WO2018052126A1 - Method for identifying heterogeneity in cells in megakaryocytic cell groups, and production method for platelets - Google Patents

Method for identifying heterogeneity in cells in megakaryocytic cell groups, and production method for platelets Download PDF

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WO2018052126A1
WO2018052126A1 PCT/JP2017/033518 JP2017033518W WO2018052126A1 WO 2018052126 A1 WO2018052126 A1 WO 2018052126A1 JP 2017033518 W JP2017033518 W JP 2017033518W WO 2018052126 A1 WO2018052126 A1 WO 2018052126A1
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cell
cells
megakaryocyte
gene
mrna
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French (fr)
Japanese (ja)
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浩之 江藤
博英 齊藤
一哉 橋本
祥彦 藤田
香倫 林
理史 松浦
芳樹 西河
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国立大学法人京都大学
株式会社メガカリオン
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to a method for identifying heterogeneity of cells in a megakaryocyte group, and a method for selecting cells having high platelet-producing ability from megakaryocytes by using miRNA expression as an index.
  • the platelet preparation is administered for the purpose of treatment and prevention of symptoms to a patient exhibiting a bleeding tendency associated with thrombocytopenia after treatment with an anticancer agent or massive bleeding during surgery or injury.
  • manufacture of platelet products relies on blood donation, but there is a need for a safer supply of platelets and a stable supply of platelets.
  • a method for producing platelets from megakaryocytes cultured in vitro has been developed today.
  • the present applicants have established a method for establishing an immortalized megakaryocyte progenitor cell lines (imMKCL) immortalized using pluripotent stem cells as a source (for example, Patent Documents 1 and 2; (See Patent Document 1).
  • the immortalized megakaryocyte progenitor cell line can be stored by freezing, and has many advantages such as the ability to promote artificial multinucleation. From this immortalized megakaryocyte progenitor cell line, platelets can be produced according to demand. Can be stably manufactured in large quantities. In search of more efficient production of platelets, the present inventors have found for the first time that the immortalized megakaryocyte progenitor cell line after establishment is heterogeneous. This is a finding that could not be obtained by conventional cell surface marker identification. For example, the megakaryocyte group has been found to contain a group of cells having high platelet-producing ability and a group of cells that are not.
  • the yield may be unstable if platelet production is performed using such a heterogeneous cell population as it is.
  • a limiting dilution method which is a classic selection method for selecting the proliferation ability, is known.
  • selection of only proliferative ability cannot select a cell group with high platelet production ability during the maturation process.
  • a method of identifying the heterogeneity of megakaryocyte cells and selecting a cell group particularly, a method of selecting a cell group having a high platelet-producing ability is desired.
  • a megakaryocyte cell group can be selected using microRNA expression as an indicator, and the present invention has been completed.
  • a method for identifying heterogeneity of cells in a megakaryocyte group comprising the following steps: (1) mRNA comprising a marker gene operably linked to a nucleic acid sequence specifically recognized by microRNA A step of introducing the cell into a megakaryocyte cell group, and (2) a step of identifying a cell group having different characteristics using the translation amount of the marker gene as an index.
  • a method for selecting a cell group having a high platelet-producing ability from a megakaryocyte group including the following steps: (1) introducing mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family into a megakaryocyte cell group; and (2) the marker gene.
  • nucleic acid sequence specifically recognized by the microRNA selected from the let-7 family and the marker gene are linked in the 5 ′ to 3 ′ direction, [2] Or the method as described in [3].
  • the selecting step selects a cell group having a low amount of translation of the marker gene from a cell group having a low microRNA activity selected from the let-7 family.
  • the method further includes the step of introducing into the cell group a control mRNA that does not have a nucleic acid sequence specifically recognized by the microRNA selected from the let-7 family and includes a marker gene different from the marker gene.
  • a megakaryocyte cell selection kit comprising mRNA comprising a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family.
  • the mRNA is a nucleic acid in which a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family and the marker gene are linked in a 5 ′ to 3 ′ direction.
  • the megakaryocyte cell sorting kit according to [12].
  • a step of inhibiting forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and encoding the Lin28A gene in the cell in the step of obtaining the immortalized megakaryocyte cells A method for producing platelets, comprising a step of reducing the amount of methylation in a CpG island region of a promoter of DNA.
  • the heterogeneity of cells in a megakaryocyte cell group can be identified by using the activity of microRNA as an index.
  • a cell group having a high resolution and a high platelet production ability can be selected. Since the method according to the present invention can be applied in a state where the cells are alive, it is particularly advantageous in that the cells after sorting can be used in medical applications.
  • the method of the present invention can be carried out by introducing mRNA into a cell group, and this mRNA is decomposed with a half-life of about 1 day and rapidly removed from the cell. The virus infection and DNA persistence in the plant will not cause problems such as damage to the genome.
  • Figure 1 shows the structure of miRNA-responsive mRNA in which the expression level of fluorescent protein is suppressed in response to let-7a-5p activity, the structure of control mRNA that expresses fluorescent protein regardless of miRNA activity, and uses this It is a figure which shows the scheme of the cell separation which had.
  • Fig. 2 shows that there are cell groups with different let-7a-5p activity under megakaryocyte growth conditions (DOX ON) using SeV2 imMKCL, and let-7a-5p under platelet production conditions (DOX OFF) from megakaryocytes A cell group with low activity indicates that there is much platelet production.
  • DOX ON megakaryocyte growth conditions
  • DOX OFF platelet production conditions
  • Figure 3 shows the platelet production conditions after separation into a cell group with low let-7a-5p activity (L) and a cell group with high activity (H) based on let-7a-5p activity.
  • Figure 4 shows that let-7a-5p activity is reduced by a let-7a-5p inhibitor (left), and a group of cells whose let-7a-5p activity is reduced by a let-7a-5p inhibitor (H + Inhibitor ) Indicates that platelet production is as high as the cell population with low let-7a-5p activity (L) under megakaryocyte growth conditions (DOX ON) (right figure).
  • Fig. 5 shows quantitative PCR for the expression levels of 3 genes (C-MYC, BMI1, and BCLXL) in a cell population with low let-7a-5p activity (L) and a high cell group (H) under megakaryocyte growth conditions (DOX ON) As a result of the confirmation, it is shown that there is no significant difference in the expression level.
  • Figure 6 shows the purpose of finding the target of let-7a-5p.
  • RNA sequencing is performed on cell populations with high and low let-7a-5p activity, gene expression is compared, and cell population with low activity.
  • FIG. 7 plots the expression levels of 18 genes, and shows that among them, the two genes LIN28A and NR6A1 are highly expressed in the cell population with low let-7a-5p activity.
  • FIG. 8 shows that the expression level of LIN28A does not change even when an inhibitor is introduced, and the expression level of NR6A1 decreases in a population with high let-7a-5p activity, and the effect is rescued by the inhibitor. Indicates that it is a 5p target. Moreover, it shows that Lin28B is also a target of let-7a-5p.
  • FIG. 9 shows that in addition to LIN28B and NR6A1, IGF2BP1 is also a target of let-7a-5p.
  • FIG. 10 is a diagram showing the experimental results of examining the effect of NR6A1 on platelet production.
  • FIG. 10 (a) shows that imMKCL in a group with high Let-7a-5p activity was infected with a virus that does not express anything.
  • FIG. 10B shows the number of platelets produced when Negative Control and NR6A1 siRNA # 1 and # 3 were introduced into SeV2VimMKCL, respectively, with Negative Control being 100%.
  • FIG. 10 (c) shows the number of platelets produced when Negative Control and NR6A1 siRNA # 1 and # 3 were introduced into cord blood-derived megakaryocytes, respectively, with Negative Control as 100%.
  • FIG. 11 is a diagram showing the experimental results of examining the effect of Lin28A, B on platelet production.
  • FIG. 11A shows the addition of Lin28A28siRNA to a cell population with low let-7a-5p activity (Group L). It is a figure which shows that let-7a-5p activity rose by this.
  • FIG. 11 (b) shows a sample in which a vehicle and a switch are co-introduced into a cell population with low let-7a-5p activity using a Let-7a-5p-responsive tagBFP mRNA switch (Let7-Low vehicle), let-7a- Sample with co-introduction of Lin28A siRNA and switch into a cell population with low 5p activity (Let7-Low Lin28A siRNA), Sample with co-introduction of Lin28B siRNA and switch into a cell population with low let-7a-5p activity (Let7-Low Lin28B siRNA) ), A graph showing the mean fluorescence intensity (MFI) of tagBFP in each of the introduced samples (Let7-High vehicle) in which vehicle and switch were co-introduced into a cell population with high let-7
  • FIG. 11 (c) shows whether the DNA encoding the Lin28A gene is methylated in each of 16 CpG sequences contained in the promoter CpG island for 7 to 8 samples of Group L and H, respectively.
  • the white circle represents unmethylated and the black circle represents methylated CpG.
  • FIG. 11 (d) shows CpG methylation% in a cell population with low let-7a-5p activity (Group (L) and a cell population with high activity (Group H).
  • Fig. 12 (a) shows an experimental scheme in which static culture (static) was changed to static culture (static) or shaking culture (flask) instead of the culture conditions in each state of DOX ON and DOX OFF.
  • FIG. 12 (a) shows an experimental scheme in which static culture (static) was changed to static culture (static) or shaking culture (flask) instead of the culture conditions in each state of DOX ON and DOX OFF.
  • FIG. 12 is a graph showing the relative amounts of LIN28A and NR6A1 when the amount of mRNA extracted from statically cultured cells is 1.
  • C is a graph showing the number of platelets produced under each of these conditions as a relative number with 100% when cultured as DOX on (Static) -DOX off (Static).
  • d) The result of analyzing the methylation of the LIN28A promoter region CpG island in DNA in cells statically or shaken (flask) for 1 week in DOX on by the same method as described above. Indicates.
  • FIG. 13 is a diagram showing the experimental results of examining the influence of iPS cell culture conditions before differentiation induction on platelet production.
  • 13 (a) is a graph showing the amount of LIN28A mRNA measured by quantitative PCR in cells at various stages induced to differentiate from MKiPS cells.
  • FIG. 13 (b) shows the results after differentiation induction from MKiPS cells. The results of measuring the Let-7a-5p activity for the 14th and 35th cells are shown, and
  • FIG. 13 (c) shows the LIN28AmRNA when the LIN28AmRNA expression level extracted from the MKiPS11 cell line cultured in Laminin511 is 1.
  • 13 (d) is a graph showing the relative amount of platelet production when the number of platelets produced by megakaryocytes differentiated from the MKiPS11 cell line cultured with Laminin511 is taken as 100%. It is a graph showing.
  • the present invention relates to a method for identifying cell heterogeneity in a megakaryocyte population comprising the following steps. (1) introducing mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by microRNA into a megakaryocyte cell group, and (2) using the amount of translation of the marker gene as an index Identifying different cell groups.
  • cell heterogeneity in a megakaryocyte group can be identified by mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by microRNA.
  • megakaryocytes can be identified, distinguished, and differentiated in the process of proliferation and maturation in addition to being identified as “megakaryocytes” by cell surface markers described in detail below. could not.
  • the present embodiment relates to a method for identifying cell heterogeneity by mRNA. As a specific example, a method for selecting a cell group having high platelet-producing ability will be described below, but the present invention is not limited to this.
  • the present invention relates to a method for selecting a cell group having high platelet-producing ability from a megakaryocyte cell group, comprising the following steps. (1) introducing an mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family into a cell group, and (2) translation of the marker gene A process of selecting a cell group having high platelet production ability using the amount as an index.
  • the megakaryocyte cell group into which mRNA is introduced refers to a cell group mainly composed of megakaryocyte cells as defined below.
  • a “megakaryocyte” is the largest cell present in the bone marrow in vivo and is characterized by releasing platelets.
  • the cell surface markers CD41a, CD42a, and CD42b are positively characterized, and in addition, a marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c May be further expressed.
  • a “megakaryocyte” has a genome 16 to 32 times that of a normal cell when multinucleated (multiploidy).
  • megakaryocyte has the above characteristics. As long as it is, it includes both multinucleated megakaryocytes and pre-multinucleated megakaryocytes. “Pre-multinucleated megakaryocyte” is also synonymous with “immature megakaryocyte” or “proliferative megakaryocyte”.
  • Megakaryocyte cells can be obtained by various known methods.
  • Non-limiting examples of the method for producing megakaryocyte cells include the method described in International Publication No. 2011/034073.
  • an immortalized megakaryocyte cell line that proliferates indefinitely can be obtained by forcibly expressing an oncogene and a polycomb gene in “an undifferentiated cell than a megakaryocyte”.
  • an immortalized megakaryocyte cell line can also be obtained by forcibly expressing an apoptosis-suppressing gene in “undifferentiated cells than megakaryocyte cells”. .
  • These immortalized megakaryocyte cell lines are polynucleated by releasing the forced expression of the gene and release platelets.
  • the methods described in the above documents may be combined.
  • the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene may be performed simultaneously or sequentially.
  • a multinucleated megakaryocyte cell may be obtained by forcibly expressing an oncogene and a polycomb gene, suppressing the forced expression, then forcibly expressing an apoptosis suppressing gene, and suppressing the forced expression.
  • multinucleated megakaryocyte cells can be obtained by forcibly expressing an oncogene, polycomb gene, and apoptosis-suppressing gene at the same time, and simultaneously suppressing the forced expression.
  • an oncogene and a polycomb gene are forcibly expressed, followed by forcibly expressing an apoptosis-inhibiting gene, and the forcible expression is simultaneously suppressed to obtain multinucleated megakaryocyte cells.
  • the step of forcibly expressing a gene may be referred to as a growth phase or a state where the gene can be proliferated, and the step of suppressing the forced expression may be referred to as a maturation phase.
  • cells that are undifferentiated from megakaryocytes means cells that have the ability to differentiate into megakaryocytes and that have various differentiation stages ranging from hematopoietic stem cell lines to megakaryocytes.
  • Non-limiting examples of cells that are undifferentiated from megakaryocytes include hematopoietic stem cells, hematopoietic progenitor cells, CD34 positive cells, megakaryocytes / erythroid progenitor cells (MEP). These cells can be obtained by isolation from, for example, bone marrow, umbilical cord blood, and peripheral blood, and further obtained by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells, which are more undifferentiated cells. You can also.
  • oncogene refers to a gene that induces canceration of a cell in a living body, such as a MYC family gene (for example, c-MYC, N-MYC, L-MYC), SRC family.
  • MYC family gene for example, c-MYC, N-MYC, L-MYC
  • protein kinase family genes such as genes, RAS family genes, RAF family genes, c-Kit, PDGFR, and Abl.
  • Polycomb gene is known as a gene that negatively regulates the CDKN2a (INK4a / ARF) gene and functions to avoid cellular senescence (Ogura et al., Regenerative Medicine vol.6, No.4, pp26 -32; Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol.7, pp667-677, 2006; Proc. Natl. Acad. Sci. USA vol.100, pp211-216, 2003).
  • Non-limiting examples of polycomb genes include BMI1, Mel18, Ring1a / b, Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HADC, Dnmt1 / 3a / 3b .
  • Apoptosis suppressor gene refers to a gene having a function of suppressing cell apoptosis, and examples thereof include BCL2 gene, BCL-xL gene, Survivin gene, and MCL1 gene.
  • the forced expression of genes and the release of forced expression are described in International Publication No. 2011/034073, International Publication No. 2012/157586, International Publication No. 2014/123242, or Nakamura S et al, Cell Stem Cell. 14, 535-548, It can be carried out by the method described in 2014, other known methods or a method analogous thereto.
  • a drug-responsive gene expression induction system such as Tet-on (registered trademark) or Tet-off (registered trademark) system
  • Corresponding agents such as tetracycline or doxycycline may be contained in the medium, and forced expression may be suppressed by removing them from the medium.
  • Culture conditions for megakaryocyte cells when forced expression of genes and suppression (cancellation) of forced expression are performed can be normal conditions.
  • the temperature can be about 35 ° C. to about 42 ° C., about 36 ° C. to about 40 ° C., or about 37 ° C. to about 39 ° C., and can be 5-15% CO 2 and / or 20% O 2. .
  • the medium for culturing megakaryocyte cells is not particularly limited, and a known medium suitable for producing platelets from megakaryocyte cells or a medium equivalent thereto can be appropriately used.
  • a medium used for animal cell culture can be prepared as a basal medium.
  • the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies) ) And mixed media thereof.
  • the culture medium may contain serum or plasma, or may be serum-free. If necessary, the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (eg L-glutamine), ascorbic acid It may also contain one or more substances such as heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like. Cytokines are proteins that promote blood cell differentiation.
  • VEGF vascular endothelial growth factor
  • TPO thrombopoietin
  • SCF Stem Cell Factor
  • ITS insulin-transferrin-selenite Supplements
  • ADAM inhibitors etc.
  • a preferable medium in the present invention is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO.
  • SCF may be included, and heparin may also be included.
  • TPO can be about 10 ng / mL to about 200 ng / mL, or about 50 ng / mL to about 100 ng / mL
  • SCF is about 10 ng / mL to about 200 ng / mL
  • Heparin can be about 10 U / mL to about 100 U / mL, or about 25 U / mL.
  • Phorbol esters eg, phorbol-12-myristate-13-acetate; PMA may be added.
  • the step of culturing megakaryocyte cells may be performed under conditions of serum-free and / or feeder cell-free.
  • the method is carried out by culturing megakaryocytes produced according to the method of the present invention in a medium containing TPO.
  • a medium containing TPO In the platelet production process, if it can be performed free of serum and feeder cells, the problem of immunogenicity hardly occurs when the obtained platelet is used clinically.
  • platelets can be produced without using feeder cells, it is not necessary to adhere feeder cells, so suspension culture can be performed in a flask or the like, which can suppress manufacturing costs and is suitable for mass production.
  • a conditioned medium may be used.
  • the conditioned medium is not particularly limited and can be prepared according to methods known to those skilled in the art. For example, the conditioned medium can be obtained by appropriately culturing feeder cells and removing the feeder cells from the culture with a filter.
  • the step of forcibly expressing the above genes in undifferentiated cells from megakaryocytes can be performed according to a conventional method of those skilled in the art.
  • vectors expressing these genes, or these genes can be achieved by introducing the protein into an undifferentiated cell from a megakaryocyte in the form of a protein or RNA that encodes.
  • it can be carried out by bringing low molecular weight compounds or the like that induce the expression of these genes into contact with undifferentiated cells from megakaryocytes.
  • vectors expressing these genes include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo) and the like can be used.
  • a retroviral vector or a lentiviral vector is preferable in that it can be carried out by single introduction.
  • promoters used in expression vectors include EF- ⁇ promoter, CAG promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia) Virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. are used.
  • the expression vector may optionally contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like.
  • Useful selection marker genes include, for example, dihydrofolate reductase gene, neomycin resistance gene, puromycin resistance gene and the like.
  • a drug-responsive vector having a tetracycline-responsive element in the promoter region may be used.
  • an expression vector in which a loxP sequence is placed so as to sandwich the gene and / or promoter region with the loxP sequence may be used.
  • cells that have been cultured with forced expression of an apoptosis-suppressing gene are treated with (a) an actomyosin complex function inhibitor, (b) a treatment with a ROCK inhibitor, c) at least one of treating with an HDAC inhibitor.
  • a person skilled in the art can determine the optimum concentration in the case of treating cells with an actomyosin complex function inhibitor, a ROCK inhibitor, an HDAC inhibitor or the like by preliminary experiments. Further, a period and a method for processing can be appropriately selected by those skilled in the art. For example, in the case of treatment with blebbistatin, which is a myosin heavy chain II ATPase inhibitor, 2 to 15 ⁇ g / ml or 5 to 10 ⁇ g / ml is added to the culture solution, and the culture period is, for example, 5 to 10 days. About 6 to 7 days is preferable.
  • RO276 inhibitor Y27632 is used at 5 to 15 ⁇ M, or 8 to 12 ⁇ M, preferably about 10 ⁇ M
  • HDAC inhibitor valproic acid is 0.1 to 1 mM, or 0.2 to 0.7 mM, preferably Can be used at about 0.5 mM.
  • the treatment time for Y27632 and valproic acid is 10 to 21 days, preferably about 14 days.
  • ROCK (Rho-associated coiled-coil forming kinase) inhibitors include [(R)-(+)-trans-N- (4-pyridyl) -4- (1-aminoethyl)- In some cases, such as cyclohexanecarboxamide, 2HCl, H 2 O] (Y27632), antibodies that inhibit Rho kinase activity, or nucleic acids (eg, shRNA) should also be used as ROCK inhibitors. Can do.
  • the HDAC inhibitor has an action of inhibiting histone deacetylase (HDAC) activity.
  • HDAC histone deacetylase
  • many HDAC inhibitors are known, and examples include valproic acid, trichostatin A, SAHA (suberoylanilide hydroxamic acid), APHA (aroylpyrrolylhydroxyamide), etc.
  • Valproic acid, trichostatin A and the like can be preferably used.
  • the drug used has a salt form, it may be used in a salt form.
  • a person skilled in the art can determine the optimum concentration in the case of treating cells with an actomyosin complex function inhibitor, a ROCK inhibitor, an HDAC inhibitor or the like by preliminary experiments. Further, a period and a method for processing can be appropriately selected by those skilled in the art. For example, in the case of treatment with blebbistatin, which is a myosin heavy chain II ATPase inhibitor, 2 to 15 ⁇ g / ml or 5 to 10 ⁇ g / ml is added to the culture solution, and the culture period is, for example, 5 to 10 days. About 6 to 7 days is preferable.
  • RO276 inhibitor Y27632 is used at 5 to 15 ⁇ M, or 8 to 12 ⁇ M, preferably about 10 ⁇ M
  • HDAC inhibitor valproic acid is 0.1 to 1 mM, or 0.2 to 0.7 mM, preferably Can be used at about 0.5 mM.
  • the treatment time for Y27632 and valproic acid is 10 to 21 days, preferably about 14 days.
  • the method further includes a step of culturing the megakaryocyte or megakaryocyte progenitor cell obtained in the step while suppressing the forced expression.
  • a method for suppressing or stopping forced expression for example, when forced expression is performed using a drug-responsive vector, it may be achieved by not contacting the corresponding drug with the cell.
  • the forced expression can be suppressed by culturing the cells in a medium from which these are removed.
  • the above-mentioned vector containing LoxP it may be achieved by introducing Cre recombinase into the cell.
  • a transient expression vector and RNA or protein introduction are used, the contact with the vector or the like may be stopped.
  • the medium used in this step can be performed using the same medium as described above.
  • the culture conditions for culturing while suppressing or stopping forced expression are as described above.
  • the culture period can be appropriately determined while monitoring the number of megakaryocytes, and is, for example, about 2 days to 10 days, preferably about 3 days to 7 days. Desirably at least 3 days.
  • a ROCK inhibitor and / or an actomyosin complex function inhibitor is added to the medium.
  • a ROCK inhibitor and an actomyosin complex function inhibitor the same thing as what was used by the manufacturing method of the multinucleated megakaryocyte mentioned above can be used.
  • the ROCK inhibitor include Y27632.
  • the actomyosin complex function inhibitor include blebbistatin, which is a myosin heavy chain II ATPase inhibitor.
  • a ROCK inhibitor may be added alone, a ROCK inhibitor and an actomyosin complex function inhibitor may be added alone, or a combination thereof may be added.
  • the ROCK inhibitor and / or actomyosin complex function inhibitor is preferably added at 0.1 ⁇ M to 30 ⁇ M, for example, 0.5 ⁇ M to 25 ⁇ M, 5 ⁇ M to 20 ⁇ M, and the like.
  • the culture period after adding the ROCK inhibitor and / or the actomyosin complex function inhibitor may be 1 to 15 days, and may be 3 days, 5 days, 7 days, or the like.
  • the megakaryocyte group into which the mRNA is introduced is preferably a megakaryocyte group in a proliferative state.
  • the megakaryocyte group in a proliferative state is selected from the group consisting of an oncogene, a polycomb gene, and an apoptosis-suppressing gene in a cell that is undifferentiated from the megakaryocyte in the method for producing a megakaryocyte described above.
  • a state in which at least one of the genes is forcibly expressed. Therefore, for example, it may be a group of megakaryocytes cultured in a growth medium containing doxycycline or tetracycline, or may be a group of megakaryocytes in which a gene is forcibly expressed by other methods.
  • time of introduction of mRNA there is no particular limitation on the time of introduction of mRNA as long as it is cultured in a growth medium, and it may be immediately after the start of the culture for growth. It may be 3 to 4 days after the start of culture. Even after the introduction of mRNA, the culture for propagation can be continued for 5 months or more over a necessary period according to a normal method.
  • the selected “cell group having high platelet-producing ability” refers to a group of cells classified as another cell group using the expression of microRNA selected from the let-7 family as an index. Say.
  • “selecting a cell group having a high platelet-producing ability” means that the platelet-producing ability is relatively selected from the megakaryocyte group found by the present inventors to be a heterogeneous cell group.
  • For a high cell group it means to present signal information that can be distinguished from other cell groups, that is, to be detectable, and in particular, to present visually recognizable information.
  • “visually recognizable information” is not limited to emitting a signal that can be directly recognized by the cell, but the signal emitted by the cell is visually expressed by a numerical value, a chart, an image, or the like. It is information that has been converted into information that can be recognized by the person, and is information that can be visually recognized by those skilled in the art.
  • the term sorting means that after the sorting, a cell group having a high platelet-producing ability is recognized, a cell group having a high platelet-producing ability is identified, a cell group having a high platelet-producing ability is identified, Classify cells with high platelet-producing ability, isolate cells with high platelet-producing ability, remove undesired cells, determine the viability of desired chondrocytes, and identify cells with high platelet-producing ability Detection or quantification, and fractionation based on a specific physical or chemical signal of a cell group having a high platelet-producing ability.
  • the indicator microRNA is a microRNA selected from the let-7 family.
  • the microRNAs belonging to the let-7 family include hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7f-5p , Hsa-let-7g-5p, hsa-let-7i-5p, hsa-let-7a-3p, hsa-let-7b-3p, hsa-let-7d-3p, hsa-let-7e-3p, hsa -let-7f-1-3p, hsa-let-7f-2-3p, hsa-let-7g-3p, hsa-let-7i-3p, hsa-let-7a-2-3p, hsa-let-7c -5p, hsa-let-7c-3p, hsa-miR-98-5p, hsa
  • let-7a-5p, let-7g-5p, and let-7i-5p are preferable as the microRNA serving as an index, but are not limited thereto.
  • microRNA selected from the let-7 family may be referred to as let-7 miRNA.
  • the messenger RNA (mRNA) introduced into the megakaryocyte population is a marker gene that is operably linked to a sequence that is specifically recognized by let-7 miRNA (also called the target sequence of let-7 miRNA). Including.
  • mRNA is also referred to as let-7 miRNA-responsive mRNA or miRNA switch. It is also referred to as let-7a-5p responsive mRNA based on the specific miRNA name.
  • let-7 miRNA is let-7a-5p
  • the target sequence and mRNA sequence can be determined and carried out in the same manner as the let-7a-5p target sequence described below.
  • a let-7a-5p is present in an mRNA containing a marker gene operably linked to a let-7a-5p target sequence
  • translation of the marker gene is controlled according to its activity.
  • marker protein the amount of protein translated from the marker gene (hereinafter referred to as marker protein) is reduced. It is mRNA that decreases quantitatively.
  • the target sequence of let-7a-5p refers to a sequence that can specifically bind to let-7a-5p.
  • the miRNA target sequence is preferably, for example, a sequence that is completely complementary to the indicator miRNA.
  • the let-7a-5p target sequence may have a mismatch (mismatch) with a completely complementary sequence as long as it can be recognized in let-7a-5p.
  • the mismatch from the sequence completely complementary to the marine RNA may be any mismatch that can be normally recognized by let-7a-5p in cells with high platelet-producing ability. It is said that there may be a discrepancy of about 40-50%. Such discrepancy is not particularly limited, class 1 base, 2 bases' 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases or 1%, 5% of all recognition sequences 10%. 20%. 30% or 40% discrepancy.
  • let-7a-5p target sequence on let-7a-5p provided by cells especially in the part other than the seed region, that is, about 16 bases on the 3 ′ side of let-7a-5p
  • the 5 ′ region in the target sequence corresponding to may contain multiple mismatches, and the seed region portion may contain no mismatches, or may contain mismatches of 1 base, 2 bases, or 3 bases.
  • a marker gene is a gene that is translated in a cell, functions as a marker, and encodes an arbitrary protein that enables selection of a cell group.
  • proteins that can be translated into cells and function as markers include, for example, proteins that can be visualized and quantified by assisting fluorescence, luminescence, coloration, or fluorescence, luminescence, or coloration. It may be.
  • fluorescent proteins blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (for example, hmAGl), ZsGreen, EmGFP, Green fluorescent proteins such as EGFP, GFP2, and HyPer; Yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, Turbo YFP, ZsYellow, mBanana; Kusabira Orange (eg, hmKO2), mOrange Color fluorescent proteins; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, etc .; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRe
  • a photoprotein can be exemplified by aequorin, but is not limited thereto.
  • proteins that assist fluorescence, luminescence, or coloration include, but are not limited to, enzymes that decompose fluorescence, luminescence, or color precursors such as luciferase, phosphatase, peroxidase, and ⁇ -lactamase.
  • enzymes that decompose fluorescence, luminescence, or color precursors such as luciferase, phosphatase, peroxidase, and ⁇ -lactamase.
  • the corresponding precursor is brought into contact with the cell, or the cell is handled. This can be done by introducing a precursor that
  • a protein that can function as a marker in a cell is a protein that directly affects the function of the cell.
  • a cell growth protein functions as a marker by proliferating only cells that express it and specifying the proliferated cells.
  • the cell killing protein causes cell death of a cell that expresses it, thereby killing the cell itself containing or not containing a specific miRNA, and functions as a marker indicating cell viability.
  • the cell signal factor functions as a marker by the cell that expresses it emits a specific biological signal and specifies this signal. Examples of the cell killing protein include Bax and Bim.
  • the marker gene may comprise a gene encoding a localization signal.
  • the localization signal include a nuclear localization signal, a cell membrane localization signal, a mitochondrial localization signal, a protein secretion signal, and more specifically, classical nuclear translocation sequences (NLS), M9 Examples include, but are not limited to, sequences, mitochondrial target sequences (MTS), and endoplasmic reticulum translocation sequences.
  • NLS classical nuclear translocation sequences
  • M9 include, but are not limited to, sequences, mitochondrial target sequences (MTS), and endoplasmic reticulum translocation sequences.
  • Such a localization signal is particularly advantageous when the screening step in the method of the present invention is performed on an image by imaging cytometry or the like described later.
  • mRNA having a second marker gene whose translation is controlled by the translation control factor is simultaneously introduced into a cell.
  • An example of mRNA having a second marker gene whose translation is controlled by such a protein is an mRNA having a second marker gene sequence operably linked to an RNA sequence to which the protein capable of controlling translation can be bound. Is done.
