WO2014168255A1 - Accélérateur de la maturation de mégacaryocytes - Google Patents

Accélérateur de la maturation de mégacaryocytes Download PDF

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WO2014168255A1
WO2014168255A1 PCT/JP2014/060590 JP2014060590W WO2014168255A1 WO 2014168255 A1 WO2014168255 A1 WO 2014168255A1 JP 2014060590 W JP2014060590 W JP 2014060590W WO 2014168255 A1 WO2014168255 A1 WO 2014168255A1
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gene
megakaryocyte
megakaryocytes
cells
inhibitor
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浩之 江藤
真治 平田
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国立大学法人京都大学
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a method for producing mature megakaryocytes from immature megakaryocytes, a megakaryocyte maturation agent, and the like.
  • platelets which are essential for blood clotting and hemostasis, are one of the most important blood cells. Platelets are in great demand for leukemia, bone marrow transplantation, anticancer treatment, etc., and the need for a stable supply is high. So far, platelets have been secured not only by a method of collecting blood by donating blood from a donor, but also by a method of administering a TPO-like structural mimetic preparation, a method of differentiating megakaryocytes from umbilical cord blood or bone marrow cells. Recently, a technique for preparing blood cells such as platelets by inducing differentiation of pluripotent stem cells such as ES cells or iPS cells in vitro has also been developed.
  • transcription factors all-trans retinoic acid (ATRA), valproic acid, known as an inhibitor of histone deacetylase, ROCK inhibitor, or blebbistatin may promote maturation of megakaryocytes. It has been reported (Non-patent document 2 and Patent document 4). Furthermore, it has also been reported that knocking down p53, which is a tumor suppressor gene product, promotes multinucleation of megakaryocytes (Non-patent Document 3).
  • Non-Patent Document 4 culturing immature megakaryocytes at 39 ° C, higher than the normal culture temperature, promotes the induction of multinucleated mature megakaryocytes and the formation of proplatelets.
  • the present inventors In order to produce a platelet preparation, the present inventors stably and more produce functional platelets (platelets that retain in vivo activity such as hemostasis and are characterized as CD42b +). We found that it was necessary to establish a megakaryocyte strain, and thought that it was necessary to further mature the megakaryocyte strain obtained by the conventional method in order to overcome this problem. Then, this invention makes it a subject to provide the substance and its use for maturing such a megakaryocyte strain.
  • the present inventors searched for a compound suitable for maturation of megakaryocytes established from hematopoietic progenitor cells prepared from pluripotent stem cells (ES cells, iPS cells, etc.). ABC transporters
  • probenecid which is an inhibitor, has the function, and has completed the present invention.
  • the present invention includes the following.
  • a method for producing mature megakaryocytes comprising a step of culturing immature megakaryocytes in a culture solution containing an ABC transporter inhibitor.
  • the immature megakaryocyte causes a pluripotent stem cell-derived hematopoietic progenitor cell to forcibly express a gene selected from the group consisting of an oncogene, a gene that suppresses the expression of p16 gene or p19 gene, and an apoptosis-suppressing gene.
  • the immature megakaryocyte is a megakaryocyte produced by forcibly expressing a gene selected from the group consisting of c-Myc, Bmi1 and Bcl-xL into hematopoietic progenitor cells derived from pluripotent stem cells.
  • FIG. 1 shows each marker gene (GATA1, NF-E2, c-MPL, ⁇ 1-tubulin, after culturing an immortalized megakaryocyte strain with addition of probenecid (provenecid) or vehicle alone. The result of having measured the expression level of MYH9 and MYH10) by quantitative PCR is shown.
  • the present invention provides a method for producing mature megakaryocytes, comprising a step of culturing immature megakaryocytes in a culture solution containing an ABC (ATP binding cassette) transporter inhibitor. Similarly, the present invention provides a method for maturation of immature megakaryocytes using an ABC transporter inhibitor.
