Method for producing megakaryocyte precursor cells or megakaryocyte cells

Abstract

The present invention provides a method for producing a megakaryocyte progenitor cell or a megakaryocytic cell, the method comprising a step of suppressing the expression of CDKN1A gene or the function of an expression product of the gene in a hematopoietic progenitor cell or a megakaryocyte progenitor cell that is not polynucleated yet.

Description

[Technical field]

[0001] The present invention broadly relates to a method for producing megakaryocyte precursor cells or megakaryocyte cells. [Background technology]

[0002] Approximately 200 billion platelets are used in each transfusion, but because platelets generally only last a few days in the body, repeated transfusions are often necessary. Due to the aging of society, the number of elderly people who require repeated platelet transfusions is steadily increasing, but the number of young donors is decreasing.

[0003] In order to meet the social demand for platelets, the inventors established a technology for stable production of platelet preparations in vitro using human iPS cells (Non-Patent Documents 1 and 2). The inventors also established a technology for mass production of platelets (human iPS cell-derived artificial platelet preparations) by simply introducing three factors (MYC / BMI1 / BCL-XL) into hematopoietic cells derived from human iPS cells to establish immortalized megakaryocyte lines (cells that become the source of platelets) and changing the culture medium (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 034073 [Patent Document 2] International Publication No. 2012 / 157586 [Non-patent literature]

[0005] [Non-Patent Document 1] Cell Stem Cell. 2014 Apr 3;14(4):535-48 [Non-Patent Document 2] Cell. 2018 Jul 26;174(3):636-648 Summary of the Invention [Problem to be solved by the invention]

[0006] 10 11 In order to supply 1,000 packs of a preparation containing 1 / 10 platelets, for example, at least 10 14-15 1000 immortalized megakaryocytes (calculated based on the number of proliferation days, this is approximately 4 to 5 months after the introduction of MYC, BMI1 and BCL-XL into hematopoietic progenitor cells on day 10-14 induced from human iPS cells). Therefore, the production of megakaryocyte progenitor cells or megakaryocyte cells with high cell proliferation ability is essential for platelet production, but it has been found that there is a limit to the cell proliferation ability of megakaryocyte progenitor cells or megakaryocyte cells obtained by introducing the above three factors (MYC / BMI1 / BCL-XL) into low-quality iPS cells or hematopoietic progenitor cells.

[0007] In view of the above circumstances, an object of the present invention is to provide a novel method for producing megakaryocyte precursor cells or megakaryocytes with high proliferation potential in order to establish a stable platelet production system. [Means for solving the problem]

[0008] As a result of extensive investigations aimed at solving the above-mentioned problems, the inventors discovered that megakaryocyte progenitor cells or megakaryocytes with high proliferation ability can be obtained by suppressing the expression of the CDKN1A gene, which encodes the cell cycle inhibitor p21, and thus completed the present invention.

[0009] That is, the present invention includes the following inventions. [1] A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising a step of suppressing expression of the CDKN1A gene or the function of its expression product in hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells. [2] The method according to [1], further comprising a step of suppressing the expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products. [3] The method according to [2], wherein the megakaryocyte cells are mature megakaryocyte cells. [4] The method according to any one of [1] to [3], wherein the megakaryocyte cells are immortalized. [5] The method according to [4], further comprising the step of forcibly expressing an oncogene selected from the MYC family of genes and the BMI1 gene in hematopoietic progenitor cells. [6] The method according to [5], further comprising a step of forcibly expressing the BCL-XL gene. [7] The method described in [6], further comprising a step of suppressing forced expression of oncogenes, BMI1 gene and BCL-XL gene. [8] The method according to any of [1] to [7], wherein suppression of expression of the CDKN1A gene or the function of its expression product is carried out simultaneously with forced expression of the BCL-XL gene. [9] The method according to any of [1] to [7], wherein the expression of the CDKN1A gene or the function of its expression product is suppressed after a decrease in cell proliferation following forced expression of the BMI1 gene and the BCL-XL gene.

[10] The method according to [9], wherein the decrease in cell proliferation occurs 30 days or more after forced expression of the BMI1 gene and the BCL-XL gene.

[11] The method according to any of [1] to

[10] , wherein the suppression step is carried out by introducing into the cell a molecule that suppresses the expression of the gene or the function of the expression product.

[12] A step of producing megakaryocyte progenitor cells or megakaryocytes by the method according to any one of [1] to

[11] ; and A method for producing platelets, comprising a step of culturing the produced megakaryocyte precursor cells or megakaryocyte cells.

[13] A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells, comprising the step of suppressing the expression of the CDKN1A gene or the function of its expression product.

[14] The method according to

[13] , further comprising a step of suppressing expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products.

[15] A proliferation promoter for megakaryocyte progenitor cells or megakaryocyte cells, comprising as an active ingredient a molecule that suppresses the expression of the CDKN1A gene or the function of its expression product.

[16] The growth-promoting agent described in

[15] , further comprising a molecule that suppresses the expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products.

[0010]

[17] A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising a step of suppressing expression of the p53 gene or the function of its expression product in hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells.

[18] The method described in

[17] , further comprising a step of suppressing the expression of the INK4A gene and / or the ARF gene and / or the CDKN1A gene, or the function of their expression products.

[19] The method according to

[17] or

[18] , wherein the megakaryocyte cells are mature megakaryocyte cells.

[20] The method according to any of

[17] to

[19] , wherein the megakaryocyte cells are immortalized.

[21] The method according to

[20] , further comprising the step of forcibly expressing an oncogene selected from the MYC family of genes and the BMI1 gene in hematopoietic progenitor cells.

[22] The method according to

[21] , further comprising a step of forcibly expressing the BCL-XL gene.

[23] The method described in

[22] , further comprising a step of suppressing forced expression of oncogenes, BMI1 gene and BCL-XL gene.

[24] The method according to any of

[17] to

[23] , wherein suppression of the expression of the p53 gene or the function of its expression product is carried out simultaneously with forced expression of the BCL-XL gene.

[25] The method described in any of

[17] to

[23] , wherein suppression of the expression of the p53 gene or the function of its expression product is carried out after a decrease in cell proliferation following forced expression of the BMI1 gene and the BCL-XL gene.

[26] The method according to

[25] , wherein the decrease in cell proliferation occurs 30 days or more after forced expression of the BMI1 gene and the BCL-XL gene.

[27] The method according to any of

[17] to

[26] , wherein the suppression step is carried out by introducing into the cell a molecule that suppresses the expression of the gene or the function of its expression product.

[28] A step of producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of

[17] to

[27] ; and A method for producing platelets, comprising a step of culturing the produced megakaryocyte precursor cells or megakaryocyte cells.

[29] A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells, comprising the step of suppressing the expression of the p53 gene or the function of its expression product.

[30] The method described in

[29] , further comprising a step of suppressing the expression of the INK4A gene and / or the ARF gene and / or the CDKN1A gene, or the function of their expression products.