  • the miRNA target sequence and the marker gene are functionally linked to each other in the 5 ′ UTR, 3 ′ UTR, and / or the open reading frame (including the start codon) that encodes the mer strength gene.
  • the open reading frame including the start codon
  • at least one let-7a-5p target sequence is provided in the open reading frame.
  • the mRNA preferably comprises a Cap structure (7 methylguanosine 5 ′ phosphate), an open reading frame encoding a marker gene, and a poly A tail in the 5 ′ to 3 ′ orientation from the 5 ′ end, Provide at least one let-7a-5p target sequence in the 'UTR, 3'UTR, and / or in the open reading frame.
  • the location of the let-7a-5p target sequence in the mRNA may be 5'UTR or 3'UTR, may be within the open reading frame (3 'to the start codon), and these All may have a let-7a-5p target sequence.
  • the number of let-7a-5p target sequences may be one, two, three, four, five, six, seven, eight or more.
  • the number of bases and the type of base between the Cap structure and the let-7a-5p target sequence are arbitrary as long as they do not contain AUG as the start codon and it is difficult to form a stem structure or a three-dimensional structure.
  • the number of bases between the Cap structure and the let-7a-5p target sequence can be designed to be 0 to 50 bases, preferably 10 to 30 bases, but is not limited thereto.
  • the number of bases and the type of base between the let-7a-5p target sequence and the start codon may be arbitrary as long as they do not easily form a stem structure or a three-dimensional structure, and the let-7a-5p target sequence
  • the number of bases between and the start codon can be designed to be 0 to 50 bases, preferably 10 to 30 bases, but is not limited thereto. It has been confirmed that even when there are four let-7a-5p target sequences in 3′UTR, it is possible to achieve translational repression.
  • the let-7a-5p-responsive mRNA preferably contains a modified base such as pseudouridine or 5-methylcytidine instead of ordinary uridine and cytidine. This is to reduce cytotoxicity.
  • the positions of the modified bases can be all or part of the uridine and cytidine independently, and if they are part of the base, they can be random positions at an arbitrary ratio.
  • Let-7a-5p-responsive mRNA can be synthesized by a person skilled in the art by any method known in genetic engineering if the sequence is determined according to the above. In particular, it can be obtained by an in vitro transcription synthesis method using a template DNA containing a promoter sequence as a template.
  • the step of introducing let-7a-5p-responsive mRNA into a megakaryocyte cell group includes lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, Let-7a-5p-responsive mRNA can be directly introduced into cells contained in a cell group using DEAE dextran method, microinjection method, gene gun method and the like.
  • the let-7a-5p-responsive mRNA can be introduced into the megakaryocyte group by introducing a vector encoding the nucleic acid sequence of the designed let-7a-5p-responsive mRNA into the megakaryocyte group.
  • let-7a-5p-responsive let-7a-5p and control mRNA (hereinafter also referred to as control mRNA) are used, it is preferable to co-introduce these mRNAs into a megakaryocyte group. This is because the activity ratio of marker proteins expressed from two or more co-introduced mRNAs is constant within the cell population.
  • the introduction amount at this time varies depending on the mRNA to be introduced, the introduction method, the kind of the introduction reagent, and the like, and those skilled in the art can appropriately select these in order to obtain a desired translation amount.
  • control mRNA refers to mRNA that does not have a let-7a-5p target site and encodes a marker gene different from the marker gene encoded by let-7a-5p-responsive mRNA.
  • the amount of control mRNA introduced can also be appropriately selected by those skilled in the art to obtain a desired translation amount.
  • Control mRNA is mRNA that expresses a marker gene regardless of the presence of let-7a-5p.
  • let-7a-5p-responsive mRNA When let-7a-5p-responsive mRNA is introduced into a cell, if the given let-7a-5p is present as RISC in the cell, the marker gene encoded by let-7a-5p-responsive mRNA The amount of translation is suppressed.
  • the amount of translation is controlled quantitatively according to the let-7a-5p activity. That is, the amount of translation of the marker gene is relatively small in cells having a large amount of let-7a-5p, and the amount of translation of a marker gene is relatively large in cells having a small amount of let-7a-5p.
  • the predetermined let-7a-5p when the predetermined let-7a-5p does not exist in the cell, or when the predetermined let-7a-5p does not exist as RISC, the translation amount of the marker gene encoded by the let-7a-5p-responsive mRNA is suppressed. None happen. Therefore, the translation amount of the marker gene differs between a plurality of cells having different abundance of let-7a-5p.
  • let-7a-5p when let-7a-5p is present as RISC is also referred to as “when let-7a-5p activity is present”, and the amount of let-7a-5p in the activated state is The case where the amount is relatively large is also referred to as “when the activity of let-7a-5p is high”.
  • control mRNA expresses the marker protein regardless of the let-7a-5p activity. This is because even if introduced, since the let-7a-5p target sequence does not exist, translation control is not performed according to the expression level of let-7a-5p.
  • a cell sorting step (hereinafter also referred to as a sorting step) is performed using the translation amount of the marker gene as an index.
  • the selection step cells are selected from the translation amount of the marker gene as described above.
  • the present inventors have reported that megakaryocyte cells with relatively high let-7a-5p activity have relatively low platelet-producing ability and megakaryocyte cells with relatively low let-7a-5p activity It has been found that the productivity is relatively high (FIGS. 1 and 2).
  • a fluorescent protein is used as a marker protein, referring to the histogram of FIG. 1, the cell group located at the lower right of the line indicated by Control is a megakaryocyte cell whose translation is suppressed in response to let-7a-5p. Represents a group.
  • a cell group having a relatively high fluorescence intensity indicated by a white arrow indicates a megakaryocyte cell group having a relatively low let-7a-5p activity.
  • a cell group having a relatively low fluorescence intensity indicated by a black arrow indicates a megakaryocyte group having a relatively high let-7a-5p activity.
  • a megakaryocyte cell group can be selected based on such let-7a-5p activity and the translational control amount of the marker gene.
  • the level of fluorescence intensity is relative and may vary depending on the characteristics of the megakaryocyte cell group to be selected and the characteristics of the marker protein, so that those skilled in the art can appropriately determine and classify the threshold value. In that case, for example, a preliminary experiment on rescue of activity using an inhibitor of let-7a-5p, which will be described later in the Examples, or a preliminary experiment on the amount of platelet production may be performed to determine the threshold value. it can.
  • the marker gene may be a drug resistance gene.
  • the “drug resistance gene” may be any gene that expresses a protein having resistance to the corresponding drug. Examples include, but are not limited to, antibiotic resistance genes. Examples of antibiotic resistance genes include kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, blasticidin resistance gene, gentamicin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, etc. . In the present invention, preferably, a puromycin resistance gene or a blasticidin resistance gene is used as the antibiotic resistance gene.
  • the marker gene is a drug resistance gene
  • the expression of the drug resistance gene is selectively suppressed in a megakaryocyte group with high let-7a-5p activity. For this reason, the megakaryocyte group with high let-7a-5p activity is killed by a decrease in resistance to the corresponding drug. Therefore, these dead cells can be selectively removed.
  • the translation amount of the second marker gene is proportional to the expression level of let-7a-5p serving as an index. That is, when the cell group with high platelet production ability is a cell with a low expression level of the indicator let-7a-5p, a cell with a low translation amount of the second marker gene is selected, and the indicator let-7a When the expression level of -5p is high, cells having a high translation amount of the second marker gene can be selected.
  • a cell killing protein is used as a marker gene, cells with a high expression level of let-7a-5p serving as an index can be specifically induced to be removed.
  • the selection step can be performed by detecting a signal from the marker protein using a predetermined detection device.
  • the detection device include, but are not limited to, a flow cytometer, an imaging cytometer, a fluorescence microscope, a light emission microscope, and a CCD force mela.
  • a detection device those suitable for those skilled in the art can be used depending on the protein and the selection mode.
  • the marker protein is a fluorescent protein or a luminescent protein
  • a marker protein quantification method using a detection device such as a luminescence microscope, a CCD camera, or a luminometer is possible.
  • a detection device such as a luminescence microscope, a CCD camera, or a luminometer
  • the marker protein is a membrane-localized protein
  • the marker protein quantification process such as a magnetic cell separation device (MACS) is not required. Cell isolation methods are possible.
  • mRNA When mRNA is introduced into a megakaryocyte cell group in the proliferative phase, after selecting a cell group having a high platelet-producing ability, if necessary, culture for proliferation of the selected megakaryocyte cell group is performed. Can continue. Subsequently, the forced expression of the gene can be suppressed (released), the megakaryocyte can be matured, and used for the production of platelets according to the normal method for producing megakaryocyte. Alternatively, even when mRNA is introduced into a megakaryocyte group other than the proliferative phase, the production of megakaryocyte cells after selection is continued according to the normal method for producing megakaryocyte cells and used for platelet production. Can do.
  • a cell group having a relatively high platelet-producing ability can be selected from a megakaryocyte cell group using miRNA expression as an index. This selection can be performed during the growth phase of megakaryocytes. Therefore, only the selected cell group having a relatively high ability to produce platelets can be easily selected in the growth phase, and only the cell population can be increased, so that only the cell population having a high ability to produce platelets is matured thereafter. Therefore, the platelet production efficiency in the final stage is increased.
  • the present invention is a method for producing platelets, comprising: (I) a step of selecting a megakaryocyte cell group having high platelet production ability by the method described in the previous embodiment; (Ii) culturing the selected cell group; (Iii) collecting platelets from the culture obtained by the culturing step.
  • Platelets are one of the cellular components in blood and are characterized by CD41a positive and CD42b positive. Platelets play an important role in thrombus formation and hemostasis, and are also involved in tissue regeneration after injury and pathophysiology of inflammation. When platelets are activated by bleeding or the like, receptors for cell adhesion factors such as Integrin ⁇ IIB ⁇ 3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on the membrane. As a result, platelets aggregate and fibrin is coagulated by various blood coagulation factors released from the platelets, thereby forming a thrombus and promoting hemostasis.
  • Integrin ⁇ IIB ⁇ 3 glycoprotein IIb / IIIa; a complex of CD41a and CD61
  • the function of platelets can be measured and evaluated by a known method.
  • the amount of activated platelets can be measured using an antibody against PAC-1 that specifically binds to Integrin ⁇ IIB ⁇ 3 on the activated platelet membrane.
  • CD62P P-selectin
  • flow cytometry can be used by gating with an antibody against the activation-independent platelet marker CD61 or CD41, and then detecting the binding of anti-PAC-1 antibody or anti-CD62P antibody. These steps may be performed in the presence of adenosine diphosphate (ADP).
  • ADP adenosine diphosphate
  • evaluation of platelet function can be performed by examining whether or not it binds to fibrinogen in the presence of ADP. Binding of platelets to fibrinogen results in integrin activation required early in thrombus formation.
  • the evaluation of platelet function can also be performed by visualizing and observing the thrombus formation ability in vivo, as shown in FIG. 6 of WO 2011/034073.
  • the method for producing platelets according to this embodiment is based on the prior art methods disclosed by the present applicants disclosed in International Publication No. 2011/034073, International Publication No. 2012/157586, and International Publication No. 2014/123242.
  • megakaryocytes are selected using let-7a-5p-responsive mRNA according to the embodiment described above, and platelets are produced in the selected megakaryocytes. Therefore, it can be carried out in the same manner as these methods except that sorting is performed at a predetermined time.
  • the step of forcibly expressing to a necessary level after the selection may be continued. it can. Then, the process of suppressing the forced expression of a gene which matures a megakaryocyte can be implemented.
  • the platelets obtained in the present invention can be administered to patients as a preparation.
  • platelets obtained by the method of the present invention are, for example, human plasma, infusion, citric acid-containing physiological saline, glucose acetated Ringer's solution, PAS (platelet additive solution) (Gulliksson, H. et al., Transfusion, 32: 435-440, (1992)) and the like.
  • the storage period is about 3 to 7 days, preferably 4 days. As storage conditions, it is desirable to store with shaking and stirring at room temperature (20-24 degrees).
  • a megakaryocyte cell selection kit which is a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family.
  • MRNA comprising.
  • the microRNA selected from the let-7 family is let-7a-5p
  • the mRNA is a let-7a-5p-responsive mRNA or miRNA switch in the previous embodiment. It may be the described mRNA.
  • the cell selection kit does not have a let-7miRNA target sequence and contains a control mRNA containing a marker gene different from let-7miRNA-responsive mRNA, and reagents necessary for introduction into these megakaryocytes, let -7 miRNA inhibitors, let-7 miRNA, and the like.
  • the present invention is a method for producing platelets, comprising: (A) in an undifferentiated cell than a megakaryocyte cell, forcibly expressing an oncogene, a polycomb gene, and an apoptosis inhibitor gene to obtain an immortalized megakaryocyte cell; (B) suppressing the forced expression and culturing megakaryocyte cells, and in the step of obtaining the immortalized megakaryocyte cells, including a step of forcibly expressing the NR6A1 gene.
  • the step (a) for obtaining immortalized megakaryocyte cells and the step (b) for culturing megakaryocyte cells are as described above.
  • the step of forcibly expressing the NR6A1 gene is included in the step of obtaining immortalized megakaryocyte cells.
  • Forced expression of the NR6A1 gene is carried out by preparing a viral vector incorporating these genes in the same manner as the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene in the process of obtaining immortalized megakaryocyte cells. can do.
  • forced expression of NR6A1 gene and cancellation of forced expression are performed in International Publication No. 2011/034073, International Publication No. 2012/157586, International Publication No. 2014/123242, or Nakamura S et al, Cell Stem Cell. 14, 535-548, 2014, other known methods, or a method analogous thereto.
  • the template sequence of the NR6A1 gene is shown in SEQ ID NO: 49.
  • the forced expression of the NR6A1 gene can be performed in the step (a) of obtaining immortalized megakaryocyte cells, and the forced expression of the R6A1 gene is almost simultaneously with the cancellation of the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene. Cancellation can also be performed.
  • an oncogene, a polycomb gene, and an apoptosis inhibitor gene are forcibly expressed to obtain an immortalized megakaryocyte, A step of inhibiting forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and encoding the Lin28A gene in the cell in the step of obtaining the immortalized megakaryocyte cells Reducing the amount of methylation in the CpG island region of the promoter of the DNA.
  • the detection of methylation in the CpG island region of the promoter of the DNA encoding the Lin28A gene in cells was performed using MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells (Oncotarget. 2016 Mar 22; 7 (12 ): 14476-14485), for example, using QIAGEN's DNA extraction kit, Zymo Research's EZ DNA Methylation-Gold Kit, or similar commercial kits, and following these kit instructions can do.
  • the term “in the cell” as used herein may be a cell in the step (a) of obtaining an immortalized megakaryocyte cell or a cell in the step (b) of culturing the megakaryocyte cell, but immortalized.
  • the step of reducing the amount of methylation can be performed by shaking and culturing the cells.
  • the step of reducing the amount of methylation can be performed in step (a) of obtaining immortalized megakaryocyte cells, step (b) of culturing megakaryocyte cells, or both of these steps.
  • the determination reference time is the step (a) of obtaining immortalized megakaryocyte cells
  • the conditions for shaking culture are, for example, 50 rpm or more, preferably 100 rpm or more, although depending on the culture scale.
  • the period of shaking culture is preferably about 3 to 10 days.
  • the amount of methylation is decreased and the amount of methylation is, for example, about 20% or less, preferably about 13% or less, the activity of let-7a-5p can be suppressed, and maturation of megakaryocytes is inhibited. It is more preferable because the factor can be removed.
  • In another aspect of the method for producing platelets Inducing differentiation of pluripotent stem cells to obtain undifferentiated cells from megakaryocytes, A step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-suppressing gene in an undifferentiated cell than the megakaryocyte cell to obtain an immortalized megakaryocyte cell; Suppressing the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, the step of obtaining undifferentiated cells from the megakaryocyte cells, the pluripotent stem cell Culturing with a substrate containing Matrigel®.
  • This embodiment includes a step of inducing differentiation of pluripotent stem cells to obtain undifferentiated cells from megakaryocytes.
  • the pluripotent stem cells are not particularly limited, and include ES cells and iPS cells, but iPS cells are preferable.
  • iPS cells can be produced by introducing a specific reprogramming factor into somatic cells in the form of DNA or protein, and have almost the same properties as ES cells, such as pluripotency and proliferation ability by self-replication, Somatic cell-derived artificial stem cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. al.
  • Such may be used.
  • it may be an iPS cell produced by the method disclosed in International Publication WO2016 / 167329, and may be an iPS cell in which an oncogene, a polycomb gene, and an apoptosis-suppressing gene are knocked in.
  • an oncogene, a polycomb gene, and an apoptosis-suppressing gene are knocked in.
  • pluripotent stem cells are selected from Matrigel (registered trademark), laminin 421 or a fragment thereof, laminin 121 or a fragment thereof, or a combination thereof. Incubate with the substrate.
  • Matrigel (registered trademark) and laminin are both one of the extracellular cell matrices constituting the basement membrane.
  • Matrigel (registered trademark) is mainly composed of laminin and contains type IV collagen, heparin sulfate proteoglycan, entactin / nidogen, and growth factor.
  • Laminin has many isoforms, and each isoform has 5 types of ⁇ chains ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5), 3 types of ⁇ chains ( ⁇ 1, ⁇ 2, ⁇ 3) and 3 types.
  • ⁇ chains ( ⁇ 1, ⁇ 2, and ⁇ 3) each as a subunit chain associate on the C-terminal side to form a coiled-coil structure, thereby forming a heterotrimeric molecule stabilized by a disulfide bond.
  • the laminin 421 used in the present embodiment is composed of an ⁇ 4 chain, a ⁇ 2 chain and a ⁇ 1 chain
  • the laminin 121 is composed of an ⁇ 1 chain, a ⁇ 2 chain and a ⁇ 1 chain.
  • Laminin may be a natural form or a modified form in which one or more amino acid residues are modified as long as the biological activity is maintained.
  • Laminin can be obtained by any known production method, and a commercially available product can also be used.
  • the method for producing laminin is not particularly limited, and examples thereof include a method for purifying from laminin-expressing cells and a method for producing as a recombinant protein, but are not limited thereto.
  • the method for producing a laminin fragment is not particularly limited, and examples thereof include a method for digesting full-length laminin with a proteolytic enzyme such as elastase, fractionating and purifying the desired fragment, and a method for producing a recombinant protein. However, it is not limited to these. Both laminin and laminin fragments are preferably produced as recombinant proteins from the viewpoint of production amount, quality uniformity, production cost, and the like.
  • a “net-like structure” prepared from iPS cells is a three-dimensional sac-like structure (with a space inside) derived from ES cells or iPS cells, which is formed by an endothelial cell population or the like and has hematopoiesis inside. Including progenitor cells.
  • the cell culture conditions suitable for preparing the net-like structure from human iPS cells vary depending on the iPS cells used. For example, as the medium, IMDM supplemented with FBS at a final concentration of 15% is used. Also in this case, those appropriately added with growth factors and supplements can be used.
  • VEGF is added in an amount of 0 to 100 ng / ml, more preferably about 20 ng / ml.
  • the culture environment varies depending on the type of ES cells or iPS cells to be used. For example, conditions of 5% CO 2 , 36 to 38 ° C., preferably 37 ° C. can be used.
  • PCR amplification was performed under the conditions of 98 ° C, 10 sec, 68 ° C, 1 min for 25 cycles.
  • the genes encoding tagBFP and hmAG1 are obtained from template plasmids (pTAP-tagBFP and pFucci-S / G2 / M Green-Hyg (Expression vector), respectively) and corresponding primers and KOD-Plus-Neo (KOD-401, TOYOBO).
  • PCR amplification was carried out under conditions of 25 cycles of 94 ° C, 2 min, and 98 ° C, 10 sec, 68 ° C, 1 min for 25 cycles.
  • Full DNA Template which is the template for IVT, is 94 ° C 2 min using the PCR product prepared above and the corresponding primer (for those with miRNA response element, use oligo DNA instead of 5 'UTR). Thereafter, PCR amplification was performed under the conditions of 25 cycles of 98 ° C for 10 sec and 68 ° C for 1 min. All PCR products were purified using MiniElute PCR purification Kit (QIAGEN). However, those using a plasmid in the PCR reaction were treated with the restriction enzyme Dpn I before purification. The primers and oligonucleotide sequences used are shown in Table 2.
  • TKDN SeV2 Sendai
  • TKDN SeV2 Sendai
  • viruses 585A1, 585B1, 606A1, 648B1, and 692D2: using episomal vectors described in Okita K, et al, Stem Cells 31, 458-66, 2012
  • Immortalized megakaryocyte progenitor cell line Cl-7 (hereinafter, imMKCL) produced by introducing c-MYC, BMI1 and BCL-XL simultaneously into hematopoietic stem cells derived from human peripheral blood mononuclear cell-derived iPS cells)
  • imMKCL Immortalized megakaryocyte progenitor cell line Cl-7
  • C3H10T1 / 2 cells are not used, and in addition to human stem cell factor (SCF) 50 ng / ml, human thrombopoietin (TPO) 50 ng / ml, ROCK (Rho associated protein kinase) inhibitor Y27632 (International Publication No. 2012/157586) 10 ⁇ M, Aryl hydrocarbon receptor antagonist, StemRegenin 1 (SR1) 0.75nM, ADAM 17 inhibitor, KP457 15 ⁇ M (Hirata et al., Stem Cell Translational Medicine, in The cells were seeded in 2 ml / well of the medium supplemented with press) at 1 * 10 5 / ml.
  • SCF stem cell factor
  • TPO human thrombopoietin
  • ROCK Rho associated protein kinase inhibitor
  • Y27632 International Publication No. 2012/157586
  • Aryl hydrocarbon receptor antagonist Aryl hydrocarbon receptor antagonist
  • DOX ON medium is described in Takayama et al, Blood. 2008 Jun 1; 111 (11): 5298-306.
  • ESC differentiation medium is SCF, TPO at the concentration described above, and doxycycline (Doxycycline) is 0.5 ⁇ g. / ml was added.
  • the DOX OFF medium was prepared by adding SCF, TPO, Y27632, KP457 at the concentrations described above.
  • miRNA switch Human stem cell factor (SCF) 50 ng / ml, human thrombopoietin (TPO) 50 ng / ml in 6-well culture plates under the condition of doxycycline DOX ON (in the presence of doxycycline)
  • SCF stem cell factor
  • TPO human thrombopoietin
  • Stemfect (Stemgent) 1 ⁇ l + Buffer 5.25 ⁇ l, 100 ng / ⁇ l of control mRNA and let-7a-5p-responsive mRNA 0.75 ⁇ l each and Buffer 4.75 ⁇ l were mixed, left at room temperature for 15 minutes, and placed in a 96-well plate 50 ⁇ l of cell suspension suspended in 1-4 * 10 6 / ml was mixed and allowed to stand at room temperature for 30 minutes, and then 150 ⁇ l of medium was added and cultured overnight. The next morning, analysis was performed using FACS Aria II under the condition of DOX ON for cell proliferation.
  • let-7a-5p inhibitor and Negetive Control were purchased from Applied Biosystems. Based on preliminary studies, imMKCL was introduced by lipofection, Stemfect (Stemgent) 1 ⁇ l + Buffer 5.25 ⁇ l, 10 ⁇ M miRNA inhibitor and negative control 0.4 ⁇ l + Buffer 5.85 ⁇ l mixed, 15 The mixture was allowed to stand at room temperature for 50 minutes, mixed with 50 ⁇ l of a cell solution suspended in 1-4 * 10 6 / ml in a 96-well plate, allowed to stand at room temperature for 30 minutes, and then 150 ⁇ l of medium was added and cultured overnight.
  • Stemfect (Stemgent) 1 ⁇ l + Buffer 5.25 ⁇ l
  • 10 ⁇ M miRNA inhibitor and negative control 0.4 ⁇ l + Buffer 5.85 ⁇ l mixed, 15 The mixture was allowed to stand at room temperature for 50 minutes, mixed with 50 ⁇ l of a cell solution suspended in 1-4 * 10 6 / ml in a 96-well plate, allowed to stand at room temperature
  • RNA extraction and cDNA synthesis were performed, and quantitative PCR was performed.
  • RNA extraction was performed using miRNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions.
  • cDNA synthesis was performed using 500 ng of total RNA and using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo) according to the manufacturer's instructions.
  • Primers used for quantitative PCR are as described previously (Nakamura et al, Cell Stem Cell. 2014 Apr 3; 14 (4): 535-48.). Detailed sequences are listed in Table 5 below.
  • SYBR PreMix ExTaq II (Takara Bio Inc.) was used to prepare a mixed solution shown in Table 4 below.
  • StepOneplus Applied Biosystems
  • PCR cycle was 95 ° C for 1 minute ⁇ (95 ° C for 10 seconds ⁇ 55 ° C for 10 seconds ⁇ 72 ° C for 30 seconds) * 40 cycles.
  • GAPDH was used for endogenous control, and ⁇ CT method was used for data analysis.
  • the cause of heterogeneity in let-7a-5p activity may be the effect of 3 genes (C-MYC, BMI1, BCLXL) that are forcibly expressed in imMKCL. Although confirmed by quantitative PCR, there was no statistically significant difference (FIG. 5).
  • let-7a-5p functionally inhibits megakaryocyte maturation and platelet production
  • the cell population with high and low let-7a-5p activity was used to find the target of let-7a-5p.
  • RNA sequencing was performed on the populations and gene expression levels were compared. A part of the RNA sample used in FIG. 5 was used for RNA sequencing.
  • cDNA synthesis and library preparation were performed according to previous papers (T Nakamura et al, Nucleic Acids Res. 2015 May 19; 43 (9): e60.).
  • MiSeq Reagent Kit v3 Illumina
  • NR6A1 is a downstream target gene of Let-7a-5p, and NR6A1 is introduced into cells of a group with high Let-7a-5p activity. It was thought that platelet production that was suppressed by overexpression was rescued. To demonstrate that, a virus was made to overexpress NR6A1.
  • PCR 94 ° C. for 1 minute ⁇ (98 ° C. for 5 seconds ⁇ 57 ° C. for 10 seconds ⁇ 72 ° C. for 1 minute)
  • SEQ ID NO: 49 3 cycles ⁇ (98 ° C.
  • the tet-dependent NR6A1 expression vector can be obtained by incorporating it into the lentiviral vector (tet-dependent, GFP expression) used in the laboratory according to the determined procedure.
  • Produced. Virus was prepared based on this plasmid. Forward Primer; CGGTACCCGGGGATCCGCCACCATGGAGCGGGACGAACCG (SEQ ID NO: 47) Reverse Primer; GCTGACTAGAGGATCCTCATTCCTTGCCCACACTGGT (SEQ ID NO: 48)
  • NR6A1 is an important gene for megakaryocyte maturation and platelet production.
  • All siRNAs were purchased from Dharmacon, and as a result of preliminary studies, it was found that introduction into imMKCL can be performed in the same manner as in the experiment using let-7a-5p inhibitor and Negative Control described above with reference to FIG.
  • AHR Aryl Hydrocarbon Receptor
  • NR6A1 siRNA was purchased for 4 types of sequence, and it was proved that sufficient knockdown efficiency was obtained with # 1 and # 3 by preliminary examination. When NR6A1 siRNA # 1, # 3 and negative control siRNA were introduced into imMKCL, platelet production ability was significantly reduced by NR6A1 knockdown (FIG. 10 (b)).
  • let-7a-5p and NR6A1 are important genes not only in imMKCL but also in cord blood-derived megakaryocytes.
  • Umbilical cord blood CD34-positive cells purchased from DS Pharma Biomedical Co., Ltd. SCF 2.5ng / ml, TPO 100ng / ml, FLT3L 2.5ng / ml, IL6 50ng in the ESC differentiation medium described in the previous cell culture and platelet production experiments Culture was performed for 7 days using a medium supplemented with / ml. Among the cells obtained after 7 days, CD41-positive cells were separated using Milteny Biotech MACS beads, and let-7a-5p, negative control, NR6A1 siRNA and negative control were introduced by lipofection method.
  • LIN28A Of the 18 genes picked up as possible targets for let-7a-5p described above, it has been reported that LIN28A is a gene that inhibits Let-7a-5p maturation. In imMKCL, LIN28A regulates Let-7a-5p activity further upstream because there is a large difference in LIN28AmRNA expression between the group with high and low Let-7a-5p activity (Fig. 8). The experiment was conducted considering the possibility.
  • PCR enzyme For PCR enzyme, Toyobo's KOD Multi & Epi was used, and the PCR product thus obtained was used with Takara's Mighty-TA. 95 ° C for 5 minutes ⁇ (95 ° C for 30 seconds ⁇ 53 ° C for 30 seconds) ⁇ 72 ° C. 30 seconds) ⁇ 40 cycles ⁇ 72 ° C. for 10 minutes.
  • This PCR product is cloned using the Takara Bio Mighty TA Cloning Kit, and a colony PCR performed using the specified primers, with a band appearing at an appropriate position, is amplified by 10 plasmids for each sample. Asked Jen for sequencing.
  • LIN28A promoter region CpG island methylation suppressed LIN28A expression, resulting in increased activity of let-7a-5p and reduced NR6A1 expression to reduce platelet production, thus demethylating the methylation It was thought that platelet production would be increased by increasing LIN28A expression.
  • a method for releasing methylation a method of shaking culture of a megakaryocyte cell line was examined. That is, a part of the cell line in the state of DOX on was statically cultured as usual, and one was put 25 ml of culture solution and cells in a 125 ml flask, and cultured with shaking at 100 rpm for one week.
  • FIG. 12 (a) RNA was extracted from the cells cultured for one week with DOX on, and the expression levels of LIN28A and NR6A1 were measured by RT-PCR.
  • FIG. 12B is a graph showing the relative amounts of LIN28A and NR6A1 when the amount of mRNA extracted from statically cultured cells is 1. As shown in FIG. 12 (b), the expression level of both genes increased when shaking culture was performed.
  • FIG. 12 (c) is a graph showing the number of platelets produced under each of these conditions as a relative number with 100% when cultured as DOX on (Static) -DOX off (Static). From FIG. 12 (c), it can be seen that the cell group shake-cultured with DOX on produced many platelets. Furthermore, methylation of the LIN28A promoter region CpG island in DNA in cells that had been allowed to stand still for one week with DOX on or shake-cultured was analyzed by the same method as described above. The analysis result is shown in FIG. From FIG. 12 (d), the methylation rate decreased by 50% or more in the shaking culture, and it was suggested that there is a shaking culture method as one of the methods for releasing the methylation.
  • MKiPS technology was used to investigate whether the culture method at the stage of iPS cells would affect the platelet production ability after the megakaryocyte cell line was created.
  • Yamanaka 4 factors OCT4, SOX2, KLF4, CMYC
  • MKiPS cells were prepared and differentiated into megakaryocytes again to make DOX on, so that imMKCL was made and culture was continued as DOX on.
  • FIG. 13 (a) is a graph showing the amount of LIN28A mRNA measured by quantitative PCR in cells at various stages induced to differentiate from MKiPS cells, and the vertical axis represents the previous items of cell culture and platelet production.
  • the relative amount when the LIN28A expression level in SeV2 imMKCL described in detail is set to 1.
  • FIG. 13 (b) shows that the Let-7a-5p-responsive tagBFP mRNA represented by SEQ ID NO: 16 and the Control hmAG1 mRNA represented by SEQ ID NO: 18 were co-expressed from MKiPS cells after 14 days and 35 days after differentiation induction. The result of introduction and measurement of Let-7a-5p activity are shown. It was confirmed that the expression level of LIN28A decreased with passage (FIG. 13 (a) and Let-7a-5p activity increased (FIG. 13 (b)).
  • MKiPS11 and 12 were cultured on Laminin511 (trade name: iMatrix-511) and Matrigel (registered trademark). Specifically, Laminin or Matrigel is coated on a 6-well plate in advance, 5 * 10 4 / well on Laminin 511, and 1: 4 to 6 so that no colonies are destroyed on Matrigel. MKiPS11 and 12 were seeded and cultured for one week. The culture medium was exchanged approximately once every two days.
  • FIG. 13 (c) is a graph showing the relative expression level of LIN28AmRNA when the expression level of LIN28AmRNA extracted from the MKiPS11 cell line cultured with Laminin511 is 1.
  • the expression level of LIN28A was higher in the Matrigel group in both strains (FIG. 13 (c)).
  • the megakaryocyte was cultured on the conditions similar to the experiment of FIG. 2, and the platelet was produced.
  • FIG. 13 (d) is a graph showing the relative amount of platelet production when the number of platelets produced by megakaryocytes differentiated from the MKiPS11 cell line cultured in Laminin511 is taken as 100%. Platelet production was also increased in the Matrigel group (FIG. 13 (d)). Although not shown, when Laminin 121 and Laminin 421 were used instead of Matrigel, the same effect as Matrigel was demonstrated. This suggests that, at the stage of iPS cells, the culture method affects platelet production after preparation of megakaryocyte cell lines.