  • ABC ATP binding cassette
  • the ABC transporter is a membrane protein having an activity of transporting a substrate from the inside of the cell to the outside of the cell using the energy of ATP.
  • 49 subtypes are roughly divided into A to G families. included.
  • Preferred ABC transporter inhibitors in the present invention are drugs that inhibit at least the C family of ABC transporters (ABCC family), probenecid, cyclosporine, glibenclamide, furosemide, indomethacin, bromosulfophthalein, benzbromarone, telmisartan , MK571 and imatinib, siRNA or shRNA against ABCC family, siRNA or shRNA expression vector against p53, antisense nucleic acid against ABCC family, ribozyme against ABCC family, etc.
  • ABC transporter inhibitors in the present invention are drugs that inhibit at least the C family of ABC transporters (ABCC family), probenecid, cyclosporine, glibenclamide, furosemide, indomethacin, bromosul
  • siRNAs, shRNAs, antisense nucleic acids, ribozymes and neutralizing antibodies can be prepared from ABCC family gene sequences or amino acid sequences by methods well known to those skilled in the art.
  • the agent that inhibits the ABCC family is preferably probenecid.
  • probenecid is a compound having the chemical name 4- (Dipropylaminosulfonyl) benzoic acid.
  • the concentration of probenecid in the medium is not particularly limited as long as it is a concentration that inhibits the ABC transporter.
  • it is 250 ⁇ M to 25 mM, more preferably 1 mM to 5 mM, and most preferably 2.5 ⁇ M. mM.
  • the culture solution used in the step of producing mature megakaryocytes is not particularly limited, but can be prepared by adding an ABC transporter inhibitor using a medium used for animal cell culture as a basal medium.
  • basal media include IMDM medium, MediumMedi199 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 Life's medium, Neurosal's medium And a mixed medium thereof.
  • the medium may contain serum or may be serum-free.
  • the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules
  • One or more substances such as compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like may also be included.
  • Cytokines are proteins that promote blood cell differentiation, and examples include VEGF, TPO, SCF, and the like.
  • a preferable medium in the present invention is an IMDM medium containing TPO, SCF, serum, insulin, transferrin, serine, thiolglycerol and ascorbic acid.
  • the period of culturing immature megakaryocytes in a culture solution containing an ABC transporter inhibitor does not prevent megakaryocyte maturation over a long period of time.
  • Examples include days, 4 days, 5 days, 6 days, 7 days or more.
  • it is 4 days or longer. More preferably, it is 4 days to 28 days, 4 days to 21 days, 4 days to 14 days, or 4 days to 7 days.
  • the culture temperature in the step of producing mature megakaryocytes is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. to 39 ° C.
  • the culture is performed in an atmosphere of CO 2 -containing air, and CO 2
  • the concentration is preferably about 2-5%.
  • “Immature megakaryocytes” in the present invention are megakaryocytes (about 2N to 8N) that have not progressed multinucleation.
  • megakaryocyte maturation-related genes GATA1, NF-E2, c-MPL, ⁇ 1-tubulin and MYH9
  • GATA1, NF-E2, c-MPL, ⁇ 1-tubulin and MYH9 are low expression levels and MYH10 expression levels are megakaryocytes. Therefore, immature megakaryocytes can be paraphrased as megakaryocyte progenitor cells. Examples of such immature megakaryocytes include immature megakaryocytes artificially produced from hematopoietic progenitor cells.
  • the immature megakaryocyte may or may not be cloned, and is not particularly limited, but the cloned one may be referred to as an immature megakaryocyte strain.
  • a “mature megakaryocyte” is a megakaryocyte with a high expression level of a megakaryocyte maturation-related gene group and a low expression level of MYH10, and a megakaryocyte (8N or more) that is becoming multinucleated.
  • “maturation” means that the maturation of megakaryocytes progresses, that is, the multinucleation progresses, the expression level of megakaryocyte maturation-related genes increases, the expression level of megakaryocyte immaturity-related genes decreases. As long as these tendencies are observed as compared with those before the maturation step, it includes cases where the result does not necessarily lead to mature megakaryocytes as defined above.
