WO2021075568A1

WO2021075568A1 - Method for producing megakaryocyte progenitor cell or megakaryocytic cell

Info

Publication number: WO2021075568A1
Application number: PCT/JP2020/039162
Authority: WO
Inventors: 直也 ▲高▼山; 浩之江藤; 正光曽根; 壮中村
Original assignee: 国立大学法人千葉大学; 国立大学法人京都大学
Priority date: 2019-10-17
Filing date: 2020-10-16
Publication date: 2021-04-22
Also published as: JPWO2021075568A1; JP7659784B2; TW202128987A

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

Method for producing megakaryocyte progenitor cells or megakaryocyte cells

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

Approximately 200 billion platelets are used for platelet transfusion each time, but since platelets generally have a short body life of several days, it is often necessary to administer them repeatedly. Although the number of elderly people who require repeated administration of platelet transfusions is steadily increasing due to the aging of society, the young donor population is declining.

In order to meet the social demand for platelets, the inventors have established a technique for stable production of platelet preparations in vitro using human iPS cells (Non-Patent Documents 1 and 2). The inventors introduced three factors (MYC / BMI1 / BCL-XL) into hematopoietic cells derived from human iPS cells to establish an immortalized megakaryocyte strain (cells that are the source of platelets), and prepared a medium. A technique for mass-producing platelets (human iPS cell-derived artificial platelet preparation) has also been established simply by changing (Patent Documents 1 and 2).

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

In ^{order to supply 1 pack of a preparation containing 10 11} cells / platelets, for example, 1000 packs, at least 10 ^14-15 cells (calculated by the number of growth days, MYC to hematopoietic progenitor cells on days 10-14 derived from human iPS cells). , BMI1 and BCL-XL (about 4-5 months after introduction)) immortalized megakaryocytic cells are required. Therefore, the production of megakaryocyte progenitor cells or megakaryocyte cells having high cell proliferation ability is indispensable for the production of platelets, but the above three factors (MYC / BMI1 / BCL-XL) are used for poor quality iPS cells and hematopoietic progenitor cells. It was found that the cell proliferation ability of megakaryocyte progenitor cells or megakaryocyte cells obtained by introducing the above was limited.

In view of such circumstances, an object of the present invention is to provide a novel method for producing megakaryocyte progenitor cells or megakaryocyte cells having high proliferative ability in order to establish a stable production system for platelets.

As a result of repeated studies to solve the above problems, the present inventors have suppressed the expression of the CDKN1A gene encoding the cell cycle inhibitor p21 to obtain megakaryocyte progenitor cells or megakaryocytes having high proliferative capacity. We have found that cells can be obtained and have completed the present invention.

That is, the invention of the present application includes the following inventions.
[1] A method for producing a megakaryocyte progenitor cell or a megakaryocyte cell, which comprises a step of suppressing the expression of the CDKN1A gene or the function of the expression product in the hematopoietic progenitor cell or the megakaryocyte progenitor cell before polynuclearization.
[2] The method according to [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 the expression product thereof.
[3] The method according to [2], wherein the megakaryocyte cell is a mature megakaryocyte cell.
[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 a step of forcibly expressing an oncogene and a BMI1 gene selected from MYC family genes 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 a 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 suppression of the function of the expression product thereof is carried out at the same time as the forced expression of the BCL-XL gene.
[9] Any of [1] to [7], wherein the expression of the CDKN1A gene or the suppression of the function of the expression product thereof is carried out after the decrease in cell proliferation after the forced expression of the BMI1 gene and the BCL-XL gene. The method described in.
[10] The method according to [9], wherein the decrease in cell proliferation is 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 performed by introducing a molecule that suppresses the expression of a gene or the function of the expression product thereof into a cell.
[12] A step of producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of [1] to [11], and a step of culturing the produced megakaryocyte progenitor cells or megakaryocyte cells. A method of producing platelets.
[13] A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells, which comprises a step of suppressing the expression of the CDKN1A gene or the function of the expression product thereof.
[14] The method according to [13], 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 the expression product thereof.
[15] An agent for promoting the growth of megakaryocyte progenitor cells or megakaryocyte cells, which comprises, as an active ingredient, a molecule that suppresses the expression of the CDKN1A gene or the function of the expression product thereof.
[16] The growth promoter 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 the expression product thereof.

[17] A method for producing megakaryocyte progenitor cells or megakaryocyte cells, which comprises a step of suppressing the expression of the p53 gene or the function of the expression product in the hematopoietic progenitor cell or the megakaryocyte progenitor cell before polynuclearization.
[18] The method according to [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 the expression product thereof.
[19] The method according to [17] or [18], wherein the megakaryocyte cell is a mature megakaryocyte cell.
[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 a step of forcibly expressing an oncogene and a BMI1 gene selected from MYC family genes 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 a 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 the expression of the p53 gene or the suppression of the function of the expression product thereof is carried out at the same time as the forced expression of the BCL-XL gene.
[25] Any of [17] to [23], wherein the expression of the p53 gene or the suppression of the function of the expression product thereof is carried out after the decrease in cell proliferation after the forced expression of the BMI1 gene and the BCL-XL gene. The method described in.
[26] The method according to [25], wherein the decrease in cell proliferation is 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 a molecule that suppresses the expression of a gene or the function of the expression product thereof into a cell.
[28] The step of producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of [17] to [27], and the step of culturing the produced megakaryocyte progenitor cells or megakaryocyte cells. A method of producing platelets.
[29] A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells, which comprises a step of suppressing the expression of the p53 gene or the function of the expression product thereof.
[30] The method according to [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 the expression product thereof.
[31] An agent for promoting the growth of megakaryocyte progenitor cells or megakaryocyte cells, which comprises, as an active ingredient, a molecule that suppresses the expression of the p53 gene or the function of the expression product thereof.
[32] The growth promoter 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 the expression product thereof.

According to the present invention, by suppressing the expression of the CDKN1A gene in the starting cells, stable platelet production can be achieved in megakaryocyte precursor cells or megakaryocyte cells having a longer growth period and higher proliferative capacity than in the prior art. It enables the production of possible megakaryocyte precursor cells or megakaryocyte cells.

By further 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 progenitor cells or megakaryocyte cells is further extended, and the proliferation ability is further increased. ..