[31] A proliferation promoter for megakaryocyte precursor cells or megakaryocyte cells, comprising as an active ingredient a molecule that suppresses the expression of the p53 gene or the function of its expression product.

[32] The growth-promoting agent described in

[31] , further comprising a molecule that suppresses the expression of the INK4A gene and / or the ARF gene and / or the CDKN1A gene, or the function of their expression products. Effect of the Invention

[0011] According to the present invention, by suppressing the expression of the CDKN1A gene in starting cells, it becomes possible to produce megakaryocyte progenitor cells or megakaryocyte cells that have a longer proliferation period and higher proliferation capacity than conventional techniques and are capable of stable platelet production.

[0012] By suppressing the expression of the tumor suppressor genes, p53 gene and / or INK4A / ARF gene, in addition to the CDKN1A gene, the proliferation period of megakaryocyte precursor cells or megakaryocyte cells can be further extended and the proliferation ability can be further increased.

[0013] By suppressing the expression of the p53 gene in the starting cells, it is also possible to produce megakaryocyte progenitor cells or megakaryocyte cells that have a longer proliferation period and higher proliferation capacity than conventional techniques and that are capable of stable platelet production.

[0014] By further suppressing the expression of the CDKN1A gene and / or the INK4A / ARF gene in addition to the p53 gene, the proliferation period of megakaryocyte progenitor cells or megakaryocyte cells can be further extended and the proliferation ability can be further increased. [Brief description of the drawings]

[0015] [Figure 1] 1 shows the results of analyzing the proliferation of megakaryocyte progenitor cells or megakaryocyte cells (MKCL1) after suppression of CDKN1A gene expression. The vertical axis shows the number of cells proliferated after suppression of gene expression, and the horizontal axis shows the number of days after suppression of expression. [Diagram 2] 2 shows the results of analyzing the proliferation of megakaryocyte progenitor cells or megakaryocyte cells (MKCL23) after suppressing the expression of CDKN1A gene, INK4A / ARF gene, and p53 gene. The vertical axis shows the number of cells proliferated after suppression of gene expression, and the horizontal axis shows the number of days after suppression of expression. [Diagram 3] 3 shows the results of analyzing the proliferation of megakaryocyte progenitor cells or megakaryocyte cells (MKCL26) after suppressing the expression of CDKN1A gene, INK4A / ARF gene, and p53 gene. The vertical axis shows the number of cells proliferated after suppression of gene expression, and the horizontal axis shows the number of days after suppression of expression. [Figure 4] 4 shows the results of analyzing the proliferation of megakaryocytes (MKCL1) after suppressing the expression of CDKN1A gene and p53 gene simultaneously with or 2 weeks after the introduction of BCL-XL. The vertical axis shows the number of cells proliferated after suppression of gene expression, and the horizontal axis shows the number of days after suppression of expression. [Diagram 5] FIG. 5 shows FACS dot plots (upper right and lower right, respectively) of megakaryocytes established by suppressing the expression of the CDKN1A gene and platelets produced therefrom (X-axis: CD41; Y-axis: CD42b). [Figure 6]Figure 6 shows the results of comparing the platelet production ability of megakaryocytic cells (MKCL21) in which expression of the CDKN1A gene was suppressed with that of a megakaryocytic cell line (SeV2) with the best platelet release ability due to forced expression of the conventional three factors (MYC / BMI1 / BCL-XL). Upper panel: The state after adding doxycycline to express the three factors MYC / BMI1 / BCL-XL. Lower panel: The state five days after removing doxycycline from the culture medium and suppressing the expression of the three factors MYC / BMI1 / BCL-XL. [Figure 7] FIG. 7 shows the results of comparing the platelet release ability of the MKCL7 strain after infection with a control vector, the MKCL21 strain after CDKN1A suppression, and the MKCL30 strain after CDKN1A and p53 suppression. [Figure 8] Figure 8 shows the results of comparing the platelet release ability of the MKCL7 strain after infection with a control vector, the MKCL21 strain after CDKN1A suppression, and the MKCL30 strain after CDKN1A and p53 suppression, respectively, without stimulation or in the presence of 0.4 μM PMA or 100 μM ADP + 40 μM TRAP-6. [Figure 9] Figure 9 shows the results of cell proliferation on days 14, 31, and 43 for the following megakaryocyte progenitor cell lines: a megakaryocyte progenitor cell line into which the c-MYC / BMI1 genes had been introduced and which was then cultured for 14 days (MB); a MB line into which the BCL-XL gene had been further introduced using a doxycycline-inducible lentiviral vector (MBX); a MB line infected with a lentiviral vector that sustains expression of sh p21 / p53 (MB-p21 / p53_KD); and a MBX line infected with a lentiviral vector that sustains expression of sh p21 / p53 (MBX-p21 / p53_KD). [Figure 10] FIG. 10 shows the results of FACS analysis of the expression of CD34 and CD41 in cells of the MB, MBX, MB-p21 / p53_KD, and MBX-p21 / p53_KD strains on days 31 and 43. [Figure 11]FIG. 11 shows the results of FACS analysis of the expression of CD34 and CD42b in cells of the MB, MBX, MB-p21 / p53_KD, and MBX-p21 / p53_KD strains on days 31 and 43. [Figure 12] FIG. 12 shows the results of FACS analysis of cellular CD34 and GPA expression on days 31 and 43 for the MB, MBX, MB-p21 / p53_KD, and MBX-p21 / p53_KD strains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] (Method of producing megakaryocyte precursor cells or megakaryocyte cells) The method for producing megakaryocyte progenitor cells or megakaryocyte cells according to the present embodiment includes a step of suppressing the expression of CDKN1A gene or the function of its expression product in hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells. As used herein, "expression of a gene" refers to the transcription of DNA encoding a gene of interest into mRNA and / or the translation of mRNA into a protein.

[0017] The CDKN1A (cyclin-dependent kinase inhibitor 1A) gene encodes the cell cycle inhibitor p21, and is also known as a downstream gene of the p53 gene, which is a tumor suppressor gene. The activated p53 protein acts as a transcription factor and increases the expression of the p53 downstream gene group. Therefore, as used herein, "suppressing the expression of a gene or the function of its expression product" may be achieved by directly suppressing the expression of the target gene or the function of its expression product (e.g., p21 in the case of the CDKN1A gene), or by controlling the expression of genes upstream of the target gene or the function of their expression products. However, in this specification, when suppressing the expression of the CDKN1A gene or the function of its expression product, the upstream gene of the target CDKN1A gene does not include the p53 gene, nor the INK4A gene and the ARF gene, which are other tumor suppressor genes upstream of the p53 gene.

[0018] The INK4 (inhibitors of CDK4) A gene and the ARF (alternative reading frame) gene are genes that exist at the same locus and are known as tumor suppressor genes. Both genes share some exons and are thought to both act to suppress proliferation. Expression of both genes can be suppressed by using molecules such as siRNA that target the common exons. As used herein, "INK4A / ARF gene" is interpreted as both genes or either one of the genes, i.e., "INK4A gene and / or ARF gene."