  • “maturation” refers to the case where the cell groups before and after the culture are compared and the maturation is progressing as a whole group, that is, the total number of nuclei of the entire cell group is increasing.
  • hematopoietic progenitor cells are cells that can differentiate into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, megakaryocytes, etc. Hematopoietic stem cells are not distinguished and indicate the same cells unless otherwise specified. Hematopoietic stem / progenitor cells can be recognized by, for example, positive surface antigens CD34 and / or CD43. In the present invention, hematopoietic stem cells can also be applied to pluripotent stem cells, hematopoietic progenitor cells derived from cord blood, bone marrow blood, peripheral blood-derived hematopoietic stem cells and progenitor cells.
  • pluripotent stem cells are cultured on C3H10T1 / 2 in the presence of VEGF according to the method described in Takayama N., et al. J Exp Med. 2817-2830 (2010) Can be prepared from a net-like structure (also referred to as ES-sac or iPS-sac).
  • the “net-like structure” is a three-dimensional sac-like structure (with space inside) derived from pluripotent stem cells, which is formed by an endothelial cell population and the like, and contains hematopoietic progenitor cells inside. It is a structure.
  • hematopoietic progenitor cells from pluripotent stem cells
  • embryoid bodies are formed and cytokines are added (Chadwick et al. Blood 2003, 102: ⁇ 906-15, Vijayaravavan et al. Cell Stem Cell 2009, 4: 248-62, Saeki et al. Stem Cells 2009, 27: 59-67) or co-culture with heterologous stromal cells (Niwa A et al. J Cell Physiol. 2009 Nov; 221 (2 ): 367-77.) And the like.
  • a pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into all cells present in a living body and also has a proliferative ability, and includes, for example, an embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic germ cells (“EG cells”), induced pluripotent stem (iPS) cells And pluripotent cells derived from cultured fibroblasts and bone marrow stem cells (Muse cells).
  • ES embryonic stem
  • ntES embryonic stem
  • GS cells sperm stem cells
  • EG cells embryonic germ cells
  • iPS induced pluripotent stem
  • pluripotent cells derived from cultured fibroblasts and bone marrow stem cells
  • the method for artificially producing immature megakaryocytes from hematopoietic progenitor cells is selected from the group consisting of genes that suppress the expression of oncogenes, p16 genes or p19 genes to hematopoietic progenitor cells, and apoptosis-suppressing genes.
  • a method of culturing by forced expression of a gene is exemplified.
  • a method for producing immature megakaryocytes by forcibly expressing at least an oncogene in hematopoietic progenitor cells is preferred.
  • More preferred is a method in which an oncogene and a gene that suppresses expression of the p16 gene or p19 gene are forcibly expressed in hematopoietic progenitor cells, and then an apoptosis-suppressing gene is forcibly expressed.
  • an apoptosis-inhibiting gene when the apoptosis-inhibiting gene is further forcibly expressed, it is preferable to forcibly express the oncogene and the gene that suppresses the expression of the p16 gene or the p19 gene for at least 14 days.
  • the term “oncogene” refers to a gene that causes canceration of normal cells due to its expression, structure or function being different from that of normal cells.
  • MYC family genes MYC family genes, Src family genes , Ras family genes, Raf family genes, protein kinase family genes such as c-Kit, PDGFR, and Abl.
  • MYC family genes include c-MYC, N-MYC, and L-MYC. More preferably, it is a c-MYC gene.
  • the c-MYC gene is, for example, a gene consisting of a nucleic acid sequence represented by NCBI accession number NM_002467.
  • the c-MYC gene may also include homologues thereof.
