JP7659784B2 - Method for producing megakaryocyte precursor cells or megakaryocyte cells - Google Patents

Description

The present invention broadly relates to a method for producing megakaryocyte progenitor cells or megakaryocyte cells.

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

To meet the societal demand for platelets, the inventors established a technology for the 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-producing 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).

International Publication No. 2011/034073 International Publication No. WO 2012/157586

Cell Stem Cell. 2014 Apr 3;14(4):535-48 Cell. 2018 Jul 26;174(3):636-648

To supply, for example, 1,000 packs of a preparation containing 10 ¹¹ cells/platelet, at least 10 ^14-15 immortalized megakaryocytes (calculated in terms of the number of days of proliferation, 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) are required. Therefore, the production of megakaryocyte progenitor cells or megakaryocyte cells having 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 poor quality iPS cells or hematopoietic progenitor cells.

In view of these circumstances, the present invention aims to provide a novel method for producing megakaryocyte precursor cells or megakaryocyte cells with high proliferation potential in order to establish a stable platelet production system.

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

That is, the present invention includes the following inventions.
[1] A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising the step of suppressing expression of 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 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 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 according to [6], further comprising the step of suppressing forced expression of an oncogene, a BMI1 gene, and a BCL-XL gene.
[8] The method according to any one of [1] to [7], 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.
[9] The method according to any one of [1] to [7], 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.
[10] The method according to [9], wherein the decrease in cell proliferation occurs 30 days or more after the forced expression of the BMI1 gene and the BCL-XL gene.
[11] The method according to any one 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 thereof.
[12] A method for producing platelets, comprising the steps of: producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of [1] to [11]; and culturing the produced megakaryocyte progenitor cells or megakaryocyte cells.
[13] A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocytes, 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 the 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 CDKN1A gene or the function of its expression product.
[16] The proliferation-promoting agent according to [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.

[17] A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising the 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 according to [17], further comprising a step of suppressing 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 one 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 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 according to [22], further comprising the step of suppressing forced expression of an oncogene, a BMI1 gene, and a BCL-XL gene.
[24] The method according to any one of [17] to [23], wherein suppression of the expression of p53 gene or the function of its expression product is carried out simultaneously with forced expression of BCL-XL gene.
[25] The method according to any one of [17] to [23], wherein the expression of the p53 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.
[26] The method according to [25], wherein the decrease in cell proliferation occurs 30 days or more after the forced expression of the BMI1 gene and the BCL-XL gene.
[27] The method according to any one 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 the expression product thereof.
[28] A method for producing platelets, comprising the steps of: producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of [17] to [27]; and culturing the produced megakaryocyte progenitor cells or megakaryocyte cells.
[29] A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocytes, comprising the step of suppressing the expression of p53 gene or the function of its expression product.
[30] The method according to [29], further comprising a step of suppressing 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 p53 gene or the function of its expression product.
[32] The proliferation-promoting agent according to [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.

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 that are capable of stable platelet production.

In addition to the CDKN1A gene, further suppressing the expression of the tumor suppressor genes p53 gene and/or INK4A/ARF gene further extends the proliferation period of megakaryocyte progenitor cells or megakaryocyte cells and further increases their proliferation ability.

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.

In addition to the p53 gene, further suppression of the expression of the CDKN1A gene and/or the INK4A/ARF gene further extends the proliferation period of megakaryocyte progenitor cells or megakaryocyte cells, and further increases their proliferation ability.