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Abstract

The present invention identifies heterogeneity in megakaryocytic cell groups and classifies cell groups. A method for identifying heterogeneity in cells in megakaryocytic cell groups. The method includes the following steps. (1) A step for introducing mRNA into megakaryocytic cell groups, the mRNA including: a nucleic acid sequence that is specifically recognized by microRNA; and a functionally linked marker gene. (2) A step for using the translated amount of the marker gene as an indicator to identify cell groups that have different characteristics.

Description

巨核球細胞群における細胞の不均質性を識別する方法及び血小板の製造方法Method for identifying cell heterogeneity in megakaryocyte cell group and method for producing platelets

　本発明は、巨核球細胞群における細胞の不均質性を識別する方法、及びmiRNAの発現を指標として巨核球細胞から血小板産生能が高い細胞を選別する方法に関する。
The present invention relates to a method for identifying heterogeneity of cells in a megakaryocyte group, and a method for selecting cells having high platelet-producing ability from megakaryocytes by using miRNA expression as an index.

　血小板製剤は、手術時や傷害時の大量出血、或いは、抗がん剤治療後の血小板減少に伴う出血傾向を呈する患者に対して、その症状の治療および予防を目的として投与される。
現在、血小板製剤の製造は、献血に依存しているが、感染症に関してより安全であり、血小板の安定供給が求められている。そのニーズに応えるべく、今日では、in vitroで培養した巨核球細胞から血小板を生産する方法が開発されている。本出願人らは、多能性幹細胞をソースとして不死化した、不死化巨核球前駆細胞株（immortalized megakaryocyte progenitor cell lines：imMKCL）の樹立方法を確立してきた（例えば、特許文献１、２、非特許文献１を参照）。
The platelet preparation is administered for the purpose of treatment and prevention of symptoms to a patient exhibiting a bleeding tendency associated with thrombocytopenia after treatment with an anticancer agent or massive bleeding during surgery or injury.
Currently, the manufacture of platelet products relies on blood donation, but there is a need for a safer supply of platelets and a stable supply of platelets. To meet these needs, a method for producing platelets from megakaryocytes cultured in vitro has been developed today. The present applicants have established a method for establishing an immortalized megakaryocyte progenitor cell lines (imMKCL) immortalized using pluripotent stem cells as a source (for example, Patent Documents 1 and 2; (See Patent Document 1).

国際公開第2012/157586号International Publication No.2012 / 157586 国際公開第2014/123242号International Publication No. 2014/123242


　不死化巨核球前駆細胞株は冷凍による保存が可能であり、人工的な多核化の促進が可能であるなど、多くの利点があり、この不死化巨核球前駆細胞株から、需要に応じて血小板を安定的に、大量に製造することが可能である。血小板のより効率的な製造を模索する中で、本発明者らは、樹立後の不死化巨核球前駆細胞株はその特性が不均一であること（heterogenuity）を初めて見出した。これは、従来の細胞表面マーカーによる識別においては、得ることができなかった知見である。例えば、巨核球細胞群には、血小板産生能の高い細胞群とそうでない細胞群が混在していることを見出した。また、このようなヘテロな細胞集団をそのまま用いて血小板産生を行うと、収量が不安定になるおそれがあることも見出した。巨核球細胞株について、増殖能を選別する古典的な選別方法である、limiting dilutionの方法が知られている。しかし、このような増殖能のみの選別では、成熟過程における血小板産生能の高い細胞群を選別することができない。　
The immortalized megakaryocyte progenitor cell line can be stored by freezing, and has many advantages such as the ability to promote artificial multinucleation. From this immortalized megakaryocyte progenitor cell line, platelets can be produced according to demand. Can be stably manufactured in large quantities. In search of more efficient production of platelets, the present inventors have found for the first time that the immortalized megakaryocyte progenitor cell line after establishment is heterogeneous. This is a finding that could not be obtained by conventional cell surface marker identification. For example, the megakaryocyte group has been found to contain a group of cells having high platelet-producing ability and a group of cells that are not. In addition, it has also been found that the yield may be unstable if platelet production is performed using such a heterogeneous cell population as it is. For megakaryocyte cell lines, a limiting dilution method, which is a classic selection method for selecting the proliferation ability, is known. However, such selection of only proliferative ability cannot select a cell group with high platelet production ability during the maturation process.

　一般的な細胞を選別する方法としては、細胞を定量的に分類する方法としては、例えば、マイクロアレイや次世代シーケンシングなどを用いた多変量の測定に基づく細胞のプロファイリングが知られている。また、近年、miRNAの発現を指標として生細胞を生きたまま、高精度で判別する方法が確立されている（国際公開第2015/105172号）。　
As a general method for sorting cells, as a method for quantitatively classifying cells, for example, cell profiling based on multivariate measurement using a microarray or next-generation sequencing is known. In recent years, a method has been established for accurately discriminating living cells using miRNA expression as an index (International Publication No. 2015/105172).

　巨核球細胞のheterogeneityを識別し、細胞群を選別する、特には、血小板産生能の高い細胞群を選別する方法が望まれている。
A method of identifying the heterogeneity of megakaryocyte cells and selecting a cell group, particularly, a method of selecting a cell group having a high platelet-producing ability is desired.

　本発明者らは上記の課題を解決すべく鋭意検討を行った結果、マイクロRNAの発現を指標として、巨核球細胞群を選別することができることを見出し、本発明を完成するに至った。　
As a result of intensive studies to solve the above problems, the present inventors have found that a megakaryocyte cell group can be selected using microRNA expression as an indicator, and the present invention has been completed.

　すなわち、本発明は次に記載の事項を提供するものである。
［１］　以下の工程を含む、巨核球細胞群における細胞の不均質性を識別する方法：（１）マイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを巨核球細胞群に導入する工程、および（２）当該マーカー遺伝子の翻訳量を指標として、特性の異なる細胞群を識別する工程。
 ［２］　以下の工程を含む、巨核球細胞群から血小板産生能の高い細胞群を選別する方法：
（１）let-7ファミリーから選択されるマイクロRNA によって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを巨核球細胞群に導入する工程、および（２）当該マーカー遺伝子の翻訳量を指標として、血小板産生能の高い細胞群を選別する工程。
［３］　前記mRNAを細胞群に導入する工程が、巨核球細胞の増殖期に実施される、［２］に記載の方法。
［４］　前記mRNAが、前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と、前記マーカー遺伝子が、5'から3'の方向に連結されている、［２］または［３］に記載の方法。
［５］　前記選別する工程が、前記let-7ファミリーから選択されるマイクロRNAの活性が低い細胞群で当該マーカー遺伝子の翻訳量が多い細胞群を選別する、［２］～［４］のいずれか１項に記載の方法。
［６］　前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列を持たず、前記マーカー遺伝子とは異なるマーカー遺伝子を含むコントロールmRNAを細胞群に導入する工程をさらに含む、［２］～［５］のいずれか１項に記載の方法。
［７］　前記選別する工程が、フローサイトメトリーを用いて行われる［１］～［６］のいずれか１項に記載の方法。
［８］　前記選別する工程が、イメージアナライザーを用いて行われる［１］～［６］のいずれか１項に記載の方法。
［９］　前記マーカー遺伝子が、薬剤耐性遺伝子である、［１］～［６］のいずれか１項に記載の方法。
［１０］　前記let-7ファミリーから選択されるマイクロRNAが、let-7a-5pである、［１］～［９］のいずれか１項に記載の方法。
［１１］　［１］～［１０］のいずれか１項に記載の方法により血小板産生能の高い細胞群を選別する工程と、
　選別した細胞群を培養する工程と、
　前記培養する工程により得られた培養物から血小板を回収する工程とを含む、血小板の製造方法。
［１２］　let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを含んでなる、巨核球細胞選別キット。
［１３］　前記mRNAが、前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と、前記マーカー遺伝子が、5'から3'の方向に連結されている核酸である、［１２］に記載の巨核球細胞選別キット。
［１４］　前記let-7ファミリーから選択されるマイクロRNAが、let-7a-5pである、［１２］または［１３］に記載の巨核球細胞選別キット。
［１５］　巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
を含み、前記不死化巨核球細胞を得る工程において、NR6A1遺伝子及び／またはLin28A遺伝子を強制発現させる工程を含む、血小板の産生方法。
［１６］　巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
を含み、前記不死化巨核球細胞を得る工程における前記細胞中の、Lin28A遺伝子をコードするDNAのプロモーターのCpGアイランド領域におけるメチル化量を減少させる工程を含む、血小板の産生方法。
［１７］　前記メチル化量を減少させる工程が、前記不死化巨核球細胞を得る工程における細胞を振盪培養する工程を含む、［１６］に記載の方法。
［１８］　多能性幹細胞を分化誘導して、巨核球細胞より未分化な細胞を得る工程と、
　前記巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
を含み、前記巨核球細胞より未分化な細胞を得る工程が、前記多能性幹細胞をMatrigel（登録商標）、ラミニン421もしくはその断片、またはラミニン121若しくはその断片、あるいはそれらの組み合わせから選択される基質を用いて培養する工程を含む、血小板の産生方法。
That is, the present invention provides the following matters.
[1] A method for identifying heterogeneity of cells in a megakaryocyte group comprising the following steps: (1) mRNA comprising a marker gene operably linked to a nucleic acid sequence specifically recognized by microRNA A step of introducing the cell into a megakaryocyte cell group, and (2) a step of identifying a cell group having different characteristics using the translation amount of the marker gene as an index.
[2] A method for selecting a cell group having a high platelet-producing ability from a megakaryocyte group including the following steps:
(1) introducing mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family into a megakaryocyte cell group; and (2) the marker gene. A process of selecting a cell group having a high platelet production ability using the translation amount of.
[3] The method according to [2], wherein the step of introducing the mRNA into a cell group is performed in the growth phase of megakaryocyte cells.
[4] The nucleic acid sequence specifically recognized by the microRNA selected from the let-7 family and the marker gene are linked in the 5 ′ to 3 ′ direction, [2] Or the method as described in [3].
[5] Any of [2] to [4], wherein the selecting step selects a cell group having a low amount of translation of the marker gene from a cell group having a low microRNA activity selected from the let-7 family. The method according to claim 1.
[6] The method further includes the step of introducing into the cell group a control mRNA that does not have a nucleic acid sequence specifically recognized by the microRNA selected from the let-7 family and includes a marker gene different from the marker gene. The method according to any one of [2] to [5].
[7] The method according to any one of [1] to [6], wherein the screening step is performed using flow cytometry.
[8] The method according to any one of [1] to [6], wherein the selecting step is performed using an image analyzer.
[9] The method according to any one of [1] to [6], wherein the marker gene is a drug resistance gene.
[10] The method according to any one of [1] to [9], wherein the microRNA selected from the let-7 family is let-7a-5p.
[11] A step of selecting a cell group having high platelet-producing ability by the method according to any one of [1] to [10];
Culturing the selected cell group;
And a step of recovering platelets from the culture obtained by the culturing step.
[12] A megakaryocyte cell selection kit comprising mRNA comprising a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family.
[13] The mRNA is a nucleic acid in which a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family and the marker gene are linked in a 5 ′ to 3 ′ direction. The megakaryocyte cell sorting kit according to [12].
[14] The megakaryocyte cell selection kit according to [12] or [13], wherein the microRNA selected from the let-7 family is let-7a-5p.
[15] A step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-inhibiting gene in an undifferentiated cell from a megakaryocyte to obtain an immortalized megakaryocyte,
And suppressing the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and in the step of obtaining the immortalized megakaryocyte cells, the NR6A1 gene and / or the Lin28A gene are forced A method for producing platelets, comprising a step of expressing.
[16] A step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-suppressing gene in an undifferentiated cell from a megakaryocyte to obtain an immortalized megakaryocyte cell;
A step of inhibiting forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and encoding the Lin28A gene in the cell in the step of obtaining the immortalized megakaryocyte cells A method for producing platelets, comprising a step of reducing the amount of methylation in a CpG island region of a promoter of DNA.
[17] The method according to [16], wherein the step of reducing the amount of methylation includes a step of shaking and culturing the cells in the step of obtaining the immortalized megakaryocyte cells.
[18] Inducing differentiation of pluripotent stem cells to obtain undifferentiated cells from megakaryocytes,
A step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-suppressing gene in an undifferentiated cell than the megakaryocyte cell to obtain an immortalized megakaryocyte cell;
Suppressing the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, the step of obtaining undifferentiated cells from the megakaryocyte cells, the pluripotent stem cell A method for producing platelets, comprising a step of culturing using a substrate selected from Matrigel®, laminin 421 or a fragment thereof, or laminin 121 or a fragment thereof, or a combination thereof.

　本発明によれば、マイクロRNAの活性を指標として利用することで、巨核球細胞群における細胞の不均質性を識別することができる。特には、巨核球細胞群から、高い分解能で血小板産生能が高い細胞群を選別することができる。本発明による方法は、細胞が生存した状態で適用することができるため、選別後の細胞を医療用途において利用することができる点で特に有利である。また、本発明の方法は、細胞群にmRNAを導入することにより実施することができ、このmRNAは約１日程度の半減期で分解されて、細胞内から速やかに除去されるため、細胞内におけるウイルス感染やDNAの残存により、ゲノムに損傷を与えるといった問題を生ずることがない。また、サイトメーターを利用した簡便な検出の方法においても有利である。本発明の方法によれば、巨核球細胞群の増殖期において、選別が可能であるため、血小板産生能が高い細胞群のみを選別して成熟させることで、効率的な血小板の産生が可能になる。
According to the present invention, the heterogeneity of cells in a megakaryocyte cell group can be identified by using the activity of microRNA as an index. In particular, from the megakaryocyte cell group, a cell group having a high resolution and a high platelet production ability can be selected. Since the method according to the present invention can be applied in a state where the cells are alive, it is particularly advantageous in that the cells after sorting can be used in medical applications. In addition, the method of the present invention can be carried out by introducing mRNA into a cell group, and this mRNA is decomposed with a half-life of about 1 day and rapidly removed from the cell. The virus infection and DNA persistence in the plant will not cause problems such as damage to the genome. It is also advantageous in a simple detection method using a cytometer. According to the method of the present invention, since selection is possible in the growth phase of megakaryocyte cells, efficient platelet production can be achieved by selecting and maturating only cells with high platelet production ability. Become.

図１は、let-7a-5p活性に応答して蛍光蛋白質の発現量が抑制されるmiRNA応答性mRNAの構成、及びmiRNA活性によらず蛍光蛋白質を発現するコントロールmRNAの構成、並びにこれを用いた細胞分離のスキームを示す図である。Figure 1 shows the structure of miRNA-responsive mRNA in which the expression level of fluorescent protein is suppressed in response to let-7a-5p activity, the structure of control mRNA that expresses fluorescent protein regardless of miRNA activity, and uses this It is a figure which shows the scheme of the cell separation which had. 図２は、SeV2 imMKCLを用いて、巨核球増殖条件（DOX ON）におけるlet-7a-5p活性が異なる細胞群があること、巨核球からの血小板産生条件（DOX OFF）におけるlet-7a-5p活性が低い細胞群は、血小板産生が多いことを示す。Fig. 2 shows that there are cell groups with different let-7a-5p activity under megakaryocyte growth conditions (DOX ON) using SeV2 imMKCL, and let-7a-5p under platelet production conditions (DOX OFF) from megakaryocytes A cell group with low activity indicates that there is much platelet production. 図３は、let-7a-5p活性に基づき、巨核球増殖条件（DOX ON）におけるlet-7a-5p活性の低い細胞集団(L)と高い細胞群（H）に分離した後、血小板産生条件（DOX OFF）とし、経時的にlet-7a-5p活性を測定した結果、let-7a-5p活性の低い細胞集団(L)の活性は低いままであり、高い細胞群（H）の活性は高いままであり、day４に至るまで活性の高さの傾向が変化しないことを示す。Figure 3 shows the platelet production conditions after separation into a cell group with low let-7a-5p activity (L) and a cell group with high activity (H) based on let-7a-5p activity. As a result of measuring let-7a-5p activity over time, the activity of the cell population with low let-7a-5p activity (L) remains low, and the activity of the high cell group (H) is It remains high, indicating that the trend of high activity does not change until day4. 図４は、let-7a-5p 阻害剤によりlet-7a-5p活性が低下すること（左図）、let-7a-5p 阻害剤によりlet-7a-5p活性が低下した細胞群(H + Inhibitor)は、巨核球増殖条件（DOX ON）におけるlet-7a-5p活性の低い細胞集団(L)と同等に血小板産生が高いことを示す（右図）。Figure 4 shows that let-7a-5p activity is reduced by a let-7a-5p inhibitor (left), and a group of cells whose let-7a-5p activity is reduced by a let-7a-5p inhibitor (H + Inhibitor ) Indicates that platelet production is as high as the cell population with low let-7a-5p activity (L) under megakaryocyte growth conditions (DOX ON) (right figure). 図５は、巨核球増殖条件（DOX ON）におけるlet-7a-5p活性の低い細胞集団(L)と高い細胞群（H）における3遺伝子（C-MYC, BMI1, BCLXLの発現量を定量PCRで確認した結果、発現量に有意差がないことを示す。Fig. 5 shows quantitative PCR for the expression levels of 3 genes (C-MYC, BMI1, and BCLXL) in a cell population with low let-7a-5p activity (L) and a high cell group (H) under megakaryocyte growth conditions (DOX ON) As a result of the confirmation, it is shown that there is no significant difference in the expression level. 図６は、let-7a-5pの標的を見つける目的で、let-7a-5p活性の高い細胞集団と低い細胞集団に対してRNAシークエンスを行い、遺伝子発現量比較を行い、活性の低い細胞集団で2倍以上発現している遺伝子は691個存在すること、さらに、miRSystem、miRDB、miRSearchの３つのmiRNA標的予測データベースを用いて、691個の遺伝子の中からlet-7a-5pの標的となりうるものを選び出したところ、18個の遺伝子がピックアップされることを示す。Figure 6 shows the purpose of finding the target of let-7a-5p. RNA sequencing is performed on cell populations with high and low let-7a-5p activity, gene expression is compared, and cell population with low activity. There are 691 genes that are expressed more than twice as much, and it can be a target of let-7a-5p among 691 genes using three miRNA target prediction databases: miRSystem, miRDB, and miRSearch After selecting one, it shows that 18 genes are picked up. 図７は、18個の遺伝子の発現量をプロットし、この中でLIN28A、NR6A1の二つの遺伝子がlet-7a-5p活性の低い細胞集団で多く発現していることを示す。FIG. 7 plots the expression levels of 18 genes, and shows that among them, the two genes LIN28A and NR6A1 are highly expressed in the cell population with low let-7a-5p activity. 図８は、LIN28Aはinhibitorを導入しても発現量は変化せず、NR6A1はlet-7a-5p活性の高い集団で発現量が下がり、inhibitorによりその効果がレスキューされることでlet-7a-5pの標的となっていることを示す。また、Lin28Ｂもlet-7a-5pの標的となっていることを示す。FIG. 8 shows that the expression level of LIN28A does not change even when an inhibitor is introduced, and the expression level of NR6A1 decreases in a population with high let-7a-5p activity, and the effect is rescued by the inhibitor. Indicates that it is a 5p target. Moreover, it shows that Lin28B is also a target of let-7a-5p. 図９は、LIN28B、NR6A1に加えて、IGF2BP1もlet-7a-5pの標的となっていることを示す。FIG. 9 shows that in addition to LIN28B and NR6A1, IGF2BP1 is also a target of let-7a-5p. 図１０は、NR6A1の血小板産生に対する影響を調べた実験結果を示す図であり、図１０（ａ）は、Let-7a-5p活性の高い群のimMKCLに、何も発現しないウイルスを感染させた場合（Mock）と、NR6A1を発現するウイルスを感染させた場合（NR6A1 O/E）の血小板産生数を、前者を１００％として示したものである。図１０（ｂ）は、SeV2 imMKCLに、Negative Control、NR6A1 siRNAの＃１と＃３をそれぞれ導入した場合の、血小板産生数を、Negative Controlを１００％として示したものである。図１０（ｃ）は、臍帯血由来巨核球細胞に、Negative Control、NR6A1 siRNAの＃１と＃３をそれぞれ導入した場合の、血小板産生数を、Negative Controlを１００％として示したものである。FIG. 10 is a diagram showing the experimental results of examining the effect of NR6A1 on platelet production. FIG. 10 (a) shows that imMKCL in a group with high Let-7a-5p activity was infected with a virus that does not express anything. The number of platelets produced in the case (Mock) and in the case of infection with a virus expressing NR6A1 (NR6A1 O / E) is shown with the former as 100%. FIG. 10B shows the number of platelets produced when Negative Control and NR6A1 siRNA # 1 and # 3 were introduced into SeV2VimMKCL, respectively, with Negative Control being 100%. FIG. 10 (c) shows the number of platelets produced when Negative Control and NR6A1 siRNA # 1 and # 3 were introduced into cord blood-derived megakaryocytes, respectively, with Negative Control as 100%. 図１１は、Lin28A,Bの血小板産生に対する影響を調べた実験結果を示す図であり、図１１（ａ）は、let-7a-5p活性の低い細胞集団(Group L)に、Lin28A siRNAを加えることにより、let-7a-5p活性が上昇したことを示す図である。図１１（ｂ）は、Let-7a-5p応答性tagBFP mRNA スイッチを用い、let-7a-5p活性の低い細胞集団にvehicleとスイッチを共導入したサンプル（Let7-Low vehicle）、let-7a-5p活性の低い細胞集団にLin28A siRNAとスイッチを共導入したサンプル（Let7-Low Lin28A siRNA）、let-7a-5p活性の低い細胞集団にLin28B siRNAとスイッチを共導入したサンプル（Let7-Low Lin28B siRNA）、let-7a-5p活性の高い細胞集団にvehicleとスイッチを共導入した導入したサンプル（Let7-High vehicle）のそれぞれにおける、tagBFPの平均蛍光強度（MFI）を示すグラフであり、let-7a-5p活性はLin28A siRNAにより上昇したが、Lin28A siRNAによっては上昇しなかったことを示す。図１１（ｃ）は、GroupL,Hそれぞれ7～8サンプルに対して前記Lin28A遺伝子をコードするDNAの、プロモーターのCpGアイランドに含まれる16個のCpG配列それぞれでメチル化しているかどうかを示した図であり、白丸が非メチル化、黒丸がメチル化したCpGを表す。図１１（ｄ）は、let-7a-5p活性の低い細胞集団(Group L)と、高い細胞集団(Group H)における、CpGのメチル化％である。FIG. 11 is a diagram showing the experimental results of examining the effect of Lin28A, B on platelet production. FIG. 11A shows the addition of Lin28A28siRNA to a cell population with low let-7a-5p activity (Group L). It is a figure which shows that let-7a-5p activity rose by this. FIG. 11 (b) shows a sample in which a vehicle and a switch are co-introduced into a cell population with low let-7a-5p activity using a Let-7a-5p-responsive tagBFP mRNA switch (Let7-Low vehicle), let-7a- Sample with co-introduction of Lin28A siRNA and switch into a cell population with low 5p activity (Let7-Low Lin28A siRNA), Sample with co-introduction of Lin28B siRNA and switch into a cell population with low let-7a-5p activity (Let7-Low Lin28B siRNA) ), A graph showing the mean fluorescence intensity (MFI) of tagBFP in each of the introduced samples (Let7-High vehicle) in which vehicle and switch were co-introduced into a cell population with high let-7a-5p activity, let-7a This shows that -5p activity was increased by Lin28A siRNA but not by Lin28A siRNA. FIG. 11 (c) shows whether the DNA encoding the Lin28A gene is methylated in each of 16 CpG sequences contained in the promoter CpG island for 7 to 8 samples of Group L and H, respectively. The white circle represents unmethylated and the black circle represents methylated CpG. FIG. 11 (d) shows CpG methylation% in a cell population with low let-7a-5p activity (Group (L) and a cell population with high activity (Group H). 図１２（ａ）は、従来、静置培養（static）としていたのを、DOX ON、DOX OFFの各状態における培養条件を、静置培養（static）または振盪培養（flask）に変更した実験スキームを示す図であり、図１２（ｂ）は、LIN28A及びNR6A1のそれぞれについて、静置培養（Static）した細胞から抽出されたmRNA量を１とした場合の相対量を示すグラフであり、図１２（ｃ）は、これらの各条件で得られた血小板産生数を、DOX on （Static）-DOX off（Static）として培養した場合を１００％とした相対数で表したグラフであり、図１２（ｄ）は、DOX onで一週間、静置培養（static）または振盪培養（flask）した細胞中のDNAにおけるLIN28Aプロモーター領域CpGアイランドのメチル化を、先に記載したのと同じ方法で解析した結果を示す。Fig. 12 (a) shows an experimental scheme in which static culture (static) was changed to static culture (static) or shaking culture (flask) instead of the culture conditions in each state of DOX ON and DOX OFF. FIG. 12 (b) is a graph showing the relative amounts of LIN28A and NR6A1 when the amount of mRNA extracted from statically cultured cells is 1. (C) is a graph showing the number of platelets produced under each of these conditions as a relative number with 100% when cultured as DOX on (Static) -DOX off (Static). d) The result of analyzing the methylation of the LIN28A promoter region CpG island in DNA in cells statically or shaken (flask) for 1 week in DOX on by the same method as described above. Indicates. 図１３は、分化誘導前のiPS細胞の培養条件が血小板産生に与える影響を調べた実験結果を示す図である。図１３（ａ）は、MKiPS細胞から、分化誘導させた各段階の細胞における定量PCRにて測定したLIN28A mRNA量を示すグラフであり、図１３（ｂ）は、MKiPS細胞から、分化誘導後、１４日、３５日の細胞について、Let-7a-5p活性を測定した結果を示し、図１３（ｃ）は、Laminin511で培養したMKiPS11細胞株から抽出したLIN28AmRNA発現量を１とした場合の、LIN28AmRNAの相対発現量を表すグラフであり、図１３（ｄ）は、Laminin511で培養したMKiPS11細胞株から分化誘導した巨核球細胞が産生した血小板数を１００％とした場合の、血小板産生数の相対量を表すグラフである。FIG. 13 is a diagram showing the experimental results of examining the influence of iPS cell culture conditions before differentiation induction on platelet production. FIG. 13 (a) is a graph showing the amount of LIN28A mRNA measured by quantitative PCR in cells at various stages induced to differentiate from MKiPS cells. FIG. 13 (b) shows the results after differentiation induction from MKiPS cells. The results of measuring the Let-7a-5p activity for the 14th and 35th cells are shown, and FIG. 13 (c) shows the LIN28AmRNA when the LIN28AmRNA expression level extracted from the MKiPS11 cell line cultured in Laminin511 is 1. 13 (d) is a graph showing the relative amount of platelet production when the number of platelets produced by megakaryocytes differentiated from the MKiPS11 cell line cultured with Laminin511 is taken as 100%. It is a graph showing.

