Method for producing megakaryocytes

Abstract

The present invention provides a method for producing megakaryocytes that retain thrombopoiesis capability, the method comprising a step for increasing, in megakaryocytes, the abundance of at least one type of protein constituting ubiquinol-cytochrome c reductase (UQCR).

Description

Method for producing megakaryocytes

[0001] The present invention relates to a method for producing megakaryocytes, megakaryocytes produced by the method, and uses of the megakaryocytes.

[0002] Many blood cells are required for the treatment of blood-related diseases and surgical procedures. Among blood cells, platelets, essential for blood clotting and hemostasis, are particularly important. Platelets are in high demand for leukemia, bone marrow transplants, anti-cancer treatments, and other treatments, making a stable supply essential. To date, platelets have been obtained by collecting them from donors, administering TPO-like mimetics, or differentiating megakaryocytes from umbilical cord blood or bone marrow cells. Recently, techniques have been developed to induce differentiation of pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), in vitro to produce blood cells such as platelets.

[0003] To meet the societal demand for platelets, the present inventors have developed a technology for the stable in vitro production of platelet preparations using human iPS cells (Non-Patent Documents 1 and 2). The present inventors established a technology for mass-producing platelets (human iPS cell-derived artificial platelet preparations) by 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 and then maturing the megakaryocyte lines (Patent Documents 1 to 4, Non-Patent Document 1). Furthermore, they have reported culture conditions (hydrodynamic requirements, particularly turbulence stimulation) that dramatically improve platelet production during the megakaryocyte maturation process (Patent Document 5, Non-Patent Document 2).

[0004] International Publication No. 2011 / 034073 International Publication No. 2012 / 157586 International Publication No. 2014 / 123242 International Publication No. 2021 / 075568 International Publication No. 2019 / 009364

[0005] Cell Stem Cell. 2014 Apr 3; 14(4): 535-548.Cell. 2018 Jul 26; 174(3): 636-648.

[0006] However, the present inventors have found that the platelet-producing ability of megakaryocyte lines induced by the above-mentioned conventional differentiation induction methods declines with passage, and the degree of decline varies significantly among the pluripotent stem cell lines from which hematopoietic progenitor cells are derived (Figure 1). Therefore, an objective of the present invention is to provide a method for proliferating megakaryocytes induced by conventional induction methods while maintaining their platelet-producing ability.

[0007] The inventors found that megakaryocytes forced to express the core proteins (UQCRC1, UQCRC2) of complex III (ubiquinol-cytochrome c reductase), which is responsible for the electron transport chain in mitochondria, maintained their platelet-producing ability even after long-term subculture.

[0008] Furthermore, we found that the addition of adipogenic factors (dexamethasone, IBMX, insulin) during the proliferation phase of megakaryocytes expressing UQCRC2 increased platelet production by approximately 1.5-fold, and that the addition of JI051 to this increased platelet production by approximately 2-fold. Furthermore, we found that the addition of an HDAC5 (histone deacetylase 5) inhibitor or an LSD1 (lysine-specific histone demethylase) inhibitor during the proliferation phase of megakaryocytes also maintained CD34 / CD41 co-positivity, and platelet production after long-term culture was also maintained.

[0009] The present inventors investigated the effect of iPS cell line origin on the proliferation potential of megakaryocytes. They found that the proliferation potential of megakaryocytes induced by the introduction of two megakaryocyte-inducing factors (i.e., the MYCdd gene and the BCL2L1 gene) differed depending on the iPS cell origin. Pursuing the cause of this difference, they found that a protein constituting the polycomb repressive complex 1.1 is involved in the proliferation potential of megakaryocytes. Knocking down this protein successfully improved the proliferation potential of megakaryocytes.

[0010] Furthermore, to further explore factors that contribute to megakaryocyte proliferation, we analyzed several transcription factors involved in the differentiation process of blood cell lineages. Surprisingly, we found that overexpression of BACH1, which contributes to erythropoiesis and lymphopoiesis, can improve megakaryocyte proliferation and platelet production. Based on these findings, the present inventors conducted further research and completed the present invention.

[0011] That is, the present invention is as follows: [1] A method for producing megakaryocytes that maintain platelet production capacity, comprising the step of increasing the abundance of at least one protein that constitutes ubiquinol cytochrome c reductase (UQCR) in megakaryocytes. [2] The method according to [1], wherein at least one of the proteins that constitute UQCR is UQCR core protein (UQCRC). [3] The method according to [2], wherein at least one of the UQCRCs is UQCRC2. [4-1] The method according to any one of [1] to [3], wherein the step of increasing the abundance of a protein that constitutes UQCR comprises the step of introducing a nucleic acid encoding the protein into megakaryocytes. [4-2] The method according to any one of [1] to [4-1], wherein the nucleic acid is introduced by a viral vector. [5-1] A method for producing megakaryocytes that maintains platelet production capacity, comprising the step of inhibiting the function of polycomb repressive complex 1.1 in megakaryocytes. [5-2] The method according to [5-1], wherein the step of inhibiting the function of polycomb repressive complex 1.1 comprises reducing the abundance of at least one protein constituting polycomb repressive complex 1.1. [6] The method according to any one of [1] to [4-2], wherein the step of inhibiting the function of polycomb repressive complex 1.1 in megakaryocytes comprises reducing the abundance of at least one protein constituting polycomb repressive complex 1.1. [7] The method according to [6], wherein the step of inhibiting the function of polycomb repressive complex 1.1 comprises reducing the abundance of at least one protein constituting polycomb repressive complex 1.1. [8] The method according to [5-2] or [7], wherein the protein constituting polycomb repressive complex 1.1 is at least one selected from the group consisting of BCOR protein, BCORL1 protein, and TRIM27 protein. [9-1] The method according to any one of [1] to [8], wherein the step of increasing the abundance of BACH1 protein in megakaryocytes. [9-2] The method according to any one of [1] to [9-1], comprising the steps of: (1) reducing the abundance of BCOR protein or BCORL1 protein, and (2) TRIM27 protein, and (3) increasing the abundance of BACH1 protein in megakaryocytes.[10-1] The method according to any one of [1] to [9-2], comprising a step of culturing megakaryocytes in a medium containing an HDAC5 inhibitor. [10-2] The method according to [10-1], wherein at least one of the HDAC5 inhibitors is LMK235. [11-1] The method according to any one of [1] to [10-2], comprising a step of culturing megakaryocytes in a medium containing an adipogenic factor. [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.

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

[0012]

[0013]

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

[12] , comprising a step of increasing the abundance of MYC protein and apoptosis inhibitor protein in megakaryocytes. [14-1] The method according to any one of [1] to

[13] , wherein the megakaryocytes are derived from pluripotent stem cells. [14-2] The method according to any one of [1] to [14-1], wherein the megakaryocytes express the CD34 gene and the CD41 gene. [14-3] The method according to any one of [1] to [14-2], wherein the megakaryocytes are derived from humans.

[15] Megakaryocytes obtained by the method according to any one of [1] to [14-3]. [16-1] Megakaryocytes having all of the following characteristics (A) to (C): (A) Having at least one protein constituting exogenous ubiquinol-cytochrome c reductase (UQCR), (B) Having platelet-producing ability, (C) Expressing the CD34 gene and the CD41 gene. [16-2] The megakaryocyte according to [16-1], further having at least one of the following characteristics (D) to (F): (D) Being derived from a pluripotent stem cell, (E) Having an exogenous megakaryocyte-inducing factor, (F) Having an expression inhibitor for the TP53 gene and / or the CDKN1A gene. [17-1] The megakaryocyte according to [16-1] or [16-2], wherein at least one protein constituting UQCR is UQCR core protein (UQCRC). [17-2] The megakaryocyte according to [17-1], wherein at least one UQCRC is UQCRC2.

[18] A method for producing platelets, comprising a step of maturing the megakaryocyte according to any one of

[15] to [17-2]. [19-1] The method according to

[18] , which comprises a shaking culture step. [19-2] The method according to

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

[20] Platelets obtained by the method according to any one of

[18] to [19-2]. [21-1] A blood product comprising the cells according to any one of

[15] to [17-2] and

[20] . [21-2] The agent according to [21-1], for treating or preventing blood diseases or bleeding. [22-1] A method for producing megakaryocytes, which comprises a step of culturing hematopoietic progenitor cells in a medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor.[22-2] The method according to [22-1], wherein the culture medium contains an HDAC5 inhibitor and an LSD1 inhibitor. [23-1] The method according to [22-1] or [22-2], wherein at least one of the HDAC5 inhibitors is LMK235. [23-2] The method according to any one of [22-1] to [23-1], wherein at least one of the LSD1 inhibitors is tranylcypromine.

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

[15] to [17-2] and

[20] into a mammal.

[25] The cells according to any one of

[15] to [17-2] and

[20] for use in treating or preventing blood disorders or bleeding.

[26] Use of the cells according to any one of

[15] to [17-2] and

[20] in the manufacture of a medicament for treating or preventing blood disorders or bleeding.

[0014] According to the present invention, it is possible to produce megakaryocytes that maintain their platelet-producing ability over a long period of time, regardless of the type of pluripotent stem cell line, which may enable a stable supply of high-quality platelets.