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. 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. 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. 4 shows the results of analyzing the proliferation of megakaryocytes (MKCL1) after suppressing the expression of CDKN1A gene and p53 gene simultaneously with or two 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 since suppression of expression. 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). 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 most excellent platelet release ability due to the conventional forced expression of the 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. 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. FIG. 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, in the absence of stimulation or in the presence of 0.4 μM PMA or 100 μM ADP + 40 μM TRAP-6. FIG. 9 shows the results of cell proliferation on days 14, 31, and 43 for the megakaryocyte progenitor cell lines (MB) into which the c-MYC/BMI1 genes had been introduced and cultured for 14 days, (MB), (MBX) into which the BCL-XL gene had been further introduced using a doxycycline-inducible lentiviral vector, (MB-p21/p53_KD) into which the MB line had been infected with a lentiviral vector that persistently expresses sh p21/p53, and (MBX-p21/p53_KD) into which the MBX line had been infected with a lentiviral vector that persistently expresses sh p21/p53. FIG. 10 shows the results of FACS analysis of cellular CD34 and CD41 expression on days 31 and 43 for the MB, MBX, MB-p21/p53_KD, and MBX-p21/p53_KD strains. 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. 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.

(Method for producing megakaryocyte precursor cells or megakaryocyte cells)
The method for producing megakaryocyte progenitor cells or megakaryocyte cells according to this 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" means that DNA encoding a gene of interest is transcribed into mRNA and/or that mRNA is translated into a protein.

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, a tumor suppressor gene. 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 genes of the target CDKN1A gene do not include the p53 gene, nor the INK4A gene and the ARF gene, which are other tumor suppressor genes upstream of the p53 gene.

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, the term "INK4A/ARF gene" is interpreted as both genes or either one of the genes, i.e., "INK4A gene and/or ARF gene."

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. A combination of the CDKN1A gene and the p53 gene is more preferable.

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

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 substantially identical to the nucleic acid sequence shown in SEQ ID NO: 1 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 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: 1, or a DNA capable of hybridizing under stringent conditions with a DNA or RNA having 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 protein encoded by this DNA inhibits the cell cycle.

In addition, the homolog 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. A cDNA having a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 2 refers to a DNA having a sequence that is 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%, and even more preferably about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99% or more, with the DNA having a sequence shown in SEQ ID NO: 2, or a DNA that can hybridize under stringent conditions with DNA or RNA having a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 2, and the 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 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: 2 have been deleted, substituted or added, and the protein encoded by this DNA suppresses cancer.

In addition, 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 having a sequence substantially identical to the nucleic acid sequence shown in SEQ ID NO: 3 refers to a DNA having a sequence that is 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, identical to the DNA having the sequence shown in SEQ ID NO: 3, or a DNA that can hybridize under stringent conditions with DNA or RNA having a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 3, and the 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.

The p53 gene is a gene whose cDNA sequence is substantially identical to the nucleic acid sequence shown in, for example, 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 that can hybridize under stringent conditions with a DNA having a sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 4, and the 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.

Here, stringent conditions refer to hybridization conditions that can be easily determined by one of ordinary skill 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 below their melting point.

Specific examples of low stringency conditions include washing the filter after hybridization in a 0.1xSSC, 0.1% SDS solution at a temperature of 37°C to 42°C. Examples of high stringency conditions include washing the filter at 65°C in 5xSSC and 0.1% SDS. By increasing the stringency of the conditions, polynucleotides with high homology can be obtained.

Suppression of gene expression or the function of its expression product can be performed 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 knocking down a gene 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.

siRNA is typically 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 sequences of these RNAs can be appropriately designed by those skilled in the art based on the sequence information of the gene whose expression is to be suppressed. shRNA can also be used instead of siRNA.

An antisense nucleic acid refers to a nucleic acid that contains a nucleotide sequence capable of specifically hybridizing with a target mRNA (mature mRNA or early transcription product) under physiological conditions in a cell expressing the target mRNA, and that, in the hybridized state, is capable of inhibiting 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 an 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.

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 as compounds that suppress the expression of the CDKN1A gene. In addition, p53 inhibitors such as pifithrin α, nutrin-3, ReACp53, and RG7388 are known.

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. Knocking out 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. Alternatively, multiple alleles may be destroyed or mutated. Knockout can be performed by known methods, such as a method of knocking out by introducing into a cell a DNA construct designed to cause genetic recombination with the target gene, or a method of knocking out by inserting, deleting, or replacing a base using genome editing techniques such as TALEN or the CRISPR-Cas system.

