successful promotion of platelet production. Therefore, any step of the preparation method of the present invention (e.g., the megakaryocyte preparation step) can be performed in the presence of adipocyte differentiation inducing factors. Examples of adipocyte differentiation inducing factors include insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, indomethacin, etc. In one embodiment, the preparation method of the present invention includes the step of culturing megakaryocytes in a culture medium containing dexamethasone, IBMX, and insulin. Furthermore, the culture medium used in the megakaryocyte preparation step preferably also contains JI051. In one embodiment, the culture medium used in the megakaryocyte preparation step contains dexamethasone, IBMX, insulin, and JI051. The structural formula of JI051 is shown below.

[0137] [Chemical Formula 2] Furthermore, in hematopoietic progenitor cells, the expression of the TP53 gene (protein name: p53) and / or the CDKN1A gene (protein name: p21) or the function of their expression products can also be inhibited. Examples of TP53 genes include those with the nucleic acid sequence shown in NCBI accession number NM_000546.6. Examples of CDKN1A genes include those with the nucleic acid sequence shown in NCBI accession number NM_001291549.3. The source of these genes is not particularly limited, but mammals (e.g., humans, mice, rats, monkeys, cattle, horses, pigs, dogs, etc.) are preferred, with genes from humans being particularly preferred. In one embodiment, these genes are human genes or homologs of human genes from other mammalian species. Genes with high identity to wild-type genes (e.g., having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are also preferred.

[0138] The megakaryocytes of the present invention are suitable as starting cells for platelet preparation because they maintain platelet-producing capacity. Therefore, in another embodiment, a method for preparing platelets (hereinafter sometimes referred to as "the platelet preparation method of the present invention") is provided, which includes the step of maturing the megakaryocytes of the present invention.

[0139] The platelet preparation method of the present invention can be carried out by known methods. For example, in the case of forced expression of megakaryocyte-inducing factors in megakaryocytes, megakaryocyte maturation can be achieved by reducing or stopping the forced expression of one or more (preferably at least the MYC gene) or all of the megakaryocyte-inducing factors. For example, in the case of forced expression of megakaryocyte-inducing factors using a drug, forced expression can be reduced or stopped by culturing megakaryocytes in the absence of the drug. Furthermore, in the case of using the above-mentioned vector containing LoxP, this can also be achieved by introducing Cre recombinase into the cells. Further, in the case of using a transient expression vector and introducing RNA or protein, this can also be achieved by stopping contact with the vector, etc.

[0140] The culture medium used in the platelet preparation method of the present invention contains any one (preferably all) of ADAM17 inhibitors, AhR (aromatic hydrocarbon receptor) inhibitors, and ROCK inhibitors. Examples of ADAM17 inhibitors include KP-457, DPC333, GW280264X, Aderbasib, TMI-1, and JG26, with KP-457 being preferred. Examples of AhR inhibitors include StemRegenin 1 (SR-1), SR-1 analogs (such as the inhibitor described in US2014 / 0369973), stilbene derivatives (such as (E)-1-(4'-trifluoromethylphenyl)-2-(3,5-bistrifluoromethylphenyl)-ethylene, (E)-1-(4'-methoxyphenyl)-2-(3,5-dichlorophenyl)-ethylene, (E)-1-(4'-chlorophenyl)-2-(3,5-dichlorophenyl)-ethylene, 3,5,4'-trihydroxystilbene, etc.), CH-223191, etc., with SR-1 being preferred. Examples of ROCK inhibitors include: Y-27632 (e.g., see Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (e.g., see Uenata et al., Nature 389: 990-994 (1997)), SR3677 (e.g., see Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (e.g., see Stavenger RA et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), GSK429286A, H1152 (e.g., see Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (for example, see Nakajima et al., Cancer Chemother Pharmacol.52(4): 319-324 (2003)), Thiazovivin and their derivatives, etc., preferably Y-27632.

[0141] The culture medium used in the platelet preparation method of the present invention may contain the aforementioned adipocyte differentiation inducing factor and / or JI051. In one embodiment, the culture medium used in the platelet preparation method of the present invention contains JI051. Specific compounds and the like are described in their entirety in the preparation method of the present invention.

[0142] Mature megakaryocytes prepared by the platelet preparation method of the present invention can be CD34-positive and CD41-positive cells. Mature megakaryocytes prepared by this method can also be CD38-negative, CD90-positive, and / or CD49f-positive.

[0143] The culture period in the platelet preparation method of the present invention is not particularly limited, as long as the platelet function is maintained, for example, 2 to 9 days (6 days in one scheme).

[0144] The platelet preparation method of the present invention typically employs suspension culture. Furthermore, it is preferable to perform the culture while agitating. Therefore, in one embodiment, the platelet preparation method of the present invention includes a shaking culture step. Shaking culture can be performed, for example, using a commercially available rotary shaking culture device (e.g., a VerMES reactor (manufactured by Satake Multimix Co., Ltd.)), by stirring the culture medium using the rotational motion of a stirrer. The stirring speed can be 50–200 rpm, for example, approximately 100 rpm.

[0145] The oscillation culture in the platelet preparation method of the present invention can also be implemented using the apparatus and method described in Patent Document 5. Specifically, for example, the following method can be listed, which includes the step of stirring the culture medium in the culture vessel using a stirring paddle, the stirring step including reciprocating motion of the stirring paddle to sufficiently satisfy one or more of the following indicators (a) to (c).

[0146] (a) Approximately 0.0005m 2 / s 2 ~ Approximately 0.02m 2 / s 2 Turbulent energy; (b) Shear stress of approximately 0.2 Pa to approximately 6.0 Pa; and (c) Kolmogorov scale, approximately 100 μm to approximately 600 μm.

[0147] In addition, as a culture device, specifically, a platelet manufacturing device can be listed as follows, which includes a container for holding a culture medium containing megakaryocytes and a stirring paddle that moves back and forth in the container. By moving the stirring paddle back and forth in the culture medium, one or more indicators selected from (a) to (c) below are sufficiently satisfied, thereby producing platelets from megakaryocytes while the culture medium is being stirred.