By suppressing the expression of the p53 gene in the starting cells, megakaryocyte precursor cells or megakaryocyte progenitor cells that have a longer proliferation period and higher proliferative capacity than the prior art and are capable of stable platelet production. Alternatively, the production of megakaryocyte cells becomes possible.

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 the megakaryocyte progenitor cell or the megakaryocyte cell is further extended, and the proliferation ability is further increased.

FIG. 1 shows the results of analyzing the proliferation of megakaryocyte progenitor cells or megakaryocyte cells (MKCL1) after suppressing the expression of the CDKN1A gene. The vertical axis shows the number of cell proliferations after suppressing gene expression, and the horizontal axis shows the number of days since expression suppression. FIG. 2 shows the results of analyzing the proliferation of megakaryocyte progenitor cells or megakaryocyte cells (MKCL23) after suppressing the expression of the CDKN1A gene, INK4A / ARF gene, and p53 gene. The vertical axis shows the number of cell proliferations after suppressing gene expression, and the horizontal axis shows the number of days since expression suppression. FIG. 3 shows the results of analyzing the proliferation of megakaryocyte progenitor cells or megakaryocyte cells (MKCL26) after suppressing the expression of the CDKN1A gene, INK4A / ARF gene, and p53 gene. The vertical axis shows the number of cell proliferations after suppressing gene expression, and the horizontal axis shows the number of days since expression suppression. FIG. 4 shows the results of analysis of the proliferation of megakaryocyte cells (MKCL1) after suppressing the expression of the CDKN1A gene and the p53 gene at the same time as the introduction of BCL-XL or 2 weeks after the introduction. The vertical axis shows the number of cell proliferations after suppressing gene expression, and the horizontal axis shows the number of days since expression suppression. FIG. 5 shows FACS dot plots (upper right and lower right, respectively) of megakaryocyte cells established by suppressing the expression of the CDKN1A gene and platelets produced from the megakaryocyte cells (X-axis: CD41; Y-axis: CD42b). FIG. 6 shows a megakaryocyte cell line having the best platelet-releasing ability by forcibly expressing the platelet-producing ability of a megakaryocyte cell (MKCL21) in which the expression of the CDKN1A gene was suppressed by the conventional three factors (MYC / BMI1 / BCL-XL). The result of comparison with (SeV2) is shown. Upper panel: A state in which doxycycline is added and the three factors of MYC / BMI1 / BCL-XL are expressed. Lower panel: The state on the 5th day 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 the control vector, the MKCL21 strain after CDKN1A suppression, and the MKCL30 strain after CDKN1A and p53 suppression. FIG. 8 shows MKCL7 strain after control vector infection, MKCL21 strain after CDKN1A suppression, and MKCL30 strain after CDKN1A and p53 suppression, respectively, with no stimulation or 0.4 μM PMA or 100 μM ADP + 40 μM TRAP-, respectively. 6 The results of comparing the platelet release ability in the presence of 6 are shown. FIG. 9 shows a strain (MB) in which a macronuclear cell progenitor cell line into which two genes of c-MYC / BMI1 were introduced was cultured as it was after 14 days, and a BCL-XL gene was further added to the MB strain using a doxycycline-induced lentiviral vector. The introduced strain (MBX), the strain infected with the sh p21 / p53 lentiviral vector persistently expressed in the MB strain (MB-p21 / p53_KD), and the sh p21 / p53 lentivirus persistently expressed in the MBX strain. For the macronuclear cell progenitor cell line of the vector-infected strain (MBX-p21 / p53_KD), the results of the cell proliferation numbers on the 14th, 31st and 43rd days are shown. FIG. 10 shows the results of FACS analysis of the expression of CD34 and CD41 in cells on the 31st and 43rd days of the MB strain, the MBX strain, the MB-p21 / p53_KD strain, and the MBX-p21 / p53_KD strain. .. FIG. 11 shows the results of FACS analysis of the expression of CD34 and CD42b in cells on the 31st and 43rd days of the MB strain, the MBX strain, the MB-p21 / p53_KD strain, and the MBX-p21 / p53_KD strain. .. FIG. 12 shows the results of FACS analysis of the expression of CD34 and GPA in cells on the 31st and 43rd days of the MB strain, the MBX strain, the MB-p21 / p53_KD strain, and the MBX-p21 / p53_KD strain. ..

(Method for producing megakaryocyte progenitor 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 the CDKN1A gene or the function of the expression product in the hematopoietic progenitor cells or the megakaryocyte progenitor cells before polynuclearization. .. As used herein, "gene expression" means that the DNA encoding the gene of interest is transcribed into mRNA and / or the mRNA is translated into 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, which is a tumor suppressor gene. The activated p53 protein acts as a transcription factor and increases the expression of p53 downstream genes. Therefore, as used herein, "suppressing gene expression or the function of an expression product thereof" means the expression of a gene of interest or the function of the expression product (for example, p21 in the case of the CDKN1A gene). It can be achieved by directly suppressing the gene, or it can be achieved by controlling the expression of genes upstream of the gene of interest and the function of those expression products. However, in the present specification, when the expression of the CDKN1A gene or the function of the expression product thereof is suppressed, the p53 gene and further the upstream gene of the p53 gene are included in the upstream gene of the target CDKN1A gene. The INK4A gene and ARF gene, which are cancer-suppressing genes, are not included.

The INK4 (inhibitors of CDK4) A gene and the ARF (alternate reading frame) gene are genes existing at the same locus and are known as tumor suppressor genes. Both genes share some exons, and both are thought to act to suppress growth. By using a molecule such as siRNA that targets a common exon, the expression of both genes can be suppressed. As used herein, "INK4A / ARF gene" is construed as both genes or 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 the expression products thereof. A combination of the CDKN1A gene and the p53 gene is more preferred.

Each gene such as CDKN1A gene, INK4A / ARF gene, p53 gene, etc. as used in the present specification means one encoded by their known nucleic acid sequence, for example, cDNA sequence. Each gene may also contain a homolog that is identified based on the homology of known nucleic acid sequences.