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 also be used.

The expression of the above genes or the suppression of the function of their expression products is carried out in cells prior to differentiation into megakaryocytes, for example, hematopoietic progenitor cells or megakaryocyte progenitor cells prior to multinucleation. As used herein, "hematopoietic progenitor cells" refer to cells of the hematopoietic system 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 referred to as 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 or the like, and containing hematopoietic progenitor cells inside.

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

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.

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

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).

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 this method, an immortalized megakaryocyte cell line that proliferates indefinitely can be obtained by forcibly expressing an oncogene and a polycomb gene in cells less differentiated than megakaryocyte cells.

In addition, 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.

The forced expression of the oncogene, polycomb gene, and/or apoptosis-suppressing 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-suppressing gene may be forcedly expressed, then the forced expression may be suppressed, to obtain multinucleated megakaryocytes. Alternatively, the oncogene, polycomb gene, and apoptosis-suppressing gene may be forcedly expressed simultaneously, and the forced expression of these may be suppressed simultaneously to obtain multinucleated megakaryocytes. The oncogene and polycomb gene may be forcedly expressed first, then the apoptosis-suppressing gene may be forcedly expressed, and the forced expression of these may be suppressed simultaneously to obtain multinucleated megakaryocytes.

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 either an oncogene, a polycomb gene, or an apoptosis-suppressing gene, preferably simultaneously with the forced expression of an 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 once a 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, for 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.

As used herein, the term "cancer gene" refers to a gene that induces canceration of cells in the body, 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.

As used herein, the term "polycomb gene" refers to a gene that functions to negatively regulate the CDKN2a (INK4A/ARF) gene and 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.

As used herein, the term "apoptosis-suppressing gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples of such genes include the BCL2 gene, the BCL-XL gene, the Survivin gene, and the MCL1 gene. Of these, the BCL-XL gene is preferred.

Forced expression of genes and release of forced expression can be performed 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.

The period of forced expression can be appropriately determined by those skilled in the art, but for forced expression of oncogenes, polycomb genes, and/or apoptosis-inhibiting 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 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.

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 during the forced expression process and removing the drug from the medium.

In a further aspect, a method for producing megakaryocyte progenitor cells or megakaryocyte cells includes 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.

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.

In this aspect, inhibition of gene expression or the function of its expression product can be carried out by the methods described above.

In this aspect, the expression of the above 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.

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, an immortalized megakaryocyte cell line that proliferates indefinitely can be obtained by forcibly expressing an oncogene and a polycomb gene in cells less differentiated than megakaryocyte cells.

In addition, 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.

In this aspect, the forced expression of the oncogene, polycomb gene, and/or apoptosis-suppressing 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-suppressing gene may be forcedly expressed, then the forced expression may be suppressed to obtain multinucleated megakaryocytes. Alternatively, the oncogene, polycomb gene, and apoptosis-suppressing gene may be forcedly expressed simultaneously, and the forced expression of these may be suppressed simultaneously to obtain multinucleated megakaryocytes. The oncogene and polycomb gene may be forcedly expressed first, then the apoptosis-suppressing gene may be forcedly expressed, and the forced expression of these may be suppressed simultaneously to obtain multinucleated megakaryocytes.

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 either an oncogene, a polycomb gene, or an apoptosis-suppressing gene, preferably simultaneously with the forced expression of an apoptosis-suppressing gene, or thereafter, for example, after a decrease in cell proliferation, for example, a state in which the proliferation rate at a certain point in time is compared 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), is confirmed to be 1/2 or less. The decrease in cell proliferation is observed, but is not intended to be limited thereto, for 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.

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

In one embodiment of this aspect, an immortalized megakaryocytic cell line is obtained by forcibly expressing an oncogene (e.g., c-MYC) and a polycomb gene (e.g., BMI1). Forcibly expressing an apoptosis-suppressing gene (e.g., BCL-XL) may or may not be performed.