　以下に本発明を、実施態様を示して詳細に説明する。しかしながら、本発明は以下の実施態様に限定されるものではない。　
Hereinafter, the present invention will be described in detail with reference to embodiments. However, the present invention is not limited to the following embodiments.

　［巨核球細胞群における細胞の不均質性を識別する方法］
　本発明は、一実施形態によれば、以下の工程を含む、巨核球細胞群における細胞の不均質性を識別する方法に関する。
（１）マイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを巨核球細胞群に導入する工程、および（２）当該マーカー遺伝子の翻訳量を指標として、特性の異なる細胞群を識別する工程。　
[Method for identifying cell heterogeneity in megakaryocyte populations]
The present invention, according to one embodiment, relates to a method for identifying cell heterogeneity in a megakaryocyte population comprising the following steps.
(1) introducing mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by microRNA into a megakaryocyte cell group, and (2) using the amount of translation of the marker gene as an index Identifying different cell groups.

　本実施形態においては、マイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAにより、巨核球細胞群における細胞の不均質性を識別することができる。従来、巨核球細胞は、以下に詳述する細胞表面マーカーにより「巨核球細胞」として識別される以外に、その増殖、成熟の過程において変化しうる特性を識別し、区別し、分別することができなかった。本実施形態は、mRNAにより、細胞の不均質性を識別する方法に関する。以下に具体的な例として、血小板産生能の高い細胞群を選別する方法について説明するが、本発明はこれに限定されるものではない。　
In the present embodiment, cell heterogeneity in a megakaryocyte group can be identified by mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by microRNA. Conventionally, megakaryocytes can be identified, distinguished, and differentiated in the process of proliferation and maturation in addition to being identified as “megakaryocytes” by cell surface markers described in detail below. could not. The present embodiment relates to a method for identifying cell heterogeneity by mRNA. As a specific example, a method for selecting a cell group having high platelet-producing ability will be described below, but the present invention is not limited to this.

　［血小板産生能の高い細胞群を選別する方法］
　本発明は、一実施形態によれば、以下の工程を含む、巨核球細胞群から血小板産生能の高い細胞群を選別する方法に関する。
（１）let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを細胞群に導入する工程、および（２）当該マーカー遺伝子の翻訳量を指標として、血小板産生能の高い細胞群を選別する工程。　
[Method for selecting cells with high platelet-producing ability]
According to one embodiment, the present invention relates to a method for selecting a cell group having high platelet-producing ability from a megakaryocyte cell group, comprising the following steps.
(1) introducing an mRNA containing a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family into a cell group, and (2) translation of the marker gene A process of selecting a cell group having high platelet production ability using the amount as an index.

　本発明の選別方法において、mRNAを導入する対象となる巨核球細胞群とは、以下に定義される巨核球細胞から主として構成される細胞群をいう。「巨核球細胞」とは、生体内においては骨髄中に存在する最大の細胞であり、血小板を放出することを特徴とする。また、細胞表面マーカーCD41a、CD42a、及びCD42b陽性で特徴づけられ、他に、CD9、CD61、CD62p、CD42c、CD42d、CD49f、CD51、CD110、CD123、CD131、及びCD203cからなる群より選択されるマーカーをさらに発現していることもある。「巨核球細胞」は、多核化（多倍体化）すると、通常の細胞の16～32倍のゲノムを有するが、本明細書において、単に「巨核球細胞」という場合、上記の特徴を備えている限り、多核化した巨核球細胞と多核化前の巨核球細胞の双方を含む。「多核化前の巨核球細胞」は、「未熟な巨核球細胞」、又は「増殖期の巨核球細胞」とも同義である。　
In the selection method of the present invention, the megakaryocyte cell group into which mRNA is introduced refers to a cell group mainly composed of megakaryocyte cells as defined below. A “megakaryocyte” is the largest cell present in the bone marrow in vivo and is characterized by releasing platelets. In addition, the cell surface markers CD41a, CD42a, and CD42b are positively characterized, and in addition, a marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c May be further expressed. A “megakaryocyte” has a genome 16 to 32 times that of a normal cell when multinucleated (multiploidy). In the present specification, the term “megakaryocyte” has the above characteristics. As long as it is, it includes both multinucleated megakaryocytes and pre-multinucleated megakaryocytes. “Pre-multinucleated megakaryocyte” is also synonymous with “immature megakaryocyte” or “proliferative megakaryocyte”.

　巨核球細胞は、公知の様々な方法で得ることができる。巨核球細胞の製造方法の非限定的な例として、国際公開第2011/034073号に記載された方法が挙げられる。同方法では、「巨核球細胞より未分化な細胞」において、癌遺伝子とポリコーム遺伝子を強制発現させることにより、無限に増殖する不死化巨核球細胞株を得ることができる。また、国際公開第2012/157586号に記載された方法に従って、「巨核球細胞より未分化な細胞」において、アポトーシス抑制遺伝子を強制発現させることによっても、不死化巨核球細胞株を得ることができる。これらの不死化巨核球細胞株は、遺伝子の強制発現を解除することにより、多核化が進み、血小板を放出するようになる。　
Megakaryocyte cells can be obtained by various known methods. Non-limiting examples of the method for producing megakaryocyte cells include the method described in International Publication No. 2011/034073. In this method, an immortalized megakaryocyte cell line that proliferates indefinitely can be obtained by forcibly expressing an oncogene and a polycomb gene in “an undifferentiated cell than a megakaryocyte”. Further, according to the method described in International Publication No. 2012/157586, an immortalized megakaryocyte cell line can also be obtained by forcibly expressing an apoptosis-suppressing gene in “undifferentiated cells than megakaryocyte cells”. . These immortalized megakaryocyte cell lines are polynucleated by releasing the forced expression of the gene and release platelets.

　巨核球細胞を得るために、上記の文献に記載された方法を組み合わせてもよい。その場合、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現は、同時に行ってもよく、順次行ってもよい。例えば、癌遺伝子とポリコーム遺伝子を強制発現させ、当該強制発現を抑制し、次にアポトーシス抑制遺伝子を強制発現させ、当該強制発現を抑制して、多核化巨核球細胞を得てもよい。また、癌遺伝子とポリコーム遺伝子とアポトーシス抑制遺伝子を同時に強制発現させ、当該強制発現を同時に抑制して、多核化巨核球細胞を得ることもできる。まず、癌遺伝子とポリコーム遺伝子を強制発現させ、続いてアポトーシス抑制遺伝子を強制発現させ、当該強制発現を同時に抑制して、多核化巨核球細胞を得ることもできる。本明細書において、遺伝子を強制発現させる工程を増殖期あるいは増殖可能な状態、強制発現を抑制する工程を成熟期ということもある。　
In order to obtain megakaryocyte cells, the methods described in the above documents may be combined. In that case, the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene may be performed simultaneously or sequentially. For example, a multinucleated megakaryocyte cell may be obtained by forcibly expressing an oncogene and a polycomb gene, suppressing the forced expression, then forcibly expressing an apoptosis suppressing gene, and suppressing the forced expression. In addition, multinucleated megakaryocyte cells can be obtained by forcibly expressing an oncogene, polycomb gene, and apoptosis-suppressing gene at the same time, and simultaneously suppressing the forced expression. First, an oncogene and a polycomb gene are forcibly expressed, followed by forcibly expressing an apoptosis-inhibiting gene, and the forcible expression is simultaneously suppressed to obtain multinucleated megakaryocyte cells. In the present specification, the step of forcibly expressing a gene may be referred to as a growth phase or a state where the gene can be proliferated, and the step of suppressing the forced expression may be referred to as a maturation phase.

　本発明において、「巨核球細胞より未分化な細胞」とは、巨核球への分化能を有する細胞であって、造血幹細胞系から巨核球細胞に至る様々な分化段階の細胞を意味する。巨核球より未分化な細胞の非限定的な例としては、造血幹細胞、造血前駆細胞、CD34陽性細胞、巨核球・赤芽球系前駆細胞（MEP）が挙げられる。これらの細胞は、例えば、骨髄、臍帯血、末梢血から単離して得ることもできるし、さらにより未分化な細胞であるES細胞、iPS細胞等の多能性幹細胞から分化誘導して得ることもできる。　
In the present invention, “cells that are undifferentiated from megakaryocytes” means cells that have the ability to differentiate into megakaryocytes and that have various differentiation stages ranging from hematopoietic stem cell lines to megakaryocytes. Non-limiting examples of cells that are undifferentiated from megakaryocytes include hematopoietic stem cells, hematopoietic progenitor cells, CD34 positive cells, megakaryocytes / erythroid progenitor cells (MEP). These cells can be obtained by isolation from, for example, bone marrow, umbilical cord blood, and peripheral blood, and further obtained by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells, which are more undifferentiated cells. You can also.

　本発明において、「癌遺伝子」とは、生体内において細胞の癌化を誘導する遺伝子のことをいい、例えば、MYCファミリー遺伝子（例えば、c-MYC、N-MYC、L-MYC）、SRCファミリー遺伝子、RASファミリー遺伝子、RAFファミリー遺伝子、c-Kit、PDGFR、Ablなどのプロテインキナーゼファミリー遺伝子が挙げられる。　
In the present invention, “oncogene” refers to a gene that induces canceration of a cell in a living body, such as a MYC family gene (for example, c-MYC, N-MYC, L-MYC), SRC family. And protein kinase family genes such as genes, RAS family genes, RAF family genes, c-Kit, PDGFR, and Abl.

　「ポリコーム遺伝子」とは、CDKN2a（INK4a/ARF）遺伝子を負に制御し、細胞老化を回避するために機能する遺伝子として知られている（小倉ら, 再生医療 vol.6, No.4, pp26-32；Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol.7, pp667-677, 2006；Proc. Natl. Acad. Sci. USA vol.100, pp211-216, 2003）。ポリコーム遺伝子の非限定的な例として、BMI1、Mel18、Ring1a/b、Phc1/2/3、Cbx2/4/6/7/8、Ezh2、Eed、Suz12、HADC、Dnmt1/3a/3bが挙げられる。　
“Polycomb gene” is known as a gene that negatively regulates the CDKN2a (INK4a / ARF) gene and functions to avoid cellular senescence (Ogura et al., Regenerative Medicine vol.6, No.4, pp26 -32; Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol.7, pp667-677, 2006; Proc. Natl. Acad. Sci. USA vol.100, pp211-216, 2003). Non-limiting examples of polycomb genes include BMI1, Mel18, Ring1a / b, Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HADC, Dnmt1 / 3a / 3b .

　「アポトーシス抑制遺伝子」とは、細胞のアポトーシスを抑制する機能を有する遺伝子をいい、例えば、BCL2遺伝子、BCL－xL遺伝子、Survivin遺伝子、MCL1遺伝子などが挙げられる。　
“Apoptosis suppressor gene” refers to a gene having a function of suppressing cell apoptosis, and examples thereof include BCL2 gene, BCL-xL gene, Survivin gene, and MCL1 gene.

　遺伝子の強制発現及び強制発現の解除は、国際公開第2011/034073号、国際公開第2012/157586号、国際公開第2014/123242、またはNakamura S et al, Cell Stem Cell. 14, 535-548, 2014に記載された方法、その他の公知の方法又はそれに準ずる方法で行うことができる。例えば、遺伝子の強制発現及びその解除のためにTet-on（登録商標）又はTet-off（登録商標）システムのような薬剤応答性の遺伝子発現誘導システムを用いる場合、強制発現する工程においては、対応する薬剤、例えば、テトラサイクリンまたはドキシサイクリンを培地に含有させ、これらを培地から除くことによって強制発現を抑制してもよい。　
The forced expression of genes and the release of forced expression are described in International Publication No. 2011/034073, International Publication No. 2012/157586, International Publication No. 2014/123242, or Nakamura S et al, Cell Stem Cell. 14, 535-548, It can be carried out by the method described in 2014, other known methods or a method analogous thereto. For example, when a drug-responsive gene expression induction system such as Tet-on (registered trademark) or Tet-off (registered trademark) system is used for forced expression of gene and its release, in the forced expression step, Corresponding agents such as tetracycline or doxycycline may be contained in the medium, and forced expression may be suppressed by removing them from the medium.

　遺伝子の強制発現及び強制発現の抑制（解除）を実施する際の巨核球細胞の培養条件は、通常の条件とすることができる。例えば、温度は約35℃～約42℃、約36℃～約40℃、又は約37℃～約39℃とすることができ、5～15%CO2及び／又は20％O2としてもよい。　
Culture conditions for megakaryocyte cells when forced expression of genes and suppression (cancellation) of forced expression are performed can be normal conditions. For example, the temperature can be about 35 ° C. to about 42 ° C., about 36 ° C. to about 40 ° C., or about 37 ° C. to about 39 ° C., and can be 5-15% CO 2 and / or 20% O 2. .

　巨核球細胞を培養する際の培地は特に限定されず、巨核球細胞から血小板が産生されるのに好適な公知の培地やそれに準ずる培地を適宜使用することができる。例えば、動物細胞の培養に用いられる培地を基礎培地として調製することができる。基礎培地としては、例えばIMDM培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium（ライフテクノロジーズ）およびこれらの混合培地が挙げられる。　
The medium for culturing megakaryocyte cells is not particularly limited, and a known medium suitable for producing platelets from megakaryocyte cells or a medium equivalent thereto can be appropriately used. For example, a medium used for animal cell culture can be prepared as a basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies) ) And mixed media thereof.

　培地には、血清又は血漿が含有されていてもよいし、あるいは無血清でもよい。必要に応じて、培地は、例えば、アルブミン、インスリン、トランスフェリン、セレン、脂肪酸、微量元素、2-メルカプトエタノール、チオールグリセロール、モノチオグリセロール（MTG）、脂質、アミノ酸（例えばL-グルタミン）、アスコルビン酸、ヘパリン、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類、サイトカインなどの1つ以上の物質も含有し得る。サイトカインとは、血球系分化を促進するタンパク質であり、例えば、血管内皮細胞増殖因子（VEGF）、トロンボポエチン（TPO）、各種TPO様作用物質、Stem Cell Factor（SCF）、ITS（インスリン－トランスフェリン－セレナイト）サプリメント、ADAM阻害剤、などが例示される。本発明において好ましい培地は、血清、インスリン、トランスフェリン、セリン、チオールグリセロール、アスコルビン酸、TPOを含むIMDM培地である。さらにSCFを含んでいてもよく、さらにヘパリンを含んでいてもよい。それぞれの濃度も特に限定されないが、例えば、TPOは、約10ng/mL～約200ng/mL、又は約50ng/mL～約100ng/mLとすることができ、SCFは、約10ng/mL～約200ng/mL、又は約50ng/mLとすることができ、ヘパリンは、約10U/mL～約100U/mL、又は約25U/mLとすることができる。ホルボールエステル（例えば、ホルボール-12-ミリスタート-13-アセタート；PMA）を加えてもよい。　
The culture medium may contain serum or plasma, or may be serum-free. If necessary, the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (eg L-glutamine), ascorbic acid It may also contain one or more substances such as heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like. Cytokines are proteins that promote blood cell differentiation. For example, vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, Stem Cell Factor (SCF), ITS (insulin-transferrin-selenite) ) Supplements, ADAM inhibitors, etc. A preferable medium in the present invention is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. Furthermore, SCF may be included, and heparin may also be included. Each concentration is not particularly limited, however, 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 is about 10 ng / mL to about 200 ng / mL. Heparin can be about 10 U / mL to about 100 U / mL, or about 25 U / mL. Phorbol esters (eg, phorbol-12-myristate-13-acetate; PMA) may be added.

　本発明に係る製造方法では、巨核球細胞の培養工程を、血清フリー及び／又はフィーダー細胞フリーの条件で行ってもよい。好ましくは、TPOを含有する培地で本発明の方法にしたがって製造された巨核球を培養することで行う方法である。血小板産生工程においては、血清フリー且つフィーダー細胞フリーで行うことができれば、得られた血小板を臨床的に用いる場合に免疫原性の問題が生じにくい。また、フィーダー細胞を用いないで血小板を産生させることができれば、フィーダー細胞を接着させる必要がないので、フラスコなどで浮遊培養することができるので、製造コストを抑制できるとともに、大量生産に適している。なお、フィーダー細胞を用いない場合は、conditioned mediumを使用してもよい。conditioned mediumは、特に限定されず、当業者が公知の方法等に従って作製することができるが、例えば、フィーダー細胞を適宜培養し、培養物からフィーダー細胞をフィルターで除去することによって得ることができる。　
In the production method according to the present invention, the step of culturing megakaryocyte cells may be performed under conditions of serum-free and / or feeder cell-free. Preferably, the method is carried out by culturing megakaryocytes produced according to the method of the present invention in a medium containing TPO. In the platelet production process, if it can be performed free of serum and feeder cells, the problem of immunogenicity hardly occurs when the obtained platelet is used clinically. In addition, if platelets can be produced without using feeder cells, it is not necessary to adhere feeder cells, so suspension culture can be performed in a flask or the like, which can suppress manufacturing costs and is suitable for mass production. . If no feeder cells are used, a conditioned medium may be used. The conditioned medium is not particularly limited and can be prepared according to methods known to those skilled in the art. For example, the conditioned medium can be obtained by appropriately culturing feeder cells and removing the feeder cells from the culture with a filter.

　具体的には、上記の遺伝子を巨核球細胞より未分化な細胞において強制発現させる工程は、当業者の常法にしたがって行うことができ、例えば、これらの遺伝子を発現するベクター、またはこれらの遺伝子をコードするタンパク質またはRNAの形態で巨核球細胞より未分化な細胞へ導入することによって成し得る。さらには、これらの遺伝子の発現を誘導する低分子化合物等を巨核球細胞より未分化な細胞と接触させることによって行うことができる。　
Specifically, the step of forcibly expressing the above genes in undifferentiated cells from megakaryocytes can be performed according to a conventional method of those skilled in the art. For example, vectors expressing these genes, or these genes Can be achieved by introducing the protein into an undifferentiated cell from a megakaryocyte in the form of a protein or RNA that encodes. Furthermore, it can be carried out by bringing low molecular weight compounds or the like that induce the expression of these genes into contact with undifferentiated cells from megakaryocytes.

　これらの遺伝子を発現するベクターとは、例えば、レトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス及びセンダイウィルスなどのウイルスベクター、動物細胞発現プラスミド（例、pA1-11，pXT1，pRc/CMV，pRc/RSV，pcDNAI/Neo）などが用いられ得る。単回導入により実施し得るという点において、好ましくは、レトロウイルスベクターまたはレンチウイルスベクターである。発現ベクターにおいて使用されるプロモーターの例としては、EF-αプロモーター、CAGプロモーター、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなどが用いられる。発現ベクターは、プロモーターの他に、所望によりエンハンサー、ポリＡ付加シグナル、選択マーカー遺伝子、SV40複製起点などを含有していてもよい。有用な選択マーカー遺伝子としては、例えば、ジヒドロ葉酸還元酵素遺伝子、ネオマイシン耐性遺伝子、ピューロマイシン耐性遺伝子等が挙げられる。　
Examples of vectors expressing these genes include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo) and the like can be used. A retroviral vector or a lentiviral vector is preferable in that it can be carried out by single introduction. Examples of promoters used in expression vectors include EF-α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia) Virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. are used. In addition to the promoter, the expression vector may optionally contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like. Useful selection marker genes include, for example, dihydrofolate reductase gene, neomycin resistance gene, puromycin resistance gene and the like.

　本発明の発現ベクターにおいて、テトラサイクリンまたはドキシサイクリンによりその遺伝子の発現を制御するため、プロモーター領域にはテトラサイクリン反応性エレメントを有している薬剤応答性ベクターであってもよい。この他にも、Cre-loxPシステムを使用して、遺伝子をベクターから切り出すため、loxP配列にて遺伝子またはプロモーター領域もしくはその両方をはさむようにloxP配列を設置された発現ベクターを用いてもよい。　
In the expression vector of the present invention, since the expression of the gene is controlled by tetracycline or doxycycline, a drug-responsive vector having a tetracycline-responsive element in the promoter region may be used. In addition, in order to cut out a gene from a vector using the Cre-loxP system, an expression vector in which a loxP sequence is placed so as to sandwich the gene and / or promoter region with the loxP sequence may be used.

　巨核球細胞の製造においては、アポトーシス抑制遺伝子を強制発現させて培養している細胞を、(a)アクトミオシン複合体機能阻害剤で処理する工程、(b)ROCK阻害剤で処理する工程、(c)HDAC阻害剤で処理する工程、の少なくとも１つを含む。これらの処理により、より安定な増殖と多核化を進めることができる。　
In the production of megakaryocytes, cells that have been cultured with forced expression of an apoptosis-suppressing gene are treated with (a) an actomyosin complex function inhibitor, (b) a treatment with a ROCK inhibitor, c) at least one of treating with an HDAC inhibitor. By these treatments, more stable proliferation and multinucleation can be promoted.

　アクトミオシン複合体機能阻害剤、ROCK阻害剤、HDAC阻害剤等で細胞を処理する場合の至適濃度などは、当業者であれば、予備的な実験によって予め決定することができる。また、処理する期間や方法なども、当業者において適宜選択することができる。例えば、ミオシン重鎖II ATPase阻害剤であるブレビスタチン処理の場合、2～15μg/ml、あるいは、5～10μg/ml程度を培養液中に添加し、培養期間としては、例えば、5～10日間程度、特に、6～7日間程度が好ましい。また、ROCK阻害剤であるY27632は、5～15μM、あるいは、8～12μM、好ましくは10μM程度で使用し、HDAC阻害剤であるバルプロ酸は、0.1～1mM、あるいは、0.2～0.7mM、好ましくは0.5mM程度で使用することができる。Y27632、バルプロ酸の処理時間としては、10～21日間、好ましくは14日間程度である。　
A person skilled in the art can determine the optimum concentration in the case of treating cells with an actomyosin complex function inhibitor, a ROCK inhibitor, an HDAC inhibitor or the like by preliminary experiments. Further, a period and a method for processing can be appropriately selected by those skilled in the art. For example, in the case of treatment with blebbistatin, which is a myosin heavy chain II ATPase inhibitor, 2 to 15 μg / ml or 5 to 10 μg / ml is added to the culture solution, and the culture period is, for example, 5 to 10 days. About 6 to 7 days is preferable. Moreover, RO276 inhibitor Y27632 is used at 5 to 15 μM, or 8 to 12 μM, preferably about 10 μM, and HDAC inhibitor valproic acid is 0.1 to 1 mM, or 0.2 to 0.7 mM, preferably Can be used at about 0.5 mM. The treatment time for Y27632 and valproic acid is 10 to 21 days, preferably about 14 days.

　ROCK（Rho-associated coiled－coil forming kinase／Rho結合キナーゼ）阻害剤としては、例えば、〔（R）－（＋）－トランス－N－（4－ピリジル）－4－（1－アミノエチル）-シクロヘキサンカルボキサミド・2HCl・H2O〕（Y27632）などを挙げることができる場合によっては、Rhoキナーゼ活性を阻害するような抗体、あるいは、核酸（例えば、shRNAなど）も、ROCK阻害剤として使用することができる。　
Examples of ROCK (Rho-associated coiled-coil forming kinase) inhibitors include [(R)-(+)-trans-N- (4-pyridyl) -4- (1-aminoethyl)- In some cases, such as cyclohexanecarboxamide, 2HCl, H 2 O] (Y27632), antibodies that inhibit Rho kinase activity, or nucleic acids (eg, shRNA) should also be used as ROCK inhibitors. Can do.

　HDAC阻害剤は、ヒストン脱アセチル化酵素（HDAC）活性を阻害する作用をもつ。これまでに、HDAC阻害剤は多数知られており、例えば、バルプロ酸、トリコスタチンA、SAHA（スベロイルアニリドヒドロキサム酸）、APHA（アロイルピロリルヒドロキシアミド）などを挙げることができ、特に、バルプロ酸、トリコスタチンAなどを好適に使用することができる。使用薬物が塩の形態を有する場合には、塩の形態で使用してもよい。　
The HDAC inhibitor has an action of inhibiting histone deacetylase (HDAC) activity. So far, many HDAC inhibitors are known, and examples include valproic acid, trichostatin A, SAHA (suberoylanilide hydroxamic acid), APHA (aroylpyrrolylhydroxyamide), etc. Valproic acid, trichostatin A and the like can be preferably used. When the drug used has a salt form, it may be used in a salt form.

　アクトミオシン複合体機能阻害剤、ROCK阻害剤、HDAC阻害剤等で細胞を処理する場合の至適濃度などは、当業者であれば、予備的な実験によって予め決定することができる。また、処理する期間や方法なども、当業者において適宜選択することができる。例えば、ミオシン重鎖II ATPase阻害剤であるブレビスタチン処理の場合、2～15μg/ml、あるいは、5～10μg/ml程度を培養液中に添加し、培養期間としては、例えば、5～10日間程度、特に、6～7日間程度が好ましい。また、ROCK阻害剤であるY27632は、5～15μM、あるいは、8～12μM、好ましくは10μM程度で使用し、HDAC阻害剤であるバルプロ酸は、0.1～1mM、あるいは、0.2～0.7mM、好ましくは0.5mM程度で使用することができる。Y27632、バルプロ酸の処理時間としては、10～21日間、好ましくは14日間程度である。　
A person skilled in the art can determine the optimum concentration in the case of treating cells with an actomyosin complex function inhibitor, a ROCK inhibitor, an HDAC inhibitor or the like by preliminary experiments. Further, a period and a method for processing can be appropriately selected by those skilled in the art. For example, in the case of treatment with blebbistatin, which is a myosin heavy chain II ATPase inhibitor, 2 to 15 μg / ml or 5 to 10 μg / ml is added to the culture solution, and the culture period is, for example, 5 to 10 days. About 6 to 7 days is preferable. Moreover, RO276 inhibitor Y27632 is used at 5 to 15 μM, or 8 to 12 μM, preferably about 10 μM, and HDAC inhibitor valproic acid is 0.1 to 1 mM, or 0.2 to 0.7 mM, preferably Can be used at about 0.5 mM. The treatment time for Y27632 and valproic acid is 10 to 21 days, preferably about 14 days.

　強制発現させる工程の後、当該工程で得られた巨核球または巨核球前駆細胞に対して、強制発現を抑制して培養する工程をさらに含む。強制発現を抑制あるいは停止する方法として、例えば、薬剤応答性ベクターを用いて強制発現をしている場合には、対応する薬剤と当該細胞と接触させないことによって達成させてもよい。具体的には、ドキシサイクリンやテトラサイクリンにより遺伝子の強制発現を行う場合には、これらを除去した培地において細胞を培養することにより、強制発現を抑制することができる。この他にも、上記のLoxPを含むベクターを用いた場合は、Creリコンビナーゼを当該細胞に導入することによって達成させてもよい。さらに、一過性発現ベクター、およびRNAまたはタンパク質導入を用いた場合は、当該ベクター等との接触を止めることによって達成させてもよい。本工程において用いられる培地は、上記と同一の培地を用いて行うことができる。　
After the forced expression step, the method further includes a step of culturing the megakaryocyte or megakaryocyte progenitor cell obtained in the step while suppressing the forced expression. As a method for suppressing or stopping forced expression, for example, when forced expression is performed using a drug-responsive vector, it may be achieved by not contacting the corresponding drug with the cell. Specifically, when the gene is forcibly expressed by doxycycline or tetracycline, the forced expression can be suppressed by culturing the cells in a medium from which these are removed. In addition, when the above-mentioned vector containing LoxP is used, it may be achieved by introducing Cre recombinase into the cell. Further, when a transient expression vector and RNA or protein introduction are used, the contact with the vector or the like may be stopped. The medium used in this step can be performed using the same medium as described above.