The homolog of the CDKN1A gene is a gene whose cDNA sequence is, for example, substantially the same as the nucleic acid sequence shown in SEQ ID NO: 1. The cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 1 is about 60% or more, preferably about 70% or more, more preferably about the DNA consisting of the sequence represented by SEQ ID NO: 1. 80% or more, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even more preferably about 90% or more, such as 91%, 92%, 93%. , 94%, 95%, 96%, 97%, 98%, most preferably a DNA consisting of a sequence having an identity of about 99% or more, or a sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 1. DNA that can be hybridized with DNA or RNA under stringent conditions, and the protein encoded by these DNAs inhibits the cell cycle. Alternatively, the cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 1 is one or more, for example, 1 to 10, preferably several, for example, in the sequence represented by SEQ ID NO: 1. DNA consisting of sequences in which 1 to 5, 1 to 4, 1 to 3, and 1 to 2 bases are deleted, substituted, or added, and the protein encoded by these DNAs is the cell cycle. It is the one that inhibits.

Further, the homolog of the INK4A gene used in the present invention is a gene whose cDNA sequence is, for example, substantially the same as the nucleic acid sequence shown in SEQ ID NO: 2. The cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 2 is about 60% or more, preferably about 70% or more, more preferably about the DNA consisting of the sequence represented by SEQ ID NO: 2. 80% or more, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even more preferably about 90% or more, such as 91%, 92%, 93%. , 94%, 95%, 96%, 97%, 98%, most preferably a DNA consisting of a sequence having an identity of about 99% or more, or a sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 2. DNA that can hybridize with DNA or RNA under stringent conditions, and the protein encoded by that DNA suppresses cancer. Alternatively, the cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 2 is one or more, for example, 1 to 10, preferably several, for example, in the sequence represented by SEQ ID NO: 2. DNA consisting of sequences in which 1 to 5, 1 to 4, 1 to 3, and 1 to 2 bases are deleted, substituted, or added, and the protein encoded by these DNAs causes cancer. It is something that suppresses.

Further, the homologue of the ARF gene used in the present invention is a gene whose cDNA sequence is, for example, substantially the same as the nucleic acid sequence shown in SEQ ID NO: 3. The cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 3 is about 60% or more, preferably about 70% or more, more preferably about the DNA consisting of the sequence represented by SEQ ID NO: 3. 80% or more, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even more preferably about 90% or more, such as 91%, 92%, 93%. , 94%, 95%, 96%, 97%, 98%, most preferably a DNA consisting of a sequence having an identity of about 99% or more, or a sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 3. DNA that can hybridize with DNA or RNA under stringent conditions, and the protein encoded by that DNA suppresses cancer. Alternatively, the cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 3 is one or more, for example, 1 to 10, preferably several, for example, in the sequence represented by SEQ ID NO: 3. DNA consisting of sequences in which 1 to 5, 1 to 4, 1 to 3, and 1 to 2 bases are deleted, substituted, or added, and the protein encoded by these DNAs causes cancer. It is something that suppresses.

The p53 gene is a gene whose cDNA sequence is substantially the same as, for example, the nucleic acid sequence shown in SEQ ID NO: 4. The cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 4 is about 60% or more, preferably about 70% or more, more preferably about the DNA consisting of the sequence represented by SEQ ID NO: 4. 80% or more, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even more preferably 90% or more, such as 91%, 92%, 93%, DNA consisting of a sequence having 94%, 95%, 96%, 97%, 98%, most preferably about 99% or more identity, or DNA consisting of a sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 4. DNA that can hybridize under stringent conditions, and the protein encoded by that DNA suppresses cancer. Alternatively, the cDNA consisting of a sequence substantially the same as the nucleic acid sequence represented by SEQ ID NO: 4 is one or more, for example, 1 to 10, preferably several, for example, in the sequence represented by SEQ ID NO: 4. DNA consisting of sequences in which 1 to 5, 1 to 4, 1 to 3, and 1 to 2 bases are deleted, substituted, or added, and the protein encoded by these DNAs causes cancer. It is something that suppresses.

Here, the stringent condition is a hybridization condition easily determined by those skilled in the art, and is generally an empirical experimental condition that depends on the base length, washing temperature, and salt concentration of nucleic acid. is there. 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 the complementary strand to reanneal in an environment slightly below its melting point.

Specifically, for example, as a low stringent condition, in the washing step of the filter after hybridization, washing is performed in a 0.1 × SSC, 0.1% SDS solution under a temperature condition of 37 ° C. to 42 ° C. Things can be raised. In addition, high stringent conditions include, for example, washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step. By increasing the stringent conditions, polynucleotides with high homology can be obtained.

Expression of a gene or suppression of the function of an expression product thereof can be performed by a known method, for example, siRNA, an antisense nucleic acid, or a nucleic acid molecule thereof that can specifically suppress the expression of each gene can be expressed. This can be done by introducing various molecules, such as expression vectors, into cells. Alternatively, the gene may be knocked down by using another technique such as genome editing technique. For example, when a gene is knocked down using the CRISPR-Cas system, a guide RNA that targets the gene and a fusion protein of inactivated Cas and a repressor domain such as dCas are used.

SiRNA is typically a double-stranded oligo RNA consisting of RNA having a sequence complementary to the nucleotide sequence of the mRNA of the target gene or a partial sequence thereof and its complementary strand. Nucleotide sequences of these RNAs can be appropriately designed by those skilled in the art based on the sequence information of genes whose expression is suppressed. shRNA can also be used instead of siRNA.

An antisense nucleic acid contains a nucleotide sequence capable of specifically hybridizing with a target mRNA under the physiological conditions of a cell expressing the target mRNA (mature mRNA or early transcript), and in a hybridized state, the target mRNA is used. Means a nucleic acid that can inhibit the translation of the encoded polypeptide. The type of antisense nucleic acid may be DNA or RNA, or may be a chimera of DNA and RNA. The nucleotide sequence of the antisense nucleic acid can be appropriately designed by those skilled in the art based on the sequence information of the gene whose expression is 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. Further, as p53 inhibitors, pifithrin α, natrin-3, ReACp53, RG7388 and the like are known.