[0019] It is preferable to suppress the expression of not only the CDKN1A gene but also the INK4A / ARF gene and / or the p53 gene, or the function of their expression products, and more preferable to suppress the combination of the CDKN1A gene and the p53 gene.

[0020] As used herein, each gene, such as CDKN1A gene, INK4A / ARF gene, p53 gene, etc., refers to those encoded by their known nucleic acid sequences, for example, cDNA sequences. Each gene may also include homologues identified based on the homology of known nucleic acid sequences.

[0021] A homolog of the CDKN1A gene is a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence shown in SEQ ID NO: 1. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 1 is a DNA consisting of a sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99% or more identity to the DNA consisting of the sequence shown in SEQ ID NO: 1, or a DNA capable of hybridizing under stringent conditions with a DNA or RNA consisting of a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 1, and a protein encoded by these DNAs inhibits the cell cycle. Alternatively, a cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 1 refers to a DNA consisting of a sequence in which one or more, for example 1 to 10, preferably several, for example 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases in the sequence shown in SEQ ID NO: 1 have been deleted, substituted or added, and the proteins encoded by these DNAs inhibit the cell cycle.

[0022] Furthermore, the homologue of the INK4A gene used in the present invention refers to a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence shown in SEQ ID NO: 2. The cDNA substantially identical to the nucleic acid sequence shown in SEQ ID NO: 2 refers to a DNA having a sequence with an identity of about 60% or more, preferably about 70% or more, more preferably about 80% or more, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99% or more, to the DNA having the sequence shown in SEQ ID NO: 2, or a DNA capable of hybridizing under stringent conditions with DNA or RNA having a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 2, and a protein encoded by the DNA suppresses cancer. Alternatively, a cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 2 is a DNA consisting of a sequence in which one or more, for example 1 to 10, and preferably several, for example 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases in the sequence shown in SEQ ID NO: 2 have been deleted, substituted or added, and the proteins encoded by these DNAs suppress cancer.

[0023] Furthermore, the homolog of the ARF gene used in the present invention refers to a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence shown in SEQ ID NO: 3. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 3 refers to a DNA consisting of a sequence having an identity of about 60% or more, preferably about 70% or more, more preferably about 80% or more, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99% or more, to the DNA consisting of the sequence shown in SEQ ID NO: 3, or a DNA that can hybridize under stringent conditions with a DNA or RNA consisting of a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 3, and a protein encoded by the DNA suppresses cancer. Alternatively, a cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 3 refers to a DNA consisting of a sequence in which one or more, for example 1 to 10, preferably several, for example 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases in the sequence shown in SEQ ID NO: 3 have been deleted, substituted or added, and the protein encoded by this DNA suppresses cancer.

[0024] The p53 gene is a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence shown in SEQ ID NO: 4. A cDNA substantially identical to the nucleic acid sequence shown in SEQ ID NO: 4 is a DNA having an identity of about 60% or more, preferably about 70% or more, more preferably about 80% or more, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99% or more, to the DNA having the sequence shown in SEQ ID NO: 4, or a DNA capable of hybridizing under stringent conditions to a DNA having a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 4, and a protein encoded by the DNA suppresses cancer. Alternatively, a cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 4 refers to a DNA consisting of a sequence in which one or more, for example 1 to 10, preferably several, for example 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases in the sequence shown in SEQ ID NO: 4 have been deleted, substituted or added, and the protein encoded by this DNA suppresses cancer.

[0025] Here, stringent conditions refer to hybridization conditions that can be easily determined by those skilled in the art, and are generally empirical experimental conditions that depend on the base length of the nucleic acid, washing temperature, and salt concentration. In general, the longer the base, the higher the temperature for proper annealing, and the shorter the base, the lower the temperature. Hybridization generally depends on the ability of complementary strands to reanneal in an environment slightly lower than their melting point.

[0026] Specifically, for example, low stringency conditions include washing the filter after hybridization in a 0.1×SSC, 0.1% SDS solution at a temperature of 37° C. to 42° C. For example, high stringency conditions include washing the filter at a temperature of 65° C., 5×SSC, and 0.1% SDS for the washing step. By increasing the stringency of the conditions, polynucleotides with high homology can be obtained.

[0027] The expression of a gene or the function of its expression product can be suppressed by known methods, for example, by introducing various molecules into cells, such as siRNA, antisense nucleic acid, or expression vectors capable of expressing these nucleic acid molecules, which can specifically suppress the expression of each gene. Alternatively, genes may be knocked down using other techniques, such as genome editing techniques. For example, when a gene is knocked down using the CRISPR-Cas system, a guide RNA targeting the gene and a fusion protein of an inactivated Cas such as dCas and a repressor domain are used.

[0028] Typically, siRNA is a double-stranded oligo-RNA consisting of an RNA having a sequence complementary to the nucleotide sequence or a partial sequence of the mRNA of a target gene and its complementary strand. The nucleotide sequence of these RNAs can be appropriately designed by those skilled in the art based on the sequence information of the gene whose expression is suppressed. shRNA can also be used instead of siRNA.

[0029] Antisense nucleic acid refers to a nucleic acid that contains a nucleotide sequence capable of specifically hybridizing with a target mRNA (mature mRNA or initial transcription product) under physiological conditions of a cell expressing the target mRNA, and that, in the hybridized state, can inhibit the translation of a polypeptide encoded by the target mRNA. The type of antisense nucleic acid may be DNA or RNA, or a chimera of DNA and RNA. The nucleotide sequence of the antisense nucleic acid can be appropriately designed by a person skilled in the art based on the sequence information of the gene whose expression is to be suppressed.

[0030] In addition to the above techniques, compounds known to suppress the expression of each gene can also be used. For example, p21 inhibitors such as UC2288, butyrolactone I, LLW10, sorafenib, and sterigmatocystin are known to suppress the expression of the CDKN1A gene. In addition, p53 inhibitors such as pifithrin α, nutrin-3, ReACp53, and RG7388 are known.

[0031] Alternatively, a target gene may be knocked out using known techniques to suppress the expression of the gene or the function of its expression product. Knockout of a gene means that all or a part of the gene is destroyed or mutated so that it does not exert its original function. A gene may be destroyed or mutated so that one allele on the genome does not function. In addition, multiple alleles may be destroyed or mutated. Knockout can be performed by known methods, such as a method of knocking out by introducing a DNA construct designed to cause genetic recombination with the target gene into a cell, or a method of knocking out by inserting, deleting, or replacing a base using genome editing techniques such as TALEN or CRISPR-Cas system.

[0032] In addition, compounds that suppress the transcription and transcription products of each gene, or inhibitors of the binding of the produced protein to its target protein (p53 binding inhibitors: pifithrin α, nutrin-3, ReACp53, RG7388, etc.; p21 binding inhibitors: UC2288, butyrolactone I, LLW10, sorafenib, sterigmatocystin, etc.) may be used.