　強制発現を抑制または停止して培養する際の培養条件は、上記のとおりである。また培養期間については、巨核球の数などをモニターしながら、適宜決定することが可能であるが、例えば、2日間～10日間、好ましくは3日間～7日間程度である。少なくとも3日以上であることが望ましい。また、培養期間中は、適宜、継代を行うことが望ましい。　
The culture conditions for culturing while suppressing or stopping forced expression are as described above. The culture period can be appropriately determined while monitoring the number of megakaryocytes, and is, for example, about 2 days to 10 days, preferably about 3 days to 7 days. Desirably at least 3 days. In addition, it is desirable to perform subculture as appropriate during the culture period.

　遺伝子の強制発現を抑制する工程においても、培地にROCK阻害剤及び／又はアクトミオシン複合体機能阻害剤を加える。ROCK阻害剤及びアクトミオシン複合体機能阻害剤としては、上述した多核化巨核球の製造方法で使用したものと同じものを使用することができる。ROCK阻害剤としては、例えばY27632が挙げられる。アクトミオシン複合体機能阻害剤としては、ミオシン重鎖II ATPase阻害剤である、ブレビスタチンが挙げられる。ROCK阻害剤を単独で加えてもよく、ROCK阻害剤とアクトミオシン複合体機能阻害剤を単独で加えてもよいし、これらを組み合わせて加えてもよい。　
Also in the step of suppressing forced gene expression, a ROCK inhibitor and / or an actomyosin complex function inhibitor is added to the medium. As a ROCK inhibitor and an actomyosin complex function inhibitor, the same thing as what was used by the manufacturing method of the multinucleated megakaryocyte mentioned above can be used. Examples of the ROCK inhibitor include Y27632. Examples of the actomyosin complex function inhibitor include blebbistatin, which is a myosin heavy chain II ATPase inhibitor. A ROCK inhibitor may be added alone, a ROCK inhibitor and an actomyosin complex function inhibitor may be added alone, or a combination thereof may be added.

　ROCK阻害剤及び／又はアクトミオシン複合体機能阻害剤は、0.1μM～30μMで加えることが好ましく、例えば0.5μM～25μM、5μM～20μM等としてもよい。ROCK阻害剤及び／又はアクトミオシン複合体機能阻害剤を加えてからの培養期間は1日～15日とすることができ、3日、5日、7日等としてもよい。ROCK阻害剤及び／又はアクトミオシン複合体機能阻害剤を加えることにより、CD42b陽性血小板の割合をさらに増加させることが可能である。　
The ROCK inhibitor and / or actomyosin complex function inhibitor is preferably added at 0.1 μM to 30 μM, for example, 0.5 μM to 25 μM, 5 μM to 20 μM, and the like. The culture period after adding the ROCK inhibitor and / or the actomyosin complex function inhibitor may be 1 to 15 days, and may be 3 days, 5 days, 7 days, or the like. By adding a ROCK inhibitor and / or an actomyosin complex function inhibitor, it is possible to further increase the proportion of CD42b positive platelets.

　なお、上述の巨核球細胞の製造方法の非限定的な例を開示するUS 2012-0238023 A1（国際公開第2011/034073号）、US 2014-0127815 A1（国際公開第2012/157586号）、US 2016-0002599 A1（国際公開第2014/123242号）は、引用することにより本明細書の一部をなすものとする。　
In addition, US 2012-0238023 A1 (International Publication No. 2011/034073), US 2014-0127815 A1 (International Publication No. 2012/157586), US, which discloses a non-limiting example of the above-described method for producing megakaryocytes 2016-0002599 A1 (International Publication No. 2014/123242) is hereby incorporated by reference.

　ｍRNAを導入する巨核球細胞群は、好ましくは、増殖可能な状態にある巨核球細胞群である。増殖可能な状態にある巨核球細胞群は、先に記載した巨核球細胞の製造方法において、巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子からなる群より選択される遺伝子の少なくとも１つを強制発現した状態をいう。したがって、例えば、ドキシサイクリンやテトラサイクリンを含む増殖用の培地で培養している状態の巨核球細胞群であってもよく、他の方法で遺伝子を強制発現した状態にある巨核球細胞群であってもよい。増殖用の培地で培養されている期間であれば、ｍRNAをの導入時期は、特に限定されることはなく、増殖のための培養開始直後であってもよく、適切な倍加速度による増殖が確認できる培養開始後、３日～４日であってもよい。なお、mRNAの導入後も、通常の方法に従って、必要な期間にわたって、増殖のための培養を５ヶ月以上継続することが可能である。　
The megakaryocyte group into which the mRNA is introduced is preferably a megakaryocyte group in a proliferative state. The megakaryocyte group in a proliferative state is selected from the group consisting of an oncogene, a polycomb gene, and an apoptosis-suppressing gene in a cell that is undifferentiated from the megakaryocyte in the method for producing a megakaryocyte described above. A state in which at least one of the genes is forcibly expressed. Therefore, for example, it may be a group of megakaryocytes cultured in a growth medium containing doxycycline or tetracycline, or may be a group of megakaryocytes in which a gene is forcibly expressed by other methods. Good. There is no particular limitation on the time of introduction of mRNA as long as it is cultured in a growth medium, and it may be immediately after the start of the culture for growth. It may be 3 to 4 days after the start of culture. Even after the introduction of mRNA, the culture for propagation can be continued for 5 months or more over a necessary period according to a normal method.

　本発明の方法において、選別される「血小板産生能が高い細胞群」とは、let-7ファミリーから選択されるマイクロRNAの発現を指標として、他の細胞群と分類されるある一群の細胞をいう。　
In the method of the present invention, the selected “cell group having high platelet-producing ability” refers to a group of cells classified as another cell group using the expression of microRNA selected from the let-7 family as an index. Say.

　本発明において、「血小板産生能が高い細胞群を選別する」とは、本発明者らによりヘテロな細胞集団であることが見いだされた巨核球細胞群の中から、相対的に血小板産生能が高い細胞群について、他の細胞群と区別可能な、すなわち検出可能な信号情報を提示することをいい、特には、視覚的に認識可能な情報を提示することをいうものとする。なお「視覚的に認識可能な構報」とは、細胞が直接的に視認しうる信号を発することに限定されるものではなく、細胞が発した信号を、数値、図表又は画像等によって視覚的に認識可能な情報に変換された情報であり、当業者が視覚的に認識可能な情報をいう。本明細書において、選別するという文言には、当該選別の後に、血小板産生能が高い細胞群を認識し、血小板産生能が高い細胞群を識別し、血小板産生能が高い細胞群を同定し、血小板産生能が高い細胞群を分類し、血小板産生能が高い細胞群を単離し、所望しない細胞群を除去し、所望の綱胞群の生死を判定し、血小板産生能が高い細胞群の特定の生物学的な信号を検出あるいは定量し、血小板産生能が高い細胞群の特定の物理的あるいは化学的な信号に基づいて分画することを含んでも良い。　
In the present invention, “selecting a cell group having a high platelet-producing ability” means that the platelet-producing ability is relatively selected from the megakaryocyte group found by the present inventors to be a heterogeneous cell group. For a high cell group, it means to present signal information that can be distinguished from other cell groups, that is, to be detectable, and in particular, to present visually recognizable information. Note that “visually recognizable information” is not limited to emitting a signal that can be directly recognized by the cell, but the signal emitted by the cell is visually expressed by a numerical value, a chart, an image, or the like. It is information that has been converted into information that can be recognized by the person, and is information that can be visually recognized by those skilled in the art. In the present specification, the term sorting means that after the sorting, a cell group having a high platelet-producing ability is recognized, a cell group having a high platelet-producing ability is identified, a cell group having a high platelet-producing ability is identified, Classify cells with high platelet-producing ability, isolate cells with high platelet-producing ability, remove undesired cells, determine the viability of desired chondrocytes, and identify cells with high platelet-producing ability Detection or quantification, and fractionation based on a specific physical or chemical signal of a cell group having a high platelet-producing ability.

　本発明の方法においては、指標となるマイクロRNAは、let-7ファミリーから選択されるマイクロRNAである。let-7ファミリーに属するマイクロRNAとしては、hsa-let-7a-5p、hsa-let-7b-5p、hsa-let-7d-5p、hsa-let-7e-5p、hsa-let-7f-5p、hsa-let-7g-5p、hsa-let-7i-5p、hsa-let-7a-3p、hsa-let-7b-3p、hsa-let-7d-3p、hsa-let-7e-3p、hsa-let-7f-1-3p、hsa-let-7f-2-3p、hsa-let-7g-3p、hsa-let-7i-3p、hsa-let-7a-2-3p、hsa-let-7c-5p、hsa-let-7c-3p、hsa-miR-98-5p、hsa-miR-98-3p、hsa-miR-99a-5p、hsa-miR-99a-3p、hsa-miR-99b-5p、hsa-miR-99b-3p、has-miR-100-5p、hsa-miR-100-3p、hsa-miR-125a-5p、hsa-miR-125a-3p、hsa-miR-125b-5p、hsa-miR-125b-1-3p、hsa-miR-125b-2-3p、hsa-miR-202-5p、hsa-miR-202-3pが挙げられるが、これらには限定されない。指標となるマイクロRNAとしては、中でも、let-7a-5p、let-7g-5p、let-7i-5pが好ましいが、これらには限定されない。なお、以下の本明細書の説明において、let-7ファミリーから選択されるマイクロRNAを、let-7 miRNAと指称する場合がある。したがって、巨核球細胞群に導入されるメッセンジャーRNA（mRNA）は、let-7 miRNAによって特異的に認識される配列（let-7 miRNAの標的配列とも指称する）と機能的に連結したマーカー遺伝子を含む。かかるmRNAを、let-7 miRNA応答性mRNA、あるいはmiRNA switchとも指称する。また、具体的なmiRNAの名称に基づいてlet-7a-5p応答性mRNAとも指称する。以下、let-7 miRNAが、let-7a-5pである場合について詳細に説明するが、本発明は、当該マイクロRNAに関するものには限定されない。他のlet-7 miRNAに関しても、下記のlet-7a-5p標的配列と同様に標的配列及びmRNAの配列を決定し、実施することができる。let-7a-5p標的配列と機能的に連結したマーカー遺伝子を含むmRNAは、let-7a-5pが存在する場合に、その活性に応じてマーカー遺伝子の翻訳が制御される。好ましくは、let-7a-5pが存在する場合に、その活性に応じてマーカー遺伝子の翻訳が抑制されることで、当該マーカー遺伝子より翻訳された蛋白質（以下、マーカー蛋白質と指称する）の量が定量的に減少するmRNAである。　
In the method of the present invention, the indicator microRNA is a microRNA selected from the let-7 family. The microRNAs belonging to the let-7 family include hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7f-5p , Hsa-let-7g-5p, hsa-let-7i-5p, hsa-let-7a-3p, hsa-let-7b-3p, hsa-let-7d-3p, hsa-let-7e-3p, hsa -let-7f-1-3p, hsa-let-7f-2-3p, hsa-let-7g-3p, hsa-let-7i-3p, hsa-let-7a-2-3p, hsa-let-7c -5p, hsa-let-7c-3p, hsa-miR-98-5p, hsa-miR-98-3p, hsa-miR-99a-5p, hsa-miR-99a-3p, hsa-miR-99b-5p , Hsa-miR-99b-3p, has-miR-100-5p, hsa-miR-100-3p, hsa-miR-125a-5p, hsa-miR-125a-3p, hsa-miR-125b-5p, hsa -miR-125b-1-3p, hsa-miR-125b-2-3p, hsa-miR-202-5p, hsa-miR-202-3p, but are not limited to these. Among them, let-7a-5p, let-7g-5p, and let-7i-5p are preferable as the microRNA serving as an index, but are not limited thereto. In the following description of the present specification, microRNA selected from the let-7 family may be referred to as let-7 miRNA. Therefore, the messenger RNA (mRNA) introduced into the megakaryocyte population is a marker gene that is operably linked to a sequence that is specifically recognized by let-7 miRNA (also called the target sequence of let-7 miRNA). Including. Such mRNA is also referred to as let-7 miRNA-responsive mRNA or miRNA switch. It is also referred to as let-7a-5p responsive mRNA based on the specific miRNA name. Hereinafter, the case where let-7 miRNA is let-7a-5p will be described in detail, but the present invention is not limited to the microRNA. For other let-7 miRNAs, the target sequence and mRNA sequence can be determined and carried out in the same manner as the let-7a-5p target sequence described below. When a let-7a-5p is present in an mRNA containing a marker gene operably linked to a let-7a-5p target sequence, translation of the marker gene is controlled according to its activity. Preferably, when let-7a-5p is present, translation of the marker gene is suppressed according to its activity, so that the amount of protein translated from the marker gene (hereinafter referred to as marker protein) is reduced. It is mRNA that decreases quantitatively.

　let-7a-5pの標的配列は、let-7a-5pに特異的に結合可能な配列をいう。miRNA標的配列は、例えば、指標となるmiRNAに完全に相補的な配列であることが好ましい。
Figure JPOXMLDOC01-appb-T000001
The target sequence of let-7a-5p refers to a sequence that can specifically bind to let-7a-5p. The miRNA target sequence is preferably, for example, a sequence that is completely complementary to the indicator miRNA.
Figure JPOXMLDOC01-appb-T000001

　あるいは、当該let-7a-5p標的配列は、let-7a-5pにおいて認識され得る限り、完全に相補的な配列との不一致（ミスマッチ）を有していても良い。当該舶RNAに完全に相補的な配列からの不一致は、血小板産生能が高い細胞において、通常に let-7a-5pが認識し得る不一致であれば良く、生体内における細胞内の本来の機能では、40～50％程度の不一致があっても良いとされている。このような不一致は、特に限定されない級1 塩基、 2塩基‘3塩基、4塩基、5 塩基、6塩基、7塩基、8塩基、9塩基、若しくは10塩基又は全認識配列の1 ％、5%、 10%. 20%. 30%、若しくは40％の不一致が例示される。また、特には、細胞が備えているlet-7a-5p 上のlet-7a-5p標的配列のように、特に、シード領域以外の部分に、すなわちlet-7a-5pの3'側16塩基程度に対応する、標的配列内の5'側の領域に、多数の不一致を含んでもよく、シード領域の部分は、不一致を含まないか、1 塩基、2塩基若しくは3塩基の不一致を含んでもよい。　
Alternatively, the let-7a-5p target sequence may have a mismatch (mismatch) with a completely complementary sequence as long as it can be recognized in let-7a-5p. The mismatch from the sequence completely complementary to the marine RNA may be any mismatch that can be normally recognized by let-7a-5p in cells with high platelet-producing ability. It is said that there may be a discrepancy of about 40-50%. Such discrepancy is not particularly limited, class 1 base, 2 bases' 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases or 1%, 5% of all recognition sequences 10%. 20%. 30% or 40% discrepancy. In particular, like let-7a-5p target sequence on let-7a-5p provided by cells, especially in the part other than the seed region, that is, about 16 bases on the 3 ′ side of let-7a-5p The 5 ′ region in the target sequence corresponding to may contain multiple mismatches, and the seed region portion may contain no mismatches, or may contain mismatches of 1 base, 2 bases, or 3 bases.

　マーカー遺伝子は、細胞内で翻訳されて、マーカーとして機能し、細胞群の選別を可能にする任意の蛋白質をコードする遺伝子である。細胞内で翻訳されてマーカーとして機能しうる蛋白質としては、一例としては、蛍光、発光、呈色、若しくは蛍光、発光又は呈色を補助することなどにより、視覚化し、定量化することができる蛋白質であってよい。蛍光蛋白質としては、Sirius、EBFPなどの青色蛍光蛋白質；mTurquoise、TagCFP、AmCyan、mTFP1、MidoriishiCyan、CFPなどのシアン蛍光蛋白質；TurboGFP、AcGFP、TagGFP、Azami-Green（例えば、hmAGl)、ZsGreen、 EmGFP、EGFP、GFP2、HyPer、などの緑色蛍光蛋白質；TagYFP、 EYFP、 Venus、YFP、PhiYFP、PhiYFP-m、Turbo YFP、ZsYellow、mBananaなどの黄色蛍光蛋白質；Kusabira Orange （例えば、hmKO2)、mOrangeなどの燈色蛍光蛋白質；TurboRFP、DsRed-Express、 DsRed2、TagRFP、DsRed-Monomer、AsRed2、mStrawberry、などの赤色蛍光蛋白質；Tur boFP602、mRFP1、JRed、KillerRed、mCherry、HcRed、KeimaRed（例えば、hd KeimaRed)、mRasberry、mPlumなどの近赤外蛍光蛋白質が挙げられるが、これらには限定されない。　
A marker gene is a gene that is translated in a cell, functions as a marker, and encodes an arbitrary protein that enables selection of a cell group. Examples of proteins that can be translated into cells and function as markers include, for example, proteins that can be visualized and quantified by assisting fluorescence, luminescence, coloration, or fluorescence, luminescence, or coloration. It may be. As fluorescent proteins, blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (for example, hmAGl), ZsGreen, EmGFP, Green fluorescent proteins such as EGFP, GFP2, and HyPer; Yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, Turbo YFP, ZsYellow, mBanana; Kusabira Orange (eg, hmKO2), mOrange Color fluorescent proteins; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, etc .; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (eg, hd KeimaRed), mRasberry And near infrared fluorescent proteins such as mPlum, but are not limited thereto.

　発光蛋白質としては、イクオリンを例示することができるが、これに限定されない。また、蛍光、発光又は呈色を補助する蛋白質として、ルシフェラーゼ、ホスフアターゼ、ペルオキシダーゼ、βラクタマーゼなどの蛍光、発光又は呈色前駆物質を分解する酵素を例示することができるが、これらには限定されない。ここで本発明において、蛍光、発光又は呈色を補助する蛋白質をマーカー遺伝子として使用する場合、血小板産生能が高い細胞の選別において、対応する前駆物質と細胞を接触させること、又は細胞内に対応する前駆物質を導入することによって行われ得る。　
A photoprotein can be exemplified by aequorin, but is not limited thereto. Examples of proteins that assist fluorescence, luminescence, or coloration include, but are not limited to, enzymes that decompose fluorescence, luminescence, or color precursors such as luciferase, phosphatase, peroxidase, and β-lactamase. Here, in the present invention, when a protein that assists fluorescence, luminescence, or coloration is used as a marker gene, in the selection of cells having high platelet-producing ability, the corresponding precursor is brought into contact with the cell, or the cell is handled. This can be done by introducing a precursor that

　また、細胞内でマーカーとして機能しうる蛋白質の別の例としては、細胞の機能に直接影響を与える蛋白質類が挙げられる。細胞増殖蛋白質、細胞死滅蛋白質、アポトーシス制御蛋白質、細胞シグナル因子、薬剤耐性遺伝子、転写制御因子、翻訳制御因子、分化制御因子、リプログラミング誘導因子、RNA結合タンパク質因子、クロマチン制御因子、膜タンパク質を例示することができるが、これらには限定されない。例えば、細胞増殖蛋白質は、それを発現した細胞のみを増殖させ、増殖した細胞を特定することでマーカーとして機能する。細胞死滅蛋白質は、それを発現した細胞の細胞死を引き起こすことで、特定のmiRNAを含有もしくは含有しない細胞自体を死滅させ、細胞の生死を示すマーカーとして機能する。細胞シグナル因子は、それを発現した細胞が、特定の生物学的信号を発し、この信号を特定することでマーカーとして機能する。細胞死滅蛋白質として、例えば、BaxまたはBimが例示される。　
Another example of a protein that can function as a marker in a cell is a protein that directly affects the function of the cell. Cell growth protein, cell death protein, apoptosis control protein, cell signal factor, drug resistance gene, transcription control factor, translation control factor, differentiation control factor, reprogramming induction factor, RNA binding protein factor, chromatin control factor, membrane protein However, it is not limited to these. For example, a cell growth protein functions as a marker by proliferating only cells that express it and specifying the proliferated cells. The cell killing protein causes cell death of a cell that expresses it, thereby killing the cell itself containing or not containing a specific miRNA, and functions as a marker indicating cell viability. The cell signal factor functions as a marker by the cell that expresses it emits a specific biological signal and specifies this signal. Examples of the cell killing protein include Bax and Bim.

　本発明において、マーカー遺伝子は、局在化シグナルをコードする遺伝子を備えていてもよい。局在化シグナルとしては、核局在化シグナル、細胞膜局在化シグナル、ミトコンドリア局在化シグナル、タンパク質分泌シグナル等を挙げることができ、具体的には、古典的核移行配列（NLS) 、M9 配列、ミトコンドリア標的配列（MTS）、小胞体移行配列を挙げることができるが、これらには限定されない。このような局在化シグナルは、後述するイメージングサイトメトリー等で、本発明の方法における選別工程を、画像上で行うときに特に有利である。　
In the present invention, the marker gene may comprise a gene encoding a localization signal. Examples of the localization signal include a nuclear localization signal, a cell membrane localization signal, a mitochondrial localization signal, a protein secretion signal, and more specifically, classical nuclear translocation sequences (NLS), M9 Examples include, but are not limited to, sequences, mitochondrial target sequences (MTS), and endoplasmic reticulum translocation sequences. Such a localization signal is particularly advantageous when the screening step in the method of the present invention is performed on an image by imaging cytometry or the like described later.

　本発明において、マーカー遺伝子として、翻訳制御因子を用いる場合、当該翻訳制御因子によって翻訳が制御される第二のマーカー遺伝子を有するmRNAを同時に細胞へ導入する。このような蛋白質によって翻訳が制御される第二のマーカー遺伝子を有するmRNAとして、当該翻訳を制御しうる蛋白質が結合できるRNA配列と機能的に連結された第ニのマーカー遺伝子配列を有するmRNAが例示される。　
In the present invention, when a translation control factor is used as a marker gene, mRNA having a second marker gene whose translation is controlled by the translation control factor is simultaneously introduced into a cell. An example of mRNA having a second marker gene whose translation is controlled by such a protein is an mRNA having a second marker gene sequence operably linked to an RNA sequence to which the protein capable of controlling translation can be bound. Is done.

　本発明においてmiRNAの標的配列とマーカー遺伝子が機能的に連結するとは、マー力一遺伝子をコードするオープンリーディングフレーム（ただし、開始コドンを含む。）の5' UTR内、3' UTR内、及び／または当該オープンリーディングフレーム内に、少なくとも１つのlet-7a-5p標的配列を備えることを意味する。 mRNAは、好ましくは、5'末端から、5' から3'、の向きに、Cap構造（7 メチルグアノシン5'リン酸）、マーカー遺伝子をコードするオープンリーディングフレーム並びに、ポリAテイルを備え、5'UTR内、3'UTR内、及び／またはオープンリーディングフレーム内に少なくとも１つのlet-7a-5p標的配列を備える。mRNAにおけるlet-7a-5p標的配列の位置は、5'UTRであっても、3'UTRであってもよく、オープンリーディングフレーム内（開始コドンの3'側）であってもよく、これらのすべてにlet-7a-5p標的配列を備えていてもよい。したがって、let-7a-5p標的配列の数は、1 つ、2つ、3つ、4つ、5つ、6つ、7つ、8つあるいはそれ以上であっても良い。　
In the present invention, the miRNA target sequence and the marker gene are functionally linked to each other in the 5 ′ UTR, 3 ′ UTR, and / or the open reading frame (including the start codon) that encodes the mer strength gene. Alternatively, it means that at least one let-7a-5p target sequence is provided in the open reading frame. The mRNA preferably comprises a Cap structure (7 methylguanosine 5 ′ phosphate), an open reading frame encoding a marker gene, and a poly A tail in the 5 ′ to 3 ′ orientation from the 5 ′ end, Provide at least one let-7a-5p target sequence in the 'UTR, 3'UTR, and / or in the open reading frame. The location of the let-7a-5p target sequence in the mRNA may be 5'UTR or 3'UTR, may be within the open reading frame (3 'to the start codon), and these All may have a let-7a-5p target sequence. Thus, the number of let-7a-5p target sequences may be one, two, three, four, five, six, seven, eight or more.

　好ましくは、let-7a-5p標的配列は、5'UTRに1つ存在する。効率的な翻訳抑制を達成することができるためである。このとき、Cap構造とlet-7a-5p標的配列との間の塩基数及び塩基の種類は、開始コドンとなるAUGを含まず、かつステム構造や立体構造を構成しにくい限り、任意であってよい。例えば、Cap 構造とlet-7a-5p標的配列と間の塩基数は、0～50塩基、好ましくは、10～30塩基となるように設計することができるが、これらには限定されない。 また、let-7a-5p標的配列と開始コドンと間の塩基数及び塩基の種類は、ステム構造や立体構造を構成しにくいものである限り、任意であってよく、let-7a-5p標的配列と開始コドンとの間の塩基数は、0～50 塩基、好ましくは、10～30塩基となるように設計することができるが、これらには限定されない。なお、let-7a-5p標的配列が3’UTRに4つ存在する場合であっても、翻訳抑制を達成することは可能であることが確認されている。　
Preferably, there is one let-7a-5p target sequence in the 5 ′ UTR. This is because efficient translation suppression can be achieved. At this time, the number of bases and the type of base between the Cap structure and the let-7a-5p target sequence are arbitrary as long as they do not contain AUG as the start codon and it is difficult to form a stem structure or a three-dimensional structure. Good. For example, the number of bases between the Cap structure and the let-7a-5p target sequence can be designed to be 0 to 50 bases, preferably 10 to 30 bases, but is not limited thereto. In addition, the number of bases and the type of base between the let-7a-5p target sequence and the start codon may be arbitrary as long as they do not easily form a stem structure or a three-dimensional structure, and the let-7a-5p target sequence The number of bases between and the start codon can be designed to be 0 to 50 bases, preferably 10 to 30 bases, but is not limited thereto. It has been confirmed that even when there are four let-7a-5p target sequences in 3′UTR, it is possible to achieve translational repression.

　let-7a-5p応答性mRNAは、通常のウリジン、シチジンに替えて、シュードウリジン、5－メチルシチジンなどの修飾塩基を含んでいることが好ましい。細胞毒性を低減させるためである。修飾塩基の位置は、ウリジン、シチジンいずれの場合も、独立に、全てあるいは一部とすることができ、一部である場合には、任意の割合でランダムな位置とすることができる。　
The let-7a-5p-responsive mRNA preferably contains a modified base such as pseudouridine or 5-methylcytidine instead of ordinary uridine and cytidine. This is to reduce cytotoxicity. The positions of the modified bases can be all or part of the uridine and cytidine independently, and if they are part of the base, they can be random positions at an arbitrary ratio.

　let-7a-5p応答性mRNAは、上記に従って配列が決定されれば、遺伝子工学的に既知の任意の方法により当業者が合成することができる。特には、プロモーター配列を含むテンプレートDNAを鋳型として用いたin vitro転写合成法により、得ることができる。　
Let-7a-5p-responsive mRNA can be synthesized by a person skilled in the art by any method known in genetic engineering if the sequence is determined according to the above. In particular, it can be obtained by an in vitro transcription synthesis method using a template DNA containing a promoter sequence as a template.