Alternatively, the gene of interest may be knocked out using a known technique in order to express the gene or suppress the function of the expression product thereof. Knockout of a gene means that all or part of the gene is disrupted or mutated so as not to perform its original function. The gene may be disrupted or mutated so that one allele on the genome does not function. In addition, a plurality of alleles may be disrupted or mutated. Knockout can be performed by a known method, for example, a method of knocking out by introducing a DNA construct designed to cause genetic recombination with a target gene into a cell, TALEN or CRISPR-Cas. A method of knocking out by inserting, deleting, or introducing a substitution using a genome editing technique such as a system can be mentioned.

In addition, compounds that suppress transcription and transcripts of each gene, or binding inhibitors of the produced proteins to target proteins (p53 binding inhibition: pifithrin α, natrin-3, ReACp53, RG7388, etc .; p21 binding inhibition: UC2288, Butyrolactone I, LLW10, sorafenib, sterigmatocystin, etc.) may be used.

Expression of the above gene or suppression of the function of the expression product thereof is performed in cells before differentiation into megakaryocyte cells, for example, hematopoietic progenitor cells or megakaryocyte progenitor cells before polynuclearization. As used herein, a "hematopoietic progenitor cell" is a hematopoietic cell characterized as a CD34-positive cell, prepared from, for example, ES cells or iPS cell-derived cells, particularly ES cells or iPS cells. Cells obtained from the net-like structure (also referred to as ES-sac or iPS-sac) to be produced (particularly, cells immediately after separation from the net-like structure) are preferable. Here, the "net-like structure" prepared from ES cells or iPS cells is a three-dimensional sac-like (with space inside) structure derived from ES cells or iPS cells, and is an endothelial cell population or the like. It is formed and contains hematopoietic progenitor cells inside.

"Megakaryocyte precursor cells before multinucleation" are cells that are more undifferentiated than multinucleated megakaryocyte cells and are CD41a-positive / CD42a-positive / CD42b-positive, which are specific markers of the megakaryocyte lineage, and are nuclear. It means mononuclear or binuclear cells that have not undergone polyploidization.

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

Megakaryocyte cells are produced by further inducing differentiation from hematopoietic progenitor cells or pre-multinucleated megakaryocyte progenitor cells 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 further express at least one marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. Sometimes. Megakaryocyte cells release platelets when they become polyploid and mature. Multinucleated megakaryocyte cells have 16 to 32 times the genome of normal cells.

In the present specification, the term "megakaryocyte cell" includes all megakaryocyte cells such as immature megakaryocyte cells and mature megakaryocyte cells (mature megakaryocyte cells) unless otherwise specified. It is used as a meaning to.

It is preferable that the megakaryocyte cells are further immortalized. Non-limiting examples of methods for producing immortalized megakaryocyte cells include those described in WO 2011/034073 (supra) and US 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 that are more undifferentiated than megakaryocyte cells.

Also, by forcibly expressing an apoptosis suppressor gene in undifferentiated cells from megakaryocyte cells according to the methods described in International Publication No. 2012/157586 (above) and US Patent Application Publication No. 2014/0127815. Immortalized megakaryocyte cells can be obtained. By releasing the forced expression of the gene, these immortalized megakaryocyte cells acquire self-renewal ability, multinucleation progresses, and platelets are released.

Forced expression of oncogene, polycomb gene, and / or apoptosis suppressor gene may be performed simultaneously or sequentially. For example, an oncogene and a polycomb gene may be forcibly expressed, then the forcible expression may be suppressed, then an apoptosis-suppressing gene may be forcibly expressed, and then this forcible expression may be suppressed to obtain polynuclear macronuclear cells. It is also possible to obtain a polynuclearized megakaryocyte cell by simultaneously forcibly expressing an oncogene, a polycomb gene, and an apoptosis suppressor gene and simultaneously suppressing these forcible expression. First, a tumor suppressor gene and a polycomb gene are forcibly expressed, and then an apoptosis suppressor gene is forcibly expressed, and these forcible expression can be suppressed at the same time to obtain a polynuclear macronuclear cell.

The expression of the CDKN1A gene, or suppression of the function of its expression product, is simultaneous with the forced expression of any of the oncogene, polycomb gene, or apoptosis suppressor gene, preferably at the same time as the forced expression of the apoptosis suppressor gene, or thereafter, for example. Decreased cell proliferation, for example, comparing the cell proliferation rate at a certain point in time with the most recent cell proliferation rate (for example, assuming that cell proliferation is confirmed weekly, the cell proliferation rate for a week is the proliferation one week before. It can be carried out after it is confirmed that the growth rate is halved or less (compared to the rate). The decrease in cell proliferation is not intended to be limited, but about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days after the forced expression of the oncogene or polycomb gene. , Or until about 90 days later.

As used herein, the term "oncogene" refers to a gene that induces canceration of cells in vivo, for example, MYC family genes (eg, c-MYC, N-MYC, L-MYC), SRC family. Examples include genes, RAS family genes, RAF family genes, c-Kit, PDGFR, Abl and other protein kinase family genes. Among these, the MYC family gene, particularly c-MYC, is preferable.

As used herein, the term "polycomb group transmitter" refers to a gene that negatively regulates the CDKN2a (INK4A / ARF) gene and functions to avoid cell senescence (Ogura et al., Regenerative Medicine, vol.6, No.). .4, pp26-32; Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol.7, pp667-677, 2006; Proc.Natur.Acad.Sci.USA, vol.2016, p. 2003). Non-limiting examples of polycomb genes include BMI1, Mel18, Ring1a / b, Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HADC, Dnmtl / 3a / 3b. .. Among these, BMI1 is preferable.

In the present specification, the "apoptosis-suppressing gene" refers to a gene having a function of suppressing cell apoptosis, and examples thereof include BCL2 gene, BCL-XL gene, Survivin gene, and MCLl gene. Of these, the BCL-XL gene is preferred.

Forcible expression of genes and release of forced expression are described in International Publication No. 2011/034073 (above), US Patent Application Publication No. 2012/0238023, International Publication No. 2012/157586 (above), and US Patent Application Publication No. 2014/0127815, International Publication No. 2014/123242 and US Patent Application Publication No. 2016/0002599, or Nakamura S et al, Cell Stem Cell. It can be carried out by the method described in 14, 535-548, 2014, other known methods, or a method equivalent thereto.