[0015] This graph shows the time course of platelet production in megakaryocytes derived from MBX cassette-introduced 15M41 human iPS cells. The horizontal axis represents the number of days since the start of culture in doxycycline (Dox)-containing medium (megakaryocyte differentiation and proliferation medium), and the vertical axis represents the number of platelets released per megakaryocyte. This graph shows the platelet production in megakaryocytes derived from MBX cassette-introduced TkDN sev2 cells after UQCRC1 and / or UQCRC2 knockdown. The horizontal axis represents the type of shRNA introduced into the megakaryocytes, and the vertical axis represents the relative platelet production compared to that of the control (megakaryocytes transduced with LacZ shRNA). Megakaryocytes induced to differentiate from MBX cassette-introduced 15M41 cells or MX cassette-introduced 15M41 cells were transduced with UQCRC1 or UQCRC2 using a lentiviral vector, and platelet production was analyzed after maturation. The horizontal axis represents the type of gene introduced using the lentiviral vector, and the vertical axis represents the number of platelets released per megakaryocyte. N=2, error bars: standard deviation. Megakaryocytes induced to differentiate from MBX cassette-introduced 15M41 cells or MX cassette-introduced 15M41 cells were transduced with UQCRC2 using a lentiviral vector, and then matured after long-term expansion culture. The horizontal axis represents the number of days since the start of culture in megakaryocyte differentiation and growth medium, and the vertical axis represents the number of platelets released per megakaryocyte. Megakaryocytes derived from MBX cassette-introduced 15M41 cells expressing exogenous UQCRC2 were expanded in differentiation and growth medium supplemented with dextran, IBMX, and insulin for more than 3 days, transferred to maturation medium, and JI051 was added 3 days later. The horizontal axis represents the compound added, and the vertical axis represents the number of platelets released per megakaryocyte. Differentiation into megakaryocytes was induced using a differentiation and growth medium in which UM729 was replaced with LMK-235, and the cell count (top graph) and CD34-positive cell rate (bottom graph) were measured by FACS three days later. The horizontal axis shows the compound used, and the number after LMK shows the concentration of LMK-235 (unit: μM).Megakaryocyte differentiation was induced using differentiation and growth medium containing either 200 nM LM-K235 (an inhibitor of HDACs 4, 5, and 6), 100 nM TMP195 (an inhibitor of HDACs 4, 5, 7, and 9), or 100 nM Tasqunimod (an inhibitor of HDAC4). CD34 positivity and CD41a positivity were measured by FACS on day 3 of expansion culture. 200 nM LMK-235, 100 nM TMP195, or 100 nM Tasqunimod have similar HDAC inhibitory effects. This graph shows platelet production performance when differentiation and expansion were induced using differentiation and growth medium containing 200 nM LMK-235 instead of 1 μM UM729, followed by static culture for maturation. The vertical axis represents the number of platelets released per megakaryocyte. A schematic diagram of the method for inducing megakaryocytes from pluripotent stem cells using UM729 is shown. The vectors outlined in the figure were introduced into iPS cell lines (15M41, MH09S01, MH15S01, MH15S01, and MH23S01) to induce imMKCL, and the CD34 and CD41a positivity rates were measured by FACS. A) FACS results 14 days after the start of SCF, TA-316, UM729, and Dox addition. The CD34 / CD41a co-positive fraction was selected and subsequently cultured in medium supplemented with SCF, TA-316, UM729, and Dox. B) After imMKCL induction (day 1 being the start of Dox addition), cells derived from all iPS cell lines maintained the CD34 / CD41a co-positive phenotype for 42 days. The vectors outlined in the figure were introduced into an iPS cell line (QHJI14s04 / AB II-KO-03) to induce imMKCL, and the CD34 and CD41a positivity rates were measured by FACS (the vertical axis of the graph represents CD34 expression levels, and the horizontal axis represents CD41a expression levels). The CD34 / CD41a co-expressing fraction was selected on day 19 after induction (day 1 was the day Dox administration began), and FACS analysis was performed on cells on days 28, 32, 36, and 42 after imMKCL induction. The results revealed the emergence of CD34-negative, CD41a-positive cells, making it difficult to maintain CD34 / CD41a expression.The vectors outlined in the figure were introduced into an iPS cell line (15M41) to induce imMKCL, and the CD34 and CD41a positivity rates were measured by FACS. 15M41 cells have been reported to have mutations in the BCOR and BRD3 genes. (A) Left: Results are shown from day 14 after the start of SCF, TA-316, UM729, and Dox addition. The CD34 / CD41a co-positive fraction was selected and subsequently cultured in medium supplemented with SCF, TA-316, UM729, and Dox. (A) Right: After imMKCL induction (day 1 is the start of Dox addition), only cells derived from the 15M41 iPS cell line maintained the CD34 / CD41a co-positive phenotype. Figure 11 shows the results of FACS analysis of CD34 and CD41a positivity in megakaryocytic cell lines derived from QHJI14s04 / AB II-KO-03 (28 days after the start of Dox administration) after lentiviral knockdown of BCOR or BCORL1. Knockdown of BCOR (BCL6 corepressor) or BCORL1 (BCL6 corepressor like 1) successfully generated megakaryocytes that maintained CD34 / CD41a co-positivity. The platelet production performance of QHJI14s04 / AB II-KO-03-derived megakaryocytes (No. 3 cells; control), No. 3 cells with BCOR knockdown (shBCOR), and No. 3 cells with BCORL1 knockdown (shBCOR) is compared. The results for No. 3 cells were obtained 29 days after the start of Dox administration, and for shBCOR and shBCORL1 cells 36 days after the start of Dox administration. shBCOR and No. 3 shBCORL1 showed 2- to 3-fold higher platelet production than No. 3 cells. Plasmids incorporating BACH1 (outlined by numbers 1 to 5 in the figure) were prepared, and combinations of the above plasmids (combination 1, 4, and 5; combination 2, 4, and 5; combination 3, 4, and 5) were introduced into iPS cell lines (QHJI14s04 / AB II-KO-03) using lipofection to induce megakaryocytes.Combinations 1, 4, and 5 are combinations of overexpressing c-MYCdd, overexpressing BACH1 and BCL-XL, and knocking down BCOR in the presence of Dox. Combinations 2, 4, and 5 are combinations of overexpressing c-MYCdd, overexpressing BACH1 and BCL-XL, and knocking down BCOR and BCORL1 in the presence of Dox. Combinations 3, 4, and 5 are combinations of overexpressing c-MYCdd, overexpressing BACH1 and BCL-XL, and knocking down BCOR and Tripartite Motif Containing 27 (TRIM27) in the presence of Dox. A) iPS cell line (QHJI14s04 / AB II-KO-03) transfected with plasmids 1, 4, and 5 in Figure 15 was induced to develop megakaryocytes, and CD34 / CD41a co-positive cells were selected. The results of FACS analysis on day 58 after induction (day 1 was the day Dox administration began). B) An iPS cell line (QHJI14s04 / AB II-KO-03) transfected with plasmids 2, 4, and 5 in Figure 15 was induced to megakaryocytes, and CD34 / CD41a co-positive cells were selected. FACS results are shown on day 58 after induction (day 1 was counted as day 1). C) An iPS cell line (QHJI14s04 / AB II-KO-03) transfected with plasmids 3, 4, and 5 in Figure 15 was induced to megakaryocytes, and CD34 / CD41a co-positive cells were selected. FACS results are shown on day 58 after induction (day 1 was counted as day 1). Expression of BACH1 successfully generated megakaryocytes that maintained CD34 / CD41a co-positive positivity. D) Comparison of platelet-producing ability of each cell line. Megakaryocytes (BCOR / TRIM27) induced with plasmids 3, 4, and 5 produced the most platelets. The graph on the far left shows the platelet production of megakaryocytes transfected with plasmids 4 and 5 (c-MYC / BCL-XL / UQCRC2). Plasmids 3, 4, and 5 outlined above were transfected into iPS cells (QHJI14s04 / AB II-KO-11) by lipofection to induce megakaryocytes, and the CD34 and CD41a positivity rates were measured by FACS.A) An iPS cell line (QHJI14s04 / AB II-KO-11) transfected with plasmids 3, 4, and 5 was induced to develop megakaryocytes, and CD34 / CD41a co-positive cells were selected. The results of FACS analysis on day 64 after induction (the day Dox addition began was counted as day 1) are shown. The platelet production capacity of QHJI14s04 / AB II-KO-3-derived megakaryocytes (No. 3 cells) and QHJI14s04 / AB II-KO-11-derived megakaryocytes (No. 11 cells) is compared. The graphs show, from left to right, the number of platelets produced by No. 3 cells (c-MYC / BCL-XL / sh p21 (p21 knocked down using shRNA) / shp53 (p53 knocked down using shRNA)) on day 40 after the start of Dox administration, the number of platelets produced by No. 3 UQ2 cells (c-MYC / BCL-XL / UQCRC2 / sh p21 / shp53) on day 36 after the start of Dox administration, the number of platelets produced by No. 3 UQ2 BACH1 cells (c-MYC / BCL-XL / UQCRC2 / BACH1 / sh BCOR / sh TRIM27 (TRIM27 knocked down using shRNA)) on days 26, 27, 33, 42, 48, and 50 after the start of Dox administration (mean platelet count ± standard deviation at each time point), and the number of platelets produced by No. 11 cells (c-MYC / BCL-XL / sh The figures show the platelet production numbers of No.11 UQ2 cells (c-MYC / BCL-XL / UQCRC2 / sh p21 / shp53) on day 40 after the start of Dox addition, the platelet production numbers of No.11 UQ2 cells (c-MYC / BCL-XL / UQCRC2 / sh p21 / shp53) on day 36 after the start of Dox addition, and the platelet production numbers of No.11 UQ2 BACH1 cells (c-MYC / BCL-XL / UQCRC2 / BACH1 / sh BCOR / sh TRIM27) on days 26 and 29 after Dox addition (mean platelet counts at each time point ± standard deviation). No.11 UQ2 BACH1 cells were shown to produce the most platelets. The plasmids outlined above were introduced into iPS cells (QHJI14s04 / AB II-KO-3, QHJI14s04 / AB II-KO-11) using lipofection to induce megakaryocytes, and the CD34 positivity rate and CD41a positivity rate were measured by FACS.A) Plasmid-transfected iPS cell lines were induced to develop megakaryocytes, and CD34 / CD41a co-positive cells were selected. The FACS results are shown on day 48 (for megakaryocytes derived from QHJI14s04 / AB II-KO-3) or day 42 (for megakaryocytes derived from QHJI14s04 / AB II-KO-11) after induction (the day Dox addition began was counted as day 1). B) Comparison of the platelet-producing ability of each megakaryocyte is shown. The graphs on the left show, from left to right, the number of platelets produced by No.3 cells (c-MYC / BCL-XL) on day 40 after the start of Dox addition, the number of platelets produced by No.3 UQ2 cells (c-MYC / BCL-XL / UQCRC2) on day 36 after the start of Dox addition, the number of platelets produced by No.3 UQ2 tet BACH1 cells (c-MYC / BCL-XL / UQCRC2 / tet BACH1) on days 33, 35, 39, 43, and 46 after the start of Dox addition (mean ± standard deviation of platelet counts at each time point), the number of platelets produced by No.11 cells (c-MYC / BCL-XL) on day 40 after the start of Dox addition, the number of platelets produced by No.11 UQ2 cells (c-MYC / BCL-XL / UQCRC2) on day 36 after the start of Dox addition, and the number of platelets produced by No.11 UQ2 tet The figures show the platelet production (mean ± standard deviation) of BACH1 cells (c-MYC / BCL-XL / UQCRC2 / tet BACH1) on days 26, 28, 32, 36, 39, and 46 after Dox addition. No. 11 UQ2 tet BACH1 cells were shown to produce the most platelets. The right panel shows the results of measuring platelet production over time in No. 11 UQ2 tet BACH1 cells. imMKCs, differentiated from MX cassette-transfected 15M41 cells, were cultured for 10 days with SCF, TA316, and Dox in combination with UM729 (1 μM), LMK235 (200 nM), 2PCPA (trans-2-phenylcyclopropylamine) (also known as tranylcypromine) (1 μM), or a combination of LMK235 (200 nM) and 2PCPA (1 μM). The cell count and FACS analysis results are shown. While LMK235 alone and 2PCPA alone were not highly effective in maintaining CD34 / CD41a expression, the combined use of LMK235 and 2PCPA maintained CD34 / CD41a expression.

[0016] 1. Method for Producing Megakaryocytes The present invention provides a method for producing megakaryocytes with platelet-producing capacity maintained. Specifically, the present invention provides a method for producing megakaryocytes with platelet-producing capacity maintained, which comprises increasing the abundance of at least one of the proteins constituting ubiquinol cytochrome c reductase (UQCR) in megakaryocytes (hereinafter, this method may be referred to as the "method of the present invention"). In another aspect, the present invention provides a method for producing megakaryocytes with platelet-producing capacity maintained, which comprises inhibiting the function of Polycomb Repressive Complex 1.1 (PRC1.1) (also referred to as non-canonical PRC1.1) in megakaryocytes (unless otherwise specified, this method is also encompassed by the "method of the present invention"). Furthermore, these methods may be combined. Specifically, the present invention also provides a method for producing megakaryocytes with platelet-producing capacity maintained, which comprises increasing the abundance 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, the steps may be performed sequentially or simultaneously. Furthermore, in this specification, the term "method for producing megakaryocytes" can also be appropriately interpreted as a "method for proliferating megakaryocytes" or a "method for expanding megakaryocytes."

[0017] As used herein, unless otherwise specified, "cells" such as megakaryocytes include "cell populations." Furthermore, unless otherwise specified, "cells" refer to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

[0018] Megakaryocytes can be characterized as CD41a-positive, CD42a-positive, and CD42b-positive. In addition to these markers, megakaryocytes can express one or more markers selected from the group consisting of CD9, CD34, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. Megakaryocytes can also express GATA1, FOG1, NF-E2, and β1-tubulin. Megakaryocytes can be multinucleated, mononucleated, or binucleated. Megakaryocytes can also be immortalized as megakaryocyte lines or cloned cell populations. Non-multinucleated megakaryocytes can be expanded by expansion culture and are sometimes referred to as immortalized megakaryocyte progenitor cell lines (imMKCL). Immortalized megakaryocytes produce functional platelets through maturation. Megakaryocyte maturation refers to the differentiation of megakaryocytes into the required multinucleated state and the ability to produce functional platelets. Functional platelets may be, for example, CD42b-positive platelets. Megakaryocyte maturation can also be confirmed by increased expression of megakaryocyte maturation-related genes, such as GATA1, FOG1, FLI1, NF-E2, and β1-tubulin. In one embodiment, the megakaryocytes used in the present invention are CD34-positive and CD41-positive cells. In another embodiment, the megakaryocytes used in the present invention are CD38-negative, CD90-positive, and / or CD49f-positive. As used herein, CD41-positive cells refer to CD41a-positive cells.

[0019] In one embodiment of the present invention, megakaryocytes have an exogenous gene encoding a MYC protein and an exogenous gene encoding an apoptosis-inhibiting protein, and preferably further have an exogenous gene encoding a polycomb group protein. In this specification, these MYC protein, apoptosis-inhibiting protein, and polycomb group protein can be collectively referred to as "megakaryocyte-inducing factor."

[0020] Examples of genes encoding MYC proteins (hereinafter also referred to as "MYC genes") include 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 the gene consisting of the nucleic acid sequence shown by NCBI accession number NM_002467.