　本発明にむいて、let-7a-5p応答性mRNAを巨核球細胞群に導入する工程（以下、導入工程と指称する）は、リポフェクション法、リポソーム法、エレクトロポレーション法、リン酸カルシウム共沈殿法、DEAEデキストラン法、マイクロインジェクション法、遺伝子銃法などを用いて、let-7a-5p応答性mRNAを直接、細胞群に含まれる細胞に導入することができる。あるいは、設計したlet-7a-5p応答性mRNAの核酸配列をコードするベクターを巨核球細胞群に導入することにより、let-7a-5p応答性mRNAを巨核球細胞群に導入することもできる。　
For the present invention, the step of introducing let-7a-5p-responsive mRNA into a megakaryocyte cell group (hereinafter referred to as the introduction step) includes lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, Let-7a-5p-responsive mRNA can be directly introduced into cells contained in a cell group using DEAE dextran method, microinjection method, gene gun method and the like. Alternatively, the let-7a-5p-responsive mRNA can be introduced into the megakaryocyte group by introducing a vector encoding the nucleic acid sequence of the designed let-7a-5p-responsive mRNA into the megakaryocyte group.

　let-7a-5p応答性let-7a-5pとコントロールとなるmRNA （以下、コントロールmRNAとも指称する）とを用いる場合には、これらのmRNAを巨核球細胞群に共導入することが好ましい。共導入した2以上のmRNAから発現するマーカー蛋白質の活性比は、細胞集団内において一定であるためである。この時の導入量は、導入するmRNA、導入方法および導入試薬の種類等により異なり、所望の翻訳量を得るために当業者は適宜これらを選択することができる。ここで、コントロールmRNAとは、let-7a-5p標的部位を有さず、let-7a-5p応答性mRNAがコードするのマーカー遺伝子とは異なるマーカー遺伝子をコードするmRNAをいう。コントロールmRNAの導入量もまた、所望の翻訳量を得るために当業者は適宜これらを選択することができる。コントロールmRNAは、let-7a-5pの存在に関わらず、マーカー遺伝子を発現するmRNAである。　
When let-7a-5p-responsive let-7a-5p and control mRNA (hereinafter also referred to as control mRNA) are used, it is preferable to co-introduce these mRNAs into a megakaryocyte group. This is because the activity ratio of marker proteins expressed from two or more co-introduced mRNAs is constant within the cell population. The introduction amount at this time varies depending on the mRNA to be introduced, the introduction method, the kind of the introduction reagent, and the like, and those skilled in the art can appropriately select these in order to obtain a desired translation amount. Here, the control mRNA refers to mRNA that does not have a let-7a-5p target site and encodes a marker gene different from the marker gene encoded by let-7a-5p-responsive mRNA. The amount of control mRNA introduced can also be appropriately selected by those skilled in the art to obtain a desired translation amount. Control mRNA is mRNA that expresses a marker gene regardless of the presence of let-7a-5p.

　let-7a-5p応答性mRNAが細胞に導入されると、細胞内では、細胞に所定のlet-7a-5pがRISCとして存在する場合、let-7a-5p応答性mRNAがコードするマーカー遺伝子の翻訳量が抑制される。そして、翻訳量の制御は、let-7a-5p活性に応じて定量的になされる。すなわち、let-7a-5pの存在量が多い細胞ではマーカー遺伝子の翻訳量が相対的に少なく、let-7a-5pの存在量が少ない細胞ではマーカー遺伝子の翻訳量が相対的に多くなる。一方、細胞に所定のlet-7a-5pが存在しない場合、もしくは所定のlet-7a-5pがRISCとして存在しない場合、let-7a-5p応答性mRNAがコードするマーカー遺伝子の翻訳量が抑制されることはない。したがって、let-7a-5pの存在量が異なる複数の細胞と間で、マーカー遺伝子の翻訳量が異なる。なお、本明細書において、let-7a-5pがRISC として存在する場合を、「let-7a-5p活性が存在する場合」とも指称し、活性化状態にあるlet-7a-5pの存在量が相対的に多い場合を「let-7a-5p活性が高い場合」とも指称する。一方、コントロールmRNAは、let-7a-5p活性に関係なくマーカー蛋白質を発現する。導入されても、let-7a-5p標的配列が存在しないため、let-7a-5p発現量に応じて翻訳制御されることがないためである。　
When let-7a-5p-responsive mRNA is introduced into a cell, if the given let-7a-5p is present as RISC in the cell, the marker gene encoded by let-7a-5p-responsive mRNA The amount of translation is suppressed. The amount of translation is controlled quantitatively according to the let-7a-5p activity. That is, the amount of translation of the marker gene is relatively small in cells having a large amount of let-7a-5p, and the amount of translation of a marker gene is relatively large in cells having a small amount of let-7a-5p. On the other hand, when the predetermined let-7a-5p does not exist in the cell, or when the predetermined let-7a-5p does not exist as RISC, the translation amount of the marker gene encoded by the let-7a-5p-responsive mRNA is suppressed. Never happen. Therefore, the translation amount of the marker gene differs between a plurality of cells having different abundance of let-7a-5p. In this specification, the case where let-7a-5p is present as RISC is also referred to as “when let-7a-5p activity is present”, and the amount of let-7a-5p in the activated state is The case where the amount is relatively large is also referred to as “when the activity of let-7a-5p is high”. On the other hand, the control mRNA expresses the marker protein regardless of the let-7a-5p activity. This is because even if introduced, since the let-7a-5p target sequence does not exist, translation control is not performed according to the expression level of let-7a-5p.

　次いで、マーカー遺伝子の翻訳量を指標として、細胞を選別する工程（以下、選別工程とも指称する）を実施する。選別工程では、上記のようなマーカー遺伝子の翻訳量から、細胞を選別する。本発明者らは、let-7a-5p活性が相対的に高い巨核球細胞群が、血小板産生能が相対的に低く、let-7a-5p活性が相対的に低い巨核球細胞群が、血小板産生能が相対的に高いことを見出している（図１、図２）。マーカー蛋白質として蛍光蛋白質を用いた場合、図１のヒストグラムを参照すると、Controlで示される線よりも右下に位置する細胞群は、let-7a-5pに応答して翻訳抑制された巨核球細胞群を表す。この中で、白色矢印が示す蛍光強度が相対的に高い細胞群は、let-7a-5p活性が相対的に低い巨核球細胞群を示している。黒色矢印が示す蛍光強度が相対的に低い細胞群は、let-7a-5p活性が相対的に高い巨核球細胞群を示している。このようなlet-7a-5p活性と、マーカー遺伝子の翻訳制御量により、巨核球細胞群を選別することができる。蛍光強度の高低は相対的なものであり、選別する対象となる巨核球細胞群の特性や、マーカー蛋白質の特性によっても異なりうるため、当業者が適宜閾値を決定し、分別することができる。その際、例えば、実施例にて後述する、let-7a-5pの阻害剤を用いた活性のレスキューに関する事前実験を行い、あるいは、血小板産生量についての事前実験を行い、閾値を決定することもできる。　
Next, a cell sorting step (hereinafter also referred to as a sorting step) is performed using the translation amount of the marker gene as an index. In the selection step, cells are selected from the translation amount of the marker gene as described above. The present inventors have reported that megakaryocyte cells with relatively high let-7a-5p activity have relatively low platelet-producing ability and megakaryocyte cells with relatively low let-7a-5p activity It has been found that the productivity is relatively high (FIGS. 1 and 2). When a fluorescent protein is used as a marker protein, referring to the histogram of FIG. 1, the cell group located at the lower right of the line indicated by Control is a megakaryocyte cell whose translation is suppressed in response to let-7a-5p. Represents a group. Among them, a cell group having a relatively high fluorescence intensity indicated by a white arrow indicates a megakaryocyte cell group having a relatively low let-7a-5p activity. A cell group having a relatively low fluorescence intensity indicated by a black arrow indicates a megakaryocyte group having a relatively high let-7a-5p activity. A megakaryocyte cell group can be selected based on such let-7a-5p activity and the translational control amount of the marker gene. The level of fluorescence intensity is relative and may vary depending on the characteristics of the megakaryocyte cell group to be selected and the characteristics of the marker protein, so that those skilled in the art can appropriately determine and classify the threshold value. In that case, for example, a preliminary experiment on rescue of activity using an inhibitor of let-7a-5p, which will be described later in the Examples, or a preliminary experiment on the amount of platelet production may be performed to determine the threshold value. it can.

　好ましい態様において、マーカー遺伝子は薬剤耐性遺伝子であってもよい。「薬剤耐性遺伝子」は、対応する薬剤に対して抵抗性を有する蛋白質を発現する遺伝子であれば何でもよい。例えば、抗生物質耐性遺伝子を含むが、これらに限定されない。抗生物質耐性遺伝子としては、例えば、カナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子、ブラストサイジン耐性遺伝子、ゲンタマイシン耐性遺伝子、カナマイシン耐性遺伝子、テトラサイクリン耐性遺伝子、クロラムフェニコール耐性遺伝子等が挙げられる。本発明において、好ましくは、ピューロマイシン耐性遺伝子またはブラストサイジン耐性遺伝子が抗生物質耐性遺伝子として用いられる。マーカー遺伝子が薬剤耐性遺伝子の場合、let-7a-5p活性が高い巨核球細胞群においては、薬剤耐性遺伝子の発現が選択的に抑制される。このため、let-7a-5p活性が高い巨核球細胞群は、対応する薬剤に対する抵抗性が低下して死滅する。したがって、これらの死細胞を選択的に除去することができる。
In a preferred embodiment, the marker gene may be a drug resistance gene. The “drug resistance gene” may be any gene that expresses a protein having resistance to the corresponding drug. Examples include, but are not limited to, antibiotic resistance genes. Examples of antibiotic resistance genes include kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, blasticidin resistance gene, gentamicin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, etc. . In the present invention, preferably, a puromycin resistance gene or a blasticidin resistance gene is used as the antibiotic resistance gene. When the marker gene is a drug resistance gene, the expression of the drug resistance gene is selectively suppressed in a megakaryocyte group with high let-7a-5p activity. For this reason, the megakaryocyte group with high let-7a-5p activity is killed by a decrease in resistance to the corresponding drug. Therefore, these dead cells can be selectively removed.
　あるいは、マーカー遺伝子として翻訳制御因子を用いた場合、同時に細胞へ導入した第二のマーカー遺伝子の翻訳量から細胞を選別することができる。上述したマーカー遺伝子により翻訳が抑制される第二のマーカー遺伝子を有するmRNAを利用することで、第二のマーカー遺伝子の翻訳量は、指標となるlet-7a-5pの発現量と比例する。すなわち、血小板産生能が高い細胞群が、指標となるlet-7a-5pの発現量が少ない細胞である場合、第二のマーカー遺伝子の翻訳量が少ない細胞を選別し、指標となるlet-7a-5pの発現量が多い細胞である場合、第二のマーカー遺伝子の翻訳量が多い細胞を選別することができる。マーカー遺伝子として細胞死滅蛋白質を用いた場合、指標となるlet-7a-5pの発現量が多い細胞は、特異的に細胞死を誘導され、取り除くことができる。 Alternatively, when a translation control factor is used as a marker gene, cells can be selected from the translation amount of the second marker gene introduced into the cell at the same time. By using mRNA having the second marker gene whose translation is suppressed by the marker gene described above, the translation amount of the second marker gene is proportional to the expression level of let-7a-5p serving as an index. That is, when the cell group with high platelet production ability is a cell with a low expression level of the indicator let-7a-5p, a cell with a low translation amount of the second marker gene is selected, and the indicator let-7a When the expression level of -5p is high, cells having a high translation amount of the second marker gene can be selected. When a cell killing protein is used as a marker gene, cells with a high expression level of let-7a-5p serving as an index can be specifically induced to be removed.
　具体的には、選別工程は、所定の検出装置を用いて、マーカー蛋白質からの信号を検出することにより実施することができる。検出装置としては、フローサイトメーター、イメージングサイトメーター、蛍光顕微鏡、発光顕微鏡、CCD力メラ等が挙げられるが、これらには限定されない。このような検出装置は、マー力一蛋白質及び選別の態様により、当業者が適したものを用いることができる。例えばマーカー蛋白質が、蛍光蛋白質又は発光蛋白質の場合には、フローサイトメーター、イメージングサイトメーター、蛍光顕微鏡、CCDカメラといった検出装遺を用いてマー力一蛋白質の定量が可能であり、マーカー蛋白質が、蛍光、発光又は呈色を補助する蛋白質の場合には、発光顕微鏡、CCDカメラ、ルミノメーターといった検出装置を用いたマーカー蛋白質の定量方法が可能であり、マーカー蛋白質が、膜局在蛋白質の場合には、抗体などの細胞表面蛋白質特異的な検出試薬と、上記の検出装置を用いたマーカー蛋白質の定量方法が可能である他、磁気細胞分離装置（MACS)といった、マーカー蛋白質の定量過程を経ない細胞の単離方法が可能である。　 Specifically, the selection step can be performed by detecting a signal from the marker protein using a predetermined detection device. Examples of the detection device include, but are not limited to, a flow cytometer, an imaging cytometer, a fluorescence microscope, a light emission microscope, and a CCD force mela. As such a detection device, those suitable for those skilled in the art can be used depending on the protein and the selection mode. For example, when the marker protein is a fluorescent protein or a luminescent protein, it is possible to quantify the protein by using a detection device such as a flow cytometer, an imaging cytometer, a fluorescence microscope, or a CCD camera. In the case of a protein that assists fluorescence, luminescence, or coloration, a marker protein quantification method using a detection device such as a luminescence microscope, a CCD camera, or a luminometer is possible. When the marker protein is a membrane-localized protein In addition to cell surface protein-specific detection reagents such as antibodies and marker protein quantification methods using the detection devices described above, the marker protein quantification process such as a magnetic cell separation device (MACS) is not required. Cell isolation methods are possible. *

　増殖期にある巨核球細胞群に対してmRNAを導入した場合には、血小板産生能の高い細胞群を選別した後、必要に応じて、選別後の巨核球細胞群について増殖のための培養を継続することができる。それに続き、通常の巨核球細胞の製造方法にしたがって、遺伝子の強制発現を抑制（解除）し、巨核球細胞を成熟させ、血小板の産生に用いることができる。
あるいは、増殖期以外の巨核球細胞群に対してmRNAを導入した場合にも、通常の巨核球細胞の製造方法にしたがって、選別後の巨核球細胞の製造を継続し、血小板の産生に用いることができる。　
When mRNA is introduced into a megakaryocyte cell group in the proliferative phase, after selecting a cell group having a high platelet-producing ability, if necessary, culture for proliferation of the selected megakaryocyte cell group is performed. Can continue. Subsequently, the forced expression of the gene can be suppressed (released), the megakaryocyte can be matured, and used for the production of platelets according to the normal method for producing megakaryocyte.
Alternatively, even when mRNA is introduced into a megakaryocyte group other than the proliferative phase, the production of megakaryocyte cells after selection is continued according to the normal method for producing megakaryocyte cells and used for platelet production. Can do.

　本実施形態によれば、miRNAの発現を指標として、巨核球細胞群から、血小板産生能が相対的に高い細胞群を選別することができる。この選別は、巨核球細胞の増殖期に実施することができる。したがって、選別された血小板産生能が相対的に高い細胞群のみを、増殖期において容易に選別し、その細胞集団のみを増やすことができることから、その後に血小板産生能の高い細胞集団のみを成熟させることから最終段階での血小板の産生効率が高くなる。
According to this embodiment, a cell group having a relatively high platelet-producing ability can be selected from a megakaryocyte cell group using miRNA expression as an index. This selection can be performed during the growth phase of megakaryocytes. Therefore, only the selected cell group having a relatively high ability to produce platelets can be easily selected in the growth phase, and only the cell population can be increased, so that only the cell population having a high ability to produce platelets is matured thereafter. Therefore, the platelet production efficiency in the final stage is increased.
　［血小板の製造方法］
　本発明は、別の実施形態によれば、血小板の製造方法であって、
　（ｉ）先の実施形態に記載の方法により血小板産生能の高い巨核球細胞群を選別する工程と、
　（ｉｉ）選別した細胞群を培養する工程と、
　（ｉｉｉ）前記培養する工程により得られた培養物から血小板を回収する工程とを含む。　 [Platelet production method]
The present invention, according to another embodiment, is a method for producing platelets, comprising:
(I) a step of selecting a megakaryocyte cell group having high platelet production ability by the method described in the previous embodiment;
(Ii) culturing the selected cell group;
(Iii) collecting platelets from the culture obtained by the culturing step.

　「血小板」は、血液中の細胞成分の一つであり、CD41a陽性及びCD42b陽性で特徴づけられる。血小板は、血栓形成と止血において重要な役割を果たすとともに、損傷後の組織再生や炎症の病態生理にも関与する。出血等により血小板が活性化されると、その膜上にIntegrin αIIBβ3（glycoprotein IIb/IIIa; CD41aとCD61の複合体）などの細胞接着因子の受容体が発現する。その結果、血小板同士が凝集し、血小板から放出される各種の血液凝固因子によってフィブリンが凝固することにより、血栓が形成され、止血が進む。　
“Platelets” are one of the cellular components in blood and are characterized by CD41a positive and CD42b positive. Platelets play an important role in thrombus formation and hemostasis, and are also involved in tissue regeneration after injury and pathophysiology of inflammation. When platelets are activated by bleeding or the like, receptors for cell adhesion factors such as Integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on the membrane. As a result, platelets aggregate and fibrin is coagulated by various blood coagulation factors released from the platelets, thereby forming a thrombus and promoting hemostasis.

　血小板の機能は、公知の方法により測定し評価することができる。例えば、活性化した血小板膜上のIntegrin αIIBβ3に特異的に結合するPAC-1に対する抗体を用いて、活性化した血小板量を測定することができる。また、同様に血小板の活性化マーカーであるCD62P（P-selectin）を抗体で検出し、活性化した血小板量を測定してもよい。例えば、フローサイトメトリーを用い、活性化非依存性の血小板マーカーCD61又はCD41に対する抗体でゲーティングを行い、その後、抗PAC-1抗体や抗CD62P抗体の結合を検出することにより行うことができる。これらの工程は、アデノシン二リン酸（ADP）存在下で行ってもよい。　
The function of platelets can be measured and evaluated by a known method. For example, the amount of activated platelets can be measured using an antibody against PAC-1 that specifically binds to Integrin αIIBβ3 on the activated platelet membrane. Similarly, CD62P (P-selectin), which is a platelet activation marker, may be detected with an antibody, and the amount of activated platelets may be measured. For example, flow cytometry can be used by gating with an antibody against the activation-independent platelet marker CD61 or CD41, and then detecting the binding of anti-PAC-1 antibody or anti-CD62P antibody. These steps may be performed in the presence of adenosine diphosphate (ADP).

　また、血小板の機能の評価は、ADP存在下でフィブリノーゲンと結合するか否かを見て行うこともできる。血小板がフィブリノーゲンと結合することにより、血栓形成の初期に必要なインテグリンの活性化が生じる。　
In addition, evaluation of platelet function can be performed by examining whether or not it binds to fibrinogen in the presence of ADP. Binding of platelets to fibrinogen results in integrin activation required early in thrombus formation.

　さらに、血小板の機能の評価は、国際公開第2011/034073号の図６に示されるように、in vivoでの血栓形成能を可視化して観察する方法で行うこともできる。　
Furthermore, the evaluation of platelet function can also be performed by visualizing and observing the thrombus formation ability in vivo, as shown in FIG. 6 of WO 2011/034073.

　本実施形態に係る血小板の製造方法は、国際公開第2011/034073号、国際公開第2012/157586号、及び国際公開第2014/123242号に開示された本出願人らによる先行技術による方法に対して、その過程で、先に記載した実施形態にしたがってlet-7a-5p応答性mRNAを用いて巨核球細胞を選別し、選別した巨核球細胞において血小板を産生させることを特徴とする。したがって、所定の時期に選別を行うことを除いては、これらの方法と同様にして実施することができる。　
The method for producing platelets according to this embodiment is based on the prior art methods disclosed by the present applicants disclosed in International Publication No. 2011/034073, International Publication No. 2012/157586, and International Publication No. 2014/123242. In the process, megakaryocytes are selected using let-7a-5p-responsive mRNA according to the embodiment described above, and platelets are produced in the selected megakaryocytes. Therefore, it can be carried out in the same manner as these methods except that sorting is performed at a predetermined time.

　例えば、好ましい実施態様において、巨核球細胞が増殖可能な状態にある所定の遺伝子を強制発現させる工程において選別方法を実施する場合には、選別後に必要な程度まで強制発現させる工程を継続することができる。その後、巨核球細胞を成熟させる、遺伝子の強制発現を抑制する工程を実施することができる。　
For example, in a preferred embodiment, when the selection method is performed in the step of forcibly expressing a predetermined gene in a state in which megakaryocyte cells can proliferate, the step of forcibly expressing to a necessary level after the selection may be continued. it can. Then, the process of suppressing the forced expression of a gene which matures a megakaryocyte can be implemented.

　本発明で得られた血小板は、製剤として患者に投与することができる。投与に当たっては、本発明の方法で得られる血小板は、例えば、ヒト血漿、輸液剤、クエン酸含有生理食塩液、ブドウ糖加アセテートリンゲル液を主剤とした液、PAS（platelet additive solution）（Gulliksson, H. et al., Transfusion, 32：435-440, (1992））等にて保存、製剤化してもよい。保存期間は、3日から7日程度で、好ましくは4日間である。保存条件として、室温（20-24度）で振盪撹拌して保存することが望ましい。　
The platelets obtained in the present invention can be administered to patients as a preparation. In administration, platelets obtained by the method of the present invention are, for example, human plasma, infusion, citric acid-containing physiological saline, glucose acetated Ringer's solution, PAS (platelet additive solution) (Gulliksson, H. et al., Transfusion, 32: 435-440, (1992)) and the like. The storage period is about 3 to 7 days, preferably 4 days. As storage conditions, it is desirable to store with shaking and stirring at room temperature (20-24 degrees).

　［巨核球細胞選別キット］
　本発明は、さらに別の実施形態によれば、巨核球細胞選別キットであって、前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを含んでなる。好ましい実施形態において、let-7ファミリーから選択されるマイクロRNA（let-7miRNA）はlet-7a-5pであり、mRNAは、先の実施形態において、let-7a-5p応答性mRNAあるいはmiRNA switchとして説明したｍRNAであってよい。このほかに、細胞選別キットは、let-7miRNA標的配列を持たず、let-7miRNA応答性mRNAとは異なるマーカー遺伝子を含むコントロールmRNAや、これらの巨核球細胞への導入に必要な試薬類、let-7miRNA阻害剤、let-7miRNAなどを含んでいてもよい。　
[Megakaryocyte sorting kit]
According to still another embodiment of the present invention, there is provided a megakaryocyte cell selection kit, which is a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family. MRNA comprising. In a preferred embodiment, the microRNA selected from the let-7 family (let-7miRNA) is let-7a-5p, and the mRNA is a let-7a-5p-responsive mRNA or miRNA switch in the previous embodiment. It may be the described mRNA. In addition to this, the cell selection kit does not have a let-7miRNA target sequence and contains a control mRNA containing a marker gene different from let-7miRNA-responsive mRNA, and reagents necessary for introduction into these megakaryocytes, let -7 miRNA inhibitors, let-7 miRNA, and the like.

　［血小板の製造方法］
　本発明は、また別の実施形態によれば、血小板の製造方法であって、
　（ａ）巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
　（ｂ）前記強制発現を抑制して、巨核球細胞を培養する工程と
を含み、前記不死化巨核球細胞を得る工程において、NR6A1遺伝子を強制発現させる工程を含む。　
[Platelet production method]
The present invention, according to another embodiment, is a method for producing platelets, comprising:
(A) in an undifferentiated cell than a megakaryocyte cell, forcibly expressing an oncogene, a polycomb gene, and an apoptosis inhibitor gene to obtain an immortalized megakaryocyte cell;
(B) suppressing the forced expression and culturing megakaryocyte cells, and in the step of obtaining the immortalized megakaryocyte cells, including a step of forcibly expressing the NR6A1 gene.

　不死化巨核球細胞を得る工程（ａ）及び巨核球細胞を培養する工程（ｂ）については、先に記載したとおりである。本実施形態においては、不死化巨核球細胞を得る工程において、NR6A1遺伝子を強制発現させる工程を含む。　
The step (a) for obtaining immortalized megakaryocyte cells and the step (b) for culturing megakaryocyte cells are as described above. In this embodiment, the step of forcibly expressing the NR6A1 gene is included in the step of obtaining immortalized megakaryocyte cells.

　NR6A1遺伝子の強制発現は、不死化巨核球細胞を得る工程における、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現と同様の方法で、これらの遺伝子を組み込んだウイルスベクターを作製することにより実施することができる。具体的には、NR6A1遺伝子の強制発現及び強制発現の解除は、国際公開第2011/034073号、国際公開第2012/157586号、国際公開第2014/123242、またはNakamura S et al, Cell Stem Cell. 14, 535-548, 2014に記載された方法、その他の公知の方法又はそれに準ずる方法で行うことができる。NR6A1遺伝子の鋳型配列を配列番号４９に示す。NR6A1遺伝子の強制発現は、不死化巨核球細胞を得る工程（ａ）において実施することができ、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現の解除と概ね同時に、R6A1遺伝子の強制発現の解除も行うことができる。　
Forced expression of the NR6A1 gene is carried out by preparing a viral vector incorporating these genes in the same manner as the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene in the process of obtaining immortalized megakaryocyte cells. can do. Specifically, forced expression of NR6A1 gene and cancellation of forced expression are performed in International Publication No. 2011/034073, International Publication No. 2012/157586, International Publication No. 2014/123242, or Nakamura S et al, Cell Stem Cell. 14, 535-548, 2014, other known methods, or a method analogous thereto. The template sequence of the NR6A1 gene is shown in SEQ ID NO: 49. The forced expression of the NR6A1 gene can be performed in the step (a) of obtaining immortalized megakaryocyte cells, and the forced expression of the R6A1 gene is almost simultaneously with the cancellation of the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene. Cancellation can also be performed.

　血小板の製造方法の別の態様においては、
　巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
を含み、前記不死化巨核球細胞を得る工程における前記細胞中の、Lin28A遺伝子をコードするDNAのプロモーターのCpGアイランド領域におけるメチル化量を減少させる工程を含む。　
In another embodiment of the method for producing platelets,
In an undifferentiated cell than a megakaryocyte, an oncogene, a polycomb gene, and an apoptosis inhibitor gene are forcibly expressed to obtain an immortalized megakaryocyte,
A step of inhibiting forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and encoding the Lin28A gene in the cell in the step of obtaining the immortalized megakaryocyte cells Reducing the amount of methylation in the CpG island region of the promoter of the DNA.

　細胞中のLin28A遺伝子をコードするDNAの、プロモーターのCpGアイランド領域におけるメチル化量の検出は、MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells（Oncotarget. 2016 Mar 22; 7(12): 14476-14485）に基づき、例えば、QIAGEN社のDNA抽出キット、ザイモリサーチ社のEZ DNA Methylation-Gold Kitを用いて、あるいは同様の市販のキットを用い、これらのキットの指示にしたがって、実施することができる。ここでいう「細胞」中とは、不死化巨核球細胞を得る工程（ａ）における細胞であってもよく、巨核球細胞を培養する工程（ｂ）における細胞であってもよいが、不死化巨核球細胞を得る工程（ａ）における細胞中の状態に基づいて判断することが好ましい。メチル化量を減少させる工程は、細胞を振盪培養することにより実施することができる。メチル化量を減少させる工程は、不死化巨核球細胞を得る工程（ａ）または巨核球細胞を培養する工程（ｂ）あるいはこれらの両方の工程において、実施することができるが、メチル化量の判断基準時を、不死化巨核球細胞を得る工程（ａ）とすることが好ましいため、不死化巨核球細胞を得る工程（ａ）において振盪培養を行うことが好ましい。振盪培養の条件は、培養規模にもよるが、例えば、５０ｒｐｍ以上であり、１００ｒｐｍ以上とすることが好ましい。また、このような条件とする場合、振盪培養の期間は、３日～１０日間程度とすることが好ましい。メチル化量を減少させて、メチル化量が、例えば、２０％以下、好ましくは１３％以下程度であると、let-7a-5pの活性を抑制することができ、巨核球の成熟を阻害する要因を除去し得るためより好ましい。　
The detection of methylation in the CpG island region of the promoter of the DNA encoding the Lin28A gene in cells was performed using MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells (Oncotarget. 2016 Mar 22; 7 (12 ): 14476-14485), for example, using QIAGEN's DNA extraction kit, Zymo Research's EZ DNA Methylation-Gold Kit, or similar commercial kits, and following these kit instructions can do. The term “in the cell” as used herein may be a cell in the step (a) of obtaining an immortalized megakaryocyte cell or a cell in the step (b) of culturing the megakaryocyte cell, but immortalized. It is preferable to judge based on the state in the cell in the step (a) of obtaining megakaryocyte cells. The step of reducing the amount of methylation can be performed by shaking and culturing the cells. The step of reducing the amount of methylation can be performed in step (a) of obtaining immortalized megakaryocyte cells, step (b) of culturing megakaryocyte cells, or both of these steps. Since it is preferable that the determination reference time is the step (a) of obtaining immortalized megakaryocyte cells, it is preferable to perform shaking culture in the step (a) of obtaining immortalized megakaryocyte cells. The conditions for shaking culture are, for example, 50 rpm or more, preferably 100 rpm or more, although depending on the culture scale. In such a condition, the period of shaking culture is preferably about 3 to 10 days. When the amount of methylation is decreased and the amount of methylation is, for example, about 20% or less, preferably about 13% or less, the activity of let-7a-5p can be suppressed, and maturation of megakaryocytes is inhibited. It is more preferable because the factor can be removed.

　血小板の製造方法のまた別の態様においては、
　多能性幹細胞を分化誘導して、巨核球細胞より未分化な細胞を得る工程と、
　前記巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
を含み、前記巨核球細胞より未分化な細胞を得る工程が、前記多能性幹細胞をMatrigel（登録商標）を含む基質を用いて培養する工程を含む。　
In another aspect of the method for producing platelets,
Inducing differentiation of pluripotent stem cells to obtain undifferentiated cells from megakaryocytes,
A step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-suppressing gene in an undifferentiated cell than the megakaryocyte cell to obtain an immortalized megakaryocyte cell;
Suppressing the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, the step of obtaining undifferentiated cells from the megakaryocyte cells, the pluripotent stem cell Culturing with a substrate containing Matrigel®.