[0148] (a) 0.0005m 2 / s 2 ~ Approximately 0.02m 2 / s 2 Turbulent energy; (b) Shear stress of approximately 0.2 Pa to approximately 6.0 Pa; and (c) Kolmogorov scale, approximately 100 μm to approximately 600 μm.

[0149] The turbulent energy, shear stress, and Kolmogorov scale in the culture medium within the container being stirred can be obtained through simulation based on the fundamental equations of turbulence. For example, calculations can be performed using the thermal fluid analysis software FLUENT (manufactured by ANSYS), but it is not limited to any particular software. More specifically, in the case of a configuration where the impeller moves in a reciprocating motion, the turbulent energy varies depending on the impeller's stroke, speed, and frequency. When multiple impellers are used, the number of impellers also becomes a variable factor. Shear stress and Kolmogorov scale also change due to the same factors.

[0150] The culture medium used in this invention is not particularly limited, and can be prepared using a culture medium for animal cell culture as the basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixtures of these media. The culture medium may contain serum or may be serum-free. Depending on the requirements, the culture medium may also contain one or more substances such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines. Cytokines are proteins that promote differentiation of the hematopoietic cell system, such as VEGF, TPO, and SCF. TA-316 ((2E)-2-[1-[5-(4-bromophenyl)-4-hydroxy-3-thienyl]ethylene]hydrazide-5-[[[[4-[[(2-hydroxyethyl)amino]carbonyl]phenyl]methyl]amino]carbonyl]-2-thiencarboxylic acid) is preferred over TPO. In one embodiment, the culture medium used in the preparation method of the present invention contains SCF and TA-316. In another embodiment, the culture medium used in the platelet preparation method of the present invention is a medium containing SCF, TA-316, SR-1, KP-457, and Y27632. Furthermore, when megakaryocytes have a drug-responsive promoter, it is preferable to contain the corresponding drug, such as tetracycline or doxycycline, in the culture medium when expressing the gene under that promoter.

[0151] In this invention, there are no particular limitations on the cell culture conditions, and conventional culture conditions can be used. As specific examples, the culture temperature is, for example, about 35–42°C, about 36–40°C, or about 37–39°C. The CO2 concentration is, for example, about 5–15%. The O2 concentration is, for example, about 15–25%.

[0152] In each step of the preparation method and platelet preparation method of the present invention, cells can be cultured under conditions without a feeder layer and / or without xenogeneic species. In the preparation method and platelet preparation method of the present invention, the entire process can also be carried out under conditions without a feeder layer and without xenogeneic species. In this specification, "without a feeder layer" means a culture medium or culture condition that does not contain other cell types (i.e., feeder layer cells) that play an auxiliary role in regulating the culture conditions of the target cells. Furthermore, "without xenogeneic species" means a culture medium or culture condition that does not contain components from organisms of a different species than the target cells.

[0153] In each step of the preparation method and platelet preparation method of the present invention, the cell seeding density is not particularly limited, as long as the cells can proliferate. A typical density is 1.0 × 10⁻⁶. 2 ~1.0×10 7 cells / cm 2 Preferably 1.0×10 3 ~1.0×10 6 cells / cm 2 More preferably 1.0×10 4 ~1.0×10 5 cells / cm 2 .

[0154] In the case of small molecule compounds used in this invention, the compounds include not only the free form but also their pharmacologically acceptable salts and hydrates. Pharmacologically acceptable salts vary depending on the type of compound, and examples include: inorganic base salts such as alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), aluminum salts, and ammonium salts; and base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, methylpyridine, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine; or inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; and acid addition salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0155] The various substances used in this invention can be synthesized by known methods or commercially available products can be used. Furthermore, when using proteins or peptides, these proteins or peptides can be obtained from cells that express them. Cells expressing the target protein or peptide can be prepared by inserting DNA encoding the target protein or peptide into a known expression vector and then introducing the resulting expression vector into a suitable host cell.

[0156] The preparation methods of the present invention and the platelet preparation methods of the present invention may include a step of recovering the target cells or tissues obtained in each step. The recovered cells can be cryopreserved using cell cryopreservation solution. In addition, the obtained cells can be counted using a cell counter, labeled with antibodies against cell surface markers, and sorted or purified by flow cytometry, mass spectrometry, or magnetic cell separation methods.

[0157] In yet another embodiment, a method for maintaining the platelet-producing capacity of megakaryocytes is provided, the method comprising the step of increasing the amount of at least one of the proteins constituting ubiquitin-cytochrome c reductase (UQCR) in megakaryocytes. Regarding this method, all matters described in relation to the preparation method of the present invention are referenced.

[0158] 2. Megakaryocytes and platelets In another aspect of the invention, megakaryocytes (hereinafter sometimes referred to as "megakaryocytes of the invention") and platelets (hereinafter sometimes referred to as "platelets of the invention") obtained by the preparation method and platelet preparation method of the invention are provided. Hereinafter, the term "cells of the invention" will sometimes be used collectively to refer to the megakaryocytes and platelets of the invention.

[0159] The megakaryocytes of the present invention typically possess all of the following features (A) to (C).

[0160] (A) Having at least one of the exogenous proteins that constitute UQCR; (B) Possesses the ability to produce platelets; (C) Expression of CD34 and CD41 genes.

[0161] In addition, the megakaryocytes of the present invention also have any one, two or three of the following features (D) to (F).

[0162] (D) Derived from pluripotent stem cells; (E) Contains exogenous megakaryocyte inducing factors; (F) Has an inhibitor of expression of the TP53 gene and / or CDKN1A gene.

[0163] In another embodiment, according to the present invention, a megakaryocyte is also provided, which has all of the above-described features (A) to (C). Additionally, a megakaryocyte is also provided, which further has at least any one, two, or three of the above-described features (D) to (F). The megakaryocyte can be obtained by the preparation method of the present invention, or by other methods. Hereinafter, the megakaryocyte is sometimes also referred to as "the megakaryocyte of the present invention".