  • the c-MYC gene homologue is a sequence whose cDNA sequence is substantially the same as the nucleic acid sequence represented by NCBI accession number NM_002467, for example. It is a gene consisting of The cDNA consisting of a sequence substantially identical to the nucleic acid sequence is about 60% or more, preferably about 70% or more, more preferably about 80%, with the DNA consisting of the sequence represented by NCBI accession number NM_002467, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, most preferably about 99% identity DNA, or DNA that can be hybridized under stringent conditions with DNA complementary to the nucleic acid sequence, Proteins encoded by DNA are those that contribute to the amplification of differentiated cells, such as hematopoietic progenitor cells.
  • stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical experimental conditions that depend on the probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally relies on the ability to reanneal in an environment where the complementary strand is slightly below its melting point.
  • low stringency conditions include washing in a 0.1 ⁇ SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. in the filter washing step after hybridization.
  • highly stringent conditions include washing in 65 ° C., 5 ⁇ SSC and 0.1% SDS in the washing step.
  • a gene that suppresses the expression of p16 gene or p19 gene is a gene that suppresses the expression of p16 gene or p19 gene by oncogene-induced cell aging caused by the introduction of an oncogene, for example, BMI1, Mel18, Ring1a / b, Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HADC and Dnmt1 / 3a / 3b are exemplified.
  • BMI1 is preferable.
  • BMI1 is, for example, a gene consisting of a nucleic acid sequence represented by NCBI accession number NM_005180.
  • the BMI1 gene may also include homologues thereof.
  • the BMI1 gene homologue is a gene whose cDNA sequence is substantially the same as the nucleic acid sequence described in NCBI accession number NM_005180. It is.
  • the cDNA consisting of a sequence substantially identical to the nucleic acid sequence represented by NCBI accession number NM_005180 is about 60% or more, preferably about 70% or more, more preferably about 80% or 81%, with the nucleic acid sequence.
  • the encoded protein is one that contributes to the differentiation of cells in the differentiation stage such as hematopoietic progenitor cells.
  • the “apoptosis-suppressing gene” is not particularly limited as long as it is a gene that suppresses apoptosis, and examples thereof include BCL2 gene, BCL-XL gene, Survivin, and MCL1.
  • BCL2 gene BCL-XL gene
  • BCL-XL gene is a gene consisting of a nucleic acid sequence represented by NCBI accession number NM_001191 or NM_138578, for example.
  • the BCL-XL gene may also include a homologue thereof.
  • the homologue of the BCL-XL gene is substantially the same as the nucleic acid sequence represented by the NCBI accession number NM_001191 or NM_138578, for example.
  • the cDNA consisting of a sequence substantially identical to the nucleic acid sequence represented by NCBI accession number NM_001191 or NM_138578 is about 60% or more, preferably about 70% or more, more preferably about 80%, with the nucleic acid sequence. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, most preferably about 99% identity DNA, or DNA that can be hybridized under stringent conditions with DNA complementary to the nucleic acid sequence, and the DNA Is a protein having an effect of suppressing apoptosis.
  • the gene forcibly expressed in hematopoietic progenitor cells may be fused with an estrogen receptor (ER) in order to control the activity by adding 17 ⁇ estradiol or 4OH-Tamoxifen.
  • ER estrogen receptor
  • the above gene can be forcibly expressed in hematopoietic progenitor cells according to the ordinary method of those skilled in the art.
  • vectors expressing these genes, or proteins or RNAs encoding these genes In the form of hematopoietic progenitor cells.
  • it can be performed by bringing a low molecular weight compound or the like that induces expression of these genes into contact with hematopoietic progenitor cells.
  • an expression vector, protein, RNA or a low molecular weight compound that induces expression should be introduced multiple times in accordance with the necessary period. Can be done.
  • 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), episomal vector (plasmid vector containing replication origin oriP and EBNA-1 gene or replication origin ori and SV40 large T antigen gene), piggyBac vector (Kaji, K.
  • artificial chromosome vectors for example, human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC, PAC)
  • HAC human artificial chromosome
  • YAC yeast artificial chromosome
  • BAC bacterial artificial chromosome
  • PAC bacterial artificial chromosome
  • 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.