[0033] The expression of the above genes or the function of the expression product thereof is suppressed in cells before differentiation into megakaryocytes, for example, hematopoietic progenitor cells or megakaryocyte progenitor cells before multinucleation. As used herein, "hematopoietic progenitor cells" refer to hematopoietic cells characterized as CD34 positive cells, and are preferably, for example, cells derived from ES cells or iPS cells, particularly cells obtained from a net-like structure (also called ES-sac or iPS-sac) prepared from ES cells or iPS cells (particularly, cells immediately after separation from the net-like structure). Here, the "net-like structure" prepared from ES cells or iPS cells refers to a three-dimensional sac-like (with a space inside) structure derived from ES cells or iPS cells, formed from an endothelial cell population, etc., and containing hematopoietic progenitor cells inside.

[0034] "Pre-multinucleated megakaryocyte progenitor cells" refer to cells that are less differentiated than multinucleated megakaryocytes, that are CD41a-positive / CD42a-positive / CD42b-positive, which are specific markers for the megakaryocyte lineage, and that are mononuclear or binuclear cells that have not undergone nuclear polyploidization.

[0035] Hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells can be obtained, for example, by isolation from bone marrow, umbilical cord blood, or peripheral blood, or can be obtained by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells, which are even more undifferentiated cells.

[0036] Megakaryocyte cells are produced by further inducing differentiation from hematopoietic progenitor cells or megakaryocyte progenitor cells before multinucleation by a known method. Megakaryocyte cells are characterized by being positive for the cell surface markers CD41a, CD42a, and CD42b. In addition to these markers, megakaryocyte cells may further express at least one marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. Megakaryocyte cells release platelets when they become multinucleated (polyploidized) and mature. Multinucleated megakaryocyte cells have a genome 16 to 32 times larger than that of normal cells.

[0037] In this specification, unless otherwise specified, the term "megakaryocytes" is used to encompass all megakaryocytes, including immature megakaryocytes and megakaryocytes that have become multinucleated and mature (mature megakaryocytes).

[0038] The megakaryocyte cells are preferably further immortalized. Non-limiting examples of methods for producing immortalized megakaryocyte cells include the methods described in WO 2011 / 034073 (see above) and U.S. Patent Application Publication No. 2012 / 0238023. In these methods, immortalized megakaryocyte cell lines that proliferate indefinitely can be obtained by forcibly expressing oncogenes and polycomb genes in cells less differentiated than megakaryocyte cells.

[0039] Immortalized megakaryocytes can also be obtained by forcibly expressing an apoptosis-suppressing gene in cells less differentiated than megakaryocytes, according to the methods described in WO 2012 / 157586 (see above) and U.S. Patent Application Publication No. 2014 / 0127815. By releasing the forced expression of the gene, these immortalized megakaryocytes acquire the ability to self-replicate, progress in multinucleation, and begin to release platelets.

[0040] The forced expression of the oncogene, polycomb gene, and / or apoptosis suppressor gene may be performed simultaneously or sequentially. For example, the oncogene and polycomb gene may be forcedly expressed, then the forced expression may be suppressed, then the apoptosis suppressor gene may be forcedly expressed, then the forced expression may be suppressed to obtain multinucleated megakaryocytes. Alternatively, the oncogene, polycomb gene, and apoptosis suppressor gene may be forcedly expressed simultaneously, and the forced expression of these may be suppressed simultaneously to obtain multinucleated megakaryocytes. First, the oncogene and polycomb gene may be forcedly expressed, then the apoptosis suppressor gene may be forcedly expressed, and the forced expression of these may be suppressed simultaneously to obtain multinucleated megakaryocytes.

[0041] The expression of the CDKN1A gene or the suppression of the function of its expression product can be performed simultaneously with the forced expression of any of the oncogene, polycomb gene, or apoptosis suppressor gene, preferably simultaneously with the forced expression of the apoptosis suppressor gene, or thereafter, for example, after a decrease in cell proliferation, for example, a state in which the proliferation rate is halved or less, is confirmed by comparing the cell proliferation rate at a certain time point with the most recent cell proliferation rate (for example, assuming that cell proliferation is confirmed every week, the cell proliferation rate in a certain week is compared with the proliferation rate from the previous week). The decrease in cell proliferation is observed, but is not intended to be limited thereto, from about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, or about 90 days after the forced expression of the oncogene or polycomb gene.

[0042] As used herein, the term "cancer gene" refers to a gene that induces canceration of cells in vivo, and examples thereof include MYC family genes (e.g., c-MYC, N-MYC, L-MYC), SRC family genes, RAS family genes, RAF family genes, and protein kinase family genes such as c-Kit, PDGFR, and Abl. Among these, MYC family genes, particularly c-MYC, are preferred.

[0043] As used herein, the term "polycomb gene" refers to a gene that negatively regulates the CDKN2a (INK4A / ARF) gene and functions to avoid cellular senescence (Ogura et al., Regenerative Medicine, vol. 6, No. 4, pp. 26-32; Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol. 7, pp. 667-677, 2006; Proc. Natl. Acad. Sci. USA, vol. 100, pp. 211-216, 2003). Non-limiting examples of polycomb genes include BMI1, Mel18, Ring1a / b, Phc1 / 2 / 3, Cbx2 / 4 / 6 / 7 / 8, Ezh2, Eed, Suz12, HADC, and Dnmtl / 3a / 3b. Among these, BMI1 is preferred.

[0044] As used herein, the term "apoptosis-suppressing gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples thereof include the BCL2 gene, the BCL-XL gene, the Survivin gene, the MCL1 gene, etc. Among these, the BCL-XL gene is preferred.

[0045] Forced expression of a gene and release of the forced expression can be carried out by the methods described in WO 2011 / 034073 (see above) and U.S. Patent Application Publication No. 2012 / 0238023, WO 2012 / 157586 (see above) and U.S. Patent Application Publication No. 2014 / 0127815, WO 2014 / 123242 and U.S. Patent Application Publication No. 2016 / 0002599, or Nakamura S et al., Cell Stem Cell. 14, 535-548, 2014, other known methods, or methods equivalent thereto.

[0046] The period of the forced expression can be appropriately determined by those skilled in the art, but for the forced expression of oncogenes, polycomb genes, and / or apoptosis-suppressing genes, it is preferable to suppress (release) the forced expression after a desired period of time has elapsed after suppressing the expression of the CDKN1A gene. After the forced expression, the cells may be subcultured, and the period from the last subculture to the day when the forced expression is released is not particularly limited, but may be, for example, 1 day, 2 days, or 3 days or more.

[0047] When a drug-responsive gene expression induction system such as the Tet-on (registered trademark) or Tet-off (registered trademark) system is used for forced gene expression and its release, the forced expression may be suppressed by adding a corresponding drug, for example, tetracycline or doxycycline, to the medium in the forced expression step and removing the drug from the medium.