[0164] In this specification, "exogenous" means that the gene is not present in megakaryocytes of mammals unless introduced from outside. Additionally, in this specification, the terms "expressed" or "positive" mean, unless otherwise stated, the production of the protein encoded by that gene. Therefore, if the target protein is detected by FACS used in the examples below, the gene can be considered expressed.

[0165] In feature (A) above, at least one of the proteins constituting the exogenous UQCR is preferably a UQCR core protein, particularly UQCRC2. The phrase "possessing platelet-producing ability" in feature (B) means that platelet production can be observed by inducing megakaryocytes into maturity. The method for inducing maturity is not limited; as long as platelet production is confirmed by at least one method, the megakaryocyte is considered to possess platelet-producing ability. In feature (C) above, the presence of at least one megakaryocyte-inducing factor is sufficient, but the presence of MYC protein (especially c-MYC) and an apoptosis-inhibiting protein (especially Bcl-xL) is preferred, or the presence of MYC protein (especially c-MYC), an apoptosis-inhibiting protein (especially Bcl-xL), and a polycomb family protein (especially BMI1). As expression inhibitors for feature (F) above, examples include antisense nucleic acids, siRNA, shRNA, and heteroduplex nucleic acids targeting the transcripts of the TP53 gene and / or the CDKN1A gene.

[0166] 3. Uses of megakaryocytes and platelets The cells of the present invention can be used in medical applications such as blood transfusions; therefore, in another embodiment, a blood preparation comprising the cells of the present invention is provided (hereinafter sometimes referred to as "the blood preparation of the present invention"). In this specification, "blood preparation" means a composition comprising blood cells such as megakaryocytes and platelets. The megakaryocytes of the present invention, for example, can be used to produce functional platelets in an organism by administration or transplantation. Additionally, the platelets of the present invention can be used, for example, to prevent bleeding caused by thrombocytopenia or platelet dysfunction, to treat bleeding, or to treat blood disorders. Furthermore, the present invention also includes a method for treating or preventing blood disorders or bleeding by administering or transplanting an effective amount of the cells of the present invention to a mammal (e.g., human, mouse, rat, monkey, cattle, horse, pig, dog, etc.) as the object of treatment or prevention. In this specification, unless otherwise stated, the treatment or prevention medicine (or treatment or prevention method) for a disease also includes a drug (or method) that can treat and prevent the disease.

[0167] Blood disorders that are the object of treatment or prevention include, for example, thrombocytopenia and platelet dysfunction. Causes of thrombocytopenia include, for example, pancytopenia due to bone marrow insufficiency; thrombocytopenia caused by platelet destruction leading to shortened platelet lifespan; thrombocytopenia caused by excessive platelet consumption; drug-induced thrombocytopenia; and thrombocytopenia caused by abnormal platelet distribution in organs. Causes of platelet dysfunction include, for example, drug-induced platelet dysfunction and hereditary platelet dysfunction (e.g., thrombocytopenia, Bernard-Soulier syndrome, MYH9 abnormality, Wiskott-Aldrich syndrome, etc.).

[0168] When using the cells of the present invention as blood preparations, from the viewpoint of avoiding rejection, it is desirable to use iPS cells derived from somatic cells with the same or substantially the same HLA genotype as the recipient individual. Here, "substantially the same" means that the degree of HLA genotype consistency is such that the immune response can be suppressed by immunosuppressants, for example, somatic cells with an HLA type consistent at three loci (HLA-A, HLA-B, and HLA-DR) or four loci (including HLA-C). In cases where sufficient cells cannot be obtained due to age or physical condition, administration or transplantation can be performed in a state of avoiding rejection by encapsulating them in capsules, porous containers, or the like, such as polyethylene glycol or silicone.

[0169] Preferably, the cells are derived from iPS cells in which the genes encoding MHC class I and II constituents (e.g., HLA, B2M, etc.) and / or the genes encoding transcription factors (e.g., CIITA, RFX5, RFXAP, RFXANK, etc.) have been disrupted or modified. Furthermore, HLA-A, HLA-B, and HLA-C proteins are closely associated with rejection responses following cell transplantation. Therefore, the cells of the present invention preferably have only the HLA-A and HLA-B genes disrupted, or all three loci (HLA-A, HLA-B, and HLA-C) disrupted, and more preferably, the HLA-E gene is further disrupted. For example, genetically modified cells can be prepared by disrupting the HLA-A and HLA-B genes, or disrupting the HLA-A, HLA-B, and HLA-C genes (optionally further disrupting the HLA-E gene), in pluripotent stem cells or cells at any stage of differentiation from pluripotent stem cells to platelets (e.g., hematopoietic progenitor cells, megakaryocytes, etc., especially megakaryocytes). These cells can then undergo differentiation induction and / or maturation steps to produce platelets, thereby eliminating HLA protein expression on the platelet surface. This is expected to reduce antigenicity during platelet transplantation. Additionally, for patients carrying anti-HLA class I antibodies, transplantable platelet preparations and other blood products can be provided.

[0170] The cells of the present invention can be mixed with pharmaceutically acceptable carriers using conventional methods to prepare parenteral preparations such as injections, suspensions, and infusions. Therefore, in one embodiment, a method for preparing a blood preparation is also provided, which includes the step of preparing the cells of the present invention into a formulation. The preparation method may include the step of preparing the cells of the present invention. Furthermore, it may also include the step of preserving the cells of the present invention.

[0171] Pharmaceutically acceptable carriers that can be included in this parenteral preparation include, for example, physiological saline, isotonic solutions containing glucose or other excipients (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and other aqueous solutions for injection. The cells of the present invention can also be used in combination with, for example, human plasma, infusion solutions, buffers (e.g., phosphate-buffered saline, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0172] The blood preparations of the present invention can also be provided frozen under conditions typically used for cell cryopreservation, and used after thawing. In this case, it may further comprise serum or a substitute thereof, an organic solvent (e.g., DMSO), etc. In this case, the concentration of serum or a substitute thereof is not particularly limited, and may be from about 1 to about 30% (v / v), preferably from about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, and may be from 0 to about 50% (v / v), preferably from about 5 to about 20% (v / v).