In this aspect, forced expression of a gene and release of forced expression can be performed by the methods described above.

In this aspect, the period of forced expression can be appropriately determined by those skilled in the art, but for 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. After 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.

The culture conditions of hematopoietic progenitor cells or megakaryocyte progenitor cells before multinucleation, and further 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, the carbon dioxide concentration can be, for example, 5% CO ₂ , and the oxygen concentration can be, for example, 20% O ₂ . Static culture or shaking culture can be used. The shaking speed in the case of shaking culture is not particularly limited, and can be, for example, 10 rpm to 200 rpm, 30 rpm to 150 rpm, etc.

The medium may be an Iscove's modified Dulbecco's medium (IMDM) medium containing serum, insulin, transferrin, serine, thiol glycerol, 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.

The cell culture process can be carried out in the presence or absence of feeder cells. As used herein, "feeder cells" refer 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. Feeder cells may be cells of the same species or different species, so long as they can be distinguished from the target cells. Feeder cells may be cells that have been treated with antibiotics or gamma rays to prevent proliferation, or may not be treated.

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, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, 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.

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).

(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.

The medium used for culturing megakaryocytes is not particularly limited, and any known medium suitable for producing platelets from megakaryocytes or a similar medium can be used as appropriate. For example, a medium used for culturing animal cells can be prepared as the 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.

From the viewpoint of producing a large amount of high-quality platelets, it is preferable to culture megakaryocytes in a culture vessel equipped with a stirring 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).

The platelets produced can be used to produce platelet preparations or blood preparations. 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 (e.g., Terumo Corporation, 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 blood preparation may also include other components that contribute to stabilizing cells.

(Method for Promoting the Proliferation of Megakaryocyte Progenitor Cells or Megakaryocyte Cells)
The method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells according to 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.

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

(Proliferation promoter of megakaryocyte precursor cells or megakaryocyte cells)
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.

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.

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.

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). That is, human iPS cell colonies (NC13X strain) 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). Culture conditions were 20% O ₂ and 5% CO ₂ .

An immortalized megakaryocytic cell line was established by introducing the three genes c-MYC/BMI1/BCL-XL into the obtained hematopoietic progenitor cells. A lentiviral vector that forcibly expresses 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 allow gene expression. After a further two weeks, a lentiviral vector that forcibly expresses BCL-XL under doxycycline control was introduced, and an immortalized megakaryocytic cell line was established.

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. Three days after infection, RFP (Red Fluorescent Protein)-positive infected cells were sorted using FACS AriaIIIu. The predicted secondary structures of clones 1 and 2 are shown below.

When the isolated cells were continued to be cultured in a culture medium containing 15% FBS, cell proliferation was confirmed for more than 300 days after suppression of CDKN1A gene expression, and megakaryocyte progenitor cells or megakaryocytes proliferated ^1066- fold compared to cells when expression was suppressed (Figure 1).

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 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.

なお、ｓｈＩＮＫ４Ａ／ＡＲＦは、ＩＮＫ４Ａ遺伝子とＡＲＦ遺伝子の共通のエクソンを標的とするものである。 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.

In addition, shINK4A/ARF targets a common exon between the INK4A gene and the ARF gene.

After sorting, the cells were continued to be cultured in a culture medium containing 15% FBS. In both MKCL23 and MKCL26 lines, the cell proliferation period was extended by 30 days or more and proliferation was increased by ¹⁰⁴ times compared to the control by suppressing the expression of CDKN1A gene (Figures 2 and 3). It was revealed that even when suppression of the expression of CDKN1A gene is insufficient, the proliferation effect can be improved by suppressing the expression of INK4A/ARF gene and p53 gene.

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.