[0021] The c-MYC gene may be a c-MYC gene encoding a protein fused with a destabilization domain. A "destabilization domain" refers to a domain that destabilizes the protein to which it is operably linked. The destabilization domain can be operably linked to the N-terminus or C-terminus of the protein. Examples of destabilization domains include ubiquitin, PEST sequences (proline, glutamic acid, serine, and threonine-rich sequences), cyclin destruction boxes, hydrophobic stretches of amino acids, Escherichia coli dihydrofolate reductase (ecDHFR), human estrogen receptor ligand binding domain (ERLBD), FK506 binding protein (FKBP12), and mutants thereof. Examples of destabilization domains include FKBP12 and mutants thereof. Examples of FKBP12 mutants used as destabilizing domains include F15S, V24A, H25R, E60G, L106P, M66T, R71G, D100G, D100N, E102G, and K105I mutants (Banaszynski et al., Cell 126:995 (2006)). Commercially available destabilizing domains may be used, for example, those available from TAKARA Bio (Clontech ProteoTuner TM It is sold by Shield System C, #631072.

[0022] The "apoptosis-inhibitory gene" is not particularly limited as long as it is a gene that inhibits apoptosis, and examples thereof include the BCL2 gene, the BCL2L1 gene (protein name: Bcl-xL), Survivin, and MCL1. The BCL2L1 gene is preferred. Examples of the BCL2L1 gene include genes consisting of the nucleic acid sequences represented by NCBI accession numbers NM_001191 and NM_138578.

[0023] As used herein, genes encoding Polycomb group proteins (hereinafter also referred to as "Polycomb genes") refer to genes known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and function to prevent cellular senescence. Specific examples of polycomb genes include BMI1 (Polycomb complex protein BMI-1, polycomb group RING finger protein 4 (PCGF4), RING finger protein 51 (RNF51)), Mel18 (Polycomb group RING finger protein 2), Ring (Ring Finger Protein) 1a / b, Phc (Polyhomeotic Homolog) 1 / 2 / 3, Cbx (Chromobox) 2 / 4 / 6 / 7 / 8, Ezh2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit), Eed (Embryonic Ectoderm Development), Suz12 (SUZ12 Polycomb Repressive Complex 2 Subunit), HADC (Histone deacetylases), and Dnmt (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b, with the BMI1 gene being preferred. An example of the BMI1 gene is a gene consisting of the nucleic acid sequence shown by NCBI accession number NM_005180.

[0024] The origin of each gene encoding a megakaryocyte-inducing factor (hereinafter also referred to as "megakaryocyte-inducing factor gene") is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.), with human-derived genes being particularly preferred. Alternatively, a homolog of a human gene in a mammalian species other than human is also preferred as a megakaryocyte-inducing factor gene. Genes with a high degree of nucleotide sequence identity to the wild-type gene (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 identity) are also preferred.

[0025] Each megakaryocyte-inducing factor gene is preferably linked under the control of a drug-responsive promoter. A drug-responsive promoter is a promoter that expresses or represses gene expression in the presence of a corresponding drug. Examples of drug-responsive promoters include the TRE promoter, which can bind to a fusion protein (rtTA) of reverse tetR (rtetR) and VP16AD, or a fusion protein (rTA) of tetR and VP16AD, in the presence or absence of a corresponding drug. Examples of such drugs include doxycycline (Dox), tetracycline, or their derivatives (abbreviated as "Dox, etc."). Other examples of drug-responsive promoters include metallothionein promoters (corresponding drug: heavy metal ions) and steroid-responsive promoters (corresponding drug: steroid hormones or their derivatives). Vectors containing these drug-responsive promoters are also referred to as drug-responsive vectors. In addition to drug-responsive promoters, light-responsive promoters (induced by light), heat shock protein promoters (induced by heat shock), etc. may also be used, and promoters that are induced by these drugs or stimuli are also called inducible promoters.

[0026] As used herein, "maintaining platelet production ability" not only means that the platelet production ability of megakaryocytes does not decrease over the course of proliferation days, but also encompasses a higher platelet production ability compared to a control (specifically, megakaryocytes cultured under the same conditions without increasing the abundance of the corresponding UQCR-constituting protein, or megakaryocytes cultured under the same conditions without inhibiting the function of PRC1.1 (in one embodiment, without reducing the abundance of the corresponding PRC1.1-constituting protein)). Furthermore, as used herein, "platelet production ability" refers to the ability to produce platelets through reticulum process formation by transitioning megakaryocytes to the maturation phase. Transition of megakaryocytes to the maturation phase can be achieved by the "step of maturing megakaryocytes" described below. Furthermore, as in the Examples described below, if megakaryocytes are transitioned to the maturation phase on day 28 after the start of megakaryocyte proliferation, and the percentage of platelets recovered after 6 days of culture (platelet count / megakaryocyte count) is higher than the percentage of platelets in the control, it can be said that the platelet production ability of megakaryocytes is maintained.

[0027] Ubiquinol-cytochrome c reductase (UQCR) is one of four complexes present in the inner mitochondrial membrane and is also known as "complex III." Within mitochondria, UQCR catalyzes the electron transfer reaction from ubiquinol to cytochrome c and simultaneously actively transports protons from the matrix to the cytoplasm. Without being bound by any theory, one possible explanation for the improved platelet production effect achieved by forced expression of the UQCR core protein is the improvement in megakaryocyte proliferation via improved mitochondrial energy production. Therefore, any protein that constitutes UQCR other than the UQCR core protein can be used in the present invention as long as it can exert such an effect. Examples of proteins that constitute UQCR include UQCRC1, UQCRC2, UQCRB, BCS1L, UQCRQ, CYC1, TTC19, LYRM7, UQCC2, and UQCC3, but UQCR core proteins (i.e., UQCRC1 and UQCRC2) are preferred, with UQCRC2 being more preferred. These may be used alone or in combination (e.g., UQCRC1 and UQCRC2 may be used in combination).

[0028] An example of the nucleotide sequence and amino acid sequence of human UQCRC1 is shown as SEQ ID NO: 1 (coding sequence (CDS) of NCBI accession number NM_003365.3) and SEQ ID NO: 2 (NCBI accession number NP_003356.2). An example of the nucleotide sequence and amino acid sequence of human UQCRC2 is shown as SEQ ID NO: 3 (CDS of NCBI accession number NM_003366.4) and SEQ ID NO: 4 (NCBI accession number NP_003357.2). An example of the nucleotide sequence and amino acid sequence of human BACH1 is shown as SEQ ID NO: 5 (CDS of NCBI accession number NM_001186.4) and SEQ ID NO: 6 (NCBI accession number NP_001177.1).

[0029] PRC1.1 is a transcriptional repression complex mediated by Polycomb group proteins, and it suppresses gene expression through ubiquitination of Lys119 on histone H2A (H2AK119). PRC1.1 is involved in a wide range of biological processes, including cell differentiation, proliferation, and tumorigenesis, through epigenetic regulation of gene expression (e.g., Nakajima-Takagi Y. et al., Elife. 12:e83004 (2023)). PRC1.1 is primarily composed of Polycomb group RING finger protein 1 (PCGF1), ring finger protein 1A (RING1A) or RING1B (RING1B), lysine demethylase 2B (KDM2B), S-phase kinase-associated protein 1 (SKP1), and BCOR or BCORL1. Other proteins that constitute PRC1.1 include TRIM27 and USP7 (ubiquitin-specific-processing protease) (Maat H., et al., iScience. 24(5): 102435 (2021)). In the production method of the present invention, the method for inhibiting the function of PRC1.1 is not particularly limited as long as it can inhibit the ubiquitination of Lys119 of H2AK119. Examples include contacting megakaryocytes with a PRC1.1 inhibitor (typically, culturing megakaryocytes in a medium containing the inhibitor), and reducing the abundance of at least one of the proteins that constitute PRC1.1.

[0030] Examples of PRC1.1 inhibitors used in the present 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 alone or in combination.

[0031] BCOR cannot accumulate in the target sequence due to frameshifts caused by partial deletion of the gene or mutations in the PCGF1 binding site. Reducing the protein levels of other PRC1.1-constituting proteins by knockdown or other methods can also inhibit PRC1.1 function. Therefore, the target protein to be reduced may be any of the PRC1.1-constituting proteins, such as PCGF1, RING1A, RING1B, KDM2B, SKP1, BCOR, BCORL1, TRIM27, and USP7, but preferably BCOR, BCORL1, or TRIM27. These may target one type of protein or multiple types of proteins.

[0032] As shown in the examples below, the method of the present invention can further improve platelet production by overexpressing BAHC1. Therefore, in one embodiment of the present invention, the method of the present invention includes a step of increasing the abundance of BACH1 protein in megakaryocytes. Therefore, the step of increasing the abundance of BACH1 protein in megakaryocytes may be combined with a step of increasing the abundance of at least one of the proteins constituting the UQCR and / or a step of inhibiting the function of PRC1.1. When these steps are combined, they may be performed sequentially or simultaneously. BACH1 is a transcription factor, and together with Bach2, it has been reported to promote erythropoiesis by regulating heme metabolism in red blood cells (e.g., Kato H., et al., Nat Immunol. 19(10):1059-1070 (2018)).

[0033] The UQCR proteins and BACH1 protein used in the present invention may be derived from any mammalian source (e.g., human, mouse, rat, monkey, bovine, equine, porcine, canine, etc.), with human-derived proteins being preferred. Furthermore, the UQCR proteins and BACH1 protein used in the present invention are preferably homologous proteins of their human counterparts in mammalian species other than humans. Alternatively, they may be paralogous proteins. Furthermore, the UQCR proteins and BACH1 proteins include not only wild-type proteins but also variants with similar functions. 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) to the amino acid sequence of a specific wild-type protein (e.g., the sequence set forth in SEQ ID NO: 2, 4, or 6). Also preferred are variants of a particular wild-type protein (e.g., the sequence shown in SEQ ID NO: 2, 4, or 6) in which one or several (2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids have been deleted, substituted, inserted, and / or added, and which have the same function as the wild-type.

[0034] As used herein, "increasing the abundance of a protein" refers to increasing the amount of a particular protein present in megakaryocytes compared to the amount of that protein present in megakaryocytes before the step of increasing the abundance of that protein was performed. Megakaryocytes may be constantly maintained in a state of high protein abundance, or may be transiently increased in protein abundance. Methods for increasing the abundance of UQCR-constituting proteins in megakaryocytes are not particularly limited, and include, for example, a method in which nucleic acid (DNA or RNA) (hereinafter also referred to as "exogenous nucleic acid") encoding a UQCR-constituting protein is exogenously introduced into megakaryocytes or cells capable of differentiating into megakaryocytes (e.g., hematopoietic progenitor cells, pluripotent stem cells, etc.) to force expression of the protein; a method in which endogenous gene encoding a UQCR-constituting protein is activated by modifying a promoter using techniques such as genome editing; and a method in which a UQCR-constituting protein is added to a culture medium and then delivered into cells. The UQCR-constituting protein or the nucleic acid encoding the protein may be introduced into cells only once or multiple times. A similar method is for increasing the abundance of BACH1 protein in megakaryocytes.

[0035] On the other hand, "reducing the abundance of a protein" means reducing the amount of a protein present in megakaryocytes compared to the amount of that protein present in megakaryocytes before the step of reducing the abundance of a specific protein is performed. This protein amount is typically the amount of wild-type (i.e., functionally normal) protein. Megakaryocytes may be constantly maintained at a low protein amount, or may be transiently reduced in protein amount. Methods for reducing the abundance of PRC1.1 proteins include, but are not limited to, methods such as introducing antisense nucleic acids (e.g., antisense oligonucleotides (ASOs)) (including nucleic acids encoding the nucleic acids), siRNA (including nucleic acids encoding the siRNA), heteroduplex oligonucleotides (HDOs), shRNA (including nucleic acids encoding the shRNA), and miRNA (microRNA) (including nucleic acids encoding the miRNA) against mRNA encoding the protein constituting PRC1.1 into megakaryocytes or cells capable of differentiating into megakaryocytes to knock down the expression of each protein. These nucleic acids may be introduced into megakaryocytes only once or multiple times. These nucleic acids may be introduced into cells multiple times. For example, when these nucleic acids are incorporated into the genome of a cell, continuous expression of the nucleic acid in the cell can be expected even with a single introduction. Furthermore, using techniques such as genome editing, mutations that result in loss of function of the protein constituting PRC1.1, or mutations that result in reduced protein function and / or reduced protein expression, may be introduced into the gene. Examples of such mutations include nonsense mutations, frameshift mutations, missense mutations, mutations associated with splicing abnormalities, mutations in gene regulatory regions (e.g., promoters, enhancers), and dominant-negative mutations.