[0173] Furthermore, the cells of the present invention can also be used in methods for screening agents, i.e., candidate agents, for the treatment or prevention of blood diseases. Therefore, in yet another aspect of the present invention, a method for screening agents for the treatment or prevention of blood diseases is provided, comprising the step of culturing the cells of the present invention in the presence or absence of a test substance. For example, using a disease model reflecting the condition of a blood disease as cells, if the pathological condition improves in the presence of the test substance, the test substance can be sorted as a candidate agent for the treatment or prevention of the blood disease. Examples of such blood diseases include those identical to those described above as being for the treatment or prevention of blood preparations of the present invention.

[0174] The step of contacting the test substance with the cells of the present invention is typically performed by adding the test substance to the culture medium in which the cells of the present invention are cultured, or by transferring the cells of the present invention to a culture medium pre-added with the test substance. The duration of the contacting step is not particularly limited, but is typically from 1 minute to 5 days, preferably from 1 hour to 1 day.

[0175] Examples of test substances used in this invention include cell extracts, cell culture supernatants, microbial fermentation products, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic small molecule compounds, and natural compounds. Test substances can be existing or candidate ingredients of pharmaceuticals or nutritional foods.

[0176] In another aspect of the invention, a kit is provided for preparing megakaryocytes and / or platelets that maintain platelet production capacity. This kit contains proteins constituting UQCRs or nucleic acids encoding such proteins. In addition to potentially containing megakaryocyte-inducing factors or nucleic acids encoding such factors, expression vectors necessary for intracellular expression of these nucleic acids, reagents, etc., it may also contain cell culture media, serum, growth factor supplements (e.g., TA-316, TPO, EPO, SCF, heparin, IL-6, IL-11, etc.), antibiotics, and initiating cells (e.g., pluripotent stem cells, hematopoietic progenitor cells, etc.). Furthermore, if cells derived from pluripotent cells are used, antibodies for identifying markers may also be included to identify the network structures prepared from these cells (e.g., antibodies against Flk1, CD31, CD34, UEA-I lectins, etc.). Moreover, the reagents, antibodies, etc., contained in the kit are supplied to any type of container, ensuring that the activity of its components remains effective over a long period, is not adsorbed by the container material, and does not deteriorate.

[0177] The following examples illustrate the present invention in more detail, but the present invention is not limited to these examples. Example

[0178] Unless otherwise stated, the following materials and methods are used in the embodiments.

[0179] 1. iPS cells The following human iPS cell lines were obtained from the Kyoto University iPS Cell Research Foundation for use: 15M41 (alias: Ff-I 01s01), Ff-MH23s01 (abbreviated: MH23S01), TkDN-sev2, QHJI14s04, QHJI14s04-AB II-KO-03, and QHJI14s04-AB II-KO-11. 15M41 and QHJI14s04 were derived from peripheral blood of the most common HLA-homozygous donors, while MH23S01 was derived from peripheral blood of the second most common HLA-homozygous donor. TkDN-sev2 was derived from human fetal skin fibroblasts established using Sendai virus. QHJI14s04-AB II-KO-03 and QHJI14s04-ABII-KO-11 are cell lines in QHJI14s04 in which HLA-A, HLA-B, and CIITA were knocked out through genome editing.

[0180] During the induction of differentiation into megakaryocytes, the following cell lines, in which megakaryocyte inducing factors were introduced into the iPS cell lines, were used. After these cell lines were induced to differentiate into hematopoietic progenitor cells, they were differentiated into megakaryocytes by adding tetracycline or its derivatives (doxycycline in this example) to the culture medium, and then removing doxycycline and the like from the culture medium to allow them to mature, thereby producing platelets.

[0181] • 15M41 cells with MBX box introduced To utilize piggyBac TM The 15M41 human iPS cell line was established by inserting a construct into the genome. This construct encodes three genes (MYCdd, BMI1, and BCL2L1) fused with a destabilization domain, under the regulation of a tetracycline-inducible promoter, and further encodes p21 and p53 shRNAs under the regulation of an H1 promoter (see Sone M. et al., Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs, Stem Cell Reports, 16 (12), 2861-2870, 2021). In this specification, this construct is sometimes referred to as the [c-MYC / BMI1 / BCLXL / p21 KD / p53KD] box or simply the MBX box. Hereinafter, the term "iPS cell line with introduced MBX box" will be used collectively to refer to iPS cell lines other than 15M41 and 15M41 cells with introduced MBX box.

[0182] • 15M41 cells from the MX box were introduced. To utilize piggyBac TM The system inserts a construct into the genome of a 15M41 human iPS cell line. This construct encodes the MYCdd and BCL2L1 genes (two factors: MYCdd / BCLxL) under the regulation of a tetracycline-inducible promoter, and encodes p21 shRNA and p53 shRNA under the regulation of an H1 promoter. In this specification, this construct is sometimes referred to as the [c-MYC / BCLXL / p21 KD / p53 KD] box or simply the MX box.

[0183] • TkDN sev2 cells with MBX box introduced The TkDN sev2 human iPS cell line with an MBX box inserted into the genome.

[0184] 2. Induction of iPS cell differentiation into hematopoietic progenitor cells The method described in Takayama N. et al., J Exp Med. 2817-2830 (2010) was modified.

[0185] iPS cells treated with the GSK-3β inhibitors CHIR-99021 and Y-27632 for 3 days were peeled into cell fragments, which were then added to feeder cells (human fibroblast cell line C3H / 10T1 / 2 cells treated with mitomycin C (ATCC number: CCL-226)).