  • the promoter region in order to control the expression of the gene by tetracycline or doxycycline, the promoter region has a tetracycline-responsive element, and a drug-responsive vector that allows gene expression in the presence of the corresponding drug
  • a vector that simultaneously expresses a fusion protein composed of a mutant Tet repressor protein (rTetR) and a VP16 activation domain (AD), or a drug reverse responsive vector that stops gene expression in the presence of the corresponding drug (Tet It may be a vector that simultaneously expresses a fusion protein composed of a presser protein (TetR) and a VP16 activation domain (AD).
  • 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.
  • the genes may be vertically linked to obtain a polycistronic vector.
  • it is ligated between 2A self-cleaving peptides of foot-and-mouth disease virus (see Science, 322, 949-953, 2008, etc.) and genes that forcefully express IRES sequences, etc. obtain.
  • a plasmid containing the nucleic acid is used as an appropriate packaging cell (eg, Plat-E cell) or a complementary cell line (eg, 293 cell). And the virus produced in the culture supernatant is collected and contacted with hematopoietic progenitor cells for infection.
  • an appropriate packaging cell eg, Plat-E cell
  • a complementary cell line eg, 293 cell
  • a plasmid vector in the case of a non-viral vector, can be introduced into a cell using a lipofection method, a liposome method, an electroporation method, a calcium phosphate coprecipitation method, a DEAE dextran method, a microinjection method, a gene gun method, or the like.
  • the feeder cell is not particularly limited as long as it can induce megakaryocytes.
  • C3H10T1 / 2 (Katagiri T, et al., Biochem Biophys Res Commun. 172, 295-299 (1990)) Can be mentioned.
  • the medium used for producing the immature megakaryocytes of the present invention is not particularly limited, but a medium used for culturing animal cells can be prepared as a basal medium.
  • basal media include IMDM medium, MediumMedi199 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 Life's medium, Neurosal's medium And a mixed medium thereof.
  • the medium may contain serum or may be serum-free.
  • the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules
  • One or more substances such as compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like may also be included.
  • Cytokines are proteins that promote blood cell differentiation, and examples include VEGF, TPO, SCF, and the like.
  • a preferable medium in the present invention is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO.
  • the undifferentiated megakaryocyte may be a megakaryocyte that maintains the expression of the gene.
  • the conditions for culturing are not particularly limited, but can be about 30 ° C. to about 42 ° C. It has been confirmed that culturing at a temperature of 37 ° C. or higher promotes the differentiation of megakaryocytes.
  • the temperature of 37 ° C. or higher is appropriate as a temperature that does not damage cells, and is preferably about 37 ° C. to about 42 ° C., preferably about 37 to about 39 ° C., for example.
  • the culture period at a temperature of 37 ° C. or higher can be appropriately determined while monitoring the number of megakaryocytes, etc., for example, 6 days to 48 days, preferably about 12 days to 36 days. is there.
  • the culture may be carried out in an atmosphere of CO 2 -containing air, in which case the CO 2 concentration is preferably about 2-5%.
  • the long culture period is not a problem in the production of megakaryocytes.
  • a substance that inhibits the expression or function of the p53 gene product (b) an actomyosin complex function inhibitor, (c) a ROCK inhibitor ( d) A culture medium further containing an HDAC inhibitor may be used.
  • PFT Pifithrin
  • WO2000 / 44364 Pifithrin
  • PFT- ⁇ Storm et al, Nat. Chem. Biol.
  • P53 dominant negative mutants such as p53P275S (de Vries, A., Proc.ciNatl. Acad. Sci. USA, 99, 2948-2953B (2002)), p53DD (Bowman, T., Genes Develop., 10 , 826-835 (1996)), siRNA or shRNA against p53, a vector expressing siRNA or shRNA against p53, an antisense nucleic acid against p53, a ribozyme against p53, and the like.
  • ROCK inhibitors include Y27632.