[0036] Nucleic acids, such as antisense nucleic acids, for inhibiting the expression of proteins constituting PRC1.1 can be appropriately designed with reference to known nucleotide sequence information. Examples of the nucleotide and amino acid sequences of human BCOR are shown as SEQ ID NO: 7 (CDS of NCBI accession number NM_001123385.2) and SEQ ID NO: 8 (NCBI accession number NP_001116857.1). Examples of the nucleotide and amino acid sequences of human BCORL1 are shown as SEQ ID NO: 9 (CDS of NCBI accession number NM_001184772.3) and SEQ ID NO: 10 (NCBI accession number NP_001171701.1). Examples of the nucleotide and amino acid sequences of human TRIM27 are shown as SEQ ID NO: 11 (CDS of NCBI accession number NM_006510.5) and SEQ ID NO: 12 (NCBI accession number NP_006501.1).

[0037] Furthermore, in this specification, increasing or reducing the abundance of a protein in megakaryocytes does not necessarily require increasing or reducing the abundance of a protein only at the megakaryocyte stage. A protein or a nucleic acid encoding the protein may be introduced into cells capable of differentiating into megakaryocytes (e.g., pluripotent stem cells, hematopoietic progenitor cells), and the abundance of the protein may already be increased or reduced in the cells before differentiation into megakaryocytes. Therefore, for example, in pluripotent stem cells, the expression level of a specific protein (i.e., BACH1 protein, any protein constituting UQCR) is increased, and the pluripotent stem cells are differentiated into megakaryocytes while maintaining this state. This also includes the step of increasing (or reducing) the abundance of a target protein in megakaryocytes. The same applies to the step of inhibiting the function of PRC1.1 or reducing the abundance of any protein constituting PRC1.1 in megakaryocytes.

[0038] Exogenous nucleic acids are typically introduced into cells (e.g., megakaryocytes, hematopoietic progenitor cells, pluripotent stem cells, etc.) in the form of an expression vector carrying the nucleic acid. Examples of vectors that can be used to express exogenous nucleic acids include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, 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 because they can be introduced in a single step.

[0039] Examples of promoters used in expression vectors include the EF-α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter. In addition to the promoter, the expression vector may optionally contain an enhancer, poly(A) addition signal, a selectable marker gene, an SV40 replication origin, and the like. Useful selectable marker genes include, for example, the dihydrofolate reductase gene, the neomycin resistance gene, and the puromycin resistance gene.

[0040] The above-mentioned inducible promoters may also be used as promoters in expression vectors. This allows for the expression of exogenous nucleic acids at the desired timing and for the desired period of time. Alternatively, an expression vector may be used in which loxP sequences are placed to flank the gene, the promoter region, or both, using the Cre-loxP system to excise the exogenous nucleic acid from the vector. This allows for the expression of the exogenous nucleic acid to be stopped when it is no longer necessary.

[0041] Exogenous nucleic acids may be introduced into cells using a transposon system. Transposon is a general term for a short gene sequence that has been conserved throughout evolution and causes gene transposition. A transposon system causes gene transposition by pairing a gene enzyme (transposase) with its specific recognition sequence. Examples of transposon systems include PiggyBac TM A transposon system can be used. TM The transposon system utilizes a transposon isolated from an insect (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), allowing highly efficient integration into mammalian chromosomes. TM Transposon systems have actually been used to introduce genes (see, for example, Nakazawa Y, et al., J. Immunother 32:826-836, 2009; Nakazawa Y et al., J. Immunother 6:3-10, 2013). The transposon system applicable to the present invention is PiggyBac. TMFor example, Sleeping Beauty (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 Acids Res 31: 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 Jun 7.), Tol2 (Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11; Johnson Hamlet MR, Yergeau DA, Kuliyev E, Takeda M, Taira M, Kawakami K, Mead PE (2006) Genesis 44: 438-445; Choo BG, Kondrichin I, Parinov S, Emelyanov A, Go W, Toh WC, Korzh V (2006) BMC Dev Biol 6: 5) or other transposon-based systems may also be employed.

[0042] The transposon system can be introduced by a conventional method, and is described in known literature (e.g., PiggyBac TMFor details of the transposon system, see 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). In one embodiment, the above-mentioned gene is PiggyBac. TM It is introduced into cells by a transposon system. Typically, it is a PiggyBac TM PiggyBac transposon system TM A vector carrying a gene encoding a transposase (transposase plasmid) and a gene encoding a target protein are inserted into PiggyBac TM Vectors containing inverted repeat sequences (transposon plasmids; PiggyBac TM The gene encoding the megakaryocyte-inducing factor may also be introduced into cells such as pluripotent stem cells and hematopoietic stem cells using a transposon system.

[0043] Alternatively, to simultaneously introduce multiple genes, genes may be tandemly linked to form a polycistronic vector. To enable polycistronic expression, the 2A self-cleaving peptide of foot-and-mouth disease virus (SFDV) (see, e.g., Science, 322, 949-953, 2008) and an IRES sequence may be ligated between the genes to be forced to express the vector.

[0044] Various techniques can be used to introduce exogenous nucleic acids into cells, including lipofection, liposomes, electroporation, nucleofection, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and gene guns. When the exogenous nucleic acid is contained in a viral vector, the exogenous nucleic acid can be introduced into cells by introducing a plasmid containing the nucleic acid into appropriate packaging cells (e.g., Plat-E cells) or complementing cell lines (e.g., 293 cells), recovering the virus produced in the culture supernatant, and contacting and infecting the cells.

[0045] Protein delivery into cells can be carried out using known methods for introducing proteins into cells. Examples of such methods include methods using protein introduction reagents, methods using protein transduction domain (PTD) or cell-penetrating peptide (CPP) fusion proteins, and microinjection. Commercially available protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systems), Pro-Ject™ Protein Transfection Reagent (PIERCE), and ProVectin (IMGENEX), lipid-based Profect-1 (Targeting Systems), membrane-penetrating peptide-based Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif), and GenomONE (Ishihara Sangyo Kaisha) that uses the HVJ envelope (inactivated Sendai virus). Introduction can be carried out according to the protocols provided with these reagents.

[0046] In one embodiment, megakaryocytes can be initiated to proliferate by increasing the abundance of MYC protein (particularly c-MYC) and apoptosis inhibitor protein (particularly Bcl-xL) (preferably, by further increasing the abundance of polycomb group proteins (particularly BMI1)). Therefore, in a preferred embodiment of the present invention, the method of the present invention comprises the step of increasing the abundance of MYC protein and apoptosis inhibitor protein in megakaryocytes (preferably, by further increasing the abundance of polycomb group proteins). Methods for increasing the abundance of these proteins in megakaryocytes are not particularly limited, and examples include increasing the expression level of nucleic acids encoding the proteins and adding proteins to culture media to deliver them into cells. Specific examples of methods for increasing the expression level of genes encoding proteins include introducing exogenous nucleic acids (DNA or RNA) encoding the proteins into megakaryocytes and culturing megakaryocytes in the presence of corresponding drugs or stimuli using the inducible promoters described above.

[0047] The culture in the method of the present invention may be either suspension culture or adherent culture, but is typically suspension culture.In addition, megakaryocytes isolated from a living body can be cultured by adherent culture.Since the method of the present invention can proliferate megakaryocytes that maintain platelet production ability for a long period of time, there is no particular limitation on the culture period, but it is typically 6 days or more, and may 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.

[0048] Furthermore, the period during which the abundance of at least one of the proteins constituting UQCR is maintained at an elevated level in megakaryocytes, the period during which the abundance of BACH1 protein is maintained at an elevated level, and the period during which the function of PRC1.1 is inhibited are not particularly limited, and may be only the period during which megakaryocytes are proliferated, or may be continued during the period during which megakaryocytes are matured. Such a period is typically 6 days or more, and may 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 efficient maturation, it is preferable that the period during which the abundance of megakaryocyte-inducing factors (particularly MYC protein) is maintained at an elevated level in megakaryocytes is only the period during which megakaryocytes are proliferated.

[0049] As used herein, "suspension culture" refers to culture carried out under conditions in which cells or cell aggregates are maintained suspended in a culture medium, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell aggregates and the culture vessel. Suspension culture may be static culture or shaking culture, and in the case of shaking culture, it can be said to be suspension culture as long as the cells are not fixed to one place.

[0050] Incubators used for suspension culture include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. For the shaking culture described below (also referred to as "agitation culture"), sealed incubators are preferred. Examples of such incubators include tissue culture flasks, culture bags, and roller bottles. Furthermore, to enable culture under non-adhesive conditions, the incubator is preferably non-cell-adhesive. Examples of non-cell-adhesive incubators include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coated with an extracellular matrix, etc.) or those whose surfaces have been artificially treated to suppress cell adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0051] The megakaryocytes used in the present invention can be obtained by known methods. Examples include isolation from biological tissues (e.g., bone marrow, umbilical cord blood, peripheral blood, etc.) using known techniques, differentiation-inducing methods for pluripotent stem cells or hematopoietic progenitor cells, and methods for obtaining megakaryocytes from companies such as ATCC. Megakaryocytes can be isolated from biological tissues, for example, using flow cytometry or mass cytometry with expressed antigens as an indicator, magnetic cell separation, or affinity columns immobilized with desired antigens. The megakaryocytes used in the present invention are preferably obtained by a method for inducing differentiation of hematopoietic progenitor cells. Therefore, the production method of the present invention may include a step of producing megakaryocytes from pluripotent stem cells or hematopoietic progenitor cells (also referred to as "differentiation induction") (hereinafter also referred to as the "megakaryocyte production step") prior to the production of megakaryocytes that maintain platelet-producing capacity. Hematopoietic progenitor cells can be obtained, for example, by isolation from biological tissues (e.g., bone marrow, umbilical cord blood, peripheral blood, etc.) or by differentiation-inducing methods from pluripotent stem cells.

[0052] The megakaryocyte production step is not particularly limited and can be carried out by a known induction method. Specific examples of megakaryocyte production include the methods described in International Publication Nos. 2011 / 034073, 2012 / 157586, etc. In one embodiment, the megakaryocyte production step includes step (A) culturing pluripotent stem cells to produce hematopoietic progenitor cells (this can also be referred to as "differentiation induction"), and / or step (B) culturing hematopoietic progenitor cells to produce megakaryocytes.

[0053] The method for differentiating pluripotent stem cells into hematopoietic progenitor cells in step (A) is not particularly limited as long as it allows differentiation into hematopoietic progenitor cells, and examples include a method for preparing hematopoietic progenitor cells from a net-like structure (also referred to as ES-sac or iPS-sac) obtained by culturing pluripotent stem cells on C3H10T1 / 2 in the presence of VEGF according to the method described in Takayama N., et al. J Exp Med. 2817-2830 (2010). Here, the term "net-like structure" refers to a three-dimensional sac-like structure (with an internal space) derived from pluripotent stem cells, formed from an endothelial cell population or the like, and containing hematopoietic progenitor cells therein. Other examples include methods involving the formation of embryoid bodies 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 co-culture with heterologous stromal cells (Niwa A et al. J Cell Physiol. 2009 Nov; 221(2): 367-77). Further examples include methods described in, for example, WO 2013 / 075222, WO 2016 / 076415, WO 2017 / 221975, and Liu S. et al., Cytotherapy, 17 (2015); 344-358.

[0054] The term "pluripotent stem cells" refers to stem cells that can differentiate into various tissues and cells with different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of the embryo. However, from an ethical standpoint, cells produced without the destruction of the embryo are preferred.

[0055] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (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 isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0056] Examples of ES cell lines that can be used in the present invention include mouse ES cell lines established by, for example, inGenious targeting laboratory, Inc., RIKEN (Riken), etc., and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, Inc. Specific examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0057] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including 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), human cell-derived iPSCs 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 selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using 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.), etc. can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 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, 795-797), or patent publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) and known in the art can be used.