　本実施形態においては、多能性幹細胞を分化誘導して、巨核球細胞より未分化な細胞を得る工程を含む。多能性幹細胞は、特には限定されず、ES細胞やiPS細胞が含まれるが、好ましくは、iPS細胞である。iPS細胞は、特定の初期化因子を、DNA又はタンパク質の形態で体細胞に導入することによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能、を有する体細胞由来の人工の幹細胞（K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M.ら,Nat. Biotechnol. 26:101-106 (2008)；国際公開WO2007/069666）であってよい。このようなであってもよい。また、特には、国際公開WO2016/167329に開示された方法により製造されたiPS細胞であって、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子がノックインされたiPS細胞であってもよく、特には限定されない。　
This embodiment includes a step of inducing differentiation of pluripotent stem cells to obtain undifferentiated cells from megakaryocytes. The pluripotent stem cells are not particularly limited, and include ES cells and iPS cells, but iPS cells are preferable. iPS cells can be produced by introducing a specific reprogramming factor into somatic cells in the form of DNA or protein, and have almost the same properties as ES cells, such as pluripotency and proliferation ability by self-replication, Somatic cell-derived artificial stem cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO2007 / 069666). Such may be used. In particular, it may be an iPS cell produced by the method disclosed in International Publication WO2016 / 167329, and may be an iPS cell in which an oncogene, a polycomb gene, and an apoptosis-suppressing gene are knocked in. Not.
　本実施形態においては、巨核球細胞より未分化な細胞を得る工程において、多能性幹細胞をMatrigel（登録商標）、ラミニン421もしくはその断片、またはラミニン121若しくはその断片、あるいはそれらの組み合わせから選択される基質を用いて培養する。Matrigel（登録商標）、ラミニンはいずれも、基底膜を構成する細胞外細胞マトリックスの1つである。Matrigel（登録商標）は、ラミニンを主成分とし、IV型コラーゲン、ヘパリン硫酸プロテオグリカン、エンタクチン／ニドゲン、成長因子を含む。ラミニンには多数のアイソフォームが存在しており、各アイソフォームは、５種類のα鎖（α１、α２、α３、α４、α５）、３種類のβ鎖（β１、β２、β３）及び３種類のγ鎖（γ１、γ２、γ３）がそれぞれ１本ずつサブユニット鎖としてＣ末端側で会合してコイルドコイル構造を作り、ジスルフィド結合によって安定化したヘテロ３量体分子を形成している。本実施形態において使用するラミニン４２１は、α４鎖、β２鎖及びγ１鎖から構成され、ラミニン１２１は、α１鎖、β２鎖及びγ１鎖から構成される。これらの断片としては、例えばＥ８断片が好ましい。ラミニンは天然型であってもよく、その生物学的活性を維持する限り、１またはそれ以上のアミノ酸残基が修飾された修飾型であってもよい。ラミニンは既知の任意の製造方法により得ることができ、市販品を用いることもできる。ラミニンの製造方法は特に限定されず、例えば、ラミニン高発現細胞から精製する方法、組換えタンパク質として製造する方法などが挙げられるが、これらには限定されない。ラミニン断片の製造方法も特に限定されず、例えば、全長ラミニンをエラスターゼ等のタンパク質分解酵素で消化し、目的の断片を分取し、精製する方法や、組換えタンパク質として製造する方法などが挙げられるが、これらには限定されない。製造量、品質の均一性、製造コスト等の観点から、ラミニンおよびラミニン断片の両者とも、組換えタンパク質として製造することが好ましい。 In the present embodiment, in the step of obtaining undifferentiated cells from megakaryocytes, pluripotent stem cells are selected from Matrigel (registered trademark), laminin 421 or a fragment thereof, laminin 121 or a fragment thereof, or a combination thereof. Incubate with the substrate. Matrigel (registered trademark) and laminin are both one of the extracellular cell matrices constituting the basement membrane. Matrigel (registered trademark) is mainly composed of laminin and contains type IV collagen, heparin sulfate proteoglycan, entactin / nidogen, and growth factor. Laminin has many isoforms, and each isoform has 5 types of α chains (α1, α2, α3, α4, α5), 3 types of β chains (β1, β2, β3) and 3 types. Γ chains (γ1, γ2, and γ3) each as a subunit chain associate on the C-terminal side to form a coiled-coil structure, thereby forming a heterotrimeric molecule stabilized by a disulfide bond. The laminin 421 used in the present embodiment is composed of an α4 chain, a β2 chain and a γ1 chain, and the laminin 121 is composed of an α1 chain, a β2 chain and a γ1 chain. As these fragments, for example, E8 fragment is preferable. Laminin may be a natural form or a modified form in which one or more amino acid residues are modified as long as the biological activity is maintained. Laminin can be obtained by any known production method, and a commercially available product can also be used. The method for producing laminin is not particularly limited, and examples thereof include a method for purifying from laminin-expressing cells and a method for producing as a recombinant protein, but are not limited thereto. The method for producing a laminin fragment is not particularly limited, and examples thereof include a method for digesting full-length laminin with a proteolytic enzyme such as elastase, fractionating and purifying the desired fragment, and a method for producing a recombinant protein. However, it is not limited to these. Both laminin and laminin fragments are preferably produced as recombinant proteins from the viewpoint of production amount, quality uniformity, production cost, and the like.

　iPS細胞などの多能性幹細胞から、分化誘導により、巨核球細胞より未分化な細胞を調製する技術は確立されており、基質以外の培養条件は、公知の技術に基づいて実施することができる。例えば、国際公開WO2011/034073において詳述した、多能性幹細胞から巨核球細胞より未分化な細胞を誘導する条件と同様であってよい。具体的には、「巨核球細胞より未分化な細胞」として、ｉＰＳ細胞から調製されるネット様構造物（ｉＰＳ－ｓａｃとも称する）から得られる細胞（特に、ネット様構造物から分離した直後の細胞）を誘導する培養条件であってよい。ｉＰＳ細胞から調製される「ネット様構造物」とは、ＥＳ細胞又はｉＰＳ細胞由来の立体的な嚢状（内部に空間を伴うもの）構造体で、内皮細胞集団などで形成され、内部に造血前駆細胞を含むもののことである。ネット様構造物の詳細については、例えば、ＴＡＫＡＹＡＭＡら，ＢＬＯＯＤ　２００８，１１１：５２９８－５３０６、を参照のこと。ネット様構造物をヒトｉＰＳ細胞から調製するために適した細胞の培養条件は、用いるｉＰＳ細胞によって異なるが、例えば、培地としては、最終濃度１５％のＦＢＳを添加したＩＭＤＭを用い、その他無血清の場合においても適宜増殖因子およびサプリメント等を加えたものを使用することができる。さらに、ネット様構造物を効率的に形成させるために、ＶＥＧＦを０～１００ｎｇ／ｍｌ、より好ましくは、２０ｎｇ／ｍｌ程度加えるのがよい。培養の環境としては、用いるＥＳ細胞又はｉＰＳ細胞の種類によって異なるが、例えば、５％　ＣＯ２、３６～３８℃、好ましくは３７℃の条件を用いることができる。
Techniques for preparing undifferentiated cells from megakaryocytes by differentiation induction from pluripotent stem cells such as iPS cells have been established, and culture conditions other than the substrate can be carried out based on known techniques . For example, the conditions described in detail in International Publication WO2011 / 034073 may be the same as the conditions for inducing undifferentiated cells from megakaryocytes from pluripotent stem cells. Specifically, cells obtained from net-like structures prepared from iPS cells (also referred to as iPS-sac) as “undifferentiated cells from megakaryocytes” (particularly immediately after separation from net-like structures) The culture conditions for inducing cells) may be used. A “net-like structure” prepared from iPS cells is a three-dimensional sac-like structure (with a space inside) derived from ES cells or iPS cells, which is formed by an endothelial cell population or the like and has hematopoiesis inside. Including progenitor cells. For details of net-like structures, see, for example, TAKAYAMA et al., BLOOD 2008, 111: 5298-5306. The cell culture conditions suitable for preparing the net-like structure from human iPS cells vary depending on the iPS cells used. For example, as the medium, IMDM supplemented with FBS at a final concentration of 15% is used. Also in this case, those appropriately added with growth factors and supplements can be used. Furthermore, in order to efficiently form a net-like structure, VEGF is added in an amount of 0 to 100 ng / ml, more preferably about 20 ng / ml. The culture environment varies depending on the type of ES cells or iPS cells to be used. For example, conditions of 5% CO 2 , 36 to 38 ° C., preferably 37 ° C. can be used.

　以下、本発明を、実施例を用いてより詳細に説明する。しかしながら、下記の実施例は、本発明を限定するものではない。　
Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples do not limit the present invention.

　IVT (in vitro transcription) に用いる鋳型DNAの作製
　5'UTRの鋳型(miRNA標的配列なし)と3'UTRの鋳型は、対応するプライマーとKOD-Plus-Neo（KOD-401,TOYOBO）を用いて、94°C 2 min後、98°C、10 sec、68°C、1 min を25サイクルという条件でPCR増幅を行い、作製した。tagBFP及びhmAG1をコードする遺伝子は、鋳型プラスミド(それぞれpTAP-tagBFP、pFucci-S/G2/M Green-Hyg (Expression vector)) から対応するプライマーとKOD-Plus-Neo（KOD-401,TOYOBO）を用いて、94°C、2 min後、98°C、10 sec、68°C、1 min を25サイクルという条件でPCR増幅を行い、作製した。　
Preparation of template DNA for IVT (in vitro transcription) 5'UTR template (no miRNA target sequence) and 3'UTR template are prepared using corresponding primers and KOD-Plus-Neo (KOD-401, TOYOBO). After 94 ° C for 2 min, PCR amplification was performed under the conditions of 98 ° C, 10 sec, 68 ° C, 1 min for 25 cycles. The genes encoding tagBFP and hmAG1 are obtained from template plasmids (pTAP-tagBFP and pFucci-S / G2 / M Green-Hyg (Expression vector), respectively) and corresponding primers and KOD-Plus-Neo (KOD-401, TOYOBO). Using, PCR amplification was carried out under conditions of 25 cycles of 94 ° C, 2 min, and 98 ° C, 10 sec, 68 ° C, 1 min for 25 cycles.

　IVTの鋳型となるFull DNA Templateは上記で作製したPCR産物と、それぞれ対応するプライマー（miRNA応答配列を持つものは、5'UTRの代わりにオリゴDNAを使用）を用いて、94°C 2 min後、98°C 10 sec、68°C 1 min を25サイクルという条件でPCR増幅を行い、作製した。全てのPCR産物は、MiniElute PCR purification Kit (QIAGEN)により精製した。但し、PCR反応でプラスミドを用いたものに関しては、精製前に制限酵素Dpn Iによる処理を施した。使用したプライマー及びオリゴヌクレオチド配列を表２に示す。　
Full DNA Template, which is the template for IVT, is 94 ° C 2 min using the PCR product prepared above and the corresponding primer (for those with miRNA response element, use oligo DNA instead of 5 'UTR). Thereafter, PCR amplification was performed under the conditions of 25 cycles of 98 ° C for 10 sec and 68 ° C for 1 min. All PCR products were purified using MiniElute PCR purification Kit (QIAGEN). However, those using a plasmid in the PCR reaction were treated with the restriction enzyme Dpn I before purification. The primers and oligonucleotide sequences used are shown in Table 2.

Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

　microRNA switchの作製と精製
　全てのmRNAは、MegaScript kit (Ambion)により作製した。この時、免疫反応を抑えるため、修飾塩基pseudouridine-5'-triphosphateと5-methylcytidine-5'-triphosphate (TriLink Bio Technologies)をUTPとCTPの代わりにそれぞれ加えた。またGTPは、Anti Reverse Cap Analog (TriLink Bio Technologies)で5倍希釈した。反応混合液を37°Cで6時間インキュベートして、TURBO DNase (Amibion)を加えた後、37°Cでさらに30分インキュベートした。得られたmRNAは、FavorPrep Blood／Cultured Cells total RNA extraction column (Favorgen Biotech)で精製し、Antarctic phosphatase (New England Biolabs)を用いて37C°で30分インキュベートした。その後、RNeasy Mini Elute Cleanup Kit (QIAGEN)により、さらに精製した。作製したmRNAの配列を表３に示す。　
Production and purification of microRNA switch All mRNAs were produced by MegaScript kit (Ambion). At this time, in order to suppress the immune reaction, modified bases pseudoridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink Bio Technologies) were added in place of UTP and CTP, respectively. GTP was diluted 5-fold with Anti Reverse Cap Analog (TriLink Bio Technologies). The reaction mixture was incubated at 37 ° C for 6 hours, TURBO DNase (Amibion) was added, and then incubated at 37 ° C for an additional 30 minutes. The obtained mRNA was purified by FavorPrep Blood / Cultured Cells total RNA extraction column (Favorgen Biotech) and incubated at 37 ° C. for 30 minutes using Antarctic phosphatase (New England Biolabs). Then, it further refine | purified by RNeasy Mini Elute Cleanup Kit (QIAGEN). The sequence of the prepared mRNA is shown in Table 3.

Figure JPOXMLDOC01-appb-T000003
  
Figure JPOXMLDOC01-appb-T000004
  
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000003
  
Figure JPOXMLDOC01-appb-T000004
  
Figure JPOXMLDOC01-appb-T000005

　細胞培養、血小板の産生
　Nakamura et al, Cell Stem Cell. 2014 Apr 3;14(4):535-48.及び国際公開第2014/123242号に記載の方法により樹立した、iPS細胞（TKDN SeV2：センダイウィルスを用いて樹立されたヒト胎児皮膚繊維芽細胞由来iPS細胞、585A1、585B1、606A1、648B1および692D2：Okita K, et al, Stem Cells 31, 458-66, 2012に記載のエピソーマルベクターを用いて樹立されたヒト末梢血単核球由来iPS細胞）由来の造血幹細胞に、c-MYC、BMI1およびBCL-XLを同時に導入して製造した不死化巨核球前駆細胞株Cl-7（以下、imMKCLともいう）を出発物質とし、Nakamura et alの12頁、Cell Cultureに記載の方法に基づいて細胞培養を行った。ただし、血小板産生条件では、C3H10T1/2細胞は用いず、フィーダーフリー条件下で6well培養プレートにヒトstem cell factor (SCF) 50ng/ml, ヒトthrombopoietin (TPO) 50 ng/mlに加えてROCK (Rho associated protein kinase)阻害剤 Y27632（国際公開第2012/157586号） 10μM、Aryl hydrocarbon receptor antagonist, StemRegenin 1 (SR1) 0.75nM、ADAM 17阻害剤、KP457 15μM（Hirata et al., Stem Cell Translational Medicine, in press）を添加した培地2ml/wellに、細胞を1*105/mlとなるよう播種した。DOX OFF （血小板産生のための培地）に変化させたday6-8後に血小板数測定をFACS Verseを用いて行った。DOX ON培地は、Takayama et al, Blood. 2008 Jun 1;111(11):5298-306に記載の、ESC differentiation mediumに、SCF、TPOを先に記載の濃度で、ドキシサイクリン（Doxycycline）を0.5μg/ml加えたものとした。DOX OFF培地は、SCF, TPO, Y27632, KP457を先に記載の濃度で加えたものとした。　
Cell culture and production of platelets iPS cells (TKDN SeV2: Sendai) established by the method described in Nakamura et al, Cell Stem Cell. 2014 Apr 3; 14 (4): 535-48. And International Publication No. 2014/123242 Human embryonic skin fibroblast-derived iPS cells established using viruses, 585A1, 585B1, 606A1, 648B1, and 692D2: using episomal vectors described in Okita K, et al, Stem Cells 31, 458-66, 2012 Immortalized megakaryocyte progenitor cell line Cl-7 (hereinafter, imMKCL) produced by introducing c-MYC, BMI1 and BCL-XL simultaneously into hematopoietic stem cells derived from human peripheral blood mononuclear cell-derived iPS cells) Cell culture was carried out based on the method described in Nakamura et al, page 12, Cell Culture. However, in the platelet production conditions, C3H10T1 / 2 cells are not used, and in addition to human stem cell factor (SCF) 50 ng / ml, human thrombopoietin (TPO) 50 ng / ml, ROCK (Rho associated protein kinase) inhibitor Y27632 (International Publication No. 2012/157586) 10μM, Aryl hydrocarbon receptor antagonist, StemRegenin 1 (SR1) 0.75nM, ADAM 17 inhibitor, KP457 15μM (Hirata et al., Stem Cell Translational Medicine, in The cells were seeded in 2 ml / well of the medium supplemented with press) at 1 * 10 5 / ml. Plate counts were measured using FACS Verse after days 6-8 when the medium was changed to DOX OFF (medium for platelet production). The DOX ON medium is described in Takayama et al, Blood. 2008 Jun 1; 111 (11): 5298-306. ESC differentiation medium is SCF, TPO at the concentration described above, and doxycycline (Doxycycline) is 0.5 μg. / ml was added. The DOX OFF medium was prepared by adding SCF, TPO, Y27632, KP457 at the concentrations described above.

　miRNA switchの導入及び解析
　巨核球細胞株が増殖を示すドキシサイクリンDOX ON (ドキシサイクリン存在下) の条件で、6well培養プレートにヒトstem cell factor (SCF) 50ng/ml, ヒトthrombopoietin (TPO) 50 ng/ml存在した状態で、miRNA switchのimMKCLへの導入はリポフェクション法で行った。Stemfect (Stemgent社)1μl+Buffer 5.25μl、および100ng/μlのコントロールmRNAとlet-7a-5p応答性ｍRNAを0.75μlずつ+Buffer 4.75μlを混ぜて15分間室温静置し、96wellプレートに入れて1-4*106/mlに懸濁した細胞液を50μl混ぜ合わせて30分間室温静置したのち、培地を150μl加えて一晩培養した。翌朝、細胞増殖するDOX ONの条件のまま、FACS AriaIIを用いて解析した。　
Introduction and analysis of miRNA switch Human stem cell factor (SCF) 50 ng / ml, human thrombopoietin (TPO) 50 ng / ml in 6-well culture plates under the condition of doxycycline DOX ON (in the presence of doxycycline) In the existing state, miRNA switch was introduced into imMKCL by the lipofection method. Stemfect (Stemgent) 1 μl + Buffer 5.25 μl, 100 ng / μl of control mRNA and let-7a-5p-responsive mRNA 0.75 μl each and Buffer 4.75 μl were mixed, left at room temperature for 15 minutes, and placed in a 96-well plate 50 μl of cell suspension suspended in 1-4 * 10 6 / ml was mixed and allowed to stand at room temperature for 30 minutes, and then 150 μl of medium was added and cultured overnight. The next morning, analysis was performed using FACS Aria II under the condition of DOX ON for cell proliferation.

　DOX ON条件のままで、let-7a-5p活性の高い群（activity High）と低い群(activity Low)をFACS AriaII(解析と同じ機種)を用いて細胞分離を実施した（フローサトメータによるソート）。分離後は再度、ドキシサイクリンを添加した培地（DOX ON）に懸濁した。一度分離した細胞は、２週間までなら分離効果が確実に残っていることは確認した（図３）。このため、実験には分離後３日から２週間までのものを使用した。それをDOX OFF(巨核球を成熟化させ、血小板が産生するための培地)に変えて６日後には、低い群(activity Low)で、有意に血小板産生数が上昇していた（図２）。　
With the DOX ON condition, cell separation was performed using FACS AriaII (same model as the analysis) for the let-7a-5p group with high activity (activity high) and low group (activity low) (sorting by flow satometer) ). After separation, the suspension was again suspended in a medium (DOX ON) supplemented with doxycycline. It was confirmed that once separated cells remained separated until 2 weeks (FIG. 3). For this reason, experiments were used from 3 days to 2 weeks after separation. 6 days after changing it to DOX OFF (medium for maturation of megakaryocytes and production of platelets), the number of platelet production was significantly increased in the low group (activity low) (Fig. 2). .

　let-7a-5p活性で分離した後にDOX OFFとし、継時的にlet-7a-5p活性を調べたが、活性の高さの傾向は保たれたままであった（図３）。　
After separating with let-7a-5p activity, DOX was turned off, and let-7a-5p activity was examined over time, but the tendency of high activity was maintained (FIG. 3).

　let-7a-5p inhibitor及びNegetive ControlはApplied Biosystems社から購入した。予備実験での検討に基づいて、imMKCLへの導入方法はリポフェクション法で行い、Stemfect (Stemgent社)1μl+Buffer 5.25μl、および10μMのmiRNA inhibitorやネガティブコントロールを0.4μl+Buffer 5.85μlを混ぜて15分間室温静置、96wellプレートに入れて1-4*106/mlに懸濁した細胞液を50μl混ぜ合わせて30分間室温静置したのち、培地を150μl加えて一晩培養した。翌朝細胞を洗浄し、一部はFACS AriaIIで解析し、残りはDOXを除いた培地に懸濁して細胞を成熟させた。Highの細胞集団へのinhibitorにより、血小板産生数が上昇し、Low細胞集団と近似した。つまり、let-7a-5p活性が高いと血小板産生放出が抑制されていることを示した（図４）。　
let-7a-5p inhibitor and Negetive Control were purchased from Applied Biosystems. Based on preliminary studies, imMKCL was introduced by lipofection, Stemfect (Stemgent) 1 μl + Buffer 5.25 μl, 10 μM miRNA inhibitor and negative control 0.4 μl + Buffer 5.85 μl mixed, 15 The mixture was allowed to stand at room temperature for 50 minutes, mixed with 50 μl of a cell solution suspended in 1-4 * 10 6 / ml in a 96-well plate, allowed to stand at room temperature for 30 minutes, and then 150 μl of medium was added and cultured overnight. The next morning, the cells were washed, a part was analyzed by FACS Aria II, and the rest was suspended in a medium excluding DOX to mature the cells. Inhibitor of high cell population increased platelet production and approximated the low cell population. That is, when let-7a-5p activity was high, it was shown that platelet production release was suppressed (FIG. 4).

　図１、２について記載した方法でlet-7a-5p活性の高い集団と低い集団を分離したのち、RNA抽出、cDNA合成を行い、定量PCRを行った。RNA抽出はmiRNeasy Mini Kit (QIAGEN社)を用い、製造者の指示に従って行った。cDNA合成は500ngのトータルRNAを用い、ReverTra Ace（登録商標） qPCR RT Master Mix (東洋紡社)を使って製造者の指示に従って行った。定量PCRに使用したプライマーは既報（Nakamura et al, Cell Stem Cell. 2014 Apr 3;14(4):535-48.）の通りである。詳細な配列は、下表５に記載する。試薬はSYBR PreMix ExTaq II (タカラバイオ社)を使用し、以下の表４に示す混合溶液を作製した。装置はStepOneplus (Applied Biosystems社)を用いた。PCRサイクルは95℃1分→（95℃10秒→55℃10秒→72℃30秒）*40サイクルで行った。内因性コントロールにはGAPDHを用い、データ解析はΔΔCT法を用いた。let-7a-5p活性に不均一性が出る原因が、imMKCLに強制発現している3遺伝子（C-MYC, BMI1, BCLXL）の影響である可能性があるため、これら3遺伝子の発現量を定量PCRで確認したが、統計学的に有意差は出なかった（図５）。　
After separating the population with high and low let-7a-5p activity by the method described in FIGS. 1 and 2, RNA extraction and cDNA synthesis were performed, and quantitative PCR was performed. RNA extraction was performed using miRNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. cDNA synthesis was performed using 500 ng of total RNA and using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo) according to the manufacturer's instructions. Primers used for quantitative PCR are as described previously (Nakamura et al, Cell Stem Cell. 2014 Apr 3; 14 (4): 535-48.). Detailed sequences are listed in Table 5 below. As a reagent, SYBR PreMix ExTaq II (Takara Bio Inc.) was used to prepare a mixed solution shown in Table 4 below. As the apparatus, StepOneplus (Applied Biosystems) was used. PCR cycle was 95 ° C for 1 minute → (95 ° C for 10 seconds → 55 ° C for 10 seconds → 72 ° C for 30 seconds) * 40 cycles. GAPDH was used for endogenous control, and ΔΔCT method was used for data analysis. The cause of heterogeneity in let-7a-5p activity may be the effect of 3 genes (C-MYC, BMI1, BCLXL) that are forcibly expressed in imMKCL. Although confirmed by quantitative PCR, there was no statistically significant difference (FIG. 5).

Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006

　let-7a-5pが機能的に巨核球成熟・血小板産生を阻害していることが判明したため、let-7a-5pの標的を見つける目的で、let-7a-5p活性の高い細胞集団と低い細胞集団に対してRNAシークエンスを行い、遺伝子発現量比較を行った。図５で用いたRNAサンプルの一部をRNAシークエンスに用いた。cDNA合成およびライブラリ作製は過去の論文通り (T Nakamura et al, Nucleic Acids Res. 2015 May 19;43(9):e60.)に行った。シークエンスはMiSeq Reagent Kit v3（イルミナ社）を用いた。活性の低い細胞集団で2倍以上発現している遺伝子は691個存在した。さらに、miRSystem、miRDB、miRSearchの３つのmiRNA標的予測データベースを用いて、691個の遺伝子の中からlet-7a-5pの標的となりうるものを選び出し、18個の遺伝子をピックアップした（図６）。これらの遺伝子のうち、Lin 28AおよびNR6A1が、let-7a-5p活性の低い細胞集団（血小板産生能力が高い細胞集団）において発現が高いことがわかった（図７）。　
Because let-7a-5p functionally inhibits megakaryocyte maturation and platelet production, the cell population with high and low let-7a-5p activity was used to find the target of let-7a-5p. RNA sequencing was performed on the populations and gene expression levels were compared. A part of the RNA sample used in FIG. 5 was used for RNA sequencing. cDNA synthesis and library preparation were performed according to previous papers (T Nakamura et al, Nucleic Acids Res. 2015 May 19; 43 (9): e60.). For the sequence, MiSeq Reagent Kit v3 (Illumina) was used. There were 691 genes that were expressed more than twice in the low activity cell population. Furthermore, using miRSystem, miRDB, and miRSearch, three miRNA target prediction databases were selected from 691 genes that could be targets of let-7a-5p, and 18 genes were picked up (FIG. 6). Among these genes, it was found that Lin 28A and NR6A1 are highly expressed in a cell population with low let-7a-5p activity (a cell population with high platelet production ability) (FIG. 7).

　図１、２及び５について記載した方法で、Lin 28A, Lin 28B, NR6A1の定量PCR（図８）、およびE2F2, TTLL4, TBKBP1, XKR8, CCNF, IGF2BP1の定量PCR（図９）を行った。各遺伝子のプライマーはRoche社Universal Probe Library Assay Design Centerで設計を行い、あらかじめ希釈系列を用いてプライマーが適正であることを確認した。配列は下表５に示す。その結果、let-7a-5p活性の高い集団にinhibitorを加えることにより発現量がレスキューされたNR6A1及びLin28Ｂが、let-7a-5pの標的となっていることが示唆された（図８）。同様に、IGF2BP1もlet-7a-5pの標的となっていることが示唆された（図９）。　
Quantitative PCR of Lin 28A, Lin 28B, and NR6A1 (FIG. 8) and quantitative PCR of E2F2, TTLL4, TBKBP1, XKR8, CCNF, and IGF2BP1 (FIG. 9) were performed by the methods described with reference to FIGS. Primers for each gene were designed at the Roche Universal Probe Library Assay Design Center, and it was confirmed beforehand that the primers were appropriate using a dilution series. The sequence is shown in Table 5 below. As a result, it was suggested that NR6A1 and Lin28B whose expression levels were rescued by adding inhibitor to a population with high let-7a-5p activity were targets of let-7a-5p (FIG. 8). Similarly, it was suggested that IGF2BP1 is also a target of let-7a-5p (FIG. 9).

Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007

　試薬の変更
　先に記載したmiRNA switchの導入及び解析に関する実験を、リポフェクション試薬をScreenfect（Wako社）に変更して行った。具体的にはScreenfect 1μl + Buffer 7μl、および100ng/μlのコントロールmRNAとlet-7a-5p応答性mRNAを0.5ulずつ+Buffer 7μlを混ぜて15分間静置し、96wellプレートに入れて2*106/mlに懸濁した細胞液を50μl混ぜ合わせて30分間室温静置したのち、培地を150μl加えて一晩培養した。また、先に記載した定量PCRのうち、NR6A1のプライマーは以下のより非特異的増幅の少ないものに変更した。変更に当たって何度か変更前後のプライマーを用いて実験を行って、結果にほとんど差がないことを証明した。
Forward Primer; CCCAGTCATACAGTCTGAT（配列番号４５）
Reverse Primer; AAGCAGGGCAAATAGTTCT（配列番号４６）　
Experiments related to introduction and analysis of miRNA switch described in the reagent change destination were performed by changing the lipofection reagent to Screenfect (Wako). Specifically, Screenfect 1 μl + Buffer 7 μl, 100 ng / μl of control mRNA and let-7a-5p-responsive mRNA in 0.5 ul increments + Buffer 7 μl, let stand for 15 minutes, put in a 96-well plate, 2 * 10 50 μl of cell suspension suspended in 6 / ml was mixed and allowed to stand at room temperature for 30 minutes, and then 150 μl of medium was added and cultured overnight. Moreover, among the quantitative PCR described above, the primer for NR6A1 was changed to one with less nonspecific amplification as described below. Several experiments were conducted using the primers before and after the change, and it was proved that there was almost no difference in the results.
Forward Primer; CCCAGTCATACAGTCTGAT (SEQ ID NO: 45)
Reverse Primer; AAGCAGGGCAAATAGTTCT (SEQ ID NO: 46)

　NR6A1の強制発現
　先に図７を参照して示した実験結果から、NR6A1はLet-7a-5pの下流標的遺伝子であることが想定され、Let-7a-5p活性の高い群の細胞にNR6A1を過剰発現させることで抑制されている血小板産生がレスキューされることが考えられた。それを実証するために、NR6A1を過剰発現させるためのウイルスを作製した。先に記載した定量PCRの実験で用いたcDNA（配列番号４９）を鋳型とし以下のプライマーを用いてPCR（94℃1分→（98℃ 5秒→57℃ 10秒→72℃ 1分）×3サイクル→（98℃ 5秒→68℃10秒→72℃1分）×40サイクル）を行ってインサートを作製した。これをクローンテック社のIn-Fusion HD cloning Kitを用いて、決められた手順通りに研究室内で用いているレンチウイルスベクター（tet依存性、GFP発現）に組み込むことでtet依存性NR6A1発現ベクターを作製した。このプラスミドをもとにウイルスを作製した。
Forward Primer; CGGTACCCGGGGATCCGCCACCATGGAGCGGGACGAACCG（配列番号４７）
Reverse Primer; GCTGACTAGAGGATCCTCATTCCTTGCCCACACTGGT（配列番号４８）
From the experimental results shown with reference to FIG. 7 as the forced expression destination of NR6A1, it is assumed that NR6A1 is a downstream target gene of Let-7a-5p, and NR6A1 is introduced into cells of a group with high Let-7a-5p activity. It was thought that platelet production that was suppressed by overexpression was rescued. To demonstrate that, a virus was made to overexpress NR6A1. PCR (94 ° C. for 1 minute → (98 ° C. for 5 seconds → 57 ° C. for 10 seconds → 72 ° C. for 1 minute)) using the cDNA (SEQ ID NO: 49) used in the quantitative PCR experiment described above as a template and the following primers: 3 cycles → (98 ° C. 5 seconds → 68 ° C. 10 seconds → 72 ° C. 1 minute) × 40 cycles) to produce an insert. Using the Clontech In-Fusion HD cloning Kit, the tet-dependent NR6A1 expression vector can be obtained by incorporating it into the lentiviral vector (tet-dependent, GFP expression) used in the laboratory according to the determined procedure. Produced. Virus was prepared based on this plasmid.
Forward Primer; CGGTACCCGGGGATCCGCCACCATGGAGCGGGACGAACCG (SEQ ID NO: 47)
Reverse Primer; GCTGACTAGAGGATCCTCATTCCTTGCCCACACTGGT (SEQ ID NO: 48)
　Let-7a-5p活性の高い群のimMKCLになにも発現しないウイルスとNR6A1を発現するウイルスをMOI=1となるように感染させ、十分に細胞数が増殖したのちにフローサイトメーターでGFP陽性細胞を分離した。そのimMKCLをDOX offとしたところ、day7で計測した血小板産生数は、NR6A1感染群で有意に多くみられた（図１０（ａ））。これにより、Let-7a-5pが機能的に血小板産生を障害した原因遺伝子の少なくとも一つはNR6A1であることが示唆された。また、NR6A1の強制発現により、血小板産生数を増加させることができることがわかった。　 A virus that does not express anything in imMKCL in the group with high Let-7a-5p activity and a virus that expresses NR6A1 are infected so that MOI = 1, and after sufficient cell growth, GFP is positive in the flow cytometer Cells were separated. When the imMKCL was DOXDoff, the number of platelet production measured on day 7 was significantly higher in the NR6A1-infected group (FIG. 10 (a)). This suggests that at least one of the causative genes that let-7a-5p functionally impaired platelet production is NR6A1. It was also found that the number of platelets produced can be increased by forced expression of NR6A1. *

　NR6A1が巨核球成熟・血小板産生に重要な遺伝子であることを証明するために、siRNAによるノックダウンを行った。siRNAはすべてDharmacon社から購入し、imMKCLへの導入は予備検討の結果、先に図４を参照して記載したlet-7a-5p inhibitor及びNegative Controlを用いる実験と同様に実施できることが判明した。さらに予備検討でAHR（Aryl Hydrocarbon Receptor）のノックダウンが血小板産生を促進することも証明し、この実験系が正常に働くことを示した。NR6A1のsiRNAは配列4種類分購入し、予備検討により＃１と＃３で十分なノックダウン効率が得られることを証明した。imMKCLに対してNR6A1 siRNA＃１、＃３およびネガティブコントロールsiRNAを導入したところ、NR6A1ノックダウンにより有意に血小板産生能が低下した（図１０（ｂ））。　
In order to prove that NR6A1 is an important gene for megakaryocyte maturation and platelet production, knockdown with siRNA was performed. All siRNAs were purchased from Dharmacon, and as a result of preliminary studies, it was found that introduction into imMKCL can be performed in the same manner as in the experiment using let-7a-5p inhibitor and Negative Control described above with reference to FIG. In addition, preliminary studies have demonstrated that knockdown of AHR (Aryl Hydrocarbon Receptor) promotes platelet production, indicating that this experimental system works normally. NR6A1 siRNA was purchased for 4 types of sequence, and it was proved that sufficient knockdown efficiency was obtained with # 1 and # 3 by preliminary examination. When NR6A1 siRNA # 1, # 3 and negative control siRNA were introduced into imMKCL, platelet production ability was significantly reduced by NR6A1 knockdown (FIG. 10 (b)).

　imMKCLだけでなく、臍帯血由来巨核球においてもlet-7a-5pやNR6A1が重要な遺伝子であるかどうか実験を行った。DSファーマバイオメディカル社から購入した臍帯血CD34陽性細胞を先の細胞培養、血小板の産生の実験に記載のESC differentiation mediumにSCF 2.5ng/ml, TPO 100ng/ml, FLT3L 2.5ng/ml, IL6 50ng/mlを加えた培地を用いて7日間培養した。7日後に得られた細胞のうち、CD41陽性細胞をミルテニーバイオテク社のMACSビーズを用いて分離し、let-7a-5pおよびネガティブコントロール、NR6A1 siRNAおよびネガティブコントロールをリポフェクション法により導入した。導入方法は予備検討の結果、Stemfect (Stemgent社)1μl+Buffer 5.25μl、および10μMのlet-7a-5pおよびネガティブコントロール、NR6A1 siRNAおよびネガティブコントロール1 μl+Buffer 5.25μlを混ぜて15分間室温静置、96 wellプレートに入れて1-4*106/mlに懸濁した細胞液を50μl混ぜ合わせて30分間室温静置したのち、imMKCLのDOX off培地と同じ培地に移し替えて7日間培養し、血小板産生数を測定した。その結果、Let-7a-5p mimicやNR6A1 siRNAは有意に血小板産生能を低下させた（図１０（ｃ））。これにより、imMKCL細胞株だけでなく、より一般的な巨核球細胞においてLet-7a-5pは成熟を阻害する因子であり、NR6A1は促進する重要な因子であることが明らかとなった。　
We examined whether let-7a-5p and NR6A1 are important genes not only in imMKCL but also in cord blood-derived megakaryocytes. Umbilical cord blood CD34-positive cells purchased from DS Pharma Biomedical Co., Ltd. SCF 2.5ng / ml, TPO 100ng / ml, FLT3L 2.5ng / ml, IL6 50ng in the ESC differentiation medium described in the previous cell culture and platelet production experiments Culture was performed for 7 days using a medium supplemented with / ml. Among the cells obtained after 7 days, CD41-positive cells were separated using Milteny Biotech MACS beads, and let-7a-5p, negative control, NR6A1 siRNA and negative control were introduced by lipofection method. As a result of preliminary studies, the introduction method was as follows: Stemfect (Stemgent) 1 μl + Buffer 5.25 μl, 10 μM let-7a-5p and negative control, NR6A1 siRNA and negative control 1 μl + Buffer 5.25 μl, and left at room temperature for 15 minutes Add 50 μl of the cell solution suspended in 1-4 * 10 6 / ml in a 96-well plate, let stand at room temperature for 30 minutes, then transfer to the same medium as imMKCL's DOX off medium and incubate for 7 days. The number of platelet production was measured. As a result, Let-7a-5p mimic and NR6A1 siRNA significantly reduced the platelet production ability (FIG. 10 (c)). This revealed that Let-7a-5p is a factor that inhibits maturation and NR6A1 is an important factor that promotes not only imMKCL cell lines but also more common megakaryocytes.

　LIN28A
　先に記載したlet-7a-5pの標的となりうるものとしてピックアップした１８の遺伝子のうち、LIN28AはLet-7a-5pの成熟を阻害する遺伝子であることは多数報告されている。imMKCLにおいても、Let-7a-5p活性の高い群と低い群とでは大きくLIN28AmRNA発現量に差がみられることから（図８）、LIN28Aがより上流でLet-7a-5p活性を調節している可能性を考えて実験を行った。Let-7a-5p活性の低い群のimMKCL（DOX ON）に、配列番号１６で示すLet-7a-5p応答性tagBFP mRNAと、配列番号１８で示すControl hmAG1 mRNAを共導入、またはこれらとLIN28A siRNAの共導入をinhibitor導入の実験と同様にリポフェクション法で行った。その結果、LIN28AノックダウンによりLet-7a-5p活性の上昇が認められた（図１１（ａ））。LIN28Bのノックダウンではそのような結果は見られなかった（図１１（ｂ））。グラフ中、"vehicle"は、siRNA negative controlのトランスフェクションを表す。imMKCLにおいてLIN28AがLet-7a-5p活性の調節因子であることが明らかとなった。　
LIN28A
Of the 18 genes picked up as possible targets for let-7a-5p described above, it has been reported that LIN28A is a gene that inhibits Let-7a-5p maturation. In imMKCL, LIN28A regulates Let-7a-5p activity further upstream because there is a large difference in LIN28AmRNA expression between the group with high and low Let-7a-5p activity (Fig. 8). The experiment was conducted considering the possibility. A group of low Let-7a-5p activity, imMKCL (DOX ON), co-introduced with the Let-7a-5p-responsive tagBFP mRNA shown in SEQ ID NO: 16 and Control hmAG1 mRNA shown in SEQ ID NO: 18, or these and LIN28A siRNA Co-introduction was carried out by the lipofection method in the same manner as the experiment of the inhibitor introduction. As a result, an increase in Let-7a-5p activity was observed by LIN28A knockdown (FIG. 11 (a)). Such a result was not seen in the knockdown of LIN28B (FIG. 11 (b)). In the graph, “vehicle” represents transfection of siRNA negative control. In imMKCL, LIN28A was found to be a regulator of Let-7a-5p activity.

　LIN28Aの発現を調節する因子を調べるために、LIN28Aプロモーター領域におけるCpGアイランドのDNAメチル化について調べた。先に細胞培養、血小板の産生について述べた条件で細胞を培養し、巨核球細胞株が増殖を示すドキシサイクリンDOX ON (ドキシサイクリン存在下) の状態において、Let-7a-5p活性の高い細胞群と低い細胞群それぞれから、QIAGEN社のDNA抽出キットを用いて決められて手順通りにDNAを抽出した。次にザイモリサーチ社のEZ DNA Methylation-Gold Kitを用いて決められた手順通りにバイサルファイト処理を行い、次にバイサルファイトPCRを行った。CpGアイランドの検索やバイサルファイトPCRのプライマーは過去の文献に基づいて行った（Oncotarget. 2016 Mar 22; 7(12): 14476-14485）。PCRの酵素には、東洋紡社のKOD Multi&Epiを用いて、このようにして得られたPCR産物をタカラ社のMighty-TAを使用し、95℃ 5分→（95℃ 30秒→53℃ 30秒→72℃ 30秒）×40サイクル→72℃ 10分でPCRを行った。このPCR産物をタカラバイオ社Mighty TAクローニングキットを用いてクローニングを行い、定められたプライマーを用いて行ったコロニーPCRで適切な位置にバンドの出るものを各サンプル当たり10個ずつプラスミド増幅させてマクロジェン社にシークエンスを依頼した。シークエンス結果はweb上無料ソフトQUMAを用いて解析を行った。その結果、Let-7a-5p活性の高い細胞群ではかなりメチル化が行われており、活性の低い細胞群の10倍近くメチル化されていることが判明した（図１１（ｃ）、（ｄ））。図１１（ｄ）中、CpG %は、全てのCpG の個数に対する、メチル化したCpGの個数を示す。LIN28Aプロモーター領域CpGアイランドのメチLIN28A及びNR6A1ル化がLIN28A発現量に大きく作用している可能性が示唆された。　
To investigate factors that regulate LIN28A expression, we investigated DNA methylation of CpG islands in the LIN28A promoter region. In the state of doxycycline DOX ON (in the presence of doxycycline) where the cells are cultured under the conditions described above for cell culture and platelet production, and the megakaryocyte cell line is proliferating, the cell group with high Let-7a-5p activity and low DNA was extracted from each cell group according to the procedure determined using the QIAGEN DNA extraction kit. Next, bisulfite treatment was performed according to the procedure determined using EZ DNA Methylation-Gold Kit of Zymo Research, and then bisulfite PCR was performed. CpG island searches and bisulfite PCR primers were based on past literature (Oncotarget. 2016 Mar 22; 7 (12): 14476-14485). For PCR enzyme, Toyobo's KOD Multi & Epi was used, and the PCR product thus obtained was used with Takara's Mighty-TA. 95 ° C for 5 minutes → (95 ° C for 30 seconds → 53 ° C for 30 seconds) → 72 ° C. 30 seconds) × 40 cycles → 72 ° C. for 10 minutes. This PCR product is cloned using the Takara Bio Mighty TA Cloning Kit, and a colony PCR performed using the specified primers, with a band appearing at an appropriate position, is amplified by 10 plasmids for each sample. Asked Jen for sequencing. The sequence results were analyzed using the free software QUMA on the web. As a result, it was found that the cell group with high Let-7a-5p activity was considerably methylated, and was nearly 10 times methylated with the cell group with low activity (FIGS. 11 (c) and (d). )). In FIG. 11D, CpG% indicates the number of methylated CpGs relative to the total number of CpGs. This suggests that the LIN28A promoter region CpG island methylation LIN28A and NR6A1 may greatly affect the expression level of LIN28A.

　LIN28Aプロモーター領域CpGアイランドのメチル化によりLIN28A発現量が抑制され、結果としてlet-7a-5pの活性を高め、NR6A1発現を抑制することで血小板産生を低下させることが判明したため、そのメチル化を解除することができればLIN28A発現を高めることで血小板産生が上がると考えられた。メチル化を解除する方法として、巨核球細胞株を振盪培養する方法について検討した。すなわち、DOX onの状態の細胞株を一部は通常通り静置培養、一つは125mlフラスコに25mlの培養液及び細胞を入れて100rpmで振盪培養を一週間行った。その後、DOX offとする際にもそれぞれの条件の細胞を静置培養と振盪培養を行い、血小板産生能を確認した（図１２（ａ））。DOX onで一週間培養した際の細胞からRNAを抽出し、LIN28A及びNR6A1発現量をRT-PCRにて測定した。図１２（ｂ）は、LIN28A及びNR6A1のそれぞれについて、静置培養（Static）した細胞から抽出されたmRNA量を１とした場合の相対量を示すグラフである。図１２（ｂ）にあるように、振盪培養を行った時のほうが両遺伝子とも発現量は上がっていた。図１２（ｃ）は、これらの各条件で得られた血小板産生数を、DOX on （Static）-DOX off（Static）として培養した場合を１００％とした相対数で表したグラフである。図１２（ｃ）から、DOX onで振盪培養した細胞群が多くの血小板を産生したことがわかる。さらに、DOX onで一週間静置あるいは振盪培養した細胞中のDNAにおけるLIN28Aプロモーター領域CpGアイランドのメチル化を先に記載したのと同じ方法で解析した。解析結果を図１２（ｄ）に示す。図１２（ｄ）から、振盪培養したほうがメチル化率は５０％以上低下しており、メチル化を解除する方法の一つとして振盪培養という方法があることが示唆された。　
LIN28A promoter region CpG island methylation suppressed LIN28A expression, resulting in increased activity of let-7a-5p and reduced NR6A1 expression to reduce platelet production, thus demethylating the methylation It was thought that platelet production would be increased by increasing LIN28A expression. As a method for releasing methylation, a method of shaking culture of a megakaryocyte cell line was examined. That is, a part of the cell line in the state of DOX on was statically cultured as usual, and one was put 25 ml of culture solution and cells in a 125 ml flask, and cultured with shaking at 100 rpm for one week. Thereafter, even when DOX was turned off, the cells under each condition were subjected to stationary culture and shaking culture, and the platelet production ability was confirmed (FIG. 12 (a)). RNA was extracted from the cells cultured for one week with DOX on, and the expression levels of LIN28A and NR6A1 were measured by RT-PCR. FIG. 12B is a graph showing the relative amounts of LIN28A and NR6A1 when the amount of mRNA extracted from statically cultured cells is 1. As shown in FIG. 12 (b), the expression level of both genes increased when shaking culture was performed. FIG. 12 (c) is a graph showing the number of platelets produced under each of these conditions as a relative number with 100% when cultured as DOX on (Static) -DOX off (Static). From FIG. 12 (c), it can be seen that the cell group shake-cultured with DOX on produced many platelets. Furthermore, methylation of the LIN28A promoter region CpG island in DNA in cells that had been allowed to stand still for one week with DOX on or shake-cultured was analyzed by the same method as described above. The analysis result is shown in FIG. From FIG. 12 (d), the methylation rate decreased by 50% or more in the shaking culture, and it was suggested that there is a shaking culture method as one of the methods for releasing the methylation.
　基質の検討
　さらに、iPS細胞の段階での培養方法が、巨核球細胞株作成後の血小板産生能に影響を与えるのかどうかを調べるために、MKiPSの技術を使用した。具体的には、引用することにより本明細書の一部をなすものとする国際公開WO2016/167329の開示に基づき、imMKCLに対して山中4因子（OCT4, SOX2, KLF4, CMYC）を導入してMKiPS細胞を作製し、それを再度巨核球に分化させてDOX onとすることでimMKCLを作製し、DOX onとして培養を続けた。図１３（ａ）は、MKiPS細胞から、分化誘導させた各段階の細胞における定量PCRにて測定したLIN28AｍRNA量を示すグラフであり、縦軸は、先の、細胞培養、血小板の産生の項目で詳述したSeV2 imMKCLにおけるLIN28A発現量を１としたときの相対量を表す。図１３（ｂ）は、MKiPS細胞から、分化誘導後、１４日、３５日の細胞について、配列番号１６で示すLet-7a-5p応答性tagBFP mRNAと、配列番号１８で示すControl hmAG1 mRNAを共導入して、Let-7a-5p活性を測定した結果を示す。継代が進むにつれてLIN28A発現量は減少し(図１３（ａ）、Let-7a-5p活性は上昇することを確認した（図１３（ｂ））。 In addition, MKiPS technology was used to investigate whether the culture method at the stage of iPS cells would affect the platelet production ability after the megakaryocyte cell line was created. Specifically, Yamanaka 4 factors (OCT4, SOX2, KLF4, CMYC) were introduced to imMKCL based on the disclosure of International Publication WO2016 / 167329, which is incorporated herein by reference. MKiPS cells were prepared and differentiated into megakaryocytes again to make DOX on, so that imMKCL was made and culture was continued as DOX on. FIG. 13 (a) is a graph showing the amount of LIN28A mRNA measured by quantitative PCR in cells at various stages induced to differentiate from MKiPS cells, and the vertical axis represents the previous items of cell culture and platelet production. The relative amount when the LIN28A expression level in SeV2 imMKCL described in detail is set to 1. FIG. 13 (b) shows that the Let-7a-5p-responsive tagBFP mRNA represented by SEQ ID NO: 16 and the Control hmAG1 mRNA represented by SEQ ID NO: 18 were co-expressed from MKiPS cells after 14 days and 35 days after differentiation induction. The result of introduction and measurement of Let-7a-5p activity are shown. It was confirmed that the expression level of LIN28A decreased with passage (FIG. 13 (a) and Let-7a-5p activity increased (FIG. 13 (b)).
　次に、MKiPS細胞を培養する基質によってその後の血小板産生に差が出るか検証した。具体的には、MKiPS11,12の二つの細胞株に対して、Laminin511（商品名：iMatrix-511）とMatrigel（登録商標）上で培養した。具体的には、6wellプレート上に予めLamininないしはMatrigelをコーティングし、そこにLaminin511上には5*104個/well、Matrigel上にはコロニーを崩さないように１：４～６となるように、MKiPS11,12の二つの細胞株を播種して一週間培養した。培養液交換は概ね二日に一回行った。その後、EB法で分化しday14で回収した細胞を一部はRNA抽出に用い、残りをDOX onで一週間培養し、その後DOX offとしてさらに一週間後に血小板産生数を測定した。Day14の段階で抽出したRNAを用いてRT-PCRを行った。図１３（ｃ）は、Laminin511で培養したMKiPS11細胞株から抽出したLIN28AmRNA発現量を１とした場合の、LIN28AmRNAの相対発現量を表すグラフである。LIN28A発現量は二株ともにMatrigel群のほうが多くなっていた (図１３（ｃ）)。さらに、これらについて、図２の実験と同様の条件で巨核球細胞を培養し、血小板を産生した。図１３（ｄ）は、Laminin511で培養したMKiPS11細胞株から分化誘導した巨核球細胞が産生した血小板数を１００％とした場合の、血小板産生数の相対量を表すグラフである。血小板産生数もMatrigel群で多くなっていた(図１３（ｄ）)。図示はしないが、Matrigelに代えて、ラミニン121、ラミニン421を用いた場合にも、Matrigelと同様の効果が実証された。このことから、iPS細胞の段階で、その培養方法が巨核球細胞株作成後の血小板産生に影響を与えることが示唆された。 Next, it was verified whether the subsequent platelet production was different depending on the substrate for culturing MKiPS cells. Specifically, two cell lines, MKiPS11 and 12, were cultured on Laminin511 (trade name: iMatrix-511) and Matrigel (registered trademark). Specifically, Laminin or Matrigel is coated on a 6-well plate in advance, 5 * 10 4 / well on Laminin 511, and 1: 4 to 6 so that no colonies are destroyed on Matrigel. MKiPS11 and 12 were seeded and cultured for one week. The culture medium was exchanged approximately once every two days. Thereafter, some of the cells differentiated by the EB method and collected on day 14 were used for RNA extraction, the rest were cultured with DOX on for one week, and then the number of platelet production was measured after another week as DOX off. RT-PCR was performed using RNA extracted at the stage of Day14. FIG. 13 (c) is a graph showing the relative expression level of LIN28AmRNA when the expression level of LIN28AmRNA extracted from the MKiPS11 cell line cultured with Laminin511 is 1. The expression level of LIN28A was higher in the Matrigel group in both strains (FIG. 13 (c)). Furthermore, about these, the megakaryocyte was cultured on the conditions similar to the experiment of FIG. 2, and the platelet was produced. FIG. 13 (d) is a graph showing the relative amount of platelet production when the number of platelets produced by megakaryocytes differentiated from the MKiPS11 cell line cultured in Laminin511 is taken as 100%. Platelet production was also increased in the Matrigel group (FIG. 13 (d)). Although not shown, when Laminin 121 and Laminin 421 were used instead of Matrigel, the same effect as Matrigel was demonstrated. This suggests that, at the stage of iPS cells, the culture method affects platelet production after preparation of megakaryocyte cell lines.

Claims (18)

  1. 　以下の工程を含む、巨核球細胞群における細胞の不均質性を識別する方法：（１）マイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを巨核球細胞群に導入する工程、および（２）当該マーカー遺伝子の翻訳量を指標として、特性の異なる細胞群を識別する工程。 A method for discriminating cell heterogeneity in a megakaryocyte group comprising the following steps: (1) megakaryocyte cells comprising an mRNA comprising a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA. And (2) identifying a group of cells having different characteristics using the translation amount of the marker gene as an index.
  2. 　以下の工程を含む、巨核球細胞群から血小板産生能の高い細胞群を選別する方法：（１）let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを巨核球細胞群に導入する工程、および（２）当該マーカー遺伝子の翻訳量を指標として、血小板産生能の高い細胞群を選別する工程。 A method for selecting a cell group having high platelet-producing ability from a megakaryocyte group including the following steps: (1) Functionally linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family A step of introducing mRNA containing the marker gene into a megakaryocyte cell group, and (2) a step of selecting a cell group having a high platelet-producing ability using the translation amount of the marker gene as an index.
  3. 　前記mRNAを細胞群に導入する工程が、巨核球細胞の増殖期に実施される、請求項２に記載の方法。 The method according to claim 2, wherein the step of introducing the mRNA into a cell group is carried out during the growth phase of megakaryocyte cells.
  4. 　前記mRNAが、前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と、前記マーカー遺伝子が、5'から3'の方向に連結されている、請求項２または３に記載の方法。 The nucleic acid sequence specifically recognized by the microRNA selected from the let-7 family and the marker gene are linked in a 5 'to 3' direction, wherein the mRNA is linked in a 5 'to 3' direction. The method described.
  5. 　前記選別する工程が、前記let-7ファミリーから選択されるマイクロRNAの活性が低い細胞群で当該マーカー遺伝子の翻訳量が多い細胞群を選別する、請求項２～４のいずれか１項に記載の方法。 5. The selection according to any one of claims 2 to 4, wherein the selecting step selects a cell group having a low amount of translation of the marker gene from a cell group having a low activity of microRNA selected from the let-7 family. the method of.
  6. 　前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列を持たず、前記マーカー遺伝子とは異なるマーカー遺伝子を含むコントロールmRNAを細胞群に導入する工程をさらに含む、請求項２～５のいずれか１項に記載の方法。 The method further comprises the step of introducing into the cell group a control mRNA that does not have a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family and includes a marker gene different from the marker gene. The method according to any one of 1 to 5.
  7. 　前記選別する工程が、フローサイトメトリーを用いて行われる請求項１～６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 6, wherein the screening step is performed using flow cytometry.
  8. 　前記選別する工程が、イメージアナライザーを用いて行われる請求項１～６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 6, wherein the selecting step is performed using an image analyzer.
  9. 　前記マーカー遺伝子が、薬剤耐性遺伝子である、請求項１～６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 6, wherein the marker gene is a drug resistance gene.
  10. 　前記let-7ファミリーから選択されるマイクロRNAが、let-7a-5pである、請求項１～９のいずれか１項に記載の方法。 The method according to any one of claims 1 to 9, wherein the microRNA selected from the let-7 family is let-7a-5p.
  11. 　請求項１～１０のいずれか１項に記載の方法により血小板産生能の高い細胞群を選別する工程と、
    　選別した細胞群を培養する工程と、
    　前記培養する工程により得られた培養物から血小板を回収する工程と
    を含む、血小板の製造方法。     Selecting a cell group having high platelet-producing ability by the method according to any one of claims 1 to 10,
    Culturing the selected cell group;
    And a step of recovering platelets from the culture obtained by the culturing step.
  12. 　let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と機能的に連結したマーカー遺伝子を含むmRNAを含んでなる、巨核球細胞選別キット。 A megakaryocyte cell selection kit comprising mRNA comprising a marker gene operably linked to a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family.
  13. 　前記mRNAが、前記let-7ファミリーから選択されるマイクロRNAによって特異的に認識される核酸配列と、前記マーカー遺伝子が、5'から3'の方向に連結されている核酸である、請求項１２に記載の巨核球細胞選別キット。 The mRNA is a nucleic acid in which a nucleic acid sequence specifically recognized by a microRNA selected from the let-7 family and the marker gene are linked in a 5 'to 3' direction. The megakaryocyte cell sorting kit described in 1.
  14. 　前記let-7ファミリーから選択されるマイクロRNAが、let-7a-5pである、請求項１２または１３に記載の巨核球細胞選別キット。 The megakaryocyte cell selection kit according to claim 12 or 13, wherein the microRNA selected from the let-7 family is let-7a-5p.
  15. 　巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
    　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
    を含み、前記不死化巨核球細胞を得る工程において、NR6A1遺伝子を強制発現させる工程を含む、血小板の産生方法。     In an undifferentiated cell than a megakaryocyte, an oncogene, a polycomb gene, and an apoptosis inhibitor gene are forcibly expressed to obtain an immortalized megakaryocyte,
    Including the step of suppressing the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and the step of forcibly expressing the NR6A1 gene in the step of obtaining the immortalized megakaryocyte cells. , Production method of platelets.
  16. 　巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
    　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
    を含み、前記不死化巨核球細胞を得る工程における前記細胞中の、Lin28A遺伝子をコードするDNAのプロモーターのCpGアイランド領域におけるメチル化量を減少させる工程を含む、血小板の産生方法。     In an undifferentiated cell than a megakaryocyte, an oncogene, a polycomb gene, and an apoptosis inhibitor gene are forcibly expressed to obtain an immortalized megakaryocyte,
    A step of inhibiting forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, and encoding the Lin28A gene in the cell in the step of obtaining the immortalized megakaryocyte cells A method for producing platelets, comprising a step of reducing the amount of methylation in a CpG island region of a promoter of DNA.
  17. 　前記メチル化量を減少させる工程が、前記不死化巨核球細胞を得る工程における細胞を振盪培養する工程を含む、請求項１６に記載の方法。 The method according to claim 16, wherein the step of reducing the amount of methylation includes a step of shaking and culturing the cells in the step of obtaining the immortalized megakaryocyte cells.
  18. 　多能性幹細胞を分化誘導して、巨核球細胞より未分化な細胞を得る工程と、
    　前記巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子を強制発現させて、不死化巨核球細胞を得る工程と、
    　前記癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子の強制発現を抑制して、巨核球細胞を培養する工程と
    を含み、前記巨核球細胞より未分化な細胞を得る工程が、前記多能性幹細胞をMatrigel（登録商標）、ラミニン421もしくはその断片、またはラミニン121若しくはその断片、あるいはそれらの組み合わせから選択される基質を用いて培養する工程を含む、血小板の産生方法。      Inducing differentiation of pluripotent stem cells to obtain undifferentiated cells from megakaryocytes,
    A step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-suppressing gene in an undifferentiated cell than the megakaryocyte cell to obtain an immortalized megakaryocyte cell;
    Suppressing the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene and culturing megakaryocyte cells, the step of obtaining undifferentiated cells from the megakaryocyte cells, the pluripotent stem cell A method for producing platelets, comprising a step of culturing using a substrate selected from Matrigel®, laminin 421 or a fragment thereof, or laminin 121 or a fragment thereof, or a combination thereof.
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