[0186] As the basal medium, IMDM (Iscove modified Dulbecco medium) (Sigma-Aldrich, Code No. I3390) was prepared, containing 15% fetal bovine serum (FBS) (Life Technologies, Code No. 10270-106), 2 mM glutamine (Life Technologies, Code No. 25030-081), 1% insulin / transferrin / selenium solution (ITS-G) (Life Technologies, Code No. 41400-045), 0.45 mM 1-thioglycerol (Sigma-Aldrich, Code No. A6145), and 50 μg / mL L-ascorbic acid (Sigma-Aldrich, Code No. A4544).

[0187] As the initial culture medium for iPS cells, a basal medium containing 20 ng / mL VEGF (Fujifilm and Kodenki Chemical, Code No. 226-01786) was used to begin culturing at 37°C, 5-8% O2, and 5% CO2.

[0188] On the 4th day of culture, the medium was replaced with a basal medium containing 20 ng / mL VEGF, 10 U / mL heparin (AY Pharma), 50 ng / mL bFGF (Wako Pure Chemical Industries, Code No. 068-04544), and 10 μM SB431542 (Wako Pure Chemical Industries, Code No. 037-24293), and cultured at 37°C, 5-8% O2, and 5% CO2.

[0189] On day 7 of the culture, the medium was replaced with a basal medium containing 20 ng / mL VEGF and 10 U / mL heparin, and cultured at 37°C, 20% O2, and 5% CO2.

[0190] On day 11 of the culture, add or replace the basal medium with 20 ng / mL VEGF and 0.75 μM SR-1 (Stemregenin-1) and culture at 37°C, 20% O2 and 5% CO2.

[0191] Suspended cells from days 7 to 14 of culture were collected as hematopoietic progenitor cells for subsequent differentiation induction experiments.

[0192] 3. Induction of differentiation from hematopoietic progenitor cells into megakaryocytes, expansion culture, and maturation. Hematopoietic progenitor cells cultured on day 7 after the start of culture in Method 2 were induced to differentiate into megakaryocytes by basal medium (sometimes called megakaryocyte differentiation and proliferation medium or differentiation and proliferation medium) containing 0.2 μg / mL TPO mimicry compound, 50 ng / mL SCF, 1 μM UM729 (MERCK ID: ATEH97ECEE61), and 1 μg / mL doxycycline, under feeder-free conditions at 37°C, 20% O2, and 5% CO2. In this example, the day on which culture with the megakaryocyte differentiation induction medium began was defined as day 0, and subsequent culture days were expressed in days. In this method, CD34-positive and CD41-positive cell populations generally began to appear around day 7. Subculture was continued using this medium to expand the megakaryocyte culture. It should be noted that in the following embodiments, "inducing differentiation of hematopoietic progenitor cells from iPS cells and inducing differentiation of megakaryocytes from the hematopoietic progenitor cells" is sometimes shortened to "inducing differentiation of megakaryocytes from iPS cells".

[0193] By replacing the culture medium with a doxycycline-free medium, the forced expression of three factors (MYCdd / BMI1 / BCLxL) or two factors (MYCdd / BCLxL) in the above expression cassette can be deactivated, thus inducing megakaryocyte maturation. The composition of the doxycycline-free medium (megakaryocyte maturation medium) used is as follows.

[0194] Megakaryocyte maturation medium (or maturation medium) The IMDM (concentration is final) contains the following components: 5% human plasma, 2 mM glutamine, 1% ITS-G, 0.45 mM 1-thioglycerol, 50 μg / mL L-ascorbic acid, 50 ng / mL SCF, 0.2 μg / mL TPO mimicry compound, 15 μM MKP-457 (research pharmaceutical), 10 μM Y-27632 (Nacalai Tesque, Code No. 18188-04), 0.75 μM SR-1 (Stemregenin-1), or 0.1 μM AhR inhibitor.

[0195] The maturation process of megakaryocytes involves seeding megakaryocytes at a density of 1×10⁶ cells / year. 5 Cells / mL, 25mL / flask, were seeded in 125mL culture flasks and cultured by shaking. Shaking culture was performed using LT-X (Lab-Therm) (Kuhner) or S41i (Eppendorf) at 37°C and 5% CO2 at a shaking speed of 100rpm.

[0196] 4. Evaluation of megakaryocyte proliferative capacity After day 0, cell counts were performed regularly, and the megakaryocyte-specific surface marker CD41 was detected by flow cytometry.

[0197] Cell counting was performed using trypan blue staining. Based on the seeded cell number, the cumulative proliferation rate from day 0 was calculated according to the calculated fold increase. Simultaneously, this was multiplied by the proportion of megakaryocytes calculated based on the CD41 positivity rate to obtain the megakaryocyte proliferation rate. The cumulative megakaryocyte proliferation rate at the nth passage was calculated using the following formula.

[0198] [Mathematical Expression 1] The inoculation density for the 0th passage is set as the inoculation density for day 0.

[0199] 5. Evaluation of platelet production capacity and platelet function After culturing megakaryocytes in megakaryocyte maturation medium with shaking for 6 days, a portion of the cell suspension was collected and analyzed using BD FACSVerse. TM (BD Biosciences) performed flow cytometry analysis, and followed the steps below to measure platelet production and evaluate platelet function.

[0200] To determine the number of platelets produced, the following antibodies were used for staining.

[0201] APC-labeled anti-CD41 antibody (BioLegend, Code No. 303710); PE-labeled anti-CD42b antibody (BioLegend, Code No. 303906); eFluor TM 450-labeled anti-CD42a antibody (Thermo Fisher, Code No. 48-0428-42).

[0202] After staining for 30 minutes, use TruCOUNT TM (BD Biosciences, Code No. 340334) Platelet count was determined (via FSC / SSC gating to microparticles, CD41 / CD42b double positive or CD41 / CD42a / CD42b positive). Flow cytometry data analysis for calculating platelet production was performed using FlowJo. TM Version 10 (BD Biosciences). Data references the number of megakaryocytes inoculated at the start of platelet production culture, expressed as the number of megakaryocytes produced per megakaryocyte.