  • actomyosin complex function inhibitor examples 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 process of stopping forced expression when immature megakaryocytes produced by forcibly expressing a gene in hematopoietic progenitor cells are used, in addition to the step of culturing in a culture solution containing ABC transporter for the megakaryocyte maturation, You may include the process of stopping forced expression.
  • a method of 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.
  • forced expression is performed using a drug reverse responsive vector, this may be achieved by bringing the corresponding drug into contact with the cell and / or stopping the contact.
  • the above-mentioned vector containing LoxP when 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.
  • maturation of megakaryocytes means that megakaryocytes are sufficiently polynucleated and can produce functional platelets. Megakaryocyte maturation can also be confirmed by, for example, increased expression of megakaryocyte maturation-related genes such as GATA1, p45pNF-E2, beta1-tubulin, c-MPL, and MYH9.
  • (Mature megakaryocyte composition) When immature megakaryocytes are treated by the method of the present invention, they can mature into mature megakaryocytes. Therefore, a cell composition having a high content of mature megakaryocytes can be obtained. Whether or not the content of mature megakaryocytes is high in a cell composition can be determined by those skilled in the art based on experience and literature.
  • the content of mature megakaryocytes is at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, more preferably 100%. It is. Therefore, according to the method of the present invention, it is possible to prepare a megakaryocyte population having a high abundance ratio of mature megakaryocytes.
  • the megakaryocytes obtained by the method of the present invention are also effective for transplanting in vivo and producing functional platelets in vivo by an appropriate method. Therefore, the therapeutic agent containing the megakaryocyte obtained by the method of this invention is provided.
  • the transplantation of megakaryocytes and the like obtained by the method of the present invention can solve the problem of insufficient donor number and donor burden in bone marrow transplantation and the problem of platelet production ability in vivo in cord blood transplantation. Compared to the transplantation method, it can be said to be a very excellent method.
  • Platelets can be produced in vitro from mature megakaryocytes obtained by the method of the present invention.
  • the method for producing platelets according to the present invention includes a step of culturing mature megakaryocytes obtained by the above-described method and collecting platelets from the culture. Culture conditions can be carried out by continuing the process of producing mature megakaryocytes.
  • 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 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 solution, citrate-containing physiological saline, a solution containing glucose-added acetate Ringer solution, PAS (platelet additive solution) (Gulliksson, H. et al., Transfusion, 32: 435-440, (1992)), etc.
  • 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).
  • An embodiment of the present invention includes a kit for producing a megakaryocyte maturation agent or mature megakaryocyte.
  • the megakaryocyte maturation agent or kit includes the ABC transporter inhibitor described above.
  • reagents that can be used for maturation of megakaryocytes such as culture broth, (a) substances that inhibit the expression or function of p53 gene product, (b) actomyosin complex function inhibitor, (c) ROCK inhibitor (d) HDAC inhibitors are mentioned.
  • the kit may further contain reagents for recognizing mature megakaryocytes (eg, PCR primers for GATA1, p45 NF-E2, beta1-tubulin, c-MPL, and MYH9).
  • the megakaryocyte maturation agent or the reagent contained in the kit must be contained in any type of container in which the components remain active for a long period of time, are not adsorbed by the container material, and do not undergo alteration. Supplied.
  • the origin of the “cell” described in the present specification is a human or non-human animal (eg, mouse, rat, cow, horse, pig, sheep, monkey, dog, cat, bird, etc.) and is not particularly limited. But. Particularly preferred are human-derived cells.
  • a human iPS cell-derived immortalized megakaryocyte strain was prepared by improving the method described in WO 2012/157586. Briefly, hematopoietic progenitor cells are prepared from iPS cells prepared from human peripheral blood, and c-Myc, Bmi1 and Bcl-xL are introduced by retrovirus method to produce an immortalized megakaryocyte strain and introduced. The cultured cells were maintained for at least 24 days with the expressed genes expressed. In addition, the introduced gene can suppress the expression of c-Myc, Bmi1 and Bcl-xL by the addition of Doxicyclin.