The period of the forced expression can be appropriately determined by those skilled in the art, but the forced expression of the oncogene, polycomb gene, and / or the apoptosis-suppressing gene is forced after the desired period has elapsed after suppressing the expression of the CDKN1A gene. It is preferable to suppress (release) the expression. The cells may be subcultured after the forced expression, and the period from the last passage 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-inducing system, such as a Tet-on® or Tet-off® system, is used for forced expression and release of a gene, the forced expression step should be addressed. Forced expression may be suppressed by containing a drug, such as tetracycline or doxycycline, in the medium and removing them from the medium.

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

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 preferred.

In this aspect, the expression of the gene or the suppression of the function of the expression product thereof can be performed by the above-mentioned method.

In this aspect, expression of the above gene or suppression of the function of the expression product thereof is performed in cells before differentiation into megakaryocyte cells, for example, hematopoietic progenitor cells or megakaryocyte progenitor cells before polynuclearization.

In this aspect, it is preferable that the megakaryocyte cells are further immortalized. Non-limiting examples of methods for producing immortalized megakaryocyte cells include those described in WO 2011/034073 (supra) and US 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 that are more undifferentiated than megakaryocyte cells.

In this aspect, the forcible expression of the oncogene, the polycomb gene, and / or the apoptosis suppressor gene may be performed simultaneously or sequentially. For example, an oncogene and a polycomb gene may be forcibly expressed, then the forcible expression may be suppressed, then an apoptosis-suppressing gene may be forcibly expressed, and then this forcible expression may be suppressed to obtain polynuclear macronuclear cells. It is also possible to obtain a polynuclearized megakaryocyte cell by simultaneously forcibly expressing an oncogene, a polycomb gene, and an apoptosis suppressor gene and simultaneously suppressing these forcible expression. First, a tumor suppressor gene and a polycomb gene are forcibly expressed, and then an apoptosis suppressor gene is forcibly expressed, and these forcible expression can be suppressed at the same time to obtain a polynuclear macronuclear cell.

In this aspect, the expression of the p53 gene, or the suppression of the function of the expression product thereof, is simultaneous with the forced expression of any of the oncogene, the polycomb gene, or the apoptosis suppressor gene, preferably at the same time as the forced expression of the apoptosis suppressor gene. Alternatively, after that, for example, a decrease in cell proliferation, for example, the cell proliferation rate at a certain point in time is compared with the latest cell proliferation rate (for example, assuming that cell proliferation is confirmed weekly, the cell proliferation rate for a certain week is one of them. It can be carried out after it is confirmed that the growth rate has been reduced to 1/2 or less (compared to the growth rate a week ago). The decrease in cell proliferation is not intended to be limited, but about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days after the forced expression of the oncogene or polycomb gene. , Or until about 90 days later.

In this aspect, the oncogene is preferably a MYC family gene, particularly c-MYC. BMI1 is preferable as the polycomb transmitter. As the tumor suppressor gene, the BCL-XL gene is preferable.

In one aspect of this aspect, an immortalized megakaryocyte cell line is obtained by forcibly expressing an oncogene (eg, c-MYC) and a polycomb gene (eg, BMI1). Forced expression of the apoptosis-suppressing gene (eg, BCL-XL) may or may not be performed.

In this aspect, the forced expression of the gene and the release of the forced expression can be performed by the method 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 the cancer gene, polycomb gene, and / or apoptosis-suppressing gene, it is desired after suppressing the expression of p53 gene. It is preferable to suppress (release) forced expression after the lapse of a period. The cells may be subcultured after the forced expression, and the period from the last passage 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. ..

Cultivation conditions for hematopoietic progenitor cells or pre-megakaryocyte progenitor cells, and further megakaryocyte progenitor cells or megakaryocyte cells can be appropriately determined by those skilled in the art according to the cell type and its state. 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 is, for example, 5% CO ₂ , and the oxygen concentration is, for example, 20. It can be% 0 _2. It may be a static culture or a shaking culture. The shaking speed in the case of shaking culture is also not particularly limited, and can be, for example, 10 rpm to 200 rpm, 30 rpm to 150 rpm, or the like.

The medium may be an Iskov modified Darbecco 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, and SCF can be about 10 g / mL to about 200 g. It can be / mL, or about 50 g / mL, and heparin can be from about 10 U / mL to about 100 U / mL, or about 25 U / mL. Phorbol ester (eg, phorbol-12-millistart-13-acetate; PMA) may be added.

The cell culture step can be carried out in the presence or absence of feeder cells. As used herein, the term "feeder cell" refers to a cell that is co-cultured with a target cell in order to prepare an environment necessary for culturing the target cell that is to be proliferated or differentiated. The feeder cell may be a cell of allogeneic origin or a cell of heterologous origin as long as it is a cell that can be distinguished from the target cell. The feeder cells may be cells that have been treated so as not to proliferate with antibiotics or gamma rays, or cells that have not been treated.

The medium may contain serum or plasma, or may be serum-free. When serum is used, human serum is preferred. If desired, the medium may be, for example, albumin, insulin, transferase, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (eg L-glutamine), ascorbic acid. , Heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like. Examples of cytokines include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like agents, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplement, and ADAM (A Discintegrin And Metalloprotase) inhibition. Examples include agents.

When the megakaryocyte cells are cultured in a medium supplemented with an aromatic hydrocarbon receptor (AhR) antagonist alone or in combination with a ROCK (Rho-associated coiled-coil forming kinase) inhibitor, the feeder cells are not used. Even in this case, a platelet production promoting effect comparable to that when feeder cells are used can be obtained (International Publication No. 2016/20456 and US Patent Application Publication No. 2019/0048317).

(Platelet manufacturing method)
The method for producing platelets according to the present embodiment includes a step of further culturing megakaryocyte progenitor cells or megakaryocyte cells produced according to the above method.

The medium for culturing megakaryocyte cells is not particularly limited, and a known medium suitable for producing platelets from megakaryocyte cells or a medium similar thereto can be appropriately used. For example, a medium used for culturing animal cells can be prepared as a basal medium. Examples of the basal medium include Iskov-modified Dalveco medium (IMDM) medium, 199 medium, Eagle's minimum essential medium (EMEM), αMEM medium, Dalveco-modified Eagle's medium (DMEM) medium, Ham F12 medium, RPMI1640 medium, Fisher medium, and neurobasal. Medium (Life Technologies) and a mixed medium thereof can be mentioned.