A megakaryocyte cell line (MKCL1 line) in which cell proliferation had decreased about 40 days after introduction of MYC/BMI1/BCL-XL 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+shARF-GFP
The shRNAs used were as described above. Then, positively infected cells were sorted using FACS AriaIIIu.

After sorting, the cells were continued to be cultured in a culture medium containing 15% FBS, and cell proliferation was significantly increased compared to the control (excluding INK4A/ARF). In particular, the cells in which the expression of all of the CDKN1A gene, INK4A gene, ARF gene, and p53 gene was suppressed showed an extension of the cell proliferation period by 20 days and a proliferation rate of ¹⁰⁴ times.

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 cell line (MKCL30 line) derived from the YZWJ516 human iPS cell line, which could not be established by conventional forced MBX expression, 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.

After sorting, the cells were continued to be cultured in a culture medium containing 15% FBS. Compared to the control, suppression of the expression of the CDKN1A gene extended the cell proliferation period by 8 weeks or more and increased the proliferation rate by 10 ¹⁵ times. The cell proliferation effect was highest when the expression of both the CDKN1A gene and the p53 gene was suppressed (2.5 x 10 ¹⁷ times in 8 weeks). The cells were proliferating as of the priority date of this application (October 17, 2019, 127 days after infection with c-MYC/BMI1). This was followed by the cell proliferation effect of cells in which the expression of all of the INK4A gene, the ARF gene, and the p53 gene was suppressed.

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

The results are shown in Figure 4. Regarding the time of infection, it was confirmed that infection at the same time as BCL-XL introduction resulted in ^104- fold or more better proliferation than infection 2 weeks after BCL-XL introduction (shCDKN1A-RFP + shp53-GFP simultaneous infection: 2.5 x ^1017- fold vs. shCDKN1A-RFP + shp53-GFP simultaneous infection: 5.8 x ^1012- fold).

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

Example 4: Investigation of platelet production by megakaryocytic cells in which expression of CDKN1A gene was suppressed Platelet release was compared between the MKCL21 line after CDKN1A suppression, whose proliferation was promoted using clone 2 (sequence number 6) in Example 1, and the MKCL21 line after infection with a control vector.

Doxycycline was removed from the culture medium of both cells to suppress the expression of the three factors MYC/BMI1/BCL-XL, and 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 number: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, catalog number: 555473). The FACS dot plot is shown in Figure 5.

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

Next, the platelet production ability of the MKCL21 line in which 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 conventional forced expression of three factors (MYC/BMI1/BCL-XL). The FACS dot plots of these results are shown in Figure 6. The antibodies used were APC anti-human CD41 Antibody (BioLegend, catalog number: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, catalog number: 555473).

The upper panel of Figure 6 shows the state after adding doxycycline and allowing the expression of the three factors MYC/BMI1/BCL-XL. As both lines are immature, there are few CD41-positive/CD42b-positive platelets (top right part of the four-part diagram). On the other hand, the lower panel of Figure 6 shows the state five days after doxycycline was removed from the culture medium and the expression of the three factors MYC/BMI1/BCL-XL was suppressed, with an increase in CD41a-positive/CD42b-positive platelets (top right part of the four-part diagram). These results show that platelet production ability was increased by suppressing expression of the CDKN1A gene.

Example 5: Investigation of platelet production by megakaryocytic cells in which expression of CDKN1A gene and p53 gene was suppressed Platelet release was compared between the MKCL7 line after infection with a control vector, the MKCL21 line after CDKN1A suppression and whose proliferation was promoted using clone 2 (sequence number 6) in Example 1, and the MKCL30 line 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.

Doxycycline was removed from the culture medium of each cell line to suppress the expression of the three factors MYC/BMI1/BCL-XL, and then 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).