[0203] To evaluate platelet function, stimulation was performed under the following two stimulation conditions. Stimulant 1: 0.02–0.4 μM PMA; Stimulant 2: 100 μM ADP / 40 μM thrombin receptor activating peptide 6 (TRAP6). After adding the stimulant, staining was performed using the following antibodies. APC-labeled anti-CD41 antibody (BioLegend, Code No. 303710); PE-labeled anti-CD62P antibody (BioLegend, Code No. 304906); FITC-labeled anti-PAC-1 antibody (BD Bioscience, Code No. 34507). After 30 minutes, use BD FACSVerse TM Conduct testing.

[0204] Example 1: Verifying the effect of megakaryocyte passage on platelet production capacity 15M41 cells introduced with the MBX box were induced to differentiate into hematopoietic progenitor cells, which were then induced to differentiate into megakaryocytes. Platelet-producing capacity analysis of cells from day 21 to day 78 of megakaryocyte induction showed... Figure 1 On day 21, each megakaryocyte has the ability to produce more than 200 platelets, but this ability declines thereafter, dropping to about 1 / 10 of the ability on day 21 after 64 days.

[0205] In addition, in megakaryocytes induced and differentiated from other human iPS cell lines (e.g., MH23S01, TkDN-sev2) using the same method, a decrease in platelet production capacity was also observed as passage culture progressed (data not disclosed).

[0206] Therefore, it was confirmed that megakaryocytes induced from human iPS cells tend to have a decreased platelet-producing capacity with passage.

[0207] Example 2: Exploration of proteins that promote platelet production As factors that, after megakaryocyte maturation under conditions that significantly enhance platelet production, are released from megakaryocytes into the culture medium, the inventors have identified six proteins, among which NRDC, possessing an endopeptidase domain, has attracted particular attention (Non-Patent Literature 2). To investigate the role of NRDC in platelet production, megakaryocyte-specific NRDC-deficient mice were created, and their platelet counts were analyzed. However, contrary to expectations, no significant reduction in platelet count was observed in these NRDC-deficient mice compared to control mice (data not disclosed).

[0208] Therefore, in order to find the factors that can truly regulate platelet production efficiency, we analyzed the proteins with increased expression levels in megakaryocytes matured through shaking culture compared to those matured through static culture. The results showed that knocking down the expression of UQCRC1 and UQCRC2, which are located in mitochondria and possess the M16 endopeptidase domain similar to NRDCs, decreased the platelet production capacity of megakaryocytes. Figure 2 Megakaryotic cells derived from TkDNsev2 cells induced to differentiate using MBX-introduced cassettes were inoculated with shRNAs of UQCRC1 and / or UQCRC2 via lentivirus on day 45, and their platelet production capacity was measured on days 66-81. Results showed... Figure 2 Compared to the control (megakaryocytes with LacZshRNA), platelet production capacity decreased to approximately 30% in megakaryocytes with either UQCRC1 shRNA or UQCRC2 shRNA, and further decreased to approximately 20% in megakaryocytes with both UQCRC1 shRNA and UQCRC2 shRNA.

[0209] These results strongly suggest that UQCRC1 and UQCRC2 play a positive role in regulating the platelet-producing capacity of megakaryocytes.

[0210] Example 3: Verification of platelet-generating capacity based on UQCRC1 and UQCRC2 The effects of forced expression of UQCRC1 and UQCRC2 on platelet production capacity were analyzed.

[0211] Using lentiviral vectors, UQCRC1 or UQCRC2 was introduced into megakaryocytes induced from differentiation of 15M41 cells with an introduced MBX box (day 21) or megakaryocytes induced from differentiation of 15M41 cells with an introduced MX box (day 28). Maturation was induced on days 47 or 56 (MBX) and 56 or 63 (MX), and platelet production capacity was analyzed. Results were shown in... Figure 3 Compared to the control group (megakaryocytes infected with a lentiviral vector encoding non-UQCRC1 / UQCRC2), megakaryocytes induced from differentiation of 15M41 cells with introduced MBX boxes produced significantly more platelets, even under forced expression of either UQCRC1 or UQCRC2. Figure 3 (Right image). By comparing with Figure 1 The comparison shows that by forcibly expressing UQCRC1 or UQCRC2, the decline in platelet production capacity associated with long-term passage culture was effectively suppressed.

[0212] Similarly, in megakaryocytes induced from differentiation of 15M41 cells with introduced MX boxes, forced expression of UQCRC1 or UQCRC2 maintained the same level of high platelet production as megakaryocytes induced from differentiation of 15M41 cells with introduced MBX boxes. Figure 3 (Left image).

[0213] Figure 4 The analysis results show the platelet-producing capacity of megakaryocytes infused with UQCRC2 via lentivirus after day 53. In megakaryocytes induced from differentiation of either 15M41 cells infused with the MX box or the MBX box, platelet-producing capacity was well maintained over extended periods, such as day 67 or day 60.

[0214] These results indicate that, at least in megakaryocytes in the proliferative phase, increasing the expression levels of UQCRC1 and / or UQCRC2 significantly improves the decline in platelet production associated with passage culture, allowing megakaryocytes to maintain platelet production capacity over a long period.

[0215] Example 4: Exploration of compounds that promote platelet production Next, we attempted to use compounds to promote platelet production. It is known that megakaryocytes in the bone marrow promote platelet production by taking up fatty acids released from adipocytes (Adipocyte Fatty Acid Transfer Supports Megakaryocyte Maturation. Cell Reports. 2020;32(1):107875). Furthermore, it is known that lipid synthesis and the uptake of fatty acids from external sources are important for megakaryocyte maturation and platelet production (Critical shifts inlipid metabolism promote megakaryocyte differentiation and proplatelet formation. Nat Cardiovasc Res. 2023 Sep;2:835-852). Based on this, we hypothesized that lipid synthesis would be more active in megakaryocytes by using drugs that induce adipocytes to perform lipid metabolism and lipid synthesis. Therefore, we verified this hypothesis by culturing megakaryocytes in the presence of adipocyte differentiation-inducing factors.