From the viewpoint of producing a large amount of high-quality platelets, it is preferable to culture megakaryocyte cells in a culture vessel equipped with a stirring blade and a stir bar (International Publication No. 2017/047942 and US Patent Application Publication No. 2018 / 0258395, WO 2017/077964 and US Patent Application Publication No. 2018/0318352).

It is also possible to further produce a platelet preparation or a blood product using the produced platelets. The method for producing a platelet preparation includes a step of collecting a platelet-rich fraction and a step of removing blood cell lineage components other than platelets from the platelet-rich fraction. The step of removing blood cell components is performed by removing blood cell components other than platelets, including megakaryocyte cells, using a leukocyte removal filter (for example, manufactured by Terumo Corporation or Asahi Kasei Medical Co., Ltd.). Can be done. More specific methods for producing platelet preparations are described, for example, in International Publication No. 2011/034073 (above) and US Patent Application Publication No. 2012/0238023. The method for producing a blood product includes a step of producing a platelet preparation and a step of mixing the platelet preparation with other components. Other components include, for example, red blood cells. Platelet and blood products may also contain other components that contribute to cell stabilization.

(Method for promoting proliferation of megakaryocyte progenitor cells or megakaryocyte cells)
The method for promoting the proliferation of megakaryocyte precursor cells or megakaryocyte cells according to the present embodiment is a step of suppressing the expression of the CDKN1A gene or the function of the expression product thereof, and optionally, the INK4A / ARF gene and / or the p53 gene. The expression of the above, or the step of suppressing the function of those expression products is included.

In a further aspect, the method of promoting the proliferation of megakaryocyte precursor cells or megakaryocyte cells is a step of suppressing the expression of the p53 gene or the function of the expression product thereof, and optionally, the INK4A / ARF gene and / or the CDKN1A gene. Includes the step of suppressing the expression of, or the function of those expression products.

(Megakaryocyte progenitor cell or megakaryocyte cell proliferation promoter)
The megakaryocyte precursor cell or the proliferation promoter of megakaryocyte cell according to the present embodiment contains a molecule that suppresses the expression of the CDKN1A gene or the function of the expression product as an active ingredient, and optionally, the INK4A / ARF gene and / Alternatively, it contains a molecule that suppresses the expression of the p53 gene or the function of those expression products.

In a further aspect, the megakaryocyte precursor cell or megakaryocyte cell proliferation promoter comprises, as an active ingredient, a molecule that suppresses the expression of the p53 gene or the function of the expression product thereof, and optionally, the INK4A / ARF gene and /. Alternatively, it contains a molecule that suppresses the expression of the CDKN1A gene or the function of those expression products.

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited thereto. One of ordinary skill 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 From human iPS cells (TKDN SeV2: human fetal skin fibroblast-derived iPS cells established using Sendai virus), Takayama N. et al. , Et al. J Exp Med. Differentiation culture into blood cell cells was carried out according to the method described in 2817-2830 (2010). That is, human iPS cell colonies (NC13X strain) were co-cultured with C3H10T1 / 2 feeder cells for 14 days in the presence of 20 ng / mL VEGF (R & D SYSTEMS) to prepare hematopoietic progenitor cells (HPC). The culture conditions were 20% O ₂ and 5% CO ₂ .

An immortalized megakaryocyte 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 was introduced into hematopoietic progenitor cells on the 14th day of culture under control of doxycycline, and 1 μg / ml of doxycycline was added 24 hours after infection to carry out gene expression. Two weeks later, a lentiviral vector that forcibly expresses BCL-XL under doxycycline control was introduced to establish an immortalized megakaryocyte cell line.

ShCDKN1A (clone 1 (SEQ ID NO: 5) or clone 2 (SEQ ID NO: 6)) was added to megakaryocyte progenitor cells or megakaryocyte strains (MKCL21 strain) with decreased cell proliferation 50 days after the introduction of MYC / BMI1 / BCL-XL. ))-The RFP lentiviral vector was infected. Three days after infection, RFP (Red Fluorescent Protein) positive infected cells were sorted using FACS Maria IIIu. The estimated secondary structures of clones 1 and 2 are shown below.

When the cells separated in the culture medium containing 15% FBS were continuously cultured, cell proliferation was confirmed for 300 days or more from the suppression of the expression of the CDKN1A gene, and the megakaryocyte precursor was compared with the cells at the time of the suppression of the expression. Cells or megakaryocyte cells ^{proliferated 1066-} fold (Fig. 1).

Example 2: Suppression of expression of CDKN1A gene, INK4A / ARF gene and p53 gene (cell proliferation reduction stage)
An immortalized megakaryocyte 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 strain) different from that used in Example 1.

The megakaryocyte strain (MKCL23 strain) whose cell proliferation decreased 60 days after the introduction of MYC / BMI1 / BCL-XL and the megakaryocyte strain (MKCL26 strain) whose cell proliferation decreased 90 days after the introduction. A lentiviral vector having the following genes was infected. 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 Maria IIIu. shCDKN1A used the above clones 1 and 2. The estimated secondary structures of the sequence of shp53 (SEQ ID NO: 7) and the sequence of shINK4A / ARF (SEQ ID NO: 8) are described below.

The shINK4A / ARF targets a common exon of the INK4A gene and the ARF gene.

After sorting, when the culture was continued in a culture medium containing 15% FBS, both the MKCL23 strain and the MKCL26 strain suppressed the expression of the CDKN1A gene in comparison with the control, resulting in a cell growth period of 30 days or more. and extension of, 10 ⁴ fold expansion was observed (Figure 2, Figure 3). It was clarified that the proliferative effect was improved by suppressing the expression of the INK4A / ARF gene and the p53 gene even when the suppression of the expression of the CDKN1A gene was insufficient.

Furthermore, an immortalized megakaryocyte 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 strain) different from that used in Example 1.

About 40 days after the introduction of MYC / BMI1 / BCL-XL, a megakaryocyte strain (MKCL1 strain) having decreased cell proliferation was infected with a lentiviral vector having 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 above shRNA was used. Then, positively infected cells were collected using FACS MariaIIIu.