[0058] Various iPS cell lines established by the NIH, RIKEN, Kyoto University, Kyoto University iPS Cell Research Foundation, and others are available as induced pluripotent stem cell lines. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain; Kyoto University's 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, and TkDN-sev2 strain; and Kyoto University iPS Cell Research Foundation's FfI-01s04 strain, Ff-MH23s01 strain, 15M41 strain (Ff-I 01s01 strain), and QHJI14s04 strain. Other established human iPS cell lines can also be used, such as the RONZA TC-1133 line. Also preferred are iPS cell lines with mutations in the proteins that make up PRC1.1 (particularly BCOR) and reduced PRC1.1 function. Furthermore, iPS cell lines that have been genetically modified, such as those that have undergone gene disruption or modification to reduce antigenicity, can also be used. Examples of such iPS cell lines include the QHJI 14s04-AB II-KO-03, QHJI14s04-AB II-KO-11, and QHJI14s04-AB II-KO-12 lines established by the Kyoto University iPS Cell Research Foundation, and the universal donor cell line established by Healios.

[0059] The induced pluripotent stem cells used in the present invention may be cells derived from patients with hereditary diseases (e.g., patients with hereditary blood disorders). Cells induced to differentiate from pluripotent stem cells derived from patients with hereditary blood disorders can serve as disease models that reflect the pathology of the disease and are therefore suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from patients with hereditary blood disorders can be genetically repaired by genome editing using the CRISPR-Cas system or the like, and then differentiated into the desired cells, making it possible to use the cells as a therapeutic agent for the disease.

[0060] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0061] As used herein, "hematopoietic progenitor cells" refers to cells capable of differentiating into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. As used herein, hematopoietic progenitor cells and hematopoietic stem cells are not distinguished from each other and refer to the same cell unless otherwise specified. Hematopoietic progenitor cells are typically cells that express the CD34 gene and / or the CD43 gene.

[0062] Those skilled in the art can appropriately determine the culture period in step (A) while monitoring the number of hematopoietic progenitor cells, etc. The number of days is not particularly limited as long as hematopoietic progenitor cells are obtained, but is typically 6 days or more, and 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 (particularly 14 days). A long culture period is not a problem in producing hematopoietic progenitor cells, but is typically 30 days or less, and may be 20 days or less.

[0063] Step (B) can also be performed by a known method (e.g., Patent Documents 1 to 4). Specific examples include a method in which an apoptosis-inhibiting gene (e.g., BCL2L1 gene) and a MYC gene (e.g., c-MYC gene) are simultaneously forcibly expressed in hematopoietic progenitor cells and the cells are cultured.

[0064] The culture period in step (B) can be appropriately determined by those skilled in the art while monitoring the number of megakaryocytes, etc. The number of days is not particularly limited as long as megakaryocytes are obtained, but is typically 5 days or more, preferably 6 days or more, and more preferably 7 days or more. The upper limit of the culture period is also not particularly limited, but is typically 20 days, preferably 17 days or less, and more preferably 14 days or less.

[0065] In one embodiment of the present invention, any step of the method of the present invention (e.g., the megakaryocyte production 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]indol-4-yl)cyclohexane-1,4-diamine) and UM729 (methyl 4-(3-(1-piperidyl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate). Pyrimidoindole derivatives are preferred, with UM171 or UM729 being more preferred. One or more pyrimidoindole derivatives may be used.

[0066] The concentration of the pyrimidoindole derivative can be appropriately selected by those skilled in the art depending on the pyrimidoindole derivative used. When the pyrimidoindole derivative is UM729, its concentration in the medium is typically 1 nM to 100 μM, preferably 10 nM to 100 μM, and more preferably 100 nM to 10 μM (in one embodiment, 1 μM).

[0067] The pyrimidoindole derivative may be added to a medium when hematopoietic progenitor cells are cultured, or may be added to a medium when pluripotent stem cells or cells at any stage of differentiation from pluripotent stem cells to hematopoietic progenitor cells are cultured. 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 a pyrimidoindole derivative.

[0068] Any step of the method of the present invention (e.g., megakaryocyte production step, megakaryocyte proliferation step) may be performed in the presence of an HDAC (histone deacetylase) 5 inhibitor and / or an LSD1 inhibitor. Thus, in one embodiment, the method of the present invention comprises a step of culturing megakaryocytes in a medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor (preferably a medium containing both an HDAC5 inhibitor (e.g., LMK235) and an LSD1 inhibitor (e.g., tranylcypromine)). In another embodiment, the present invention also provides a method for producing megakaryocytes, comprising a step of culturing hematopoietic progenitor cells in a medium containing an HDAC5 inhibitor and / or an LSD1 inhibitor.

[0069] The HDAC5 inhibitor used in the present invention is not limited to HDAC5-specific inhibitors and may also have inhibitory activity against other molecules (typically, other HDACs). Examples of HDAC5 inhibitors include LMK235, TMP195, Quisinostat (JNJ-26481585) 2HCl, CUDC-101, Pracinostat (SB939), TMP269, and Domatinostat (4SC-202). Preferred are LMK235 and TMP195, with LMK235 being more preferred. Antibodies, peptides, or aptamers against HDAC5 are also preferred. Alternatively, the HDAC5 inhibitor may be an HDAC5 expression inhibitor, such as an antisense nucleic acid, siRNA, shRNA, or heteroduplex nucleic acid, which inhibits HDAC5 expression. A single HDAC5 inhibitor may be used, or multiple HDAC5 inhibitors may be used. It is also preferred to combine an HDAC5 inhibitor with a pyrimidoindole derivative.

[0070] The concentration of the HDAC5 inhibitor can be appropriately selected by those skilled in the art depending on the HDAC5 inhibitor used. When the HDAC5 inhibitor is LMK235, its concentration in the medium is typically 1 nM to 100 μM, preferably 10 nM to 100 μM, and more preferably 20 nM to 1 μM (in one embodiment, 50 nM to 200 nM).

[0071] The LSD1 inhibitor is not particularly limited as long as it can inhibit the enzymatic activity of LSD1 (i.e., the demethylation activity of histone H3K4me1 / 2 and H3K9me1 / 2). Examples of the LSD1 inhibitor include tranylcypromine (TCP), ORY-1001 (Iadademstat), GSK2879552, IMG-7289 (Bomedemstat), SP-2509, HCI-2509, RN-1, MC3324, SP-2577 (Seclidemstat), and Corin, with tranylcypromine being preferred. Antibodies, peptides, or aptamers against LSD1 are also preferred. Alternatively, the LSD1 inhibitor may be an LSD1 expression inhibitor, such as an antisense nucleic acid, siRNA, shRNA, or heteroduplex nucleic acid, which inhibits LSD1 expression. A single LSD1 inhibitor may be used, or multiple LSD1 inhibitors may be used. It is also preferred to combine an LSD1 inhibitor with a pyrimidoindole derivative.

[0072] The concentration of the LSD1 inhibitor can be appropriately selected by those skilled in the art depending on the LSD1 inhibitor used. When the LSD1 inhibitor is tranylcypromine, its concentration in the medium is typically 5 nM to 500 μM, preferably 50 nM to 500 μM, and more preferably 500 nM to 5 μM (in one embodiment, 1 μM).

[0073] As shown in the Examples below, inspired by the phenomenon occurring in bone marrow, culturing megakaryocytes in the presence of an adipogenic factor successfully promoted platelet production. Therefore, any step of the method of the present invention (e.g., the megakaryocyte production step) may be performed in the presence of an adipogenic factor. Examples of adipogenic factors include insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and indomethacin. In one embodiment, the method of the present invention comprises culturing megakaryocytes in a medium containing dexamethasone, IBMX, and insulin. It is also preferred that the medium used in the megakaryocyte production step contains JI051. In one embodiment, the medium used in the megakaryocyte production step contains dexamethasone, IBMX, insulin, and JI051. The structural formula of JI051 is shown below.

[0074]

[0075] Furthermore, the expression of the TP53 gene (protein name: p53) and / or the CDKN1A gene (protein name: p21) or the function of its expression product may be suppressed in hematopoietic progenitor cells. Examples of the TP53 gene include a gene consisting of the nucleic acid sequence represented by NCBI accession number NM_000546.6. Examples of the CDKN1A gene include a gene consisting of the nucleic acid sequence represented by NCBI accession number NM_001291549.3. The origin of these genes is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, bovine, equine, porcine, canine, etc.), with human origin being particularly preferred. In one embodiment, these genes are human genes or homologs of human genes in other mammalian species. Genes that have a high degree of identity in nucleotide sequence to the wild-type gene (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 identity) are also preferred.

[0076] The megakaryocytes of the present invention maintain their platelet-producing ability and are therefore suitable as starting cells for platelet production. Therefore, in another aspect, there is provided a method for producing platelets (hereinafter, sometimes referred to as the "method for producing platelets of the present invention"), which comprises a step of maturing the megakaryocytes of the present invention.

[0077] The method for producing platelets of the present invention can be carried out by known methods. For example, when megakaryocytes are forced to express megakaryocyte-inducing factors, the forced expression of one or more (preferably at least the MYC gene) or all of the megakaryocyte-inducing factors can be reduced or stopped to mature the megakaryocytes. For example, when the megakaryocyte-inducing factors are forced to be expressed using a drug, the forced expression can be reduced or stopped by culturing the megakaryocytes in the absence of the drug. Alternatively, when a vector containing the above-mentioned LoxP is used, this can be achieved by introducing Cre recombinase into the cells. Furthermore, when a transient expression vector or RNA or protein introduction is used, this can be achieved by terminating contact with the vector, etc.

[0078] In a preferred embodiment, the medium used in the platelet production method of the present invention contains one or more (preferably all) of an ADAM17 inhibitor, an AhR (aryl hydrocarbon receptor) inhibitor, and a ROCK inhibitor. 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 (e.g., those described in US2014 / 0369973, etc.), stilbene derivatives (e.g., (E)-1-(4'-trifluoromethylphenyl)-2-(3,5-ditrifluoromethylphenyl)-ethene, (E)-1-(4'-methoxyphenyl)-2-(3,5-dichlorophenyl)-ethene, (E)-1-(4'-chlorophenyl)-2-(3,5-dichlorophenyl)-ethene, 3,5,4'-trihydroxystilbene, etc.), and CH-223191, with SR-1 being preferred. Examples of ROCK inhibitors include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), SR3677 (see, e.g., Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (see, e.g., Stavenger RA et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), GSK429286A, H1152 (see, e.g., Sasaki et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), and the like. al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), thiazovivin and derivatives thereof, with Y-27632 being preferred.

[0079] The medium used in the method for producing platelets of the present invention may contain the above-mentioned adipogenic factor and / or JI051. In one embodiment, the medium used in the method for producing platelets of the present invention contains JI051. Specific types of compounds and the like are all incorporated by reference in the description of the method for producing platelets of the present invention.

[0080] Mature megakaryocytes produced by the platelet production method of the present invention can be CD34+CD41+ cells. Mature megakaryocytes produced by this method can also be CD38-negative, CD90-positive, and / or CD49f-positive.

[0081] The culture period in the method for producing platelets of the present invention is not particularly limited as long as the function of the platelets is maintained, but is, for example, 2 to 9 days (6 days in one embodiment).

[0082] The culture in the platelet production method of the present invention is typically suspension culture. Furthermore, the culture is preferably performed with shaking. Therefore, in one embodiment, the platelet production method of the present invention includes a shaking culture step. Shaking culture can be performed by stirring the culture solution with the rotating motion of a stirring blade, using, for example, a commercially available rotary shaking culture machine (e.g., VerMES Reactor (manufactured by Satake Multimix)). The stirring speed can be 50 to 200 rpm, for example, about 100 rpm.

[0083] The shaking culture in the platelet production method of the present invention may be carried out using the device and method described in Patent Document 5. Specific examples include a method comprising a step of stirring the medium in a culture vessel using a stirring blade, the stirring step comprising reciprocating the stirring blade so as to satisfy one or more indices selected from the following: (a) turbulence energy of about 0.0005 m / s to about 0.02 m / s; (b) shear stress of about 0.2 Pa to about 6.0 Pa; and (c) Kolmogorov scale of about 100 μm to about 600 μm.

[0084] Specifically, the culture device includes, for example, a container for accommodating a medium containing megakaryocytes, and a stirring blade that reciprocates within the container, and the following is present in the medium: 2 / s 2 ~ approx. 0.02m2 / s 2 (b) a turbulence energy of about 0.2 Pa to about 6.0 Pa; and (c) a Kolmogorov scale of about 100 μm to about 600 μm.