[0048] In a further aspect, the method for producing megakaryocyte progenitor cells or megakaryocyte cells comprises the step of suppressing the expression of the p53 gene or the function of its expression product in hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells.

[0049] In this aspect, it is preferable to suppress the expression of not only the p53 gene, but also the INK4A / ARF gene and / or the CDKN1A gene, or the function of their expression products. A combination of the CDKN1A gene and the p53 gene is more preferable.

[0050] In this aspect, the expression of a gene or the function of its expression product can be suppressed by the methods described above.

[0051] In this aspect, the expression of the above-mentioned gene or the function of its expression product is suppressed in cells prior to differentiation into megakaryocytes, such as hematopoietic progenitor cells or megakaryocyte progenitor cells prior to multinucleation.

[0052] In this aspect, it is preferable that the megakaryocyte cells are further immortalized. Non-limiting examples of methods for producing immortalized megakaryocyte cells include the methods described in WO 2011 / 034073 (see above) and U.S. Patent Application Publication No. 2012 / 0238023. In these methods, immortalized megakaryocyte cell lines that proliferate indefinitely can be obtained by forcibly expressing oncogenes and polycomb genes in cells less differentiated than megakaryocyte cells.

[0053] Immortalized megakaryocytes can also be obtained by forcibly expressing an apoptosis-suppressing gene in cells less differentiated than megakaryocytes, according to the methods described in WO 2012 / 157586 (see above) and U.S. Patent Application Publication No. 2014 / 0127815. By releasing the forced expression of the gene, these immortalized megakaryocytes acquire the ability to self-replicate, progress in multinucleation, and begin to release platelets.

[0054] In this aspect, the forced expression of the oncogene, polycomb gene, and / or apoptosis suppressor gene may be performed simultaneously or sequentially. For example, the oncogene and polycomb gene may be forcedly expressed, then the forced expression may be suppressed, then the apoptosis suppressor gene may be forcedly expressed, then the forced expression may be suppressed to obtain multinucleated megakaryocytes. Alternatively, the oncogene, polycomb gene, and apoptosis suppressor gene may be forcedly expressed simultaneously, and the forced expressions may be suppressed simultaneously to obtain multinucleated megakaryocytes. First, the oncogene and polycomb gene may be forcedly expressed, then the apoptosis suppressor gene may be forcedly expressed, and the forced expressions may be suppressed simultaneously to obtain multinucleated megakaryocytes.

[0055] In this aspect, the expression of the p53 gene or the suppression of the function of its expression product can be performed simultaneously with the forced expression of any of the oncogene, polycomb gene, or apoptosis-suppressing gene, preferably simultaneously with the forced expression of the apoptosis-suppressing gene, or thereafter, for example, after a decrease in cell proliferation, for example, a state in which the proliferation rate is confirmed to be 1 / 2 or less, by comparing the cell proliferation rate at a certain time point with the most recent cell proliferation rate (for example, assuming that cell proliferation is confirmed every week, the cell proliferation rate in a certain week is compared with the proliferation rate from the week before). The decrease in cell proliferation is observed, but is not intended to be limited thereto, from about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, or about 90 days after the forced expression of the oncogene or polycomb gene.

[0056] In this aspect, the oncogene is preferably a MYC family gene, particularly c-MYC, the polycomb gene is preferably BMI1, and the apoptosis-suppressing gene is preferably BCL-XL gene.

[0057] In one embodiment of this aspect, an immortalized megakaryocytic cell line is obtained by forced expression of an oncogene (e.g., c-MYC) and a polycomb gene (e.g., BMI1), with or without forced expression of an apoptosis-suppressing gene (e.g., BCL-XL).

[0058] In this aspect, forced expression of a gene and release of the forced expression can be carried out by the methods described above.

[0059] In this aspect, the period of the forced expression can be appropriately determined by those skilled in the art, but for the forced expression of oncogenes, polycomb genes, and / or apoptosis-suppressing genes, it is preferable to suppress (release) the forced expression after a desired period of time has elapsed after suppressing the expression of the p53 gene. Note that, after the forced expression, the cells may be subcultured, and the period from the last subculture to the day when the forced expression is released is not particularly limited, but may be, for example, 1 day, 2 days, or 3 days or more.

[0060] The culture conditions for hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells, and further for megakaryocyte progenitor cells or megakaryocyte cells, can be appropriately determined by those skilled in the art depending on the type and state of the cells. For example, the culture temperature can be about 35°C to about 42°C, about 36°C to about 40°C, or about 37°C to about 39°C, and the carbon dioxide concentration can be, for example, 5% CO 2 , the oxygen concentration is, for example, 20% 2 The culture may be static culture or shaking culture. In the case of shaking culture, the shaking speed is not particularly limited, and may be, for example, 10 rpm to 200 rpm, 30 rpm to 150 rpm, etc.

[0061] The medium may be an Iscove's modified Dulbecco's medium (IMDM) medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. In this case, the IMDM medium may further contain SCF, and may further contain heparin. The respective concentrations are not particularly limited, but for example, TPO can be about 10 ng / mL to about 200 ng / mL, or about 50 ng / mL to about 100 ng / mL, SCF can be about 10 g / mL to about 200 g / mL, or about 50 g / mL, and heparin can be about 10 U / mL to about 100 U / mL, or about 25 U / mL. Phorbol ester (e.g., phorbol-12-myristate-13-acetate; PMA) may be added.

[0062] The cell culture step can be carried out in the presence or absence of feeder cells. As used herein, the term "feeder cells" refers to cells that are co-cultured with target cells to provide the environment necessary for the culture of the target cells to be proliferated or differentiated. The feeder cells may be cells of the same species or different species, so long as they can be distinguished from the target cells. The feeder cells may be cells that have been treated with antibiotics or gamma rays to prevent proliferation, or may not be treated.

[0063] The medium may contain serum or plasma, or may be serum-free. When serum is used, human serum is preferred. If necessary, the medium may also contain one or more substances such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines. Examples of cytokines include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplements, and ADAM (A Disintegrin And Metalloprotease) inhibitors.

[0064] When megakaryocytes are cultured in a medium containing an aryl hydrocarbon receptor (AhR) antagonist alone or in combination with a ROCK (Rho-associated coiled-coil forming kinase) inhibitor, a platelet production-promoting effect comparable to that achieved when feeder cells are used can be obtained even without the use of feeder cells (WO 2016 / 204256 and U.S. Patent Application Publication No. 2019 / 0048317).

[0065] (Method of producing platelets) The method for producing platelets according to this embodiment includes a step of further culturing the megakaryocyte precursor cells or megakaryocyte cells produced according to the above-mentioned method.

[0066] The medium for culturing megakaryocytes is not particularly limited, and a known medium suitable for producing platelets from megakaryocytes or a medium similar thereto can be used as appropriate. For example, a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include Iscove's modified Dulbecco's medium (IMDM) medium, 199 medium, Eagle's minimum essential medium (EMEM), αMEM medium, Dulbecco's modified Eagle's medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fisher's medium, Neurobasal medium (Life Technologies), and mixed media thereof.