  • ⁇ Maturation of immortalized megakaryocyte strain > C3H10T1 / 2 cells previously inactivated with mitomycin C were seeded at 1 ⁇ 10 4 cells / well (96 well plate) and cultured overnight or more to prepare feeder cells. On this feeder cell, the above immortalized megakaryocyte strain is seeded at 3 ⁇ 10 4 cells / well (96 well plate) and 50 ng / mL SCF, 50 ng / mL in the presence or absence of 2.5 mM Probenecid (addition of base alone) Basal medium (15% Fetal Bovine Serum, 1% Penicillin-Streptomycin-Glutamine, 1% Insulin, Transferrin, Selenium Solution, 0.45 mM 1-Thioglycerol and 50 ⁇ g / mL L-Ascorbic Acid containing mL TPO and 0.5 ⁇ g / mL Doxicyclin The cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium
  • immortalized megakaryocyte strains cultured in the presence of 2.5 mM Probenecid were compared with immortalized megakaryocyte strains cultured with only the base added (GATA1, NF-E2).
  • C-MPL, ⁇ 1-tubulin, and MYH9 were increased about 2-3 times, and MYH10, a megakaryocyte immaturity-related gene, was reduced to about 1/2. From the above, it was confirmed that the maturation of human iPS cell-derived immortalized megakaryocyte strain was promoted by adding Probenecid and culturing.

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Abstract

La présente invention aborde le problème de fournir un procédé de production pour des mégacaryocytes matures. La présente invention concerne un procédé de production pour des mégacaryocytes matures qui comprend une étape dans laquelle les mégacaryocytes immatures sont mis en culture dans une solution de culture qui contient un inhibiteur de transporteur ABC.
PCT/JP2014/060590 2013-04-12 2014-04-14 Accélérateur de la maturation de mégacaryocytes WO2014168255A1 (fr)

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WO2017131230A1 (fr) * 2016-01-29 2017-08-03 国立大学法人京都大学 Promoteur de production plaquettaire et procédé de production plaquettaire au moyen de celui-ci
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017131228A1 (fr) * 2016-01-29 2017-08-03 国立大学法人京都大学 Procédé de criblage d'un promoteur de production plaquettaire
WO2017131230A1 (fr) * 2016-01-29 2017-08-03 国立大学法人京都大学 Promoteur de production plaquettaire et procédé de production plaquettaire au moyen de celui-ci
JPWO2017131230A1 (ja) * 2016-01-29 2018-11-22 国立大学法人京都大学 血小板産生促進剤及びそれを用いた血小板の製造方法
JPWO2017131228A1 (ja) * 2016-01-29 2018-11-22 国立大学法人京都大学 血小板産生促進剤のスクリーニング方法
EP3409774A4 (fr) * 2016-01-29 2019-10-23 Kyoto University Procédé de criblage d'un promoteur de production plaquettaire
US10941382B2 (en) 2016-01-29 2021-03-09 Kyoto University Platelet production promoter and method of producing platelets using same
US11016083B2 (en) 2016-01-29 2021-05-25 Kyoto University Method for screening for platelet production promoters
US20210239682A1 (en) * 2016-01-29 2021-08-05 Kyoto University Method for Screening for Platelet Production Promoters
JP7350246B2 (ja) 2016-01-29 2023-09-26 国立大学法人京都大学 血小板産生促進剤のスクリーニング方法
US11977071B2 (en) 2016-01-29 2024-05-07 Kyoto University Method for screening for platelet production promoters
US11116737B1 (en) 2020-04-10 2021-09-14 University Of Georgia Research Foundation, Inc. Methods of using probenecid for treatment of coronavirus infections
US11903916B2 (en) 2020-04-10 2024-02-20 University Of Georgia Research Foundation, Inc. Methods of using probenecid for treatment of coronavirus infections

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