[0085] The turbulence energy, shear stress, and Kolmogorov scale of the medium in the vessel being stirred can be calculated by simulation based on the basic equations of turbulence. For example, calculations can be performed using the thermal fluid analysis software FLUENT (manufactured by ANSYS), but the software is not limited to this. More specifically, when the stirring blade is configured to reciprocate, the turbulence energy varies depending on the reciprocating stroke, reciprocating speed, and reciprocating frequency of the stirring blade. When multiple stirring blades are used, the number of stirring blades can also be a factor that causes changes. Shear stress and Kolmogorov scale also vary with changes in the same factors.

[0086] The medium used in the present invention is not particularly limited, but a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixtures thereof. The medium may contain serum or may be serum-free. If necessary, the 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 molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines. Cytokines are proteins that promote hematopoietic differentiation, such as VEGF, TPO, and SCF. Instead of TPO, TA-316 ((2E)-2-[1-[5-(4-Bromophenyl)-4-hydroxy-3-thienyl]ethylidene]hydrazide-5-[[[[4-[[(2-hydroxyethyl)amino]carbonyl]phenyl]methyl]amino]carbonyl]-2-thiophenecarboxylic Acid) is also preferably used. In one embodiment, the medium used in the method of the present invention contains SCF and TA-316. In another embodiment, the medium used in the method of the present invention for producing platelets contains SCF, TA-316, SR-1, KP-457, and Y27632. When megakaryocytes have a drug-responsive promoter, it is preferable to add the corresponding drug, such as tetracycline or doxycycline, to the medium when expressing a gene under the promoter.

[0087] In the present invention, the cell culture conditions are not particularly limited, and conventional culture conditions can be used. Specific examples of the culture temperature include about 35 to about 42°C, about 36 to about 40°C, and about 37 to about 39°C. The CO2 concentration is, for example, about 5 to about 15%. The O2 concentration is, for example, about 15 to about 25%.

[0088] In each step of the production method of the present invention and the platelet production method of the present invention, cells may be cultured under feeder-free conditions and / or xeno-free conditions. In the production method of the present invention and the platelet production method, all steps may be performed under feeder-free and xeno-free conditions. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to establish culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms other than the biological species of the cells to be cultured.

[0089] The seeding density of cells in each step of the production method of the present invention and the method for producing platelets of the present invention is not particularly limited as long as the cells can grow. 2 ~1.0×10 7 cells / cm 2 , preferably 1.0 x 10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0 × 10 4 ~1.0×10 5 cells / cm 2 is.

[0090] When the substance used in the present invention is a low molecular weight compound, the compound includes not only the free form but also its pharmacologically acceptable salt and hydrate.Pharmacologically acceptable salts vary depending on the type of compound, but include, for example, inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salt, ammonium salt, etc., and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc., base addition salts, or inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc., and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc., acid addition salts.

[0091] Each substance used in the present invention may be synthesized by a known method or may be a commercially available product. Furthermore, when a protein or peptide is used, the protein or peptide may be obtained from a cell that expresses it. Cells that express the protein or peptide of interest can be produced by inserting DNA encoding the protein or peptide of interest into a known expression vector and then introducing the resulting expression vector into an appropriate host cell.

[0092] The production methods of the present invention and the platelet production methods of the present invention may include a step of recovering the target cells or tissues obtained in each step. The recovered cells may be cryopreserved using a cell cryopreservation solution. The obtained cells may be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and then selected or purified by flow cytometry, mass cytometry, magnetic cell sorting, or the like.

[0093] In yet another embodiment, there is provided a method for maintaining the platelet-producing ability of megakaryocytes, comprising the step of increasing the abundance of at least one protein constituting ubiquinol-cytochrome c reductase (UQCR) in megakaryocytes. With regard to this method, all of the descriptions regarding the production method of the present invention are incorporated by reference.

[0094] 2. Megakaryocytes and Platelets In another aspect of the present invention, megakaryocytes (hereinafter sometimes referred to as "megakaryocytes of the present invention") and platelets (hereinafter sometimes referred to as "platelets of the present invention") obtained by the production method of the present invention and the method for producing platelets of the present invention ("obtained" can be read as "obtained" as appropriate) are provided. Hereinafter, the megakaryocytes and platelets of the present invention may be collectively referred to as "cells of the present invention."

[0095] The megakaryocytes of the present invention typically have all of the following characteristics (A) to (C): (A) they contain at least one protein constituting exogenous UQCR, (B) they have the ability to produce platelets, and (C) they express the CD34 gene and the CD41 gene.

[0096] Furthermore, the megakaryocytes of the present invention further have any one, two, or three of the following characteristics (D) to (F): (D) are derived from pluripotent stem cells, (E) contain an exogenous megakaryocyte-inducing factor, and (F) contain an expression inhibitor for the TP53 gene and / or the CDKN1A gene.

[0097] In another aspect, the present invention also provides megakaryocytes having all of the above characteristics (A) to (C). Furthermore, the present invention also provides megakaryocytes having at least one, two, or three of the above characteristics (D) to (F). Such megakaryocytes may be obtained by the production method of the present invention, or may be obtained by another method. Hereinafter, the above megakaryocytes may also be referred to as "megakaryocytes of the present invention."

[0098] As used herein, "exogenous" means that megakaryocytes present in a mammalian organism do not exist unless introduced from the outside. Furthermore, as used herein, "expressing" or "positive" a gene means that the protein encoded by the gene is produced, unless otherwise specified. Therefore, if the target protein is detected by FACS as used in the examples below, it can be said that the gene is expressed.

[0099] In the above characteristic (A), at least one of the proteins constituting the exogenous UQCR is preferably a UQCR core protein, and UQCRC2 is particularly preferred. In the above characteristic (B), "having platelet-producing ability" means that platelet production is confirmed by transitioning megakaryocytes to the maturation stage. The method for transitioning to the maturation stage is not limited, and megakaryocytes can be said to have "platelet-producing ability" as long as platelet production is confirmed by at least one method. In the above characteristic (C), it is sufficient for the megakaryocyte to have at least one megakaryocyte-inducing factor, but it is preferable to have a MYC protein (particularly c-MYC) and an apoptosis-inhibiting protein (particularly Bcl-xL), or a MYC protein (particularly c-MYC), an apoptosis-inhibiting protein (particularly Bcl-xL), and a polycomb group protein (particularly BMI1). Examples of expression inhibitors for the above characteristic (F) include antisense nucleic acids, siRNAs, shRNAs, and heteroduplex nucleic acids against transcription products of the TP53 gene and / or CDKN1A gene.

[0100] 3. Uses of Megakaryocytes and Platelets Because the cells of the present invention can be used in medical treatments such as blood transfusions, in another embodiment, a blood product containing the cells of the present invention (hereinafter, sometimes referred to as the "blood product of the present invention") is provided. As used herein, "blood product" refers to a composition containing blood cells such as megakaryocytes and platelets. The megakaryocytes of the present invention can be used, for example, by administration or transplantation into a living body to produce functional platelets in the living body. Furthermore, the platelets of the present invention can be used, for example, to prevent bleeding due to thrombocytopenia or platelet dysfunction, to treat bleeding, or to treat blood diseases. The present invention also encompasses methods for treating or preventing blood diseases or bleeding, in which an effective amount of the cells of the present invention is administered or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) to be treated or prevented. Unless otherwise specified herein, the term "drug for treating or preventing a disease (or method for treating or preventing a disease)" also encompasses pharmaceuticals (or methods) that can both treat and prevent the disease.

[0101] Examples of blood diseases to be treated or prevented include thrombocytopenia and platelet dysfunction. Causes of thrombocytopenia include pancytopenia due to bone marrow failure, thrombocytopenia due to shortened platelet lifespan due to platelet destruction, thrombocytopenia due to increased platelet consumption, drug-induced thrombocytopenia, and thrombocytopenia due to abnormal platelet distribution in organs. Causes of platelet dysfunction include drug-induced platelet dysfunction and hereditary platelet dysfunction (e.g., thrombasthenia, Bernard-Soulier syndrome, MYH9 abnormality, Wiskott-Aldrich syndrome, etc.).

[0102] When the cells of the present invention are used as blood products, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" refers to HLA genotypes that are sufficiently identical to those of the administered or transplanted cells that immune responses can be suppressed with immunosuppressants. For example, somatic cells with HLA types that match the HLA-A, HLA-B, and HLA-DR loci or the HLA-C locus (all loci). If sufficient cells cannot be obtained due to age or constitution, they can be administered or transplanted in capsules or porous containers made of polyethylene glycol or silicone to avoid rejection. Cells derived from iPS cells in which genes encoding MHC class I and II components (e.g., HLA, B2M, etc.) and / or genes encoding their transcription factors (e.g., CIITA, RFX5, RFXAP, RFXANK, etc.) have been disrupted or modified are also preferred. Furthermore, HLA-A, HLA-B, and HLA-C proteins are highly associated with rejection reactions during cell transplantation. Therefore, it is preferred that only the HLA-A and HLA-B genes of the cells of the present invention be disrupted, or that all three loci (HLA-A, HLA-B, and HLA-C) be disrupted. Furthermore, it is even more preferred that the HLA-E gene be disrupted. For example, genetically modified cells can be produced by disrupting the HLA-A and HLA-B genes, or the HLA-A, HLA-B, and HLA-C genes (and optionally the HLA-E gene) in pluripotent stem cells or in any cell at the differentiation stage from pluripotent stem cells to platelets (e.g., hematopoietic progenitor cells, megakaryocytes, etc., particularly megakaryocytes), and then producing platelets from these cells through a differentiation induction process and / or maturation process, thereby eliminating the expression of HLA proteins on the platelet surface. This is expected to reduce antigenicity during platelet transplantation. Furthermore, transplantable blood products such as platelet products can be provided to patients with antibodies against HLA class 1.

[0103] The cells of the present invention are prepared as parenteral preparations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Thus, in one embodiment, there is provided a method for producing a blood product, which includes a step of formulating the cells of the present invention. Such a method may include a step of preparing the cells of the present invention. Furthermore, it may also include a step of preserving the cells of the present invention.

[0104] Pharmaceutically acceptable carriers that can be included in such parenteral formulations include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The cells of the present invention may be formulated with, for example, human plasma, infusion agents, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0105] The blood products of the present invention are provided in a frozen state preserved under conditions typically used for cryopreserving cells and can be thawed immediately before use. In this case, they may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be about 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0106] The cells of the present invention can also be used in methods for screening candidate drugs useful for treating or preventing blood diseases. Thus, in yet another aspect of the present invention, a method for screening therapeutic or preventive drugs for blood diseases is provided, comprising culturing the cells of the present invention in the presence or absence of a test substance. For example, a disease model reflecting the pathology of a blood disease can be used as the cells. If the pathology improves in the presence of the test substance, the test substance can be selected as a candidate drug for treating or preventing a blood disease. Examples of such blood diseases include those similar to those described above that are the target of treatment or prevention using the blood products of the present invention.

[0107] The step of contacting the test substance with the cells of the present invention can typically be carried out by adding the test substance to a medium in which the cells of the present invention are cultured, or by transferring the cells of the present invention to a medium to which the test substance has been added in advance. The period of time for the step of contacting the test substance with the cells of the present invention is not particularly limited, but is typically 1 minute to 5 days, preferably 1 hour to 1 day.

[0108] Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular-weight compounds, natural compounds, etc. Test substances may also be existing or candidate components of pharmaceuticals, nutritional foods, etc.

[0109] In yet another embodiment of the present invention, a kit for producing megakaryocytes and / or platelets that maintain platelet-producing capacity is provided. The kit includes a protein constituting UQCR or a nucleic acid encoding the protein. In addition, the kit may also include a megakaryocyte-inducing factor or a nucleic acid encoding the factor, an expression vector necessary for expressing the nucleic acid in cells, reagents, and the like, as well as cell culture media, serum, supplements such as growth factors (e.g., TA-316, TPO, EPO, SCF, heparin, IL-6, IL-11, etc.), antibiotics, and starting cells (e.g., pluripotent stem cells, hematopoietic progenitor cells, etc.). For example, when using cells derived from pluripotent cells, the kit may also include antibodies for detecting markers (e.g., antibodies against Flk1, CD31, CD34, UEA-I lectin, etc.) to identify net-like structures prepared from these cells. Furthermore, the reagents, antibodies, etc. contained in the kit are supplied in a container of any type that allows the components to maintain their activity for a long period of time, is not adsorbed by the container material, and is not subject to deterioration.