[0067] From the viewpoint of producing a large amount of high-quality platelets, it is preferable to culture megakaryocytes in a culture vessel equipped with an agitating blade or stirrer (WO 2017 / 047492 and U.S. Patent Application Publication No. 2018 / 0258395, WO 2017 / 077964 and U.S. Patent Application Publication No. 2018 / 0318352).

[0068] The produced platelets can be used to produce a platelet preparation or a blood preparation. The method for producing a platelet preparation includes a step of recovering a platelet-rich fraction and a step of removing blood cell components other than platelets from the platelet-rich fraction. The step of removing blood cell components can be performed by removing blood cell components other than platelets, including megakaryocytes, using a leukocyte removal filter (for example, manufactured by Terumo Corporation or Asahi Kasei Medical Corporation). More specific methods for producing platelet preparations are described, for example, in International Publication No. 2011 / 034073 (above) and U.S. Patent Application Publication No. 2012 / 0238023. The method for producing a blood preparation includes a step of producing a platelet preparation and a step of mixing the platelet preparation with other components. Examples of other components include red blood cells. The platelet preparation and the blood preparation may also contain other components that contribute to stabilizing cells.

[0069] (Method for promoting proliferation of megakaryocyte precursor cells or megakaryocyte cells) The method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells in this embodiment includes a step of suppressing the expression of the CDKN1A gene or the function of its expression product, and, optionally, a step of suppressing the expression of the INK4A / ARF gene and / or the p53 gene, or the function of their expression products.

[0070] In a further aspect, a method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells comprises the step of inhibiting the expression of the p53 gene or the function of its expression product, and, optionally, the step of inhibiting the expression of the INK4A / ARF gene and / or the CDKN1A gene, or the function of their expression products.

[0071] (Megakaryocyte precursor cell or megakaryocyte cell proliferation promoter) The proliferation promoter of megakaryocyte progenitor cells or megakaryocyte cells in this embodiment contains as an active ingredient a molecule that suppresses the expression of the CDKN1A gene or the function of its expression product, and optionally further contains a molecule that suppresses the expression of the INK4A / ARF gene and / or the p53 gene, or the function of their expression products.

[0072] In a further aspect, the proliferation promoter of megakaryocyte progenitor cells or megakaryocyte cells contains as an active ingredient a molecule that suppresses the expression of the p53 gene or the function of its expression product, and optionally further contains a molecule that suppresses the expression of the INK4A / ARF gene and / or the CDKN1A gene, or the function of their expression products.

[0073] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Those skilled in the art can modify the present invention in various aspects without departing from the meaning of the present invention, and such modifications are also included in the scope of the present invention. EXAMPLES

[0074] Example 1: Suppression of CDKN1A gene expression Human iPS cells (TKDN SeV2: iPS cells derived from human fetal skin fibroblasts established using Sendai virus) were cultured to differentiate into blood cells according to the method described in Takayama N., et al. J Exp Med. 2817-2830 (2010). Specifically, human iPS cell colonies (NC13X line) were cocultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / mL VEGF (R&D SYSTEMS) for 14 days to produce hematopoietic progenitor cells (HPCs). The culture conditions were 20% O 2 , 5% CO 2 It was carried out in.

[0075] An immortalized megakaryocytic cell line was established by introducing the three genes c-MYC / BMI1 / BCL-XL into the hematopoietic progenitor cells obtained. A lentiviral vector that forced expression of c-MYC / BMI1 under doxycycline control was introduced into the hematopoietic progenitor cells on the 14th day of culture, and 1 μg / ml of doxycycline was added 24 hours after infection to express the genes. After another two weeks, a lentiviral vector that forced expression of BCL-XL under doxycycline control was introduced, and an immortalized megakaryocytic cell line was established.

[0076] Megakaryocyte progenitor cells or megakaryocyte line (MKCL21 line) with reduced cell proliferation 50 days after introduction of MYC / BMI1 / BCL-XL were infected with shCDKN1A (clone 1 (SEQ ID NO: 5) or clone 2 (SEQ ID NO: 6))-RFP lentiviral vector. RFP (Red Fluorescent Protein)-positive infected cells were sorted using FACS AriaIIIu 3 days after infection. The predicted secondary structures of clones 1 and 2 are shown below. [ka]

[0077] When the cells were cultured in a culture medium containing 15% FBS, cell proliferation was confirmed for more than 300 days after suppression of CDKN1A gene expression, and the number of megakaryocyte progenitor cells or megakaryocytes increased by 10 66 It multiplied by 2-fold (Figure 1).

[0078] Example 2: Suppression of expression of CDKN1A gene, INK4A / ARF gene, and p53 gene (cell proliferation reduction stage) An immortalized megakaryocytic cell line was established by introducing the three genes MYC / BMI1 / BCL-XL into hematopoietic progenitor cells derived from a human iPS cell line (YZWJ line) different from that used in Example 1.

[0079] A megakaryocyte line (MKCL23 line) in which cell proliferation was reduced 60 days after introduction of MYC / BMI1 / BCL-XL, and a megakaryocyte line (MKCL26 line) in which cell proliferation was reduced 90 days after introduction were infected with lentivirus vectors carrying the following genes. The procedure was the same as in Example 1. 1: Control-RFP (LacZ) 2:shCDKN1A-RFP+shp53-GFP+shINK4A / ARF-GFP Three days after infection, RFP / GFP-positive infected cells were sorted using FACS AriaIIIu. For shCDKN1A, clones 1 and 2 were used. The predicted secondary structures of the shp53 sequence (SEQ ID NO: 7) and the shINK4A / ARF sequence (SEQ ID NO: 8) are shown below. [ka] In addition, shINK4A / ARF targets a common exon between the INK4A gene and the ARF gene.

[0080] After sorting, the cells were continuously cultured in a culture medium containing 15% FBS. In both MKCL23 and MKCL26, the cell proliferation period was extended by more than 30 days and the cell proliferation period was increased by 10 days by suppressing the expression of CDKN1A gene compared to the control. 4A 2-fold increase in proliferation was confirmed (Figures 2 and 3). It was revealed that even when suppression of CDKN1A gene expression was insufficient, the proliferation effect was improved by suppressing the expression of INK4A / ARF gene and p53 gene.

[0081] Furthermore, an immortalized megakaryocytic cell line was established by introducing the three genes MYC / BMI1 / BCL-XL into hematopoietic progenitor cells derived from a human iPS cell line (NIH5 line) different from that used in Example 1.

[0082] A megakaryocytic cell line (MKCL1 line) in which cell proliferation had decreased about 40 days after introduction of MYC / BMI1 / BCL-XL was infected with a lentiviral vector carrying the following genes. The procedure was the same as in Example 1. 1: Control-RFP (LacZ) 2:shCDKN1A-RFP 3:shp53-GFP 4:shINK4A / ARF-GFP 5:shCDKN1A-RFP+shp53-GFP+shARF-GFP The shRNAs used were as described above. Then, positively infected cells were sorted using FACS AriaIIIu.