After sorting, when the culture was continued in the culture medium containing 15% FBS, the cell proliferation was significantly increased as compared with the control (excluding INK4A / ARF). In particular, CDKN1A gene, INK4A gene, an extension of all the cells that suppress the expression of cell growth period of 20 days of ARF gene and p53 gene, 10 ⁴ fold expansion was observed.

Example 3: Suppression of expression of CDKN1A gene, INK4A / ARF gene and p53 gene (at the time of introduction of BCL-XL)
At the same time that BCL-XL was introduced in Example 2, a megakaryocyte strain (MKCL30 strain) derived from the YZWJ516 human iPS cell line, which could not be established by conventional forced expression of MBX, was infected with a wrench viral vector having 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: shRNA1A-RFP + hep53-GFP + shRNA-GFP Each shRNA used as described above. Then, positively infected cells were collected using FACS MariaIIIu.

After sorting, when the culture was continued in a culture medium containing 15% FBS, the cell proliferation period was extended by 8 weeks or more and the proliferation was 10 to ¹⁵ times by suppressing the expression of the CDKN1A gene as compared with the control. Was confirmed. The cell proliferation effect was highest when the expression of both the CDKN1A gene and the p53 gene was suppressed (2.5x10 ¹⁷ times in 8 weeks). It is growing on the priority date of the present application (October 17, 2019, 127 days after c-MYC / BMI1 infection). Following this, the cell proliferation effect of the cells in which the expression of all the INK4A gene, ARF gene and p53 gene was suppressed was high.

Furthermore, in order to verify the timing of knockdown, the following knockdown experiments were performed at the same time as the introduction of BCL-XL and 2 weeks after the introduction.
9: Control-RFP (LacZ)
10: shCDKN1A-RFP + shp53-GFP

The results are shown in FIG. The time of infection, BCL-XL towards the introduction and simultaneous infection, BCL-XL of introduction 10 ⁴ times more than the infection after two weeks of growth is good (shCDKN1A-RFP + shp53-GFP simultaneous 2.5x10 ¹⁷ times vs shCDKN1A -RFP + shp53-GFP simultaneous 5.8x10 ¹² times) was confirmed.

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

Example 4: Examination of platelet production by megakaryocyte cells in which the expression of the CDKN1A gene was suppressed MKCL21 strain after CDKN1A suppression and control vector infection in which proliferation was promoted using clone 2 (SEQ ID NO: 6) in Example 1 Platelet release in the MKCL21 strain was compared.

Doxycycline was removed from the culture mediums of both cells, and after suppressing the expression of the three factors MYC / BMI1 / BCL-XL, megakaryocytes and platelets released into the culture supernatant were analyzed by FACS on the 5th day. As the antibody, APC anti-human CD41 Antibody (BioLegend, catalog number: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, catalog number: 555473) were used. The FACS dot plot is shown in FIG.

Moreover, when the number of platelets counted using FACS was compared, it was clarified that the platelet production was enhanced by suppressing the expression of the CDKN1A gene.

Subsequently, the platelet-producing ability of the MKCL21 strain in which the expression of the CDKN1A gene was suppressed in Example 2 was changed to the megakaryocyte strain having the best platelet-releasing ability by the conventional forced expression of three factors (MYC / BMI1 / BCL-XL). Compared with SeV2). The FACS dot plots are shown in FIG. As the antibody, APC anti-human CD41 Antibody (BioLegend, catalog number: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, catalog number: 555473) were used.

The upper panel of FIG. 6 shows a state in which doxycycline is added to express the three factors of MYC / BMI1 / BCL-XL. Since both strains are immature, there are few CD41-positive / CD42b-positive platelets (upper right part of the figure divided into four). On the other hand, the lower panel of FIG. 6 shows the state on the 5th day when doxycycline was removed from the culture medium and the expression of the three factors MYC / BMI1 / BCL-XL was suppressed, and the platelets were CD41a-positive / CD42b-positive (4). The upper right part of the divided figure) is increasing. From these results, it can be seen that the platelet-producing ability was increased by suppressing the expression of the CDKN1A gene.

Example 5: Examination of platelet production by macronuclear cells that suppressed the expression of CDKN1A gene and p53 gene MKCL7 strain after control vector infection and clone 2 (SEQ ID NO: 6) in Example 1 were used to promote proliferation. CDKN1A and p53 whose proliferation was promoted using "5: shCDKN1A-RFP + shp53-GFP" of Example 3 at the same time as the introduction of the MKCL21 strain after CDKN1A suppression and clone 2 (SEQ ID NO: 6) of Example 1. Platelet release with the suppressed MKCL30 strain was compared.

After removing doxycycline from the culture medium of each cell and suppressing the expression of the three factors of MYC / BMI1 / BCL-XL, megakaryocytes and platelets released into the culture supernatant were analyzed by FACS on the 5th day. As the antibody, APC anti-human CD41 Antibody (BioLegend, catalog number: 303710) and PE Mouse Anti-Human CD42b Clone HIP1 (BD, catalog number: 555473) were used.
However, for the MKCL7 strain, a sample in which doxycycline was not removed and the expression of the three factors MYC / BMI1 / BCL-XL was not suppressed was also prepared (MBX ON). On the 5th day, megakaryocytes and platelets released into the culture supernatant were analyzed by FACS in the same manner as described above.
The FACS dot plot is shown in FIG. 7 (A). In addition, the result of the platelet count counted using FACS is shown in FIG. 7 (B).