[0110] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0111] Unless otherwise specified, the following materials and methods were used in the Examples. 1. iPS Cells The following human iPS cell lines were obtained from the Kyoto University iPS Cell Research Foundation and used: 15M41 (also known as Ff-I 01s01), Ff-MH23s01 (abbreviated as MH23S01), TkDN-sev2, QHJI14s04, QHJI 14s04-AB II-KO-03, and QHJI14s04-AB II-KO-11. Of these, 15M41 and QHJI14s04 were derived from peripheral blood of the most frequently occurring HLA-homologous donor, and MH23S01 was derived from peripheral blood of the second most frequently occurring HLA-homologous donor. TkDN-sev2 was derived from human fetal skin fibroblasts established using Sendai virus. QHJI 14s04-AB II-KO-03 and QHJI14s04-AB II-KO-11 are strains in which HLA-A, HLA-B, and CIITA were knocked out in QHJI14s04 by genome editing. To induce differentiation into megakaryocytes, the following cell lines were used, in which megakaryocyte-inducing factors were introduced into the iPS cell line. These cell lines were induced to differentiate into hematopoietic progenitor cells, and then differentiated into megakaryocytes by adding tetracycline or its derivative (doxycycline in this example) to the culture medium. Removing doxycycline or other agents from the culture medium allowed the cells to mature and produce platelets.

[0112] ・MBX cassette-transfected 15M41 cells. A construct encoding three genes (MYCdd / BMI1 / BCLxL) fused with a destabilization domain, the c-MYC gene (MYCdd gene), the BMI1 gene, and the BCL2L1 gene under the control of a tetracycline-inducible promoter, and encoding p21 shRNA and p53 shRNA under the control of the H1 promoter, was transfected into piggyBac TMThe 15M41 human iPS cell line was engineered using a system to integrate the MBX cassette into its genome (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). Herein, this construct is sometimes referred to as the [c-MYC / BMI1 / BCLXL / p21 KD / p53 KD] cassette or simply the MBX cassette. Hereinafter, the term "MBX cassette-introduced iPS cell line" will be used to collectively refer to iPS cell lines other than the 15M41 line that have the MBX cassette introduced, as well as 15M41 cells with the MBX cassette introduced.

[0113] MX cassette-transfected 15M41 cells: A construct encoding two genes, MYCdd and BCL2L1 (two factors: MYCdd / BCLxL), under the control of a tetracycline-inducible promoter, and encoding p21 shRNA and p53 shRNA under the control of the H1 promoter, was introduced into piggyBac TM The 15M41 human iPS cell line was constructed using the MX cassette insertion system.

[0114] - MBX cassette-introduced TkDN sev2 cells This is the TkDN sev2 human iPS cell line in which the MBX cassette has been inserted into the genome.

[0115] 2. Differentiation of iPS cells into hematopoietic progenitor cells This was performed using a modified version of the method described in Takayama N., et al. J Exp Med. 2817-2830 (2010). iPS cells were treated with the GSK-3β inhibitors CHIR-99021 and Y-27632 for 3 days, then detached as small cell clumps. These small iPS cell clumps were then added to feeder cells (mitomycin-treated human fibroblast cell line C3H / 10T1 / 2 cells (ATCC number: CCL-226)).

[0116] The basal medium was prepared as IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich, Code No. I3390) 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 l-Thioglycerol (Sigma-Aldrich, Code No. A6145), and 50 μg / mL L-Ascorbic Acid (Sigma-Aldrich, Code No. A4544).

[0117] The iPS cell culture medium used at the start of the culture was a basal medium containing 20 ng / mL VEGF (Fujifilm Wako Pure Chemical Industries, Ltd., Code No. 226-01786), and the culture was initiated under the conditions of 37°C, 5-8% O2, and 5% CO2.

[0118] On day 4, the medium was replaced with basal medium containing 20 ng / mL VEGF, 10 U / mL heparin (AY Pharmaceuticals), 50 ng / mL bFGF (Wako Pure Chemical Industries, Ltd., Code No. 068-04544), and 10 μM SB431542 (Wako Pure Chemical Industries, Ltd., Code No. 037-24293) and cultured at 37°C, 5-8% O2, and 5% CO2. On day 7, the medium was replaced with basal medium containing 20 ng / mL VEGF and 10 U / mL heparin and cultured at 37°C, 20% O2, and 5% CO2. On day 11, the medium was replaced with basal medium containing 20 ng / mL VEGF and 0.75 μM SR-1 (Stemregenin-1) and cultured at 37°C, 20% O2, and 5% CO2. Floating cells on days 7-14 after the start of culture were collected as hematopoietic progenitor cells and used in the subsequent differentiation induction experiments.

[0119] 3. Induction of Hematopoietic Progenitor Cell Differentiation into Megakaryocytes, Expansion, and Maturation Hematopoietic progenitor cells on day 7 of culture in Method 2 were cultured in a basal medium containing 0.2 μg / mL TPO mimetic compound, 50 ng / mL SCF, 1 μM UM729 (Merck Code No. ATEH97ECEE61), and 1 μg / mL doxycycline (sometimes referred to as megakaryocyte differentiation and growth medium) under feeder-free conditions at 37°C, 20% O2, and 5% CO2, to induce differentiation into megakaryocytes. In the examples, the day of initiation of culture in the megakaryocyte differentiation medium is designated Day 0, and subsequent days of culture are indicated as Days. In this method, a CD34-positive / CD41-positive cell population generally appears around Day 7. Megakaryocytes were expanded by continuing subculture using the same medium. In the following examples, "inducing differentiation of iPS cells into hematopoietic progenitor cells, and inducing differentiation of the hematopoietic progenitor cells into megakaryocytes" may be shortened to "inducing differentiation of iPS cells into megakaryocytes."

[0120] By switching from a doxycycline-containing medium to a doxycycline-free medium, the forced expression of the three factors (MYCdd / BMI1 / BCLxL) or two factors (MYCdd / BCLxL) in the cassette was released, and megakaryocyte maturation was induced. The composition of the doxycycline-free medium (megakaryocyte maturation medium) used was as follows: Megakaryocyte maturation medium (or maturation medium): IMDM containing 5% human plasma, 2 mM glutamine, 1% ITS-G, 0.45 mM l-thioglycerol, 50 μg / mL L-ascorbic acid, 50 ng / mL SCF, 0.2 μg / mL TPO mimetic compound, 15 μM KP-457 (Kaken Pharmaceutical), 10 μM Y-27632 (Nacalai Tesque, Code No. 18188-04), 0.75 μM SR-1 (Stemregenin-1), or 0.1 μM AhR inhibitor (final concentrations). The megakaryocyte maturation process involves seeding megakaryocytes at a density of 1 x 10 in a 125 mL culture flask. 5 The cells were seeded at 25 mL / flask and cultured with shaking at 100 rpm in a LT-X (Lab-Therm) (Kuhner) or S41i (Eppendorf) shaker at 37°C and 5% CO2.

[0121] 4. Evaluation of megakaryocyte proliferation From Day 0 onwards, cell counts were periodically measured and the megakaryocyte-specific surface marker CD41 was detected by flow cytometry. Cell counts were performed using trypan blue staining, and the cumulative proliferation rate from Day 0 was calculated based on the proliferation fold calculated based on the number of seeded cells. At the same time, this was multiplied by the megakaryocyte abundance ratio calculated from the CD41 positivity measurement results to determine the megakaryocyte proliferation rate. The cumulative proliferation rate of megakaryocytes at the nth passage was calculated using the following formula.

[0122]

[0123] However, the seeding density at passage 0 is the seeding density on Day 0.

[0124] 5. Evaluation of platelet production and platelet function Megakaryocytes were cultured in megakaryocyte maturation medium with shaking for 6 days, and then a portion of the cell suspension was collected and analyzed using a BD FACSVerse. TM Flow cytometry analysis was performed using a flow cytometry system (BD Biosciences), and the number of produced platelets was measured and platelet function was evaluated according to the following procedures.

[0125] To measure the number of platelets produced, staining was performed with the following antibodies: APC-labeled anti-CD41 antibody (BioLegend, Code No. 303710), PE-labeled anti-CD42b antibody (BioLegend, Code No. 303906), and eFluor TM 450-labeled anti-CD42a antibody (Thermo Fisher, Code No. 48-0428-42) After 30 minutes of staining, TruCOUNT TM Platelet counts (CD41 / CD42b positive or CD41 / CD42a / CD42b positive, gated as small particles by FSC / SSC) were measured using a flow cytometry system (BD Biosciences, Code No. 340334). FlowJoint was used for flow cytometry data analysis to calculate the number of platelets produced. TM Version 10 (BD Biosciences) was used. Data were expressed as the number of megakaryocytes produced per megakaryocyte, based on the number of megakaryocytes seeded at the start of platelet production culture.

[0126] To evaluate platelet function, the cells were stimulated under the following two conditions: Stimulator 1: 0.02-0.4 μM PMA Stimulator 2: 100 μM ADP / 40 μM Thrombin Receptor Activator Peptide 6 (TRAP6). After addition, the cells were stained with the following antibodies: APC-labeled anti-CD41 antibody (BioLegend, Code No. 303710), PE-labeled anti-CD62P antibody (BioLegend, Code No. 304906), and FITC-labeled anti-PAC-1 antibody (BD Bioscience, Code No. 34507). After 30 minutes, the cells were analyzed using BD FACSVerse. TM Measurements were carried out.

[0127] Example 1: Verification of the effect of megakaryocyte passaging on platelet production. MBX cassette-introduced 15M41 cells were induced to differentiate into hematopoietic progenitor cells, and these progenitor cells were then induced to differentiate into megakaryocytes. The platelet production capacity of the cells analyzed on days 21 (Day 21) to 78 (Day 78) of megakaryocyte induction is shown in Figure 1. On Day 21, megakaryocytes were capable of producing more than 200 platelets per cell, but this capacity subsequently declined, reaching approximately one-tenth of the Day 21 level by Day 64. Furthermore, a decrease in platelet production capacity with passaging was also observed in megakaryocytes induced to differentiate from other human iPS cell lines (e.g., MH23S01 and TkDN-sev2) using a similar method (data not shown). Therefore, it was confirmed that megakaryocytes induced to differentiate from human iPS cells tend to exhibit a decrease in platelet production capacity with increasing passaging.

[0128] Example 2: Search for Proteins Promoting Platelet Production We have previously identified six proteins as factors released into the culture medium from megakaryocytes matured by shaking culture under conditions that dramatically enhance platelet production. Among these, we focused on NRDC, which contains an endopeptidase domain (Non-Patent Document 2). To investigate the role of NRDC in platelet production, we generated megakaryocyte-specific NRDC-deficient mice and analyzed their in vivo platelet mass. However, contrary to expectations, the NRDC-deficient mice did not exhibit a significant decrease in platelet mass compared with control mice (data not shown). Therefore, to identify factors that truly regulate platelet production efficiency, we analyzed proteins whose expression levels were increased in megakaryocytes matured by shaking culture compared with megakaryocytes matured by static culture. We found that knockdown of UQCRC1 and UQCRC2, which are localized in mitochondria and share the M16 endopeptidase domain with NRDC, reduced the platelet production capacity of megakaryocytes (Figure 2). Megakaryocytes differentiated from MBX cassette-transfected TkDN sev2 cells were transfected with UQCRC1 and / or UQCRC2 shRNAs using lentivirus on Day 45, and platelet production was measured on Days 66-81. The results are shown in Figure 2. Compared to control (LacZ shRNA-transfected megakaryocytes), platelet production was reduced to approximately 30% in megakaryocytes transfected with UQCRC1 shRNA or UQCRC2 shRNA. Furthermore, platelet production was reduced to approximately 20% in megakaryocytes transfected with both UQCRC1 and UQCRC2 shRNAs. These results strongly suggest that UQCRC1 and UQCRC2 play a role in positively regulating megakaryocyte platelet production.