[0216] In the culture medium (differentiation and proliferation medium) of megakaryocytes expressing the exogenous UQCRC2 (derived from 15M41 cells introduced with an MBX cassette), dextran (1 μM), IBMX (50 μM), and insulin (10 μg / ml) were added from day 1 to day 3. The concentrations in parentheses are final concentrations. After transfer to megakaryocyte maturation medium, JI051 (10 μM) was added after 3 days, and platelet production capacity was analyzed. The results are shown in […]. Figure 5 Compared to the control group without JI051, megakaryocytes with JI051 showed approximately 2-fold increased platelet production.

[0217] Therefore, it can be seen that if glucan, IBMX and insulin are added to expand the culture of megakaryocytes, and JI051 is added to mature them in the subsequent maturation stage, the platelet production capacity of megakaryocytes will be further improved.

[0218] Furthermore, LMK-235 was discovered as a substitute compound for UM729 in megakaryocyte differentiation and proliferation media. Megakaryocyte differentiation was induced using differentiation and proliferation media in which UM729 was replaced with LMK-235, and the cell number was analyzed after 3 days. Figure 6 (as shown in the image above) and CD34 positive cell rate ( Figure 6 (See the image below), the results are shown in Figure 6It should be noted that forced expression of UQCRC2 was not performed in this experiment. When using LMK-235 at a final concentration of 0.2 μM, both the cell number and the CD34-positive cell rate increased compared to the case using UM729.

[0219] LMK-235 is known as an inhibitor of HDAC4, 5, and 6, so the same analysis was performed on other HDAC inhibitors as well. Figure 7 The analysis shows the percentage of CD34 and CD41a positive cells in megakaryocytes from 15M41 cells introduced with the MBX box or the MX box, which were forced to express UQCRC2. CD34 expression was maintained in LMK-235 and TMP195 (HDAC4, 5, 7, 9 inhibitors), while it was decreased in taquimod (HDAC4 inhibitor). These results suggest that HDAC5 helps maintain CD34 expression in proliferating megakaryocytes.

[0220] To date, although shaking culture was performed during the maturation process, the effects of switching to static culture on UM729 and LMK-235 have been analyzed. Megakaryotic cells derived from 15M41 cells with an introduced MX box and forced expression of UQCRC2 were induced for differentiation and expansion using either standard differentiation and proliferation medium containing 1 μM UM729 or differentiation and proliferation medium with UM729 replaced by 200 nM LMK-235. These cells were then matured under static culture, and their platelet production capacity was compared. Figure 8 Since maturation is achieved through static culture, the number of platelets produced is less compared to maturation through shaking culture. However, even when expanded culture is carried out in a medium containing LMK-235, the number of platelets produced is not significantly different from that when expanded culture is carried out in a medium containing UM729.

[0221] Therefore, as one aspect of the present invention, it is clarified that the pyridoindole derivatives (UM729, etc.) added to the culture medium during the induction of differentiation from hematopoietic progenitor cells to megakaryocytes and the subsequent expansion culture steps can be replaced by HDAC5 inhibitors such as LMK-235 or TMP195.

[0222] Example 5: Validation of the proliferative capacity of megakaryocytes derived from iPS cell lines This study verified whether the induced megakaryocyte proliferation capacity differed depending on the iPS cell line source. First, MBX boxes were introduced into various iPS cell lines (15M41, MH09S01, MH15S01, MH15S01, and MH23S01) to prepare MBX-boxed iPS cell lines (introducing three megakaryocyte inducing factors). These MBX-boxed iPS cell lines were induced into imMKCL, sorted into CD34 / CD41a co-positive fractions, and cultured further. CD34 and CD41 expression was evaluated using FACS. The results showed that imMKCL from any iPS cell line maintained a CD34 / CD41a co-positive expression system for 42 days. Figure 10 ).

[0223] Next, validation was performed using two, rather than three, megakaryocyte inducing factors. A construct encoding two genes (MYCdd under a tetracycline-inducible promoter and BCL2L1 under a UbiC promoter) was introduced into various iPS cells (15M41 and QHJI14s04-AB II-KO-03). This construct encodes p21 shRNA and p53 shRNA under the H1 promoter. The iPS cells were induced to differentiate into megakaryocytes, and CD34 / CD4a co-positive fractions were sorted and cultured. CD34 and CD41 expression was evaluated using FACS. The results showed that a large number of cells from the QHJI14s04-AB II-KO-03 megakaryocyte population were unable to maintain CD34 / CD41a co-expression. Figure 11 On the other hand, megakaryocytes derived from 15M41 maintained CD34 / CD41a co-expression. Figure 12 ).

[0224] Example 6: Verification of the correlation between multicomb complex 1.1 and megakaryocyte proliferation capacity It has been reported that 15M41 carries mutations in the BCOR (BCL6 co-repressor) and BRD3 genes, while QHJI14s04 does not. Therefore, the difference in mutations in the BCOR or BRD3 genes is thought to be the reason why the maintenance effect of CD34 / CD41a co-expression varies depending on the iPS cell origin. First, we focus on the BCOR gene and its homolog, BCORL1 (BCL6 co-repressor-like protein 1).

[0225] Therefore, by inhibiting the expression of BCOR or BCORL1, we investigated whether megakaryocytes from the QHJI14s04 / ABII-KO-03 strain exhibited an expression system equivalent to that from megakaryocytes from the 15M41 strain. In megakaryocytes from the QHJI14s04 / ABII-KO-03 strain, starting on day 28 from the initial addition of Dox, BCOR or BCORL1 expression was knocked down using lentivirus with shRNAs (also known as sh BCOR and sh BCORL1, respectively) targeting the transcripts of these genes. The results successfully yielded a megakaryocyte cell line maintaining CD34 / CD41a co-expression. Figure 13 Furthermore, it was confirmed that megakaryocytes with knocked-down BCOR or BCORL1 expression had higher platelet-producing capacity compared to unknocked megakaryocytes. Figure 14 ).