[0083] After sorting, the cells were cultured in a culture medium containing 15% FBS. Compared to the control, cell proliferation was significantly increased (excluding INK4A / ARF). In particular, the cells in which the expression of CDKN1A, INK4A, ARF and p53 genes was suppressed showed an extension of the cell proliferation period by 20 days and an increase of 10 days. 4 A two-fold increase was observed.

[0084] Example 3: Suppression of expression of CDKN1A gene, INK4A / ARF gene, and p53 gene (when BCL-XL is introduced) At the same time as introducing BCL-XL in Example 2, a megakaryocytic line (MKCL30 line) derived from the YZWJ516 human iPS cell line, which could not be established by conventional forced expression of MBX, was infected with a lentivirus vector carrying the following genes. The procedure was the same as in Example 1. 1: Control-RFP (LacZ) 2:shCDKN1A-RFP 3:shp53-GFP 4:shINK4A / ARF-GFP 5: shCDKN1A-RFP+shp53-GFP 6: shCDKN1A-RFP+shARF-GFP 7:shp53-GFP+shARF-GFP 8: shCDKN1A-RFP+shp53-GFP+shARF-GFP. The shRNAs used were as described above. Then, positively infected cells were sorted using FACS AriaIIIu.

[0085] After sorting, the cells were cultured in a culture medium containing 15% FBS. Compared to the control, the suppression of CDKN1A gene expression extended the cell proliferation period by more than 8 weeks and increased the cell proliferation period by 10 15 The cell proliferation effect was highest when both CDKN1A and p53 gene expression was suppressed (2.5x10 17 fold). As of the priority date of this application (October 17, 2019, 127 days after c-MYC / BMI1 infection), the cells were proliferating. Subsequently, the cells in which the expression of the INK4A gene, the ARF gene, and the p53 gene was all suppressed showed high cell proliferation effects.

[0086] Furthermore, to verify the timing of knockdown, the following knockdown experiment was performed simultaneously with and 2 weeks after BCL-XL introduction. 9: Control-RFP (LacZ) 10:shCDKN1A-RFP+shp53-GFP

[0087] The results are shown in Figure 4. In terms of the timing of infection, infection at the same time as BCL-XL administration was 10 times longer than infection 2 weeks after BCL-XL administration. 4 More than 2.5x10 17 Double vs shCDKN1A-RFP+shp53-GFP simultaneous 5.8x10 12 It was confirmed that the

[0088] Although the proliferation effect was inferior to the combination of CDKN1A gene regulation and p53 gene regulation, it was found that knocking down CDKN1A gene regulation alone or p53 gene regulation alone at the same time as introducing BCL-XL increased the cell proliferation ability.

[0089] Example 4: Examination of platelet production by megakaryocyte cells in which expression of the CDKN1A gene is suppressed Platelet release was compared between the MKCL21 line after CDKN1A suppression, whose proliferation was promoted using clone 2 (SEQ ID NO: 6) in Example 1, and the MKCL21 line after infection with a control vector.

[0090] Doxycycline was removed from the culture medium of both cells, and the expression of the three factors MYC / BMI1 / BCL-XL was suppressed. On the fifth day, megakaryocytes and platelets released into the culture supernatant were analyzed by FACS. The antibodies used were APC anti-human CD41 Antibody (BioLegend, Catalog No.: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, Catalog No.: 555473). The FACS dot plot is shown in Figure 5.

[0091] In addition, a comparison of platelet counts using FACS revealed that platelet production was enhanced by suppressing CDKN1A gene expression.

[0092] Next, the platelet production ability of the MKCL21 line in which the expression of the CDKN1A gene was suppressed in Example 2 was compared with that of a megakaryocytic line (SeV2) with the best platelet release ability due to the forced expression of the conventional three factors (MYC / BMI1 / BCL-XL). The FACS dot plots are shown in Figure 6. The antibodies used were APC anti-human CD41 Antibody (BioLegend, Catalog No.: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, Catalog No.: 555473).

[0093] The upper panel of Figure 6 shows the state after adding doxycycline and expressing the three factors MYC / BMI1 / BCL-XL. Both lines are immature, so there are few CD41-positive / CD42b-positive platelets (upper right part of the four-part figure). On the other hand, the lower panel of Figure 6 shows the state five days after removing doxycycline from the culture medium and suppressing the expression of the three factors MYC / BMI1 / BCL-XL, and there is an increase in CD41a-positive / CD42b-positive platelets (upper right part of the four-part figure). These results show that platelet production ability is increased by suppressing the expression of the CDKN1A gene.

[0094] Example 5: Examination of platelet production by megakaryocyte cells in which expression of CDKN1A gene and p53 gene is suppressed Platelet release was compared between the MKCL7 strain after infection with a control vector, the MKCL21 strain after CDKN1A suppression and whose proliferation was promoted using clone 2 (sequence number 6) in Example 1, and the MKCL30 strain after CDKN1A and p53 suppression and whose proliferation was promoted using "5: shCDKN1A-RFP+shp53-GFP" in Example 3 at the same time as the introduction of clone 2 (sequence number 6) in Example 1.

[0095] Doxycycline was removed from the culture medium of each cell line, and the expression of the three factors MYC / BMI1 / BCL-XL was suppressed. Megakaryocytes and platelets released into the culture supernatant were analyzed by FACS on day 5. The antibodies used were APC anti-human CD41 Antibody (BioLegend, Catalog No.: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, Catalog No.: 555473). However, for the MKCL7 line, a sample was also prepared in which doxycycline was not removed and the expression of the three factors MYC / BMI1 / BCL-XL was not suppressed (MBX ON). On the fifth day, megakaryocytes and platelets released into the culture supernatant were analyzed by FACS in the same manner as above. The FACS dot plot is shown in Figure 7(A), and the platelet count results counted using FACS are shown in Figure 7(B).