Next, for the MKCL7, MKCL21, and MKCL30 strains in which the expression of the MYC/BMI1/BCL-XL three factors was not suppressed (ON), and the MKCL7, MKCL21, and MKCL30 strains in which the expression of the MYC/BMI1/BCL-XL three factors was suppressed, megakaryocytes and platelets released into the culture supernatant were collected on the fifth day, and the cells were cultured without stimulation, or with 0.4 μM PMA (phorbol myristate acetate, Sigma Aldrich Cat#P1585) (PMA), or with 100 μM ADP (adenosine diphosphate, Sigma Aldrich Cat#A-2754) + 40 μM TRAP-6 (thrombin receptor activator The cells were stained with P-selectin antibody (Bio Legend/#304910) and PAC-1 antibody (BD Bioscience/#34507) in the presence of peptide 6, BACHEM Cat#H-8365.0005) (AT) and analyzed by FACS. The results with P-selectin and PAC-1 antibodies are shown in Figures 8(D) and (E), respectively.
The explanation in the table is as follows:
No stimulation-ON: Results without stimulation using a strain that did not suppress the expression of the three factors MYC/BMI1/BCL-XL. PMA-ON: Results under PMA using a strain that did not suppress the expression of the three factors MYC/BMI1/BCL-XL. AT-ON: Results under AT using a strain that did not suppress the expression of the three factors MYC/BMI1/BCL-XL. No stimulation_MKCL7: Results without stimulation using the MKCL7 strain. 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 MKCL21 strain PMA_MKCL21#: Results under PMA using MKCL21 strain AT_MKCL21#: Results under AT using MKCL21 strain No stimulation_MKCL30: Results without stimulation using MKCL30 strain PMA_MKCL30: Results under PMA using MKCL30 strain AT_MKCL30: Results under AT using MKCL30 strain

These results demonstrate that suppressing the expression of the CDKN1A gene and p53 gene produces functional platelets that respond well to stimulation and do not inhibit platelet function. On the other hand, in the group in which doxycycline was not removed and the expression of the three factors MYC/BMI1/BCL-XL was not suppressed (MBX ON), megakaryocyte maturation did not progress and almost no platelets were produced.

Example 6: Expression of BCL-XL gene and suppression of p21/p53 gene expression 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)).

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 sh p21/p53 lentiviral vector that continuously expresses (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 sh p21/p53 lentiviral vector that continuously expresses (MBX-p21/p53_KD). The results of confirming the proliferation of cells on days 14, 31, and 43 for each megakaryocyte progenitor cell line are shown in Figure 9.

Furthermore, the results of FACS analysis of cellular CD34 and CD41 expression on days 31 and 43 for the MB, MBX, MB-p21/p53_KD, and MBX-p21/p53_KD strains are shown as FACS dot plots in Figure 10, Figure 11, and Figure 12, respectively. 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).

Under all conditions, megakaryocyte progenitor cells positive for CD41 and weakly positive for CD42b were obtained. On the 43rd day of culture, the results of the MB-p21/p53_KD line (white circles in FIG. 9) indicate a significantly higher proliferation rate than the MB line (black circles). The results of the MBX-p21/p53_KD line (black triangles) indicate a proliferation rate 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 megakaryocyte progenitor cell lines obtained by introducing two genes, c-MYC/BMI1, is promoted by p21/p53KD even without the introduction of the BCL-XL gene, i.e., BCL-XL can be replaced by p21/p53KD.

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

Claims (16)

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. The method according to claim 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. The method according to claim 2, wherein the megakaryocyte cells are mature megakaryocyte cells. The method according to any one of claims 1 to 3, wherein the megakaryocyte cells are immortalized. The method according to any one of claims 1 to 4, further comprising a step of forcibly expressing the BCL-XL gene. The method according to claim 5, further comprising a step of suppressing the forced expression of an oncogene, a BMI1 gene, and a BCL-XL gene. 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. 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. 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. 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. 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. The method according to claim 11, wherein the megakaryocyte cells are mature megakaryocyte cells. The method of claim 10 or 11, wherein the megakaryocyte cells are immortalized. 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. 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 functions of their expression products, in pre-multinucleated megakaryocyte progenitor cells in vitro. 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.