[0226] BCOR and BCORL1 are proteins that constitute the polycomb repressor complex 1.1 (PRC1.1) and are known as transcription factors involved in embryogenesis, mesenchymal stem cell function, hematopoiesis, and the regulation of lymphatic system development (e.g., Sportoletti P., Sorcini D., and Falini B., Blood. 138(24):2455-2468 (2021)). Furthermore, the inventors previously identified TRIM27 (containing triple motif 27) as one of the genes with significantly different methylation levels among iPS cells with vastly different induction efficiencies when inducing megakaryocytes using three factors (MYCdd / BMI1 / BCLxL) (data not publicly available). TRIM27 has also been reported as a component of PRC1.1 (Maat H. et al., iScience. 24(5):102435 (2021)). Additionally, the inventors believe that among the transcription factors involved in hematopoietic lineage differentiation, there may be factors involved in megakaryocyte proliferation. Subsequently, further analysis was conducted on the results of in-depth research, focusing on BACH1, which contributes to the formation of erythrocytes and lymphocytes.

[0227] Starting with iPS cells, introduce Figure 15 When the vector combinations shown were used to induce megakaryocytes, it was demonstrated that by overexpressing UQCRC2 and BACH1 and knocking down BCOR, megakaryocytes co-expressing CD34 / CD41a could be maintained, and platelet production could also be maintained at a high level; by further knocking down BCORL1 or TRIM27, platelet production was further enhanced (Figs. 16 and 17). In addition, it was also shown that even without knocking down any of BCOR, BCORL1, and TRIM27, platelet production could be maintained at a high level simply by overexpressing BACH1 in megakaryocytes (Fig. 18).

[0228] Example 7: Exploration of compounds that help maintain megakaryocytes In addition to HDAC5 inhibitors, compounds that promote megakaryocyte maintenance or platelet production were explored, showing that a lysine-specific histone demethylase 1 (LSD1) inhibitor (transphenylcyclopropane) effectively maintains CD34 / CD41a co-expression in megakaryocytes. It was demonstrated that, particularly by combining HDAC5 and LSD1 inhibitors, high levels of CD34 / CD41a co-expression were maintained in megakaryocytes. Figure 19 ).

[0229] Industrial practicality According to the present invention, regardless of the type of iPS cell line, megakaryocytes (high-quality chief cells) that can be maintained at a high quality over a long period can be prepared. This enables a stable supply of high-quality platelets (platelets that maintain hemostasis and circulation). Since the above objectives can be achieved easily and at low cost, this invention is extremely useful from the viewpoint of the social application of artificial platelets.

[0230] This application is based on Japanese Special Application 2024-020854 (application date: February 15, 2024), the entire contents of which are contained in this specification.

Claims

1. A method for preparing megakaryocytes with platelet-producing capacity, comprising the step of increasing the amount of at least one of the proteins constituting ubiquitin-cytochrome c reductase (UQCR) in the megakaryocytes.

2. The method according to claim 1, wherein, At least one of the proteins that make up UQCR is the UQCR core protein (UQCRC).

3. The method according to claim 2, wherein, At least one of the UQCRCs is UQCRC2.

4. The method according to any one of claims 1 to 3, wherein, The steps to increase the amount of the protein that makes up the UQCR include introducing the nucleic acid encoding the protein into megakaryocytes.

5. A method for preparing megakaryocytes with maintained platelet-producing capacity, comprising the step of inhibiting the function of polycomb inhibitory complex 1.1 in the megakaryocytes.

6. The method according to any one of claims 1 to 4, comprising the step of inhibiting the function of the multicomb inhibitory complex 1.1 in megakaryocytes.

7. The method according to claim 6, wherein, The steps of inhibiting the function of the polycomb repressor complex 1.1 include reducing the amount of at least one of the proteins constituting the polycomb repressor complex 1.

1.

8. The method according to claim 7, wherein, The protein constituting the polycomb inhibitory complex 1.1 is at least one selected from BCOR protein, BCORL1 protein and TRIM27 protein.

9. The method according to any one of claims 1 to 8, comprising the step of increasing the amount of BACH1 protein present in megakaryocytes.

10. The method according to any one of claims 1 to 9, comprising the step of culturing megakaryocytes in a culture medium containing an HDAC5 inhibitor.

11. The method according to any one of claims 1 to 10, comprising the step of culturing megakaryocytes in a culture medium containing adipocyte differentiation inducing factor.

12. The method according to any one of claims 1 to 11, wherein, The culture medium contains compounds represented by the following structural formulas. [Chemical Formula 1] 。 13. The method according to any one of claims 1 to 12, comprising the step of increasing the amount of MYC protein and apoptosis inhibitor protein present in megakaryocytes.

14. The method according to any one of claims 1 to 13, wherein, Megakaryocytes are derived from pluripotent stem cells.

15. A megakaryocyte obtained by the method according to any one of claims 1 to 14.

16. A megakaryocyte possessing all of the following characteristics (A) to (C): (A) Having at least one of the exogenous proteins that constitute ubiquitin-cytochrome c reductase (UQCR); (B) Possesses the ability to produce platelets; (C) Expression of CD34 and CD41 genes.

17. The megakaryocyte according to claim 16, wherein, At least one of the proteins that make up UQCR is a UQCR core protein.

18. A method for preparing platelets, comprising the step of maturing megakaryocytes according to any one of claims 15 to 17.

19. The method of claim 18, further comprising a shaking culture step.

20. A platelet obtained by the method of claim 18 or 19.

21. A blood preparation comprising the cells according to any one of claims 15-17 and 20.

22. A method for preparing megakaryocytes, comprising the step of culturing hematopoietic progenitor cells in a culture medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor.

23. The method according to claim 22, wherein, At least one of the HDAC5 inhibitors is LMK235.

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