[0096] Next, megakaryocytes and platelets released into the culture supernatant were collected on the 5th day from each of the MKCL7, MKCL21, and MKCL30 lines, which did not suppress the expression of the MYC / BMI1 / BCL-XL three factors (ON) and had the expression of the MYC / BMI1 / BCL-XL three factors suppressed. They were then subjected to stimulation with P-selectin antibody (Bio Legend / #304910) and PAC-1 antibody (BD Pharma) in the presence of no stimulation, 0.4 μM PMA (Phorbol Myristate Acetate, Sigma Aldrich Cat#P1585) (PMA), or 100 μM ADP (Adenosine diphosphate, Sigma Aldrich Cat#A-2754) + 40 μM TRAP-6 (Thrombin receptor activator peptide 6, BACHEM Cat#H-8365.0005) (AT). The cells were stained with ELISA kit (Bioscience / #34507) and analyzed by FACS. The results using P-selectin and PAC-1 antibodies are shown in Figure 8 (D) and (E), respectively. The explanation in the table is as follows: No stimulation-ON: Results without stimulation using a line that did not suppress the expression of the three factors MYC / BMI1 / BCL-XL PMA-ON: Results under PMA using a line that did not suppress the expression of the three factors MYC / BMI1 / BCL-XL AT-ON: Results under AT using a line that did not suppress the expression of the three factors MYC / BMI1 / BCL-XL No stimulation_MKCL7: Results without stimulation using the MKCL7 line PMA_MKCL7: Results under PMA using the MKCL7 strain AT_MKCL7: Results under AT using the MKCL7 strain No stimulation_MKCL21#: Results without stimulation using the MKCL21 line PMA_MKCL21#: Results under PMA using the MKCL21 strain AT_MKCL21#: Results under AT using the MKCL21 strain No stimulation_MKCL30: Results without stimulation using the MKCL30 line PMA_MKCL30: Results under PMA using the MKCL30 strain AT_MKCL30: Results under AT using the MKCL30 strain

[0097] These results demonstrated that inhibition of CDKN1A and p53 gene expression produced functional platelets that showed good activation responses to stimuli and did not inhibit platelet function. On the other hand, in the group without doxycycline removal and without inhibition of the expression of the three factors MYC / BMI1 / BCL-XL (MBX ON), megakaryocyte maturation did not progress and almost no platelets were produced.

[0098] Example 6: Expression of BCL-XL gene and suppression of expression of p21 / p53 gene Hematopoietic progenitor cells were produced from iPS cells derived from human skin fibroblasts by culturing for 14 days according to the iPS-Sac method (the method described in Takayama N., et al. J Exp Med. 2817-2830 (2010)).

[0099] The obtained hematopoietic progenitor cells were introduced with two genes (MB), c-MYC / BMI1, using a doxycycline-inducible lentiviral vector, to establish megakaryocyte progenitor cell lines. 14 days later, the following megakaryocyte progenitor cell lines were produced: a line cultured as is (MB), a line in which the BCL-XL gene was further introduced using a doxycycline-inducible lentiviral vector in addition to MB introduction (MBX), a line in which the BCL-XL gene was further introduced using a doxycycline-inducible lentiviral vector in addition to MB introduction and further infected with a persistently expressed sh p21 / p53 lentiviral vector (MB-p21 / p53_KD), and a line in which the BCL-XL gene was introduced using a doxycycline-inducible lentiviral vector in addition to MB introduction and infected with a persistently expressed sh p21 / p53 lentiviral vector (MBX-p21 / p53_KD). The results of confirming the proliferation of cells on the 14th, 31st, and 43rd days for each megakaryocyte progenitor cell line are shown in FIG. 9.

[0100] In addition, the results of FACS analysis of the expression of CD34 and CD41 of the cells of the MB strain, MBX strain, MB-p21 / p53_KD strain, and MBX-p21 / p53_KD strain on days 31 and 43 are shown in Fig. 10, the results of FACS analysis of the expression of CD34 and CD42b of the cells are shown in Fig. 11, and the results of FACS analysis of the expression of CD34 and GPA of the cells are shown in Fig. 12, which are shown as FACS dot plots. The antibodies used were APC anti-human CD41 Antibody (BioLegend, catalog number: 303710), PE Mouse Anti-Human CD42b Clone HIP1 (BD, catalog number: 555473), GPA (Glycophorin A) antibody (BioLegend, catalog number: 306612), and CD34 antibody (BioLegend, catalog number: 343514).

[0101] Under all conditions, megakaryocyte progenitor cells positive for CD41 and weakly positive for CD42b were obtained. At the stage of 43 days of culture, the results of the MB-p21 / p53_KD line (white circles in FIG. 9) show that the proliferation is considerably superior to that of the MB line (black circles). Moreover, the results of the MBX-p21 / p53_KD line (black triangles) show that the proliferation is equal to or higher than that of the MBX line (black squares) or the MB-p21 / p53_KD line (white circles). These results suggest that the proliferation of the megakaryocyte progenitor cell line obtained by introducing two genes, c-MYC / BMI1, is promoted by p21 / p53KD even without the introduction of the BCL-XL gene, that is, BCL-XL can be replaced by p21 / p53KD.

[0102] All references cited herein, including publications, patent documents, and the like, are hereby incorporated by reference to the same extent as if each was individually and specifically incorporated by reference and the contents of which were specifically set forth in their entirety.

Claims

1. A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising the steps of forcibly expressing an oncogene selected from the MYC family genes and the BMI1 gene in pre-multinucleated megakaryocyte progenitor cells in vitro, and suppressing the expression of the CDKN1A gene or the function of its expression product.

2. The method of claim 1, further comprising the step of suppressing the expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products.

3. The method of claim 2 , wherein the megakaryocyte cells are mature megakaryocyte cells.

4. The method of any one of claims 1 to 3, wherein the megakaryocyte cells are immortalized.

5. The method according to any one of claims 1 to 4, further comprising a step of forcibly expressing the BCL-XL gene.

6. The method according to claim 5, further comprising the step of suppressing the forced expression of an oncogene, a BMI1 gene and a BCL-XL gene.

7. The method according to claim 5 or 6, wherein the expression of the CDKN1A gene or the function of its expression product is suppressed simultaneously with the forced expression of the BCL-XL gene.

8. The method according to claim 5, wherein the expression of the CDKN1A gene or the function of its expression product is suppressed after the reduction in cell proliferation following the forced expression of the BMI1 gene and the BCL-XL gene.

9. The method according to claim 8, wherein the decrease in cell proliferation occurs 30 days or more after the forced expression of the BMI1 gene and the BCL-XL gene.

10. The method according to claim 1 , wherein the suppression step is carried out by introducing into the cell a molecule that suppresses the expression of the gene or the function of its expression product.

11. A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising the steps of suppressing the expression of the CDKN1A gene or the function of its expression product, and suppressing the expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products, in pre-multinucleated megakaryocyte progenitor cells in vitro.

12. The method of claim 11 , wherein the megakaryocyte cells are mature megakaryocyte cells.

13. The method of claim 10 or 11, wherein the megakaryocyte cells are immortalized.

14. A method for producing platelets, comprising the steps of producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of claims 1 to 13, and culturing the produced megakaryocyte progenitor cells or megakaryocyte cells.

15. A method for promoting the proliferation of pre-multinucleated megakaryocyte progenitor cells, comprising the steps of suppressing the expression of the CDKN1A gene or the function of its expression product, and suppressing the expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products, in pre-multinucleated megakaryocyte progenitor cells in vitro.

16. A proliferation promoter for pre-multinucleated megakaryocyte progenitor cells, comprising as active ingredients a molecule that suppresses the expression of the CDKN1A gene or the function of its expression product, and a molecule that suppresses the expression of the INK4A gene and / or the ARF gene and / or the p53 gene, or the function of their expression products.

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