Subsequently, the strain (ON) that did not suppress the expression of the three factors of MYC / BMI1 / BCL-XL and the MKCL7 strain, the MKCL21 strain, and the MKCL30 after suppressing the expression of the three factors of MYC / BMI1 / BCL-XL. For each of the strains, megakaryocytes and platelets released into the culture supernatant were collected on day 5, and were unstimulated or 0.4 μM PMA (Thrombol Myristate Antibody, 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, in the presence of BACHEM Cat # H-8365.0005) antibody (AT Staining with Legend / # 304910) and PAC-1 antibody (BD Bioscience / # 34507) was performed and analyzed by FACS. The results with P-selectin and PAC-1 antibodies are shown in FIGS. 8 (D) and 8 (E), respectively.
The explanations in the table are as follows.
No stimulation-ON: Results in no stimulation using a strain that did not suppress the expression of 3 factors of MYC / BMI1 / BCL-XL PMA-ON: Suppressed the expression of 3 factors of MYC / BMI1 / BCL-XL Results under PMA using a strain that did not suppress AT-ON: Results under AT using a strain that did not suppress the expression of the three factors AT-ON: MYC / BMI1 / BCL-XL. Results in non-stimulation PMA_MKCL7: MKCL7 strain was used, results under PMA AT_MKCL7: MKCL7 strain was used, results under AT No simulation_MKCL21 #: MKCL21 strain was used, results in no stimulation PMA_MKCL21 #: MKCL21 strain was used. , PMA result AT_MKCL21 #: MKCL21 strain was used, AT result No simulation_MKCL30: MKCL30 strain was used, no stimulation result PMA_MKCL30: MKCL30 strain was used, PMA result AT_MKCL30: MKCL30 strain was used. , Results under AT

From the above results, it was clarified that even if the expression of the CDKN1A gene or the p53 gene is suppressed, functional platelets showing a good activation reaction to the stimulus are produced, and the function of the platelets is not inhibited. On the other hand, in the group (MBX ON) in which the expression of the three factors MYC / BMI1 / BCL-XL was not suppressed without removing doxycycline, it was confirmed that the maturation of megakaryocytes did not proceed and almost no platelets were produced.

Example 6: Expression of BCL-XL gene and suppression of expression of p21 / p53 gene From iPS cells derived from human skin fibroblasts, iPS-Sac method (Takayama N., et al. J Exp Med. 2817-2830 (2010) )), The cells were cultured for 14 days to prepare hematopoietic progenitor cells.

A megakaryocyte progenitor cell line was established by introducing two genes (MB) of c-MYC / BMI1 into the obtained hematopoietic progenitor cells using a doxycycline-induced lentiviral vector. 14 days later, the strain (MB) cultured as it was, the strain (MBX) in which the BCL-XL gene was further introduced using a doxycycline-induced lentiviral vector in addition to the MB introduction, and the sh that was continuously expressed in addition to the MB introduction. A strain further infected with a p21 / p53 lentiviral vector (MB-p21 / p53_KD), and a BCL-XL gene introduced using a doxycycline-induced lentiviral vector in addition to MB introduction, and continuously expressed sh p21 / A giant nuclear progenitor cell line of a strain infected with the p53 lentiviral vector (MBX-p21 / p53_KD) was prepared. The results of confirming the proliferative properties of the cells on the 14th, 31st, and 43rd days of each megakaryocyte progenitor cell line are shown in FIG.

The results of FACS analysis of the expression of CD34 and CD41 in cells on the 31st and 43rd days of the MB strain, MBX strain, MB-p21 / p53_KD strain, and MBX-p21 / p53_KD strain are shown in FIG. The results of FACS analysis of the expression of CD34 and CD42b in cells are shown in FIG. 11, and the results of FACS analysis of the expression of CD34 and GPA in cells are shown in FACS dot plot. Antibodies are 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: 555473), GPA (Glycophorin A) antibody (Bio A CD34 antibody (BioLegend, Catalog No .: 343514) was used.

Under all conditions, CD41-positive and CD42b-weakly positive megakaryocyte progenitor cells were obtained. At the stage of the 43rd day of culture, the results of the MB-p21 / p53_KD strain (white circles in FIG. 9) show that the results of the MB strain (black circles) are considerably superior to the results of the MB strain (black circles). In addition, the result of the MBX-p21 / p53_KD strain (black triangle) indicates that the growth is equal to or higher than the result of the MBX strain (black square) or the result of the MB-p21 / p53_KD strain (white circle). From these results, the proliferation of the megakaryocyte progenitor cell line obtained by introducing the two genes of c-MYC / BMI1 is promoted by p21 / p53KD even without the introduction of the BCL-XL gene, that is, It was suggested that BCL-XL could be replaced by p21 / p53KD.

All references cited in this specification, including publications, patent documents, etc., are individually and specifically incorporated as references and the entire contents are specifically described. To the same extent, incorporated herein.

Claims

A method for producing a megakaryocyte progenitor cell or a megakaryocyte cell, which comprises a step of suppressing the expression of the CDKN1A gene or the function of the expression product in the hematopoietic progenitor cell or the megakaryocyte progenitor cell before polynuclearization.
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 the expression product thereof.
The method according to claim 2, wherein the megakaryocyte cell is a mature megakaryocyte cell.
The method according to any one of claims 1 to 3, wherein the megakaryocyte cells are immortalized.
The method according to claim 4, further comprising a step of forcibly expressing an oncogene and a BMI1 gene selected from MYC family genes in hematopoietic progenitor cells.
The method according to claim 5, further comprising a step of forcibly expressing the BCL-XL gene.
The method according to claim 6, further comprising a step of suppressing forced expression of an oncogene, a BMI1 gene and a BCL-XL gene.
The method according to any one of claims 1 to 7, wherein the expression of the CDKN1A gene or the suppression of the function of the expression product thereof is carried out at the same time as the forced expression of the BCL-XL gene.
The expression according to any one of claims 1 to 7, wherein the expression of the CDKN1A gene or the suppression of the function of the expression product thereof is carried out after the decrease in cell proliferation after the forced expression of the BMI1 gene and the BCL-XL gene. Method.
The method according to claim 9, wherein the decrease in cell proliferation is 30 days or more after the forced expression of the BMI1 gene and the BCL-XL gene.
The method according to any one of claims 1 to 10, wherein the suppression step is performed by introducing a molecule that suppresses the expression of a gene or the function of the expression product into a cell.
Producing platelets, which comprises the step of producing megakaryocyte progenitor cells or megakaryocyte cells by the method according to any one of claims 1 to 11 and the step of culturing the produced megakaryocyte progenitor cells or megakaryocyte cells. how to.
A method for promoting the proliferation of megakaryocyte progenitor cells or megakaryocyte cells, which comprises a step of suppressing the expression of the CDKN1A gene or the function of the expression product thereof.
The method according to claim 13, 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 the expression product thereof.
A growth promoter for megakaryocyte progenitor cells or megakaryocyte cells, which comprises a molecule that suppresses the expression of the CDKN1A gene or the function of the expression product as an active ingredient.
The growth promoter according to claim 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 the expression product thereof.