[0129] Example 3: Verification of platelet production by UQCRC1 and UQCRC2. The effect of forced expression of UQCRC1 and UQCRC2 on platelet production was analyzed. Megakaryocytes induced to differentiate from MBX cassette-introduced 15M41 cells (Day 21) or from MX cassette-introduced 15M41 cells (Day 28) were transfected with UQCRC1 or UQCRC2 using a lentiviral vector. Maturation was induced on Day 47 or Day 56 (MBX) and Day 56 or Day 63 (MX), and platelet production was analyzed. The results are shown in Figure 3. Megakaryocytes induced to differentiate from MBX cassette-introduced 15M41 cells produced significantly more platelets than controls (megakaryocytes infected with a non-UQCRC1 / UQCRC2-encoding lentiviral vector) regardless of whether UQCRC1 or UQCRC2 was forced to be expressed (Figure 3, right graph). Comparison with Figure 1 reveals that forced expression of UQCRC1 or UQCRC2 significantly suppressed the decline in platelet production associated with long-term subculture. Similarly, forced expression of UQCRC1 or UQCRC2 maintained the same high platelet production capacity as that of megakaryocytes induced to differentiate from MX cassette-transfected 15M41 cells (Figure 3, left graph). Figure 4 shows the results of analyzing the platelet production capacity of megakaryocytes transfected with UQCRC2 using lentivirus from Day 53 onward. Megakaryocytes induced to differentiate from either MX cassette-transfected 15M41 cells or MBX cassette-transfected 15M41 cells maintained platelet production well over the long term, up to Day 67 or Day 60. These results demonstrated that increasing the expression levels of UQCRC1 and / or UQCRC2 in megakaryocytes, at least in the proliferative phase, significantly improved the decline in platelet production ability associated with subculture, resulting in megakaryocytes that maintain platelet production ability for a long period of time.

[0130] Example 4: Search for Compounds Promoting Platelet Production Next, we attempted to promote platelet production using compounds. It is known that megakaryocytes in bone marrow promote platelet production by uptake of fatty acids released from adipocytes (Adipocyte Fatty Acid Transfer Supports Megakaryocyte Maturation. Cell Reports. 2020;32(1):107875). It is also known that lipid synthesis and the uptake of exogenous fatty acids are important for megakaryocyte maturation and platelet formation (Critical Shifts in Lipid Metabolism Promote Megakaryocyte Differentiation and Proplatelet Formation Nat Cardiovasc Res. 2023 Sep;2:835-852). Based on these findings, we hypothesized that the use of a drug that induces lipid metabolism and synthesis in adipocytes might promote lipid synthesis in megakaryocytes. Therefore, we tested this hypothesis by culturing megakaryocytes in the presence of an adipocyte differentiation-inducing factor. Dextran (1 μM), IBMX (50 μM), and insulin (10 μg / ml) were added to the culture medium (differentiation and proliferation medium) for megakaryocytes expressing exogenous UQCRC2 (megakaryocytes derived from MBX cassette-transfected 15M41 cells) from Day 1 to Day 3. The final concentrations are shown in parentheses. JI051 (10 μM) was added 3 days after transfer to megakaryocyte maturation medium, and platelet production ability was analyzed. The results are shown in Figure 5. Compared to the control without JI051, the platelet production ability of megakaryocytes treated with JI051 was approximately 2-fold increased. Therefore, it was revealed that megakaryocyte expansion with the addition of dextran, IBMX, and insulin, followed by the subsequent maturation step with the addition of JI051, further improved the platelet production ability of megakaryocytes.

[0131] Furthermore, we identified LMK-235 as a compound that can replace UM729 in megakaryocyte differentiation and growth medium. Megakaryocyte differentiation was induced using a differentiation and growth medium in which UM729 was replaced with LMK-235. The cell number (top graph in Figure 6) and CD34-positive cell percentage (bottom graph in Figure 6) were analyzed after 3 days. Note that forced expression of UQURC2 was not performed in this experiment. When LMK-235 was used at a final concentration of 0.2 μM, both the cell number and CD34-positive cell percentage were increased compared to when UM729 was used. Since LMK-235 is known to be an inhibitor of HDACs 4, 5, and 6, similar analysis was performed with other HDAC inhibitors. Figure 7 shows the results of analyzing the CD34- and CD41a-positive cell percentages in megakaryocytes derived from MBX cassette-transfected 15M41 cells or MX cassette-transfected 15M41 cells overexpressing UQURC2. LMK-235 and TMP195 (inhibitors of HDACs 4, 5, 7, and 9) maintained CD34 expression, whereas Tasqunimod (an HDAC4 inhibitor) reduced CD34 expression. These results suggest that HDAC5 contributes to the maintenance of CD34 expression in megakaryocytes during the proliferative phase. We analyzed the effects of UM729 and LMK-235 on megakaryocytes derived from MX cassette-transfected 15M41 cells overexpressing UQCRC2. Megakaryocytes were differentiated and expanded in standard differentiation and expansion medium containing 1 μM UM729 or in differentiation and expansion medium in which UM729 was replaced with 200 nM LMK-235. Then, they were matured in static culture and their platelet-producing activity was compared (Figure 8). Although the number of platelets produced was lower when maturation was performed in static culture compared to when maturation was performed in shaking culture, the number of platelets produced when expansion culture was performed in an LMK-235-containing medium was not significantly different from that when expansion culture was performed in an UM729-containing medium. Thus, it was revealed that the pyrimidoindole derivatives (e.g., UM729) added to the medium in one embodiment of the present invention during the differentiation induction of hematopoietic progenitor cells into megakaryocytes and the subsequent expansion culture process can be replaced with HDAC5 inhibitors such as LMK-235 and TMP195.

[0132] Example 5: Examination of megakaryocyte proliferation potential depending on the origin of iPS cell lines. We investigated whether differences in the proliferation potential of induced megakaryocytes exist depending on the origin of the iPS cell line. First, MBX cassette-introduced iPS cell lines were generated by introducing an MBX cassette into various iPS cell lines (15M41, MH09S01, MH15S01, MH15S01, and MH23S01) (introducing three megakaryocyte-inducing factors). These MBX cassette-introduced iPS cell lines were induced into imMKCL, and the CD34 / CD41a co-positive fraction was selected and cultured. The expression of CD34 and CD41 was evaluated by FACS. The results showed that imMKCL derived from all iPS cell lines maintained the CD34 / CD41a co-positive phenotype for 42 days (Figure 10).

[0133] Next, we tested two megakaryocyte-inducing factors instead of three. Various iPS cells (15M41 and QHJI14s04-AB II-KO-03) were transfected with constructs encoding two megakaryocyte-inducing factors (MYCdd / BCLxL)—MYCdd under the control of a tetracycline-inducible promoter and BCL2L1 under the control of the UbiC promoter—and p21 shRNA and p53 shRNA under the control of the H1 promoter. Megakaryocyte differentiation was then induced from these iPS cells. CD34 / CD4a co-positive fractions were selected and cultured, and CD34 and CD41 expression was assessed by FACS. The QHJI14s04-AB II-KO-03-derived megakaryocyte population exhibited a high failure to maintain CD34 / CD41a co-expression (Figure 11). In contrast, 15M41-derived megakaryocytes maintained CD34 / CD41a co-expression (Figure 12).

[0134] Example 6: Verification of the Relationship between Polycomb Complex 1.1 and Megakaryocyte Proliferation Potential. 15M41 has been reported to have mutations in the BCOR (BCL6 corepressor) gene and the BRD3 gene, whereas QHJI14s04 does not. Therefore, we suspected that differences in BCOR or BRD3 mutations could be the cause of differences in the maintenance effect of CD34 / CD41a coexpression depending on the iPS cell origin. We first focused on the BCOR gene and its homolog, BCORL1 (BCL6 corepressor like 1) gene.

[0135] Therefore, we investigated whether suppressing BCOR or BCORL1 expression in megakaryocytes derived from QHJI14s04 / AB II-KO-03 would result in a phenotype equivalent to that of megakaryocytes derived from the 15M41 line. In QHJI14s04 / AB II-KO-03-derived megakaryocytes, we knocked down BCOR or BCORL1 expression using lentiviral shRNAs targeting the transcripts of these genes (also referred to as shBCOR and shBCORL1, respectively) on day 28 after the start of Dox administration. As a result, we successfully obtained megakaryocyte lines that maintained CD34 / CD41a coexpression (Figure 13). Furthermore, we demonstrated that megakaryocytes with BCOR or BCORL1 knockdown exhibited higher platelet production capacity than those without knockdown (Figure 14).

[0136] BCOR and BCORL1 are proteins that constitute the polycomb repressive complex 1.1 (PRC1.1) and are known as transcription factors involved in the regulation of embryogenesis, mesenchymal stem cell function, hematopoiesis, and lymphoid development (e.g., Sportoletti P., Sorcini D. and Falini B., Blood. 138(24):2455-2468 (2021)). Furthermore, the present inventors previously identified TRIM27 (tripartite motif containing 27) as one of the genes whose methylation levels were significantly different between iPS cells with significantly different induction efficiencies in megakaryocyte induction using the three factors (MYCdd / BMI1 / BCLxL) (data not shown). TRIM27 has also been reported to be a component of PRC1.1 (Maat H., et al., iScience. 24(5):102435). (2021)). The inventors also considered the possibility that among the transcription factors involved in the differentiation process of blood cell lineages, there may be one involved in the proliferation of megakaryocytes. After extensive investigation, they focused on BACH1, which contributes to erythropoiesis and lymphopoiesis, and conducted further analysis.

[0137] Megakaryocytes were induced from iPS cells by introducing the vector combinations shown in Figure 15. Overexpression of UQCRC2 and BACH1 and knockdown of BCOR maintained megakaryocytes co-expressing CD34 / CD41a and maintained high platelet production. Furthermore, knockdown of BCORL1 or TRIM27 further enhanced platelet production (Figures 16 and 17). Furthermore, overexpression of BACH1 in megakaryocytes alone maintained high platelet production even without knockdown of BCOR, BCORL1, or TRIM27 (Figure 18).

[0138] Example 7: Search for compounds that contribute to megakaryocyte maintenance. Searching for compounds other than HDAC5 inhibitors that promote megakaryocyte maintenance and platelet production, we found that a lysine-specific histone demethylase 1 (LSD1) inhibitor (tranylcypromine) maintained CD34 / CD41a co-expression in megakaryocytes. In particular, the combination of an HDAC5 inhibitor and an LSD1 inhibitor maintained high levels of CD34 / CD41a co-expression in megakaryocytes (Figure 19).

[0139] This invention makes it possible to produce megakaryocytes (high-quality master cells) that maintain high quality for a long period of time, regardless of the type of iPS cell line. This may enable a stable supply of high-quality platelets (platelets that maintain hemostatic and circulatory capabilities). Because this can be achieved easily and at low cost, it may be extremely useful from the perspective of social implementation of artificial platelets.

[0140] This application is based on patent application No. 2024-020854 filed in Japan (filing date: February 15, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing megakaryocytes that maintains platelet production ability, comprising the step of increasing the abundance of at least one of the proteins that constitute ubiquinol-cytochrome c reductase (UQCR) in megakaryocytes.

2. The method according to claim 1, wherein at least one of the proteins constituting UQCR is UQCR core protein (UQCRC).

3. The method of 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 step of increasing the abundance of a protein constituting UQCR comprises the step of introducing a nucleic acid encoding said protein into megakaryocytes.

5. A method for producing megakaryocytes that maintains platelet production ability, comprising a step of inhibiting the function of polycomb repressive complex 1.1 in megakaryocytes.

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

7. The method of claim 6, wherein the step of inhibiting the function of polycomb repressive complex 1.1 comprises a step of reducing the abundance of at least one of the proteins that constitute polycomb repressive complex 1.

1.

8. The method according to claim 7, wherein the protein constituting the polycomb repressive complex 1.1 is at least one selected from the group consisting of 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 abundance of BACH1 protein in megakaryocytes.

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

11. The method according to any one of claims 1 to 10, which comprises a step of culturing megakaryocytes in a medium containing an adipogenic factor.

12. The method according to any one of claims 1 to 11, wherein the medium contains a compound represented by the following structural formula:

13. The method according to any one of claims 1 to 12, comprising the step of increasing the abundance of MYC protein and apoptosis inhibitor protein in megakaryocytes.

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

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

16. Megakaryocytes that have all of the following characteristics (A) to (C): (A) They contain at least one protein that constitutes exogenous ubiquinol-cytochrome c reductase (UQCR); (B) They have the ability to produce platelets; and (C) They express the CD34 gene and the CD41 gene.

17. The megakaryocyte according to claim 16, wherein at least one of the proteins constituting UQCR is a UQCR core protein.

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

19. The method according to claim 18, which comprises a shaking culture step.

20. Platelets obtained by the method of claim 18 or 19.

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

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

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

Images