WO2021060467A1 - Procédé de production de cellule progénitrice de mégacaryocyte et de cellule mégacaryocyte, et cellule progénitrice de mégacaryocyte et cellule mégacaryocyte ainsi obtenue - Google Patents

Procédé de production de cellule progénitrice de mégacaryocyte et de cellule mégacaryocyte, et cellule progénitrice de mégacaryocyte et cellule mégacaryocyte ainsi obtenue Download PDF

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WO2021060467A1
WO2021060467A1 PCT/JP2020/036272 JP2020036272W WO2021060467A1 WO 2021060467 A1 WO2021060467 A1 WO 2021060467A1 JP 2020036272 W JP2020036272 W JP 2020036272W WO 2021060467 A1 WO2021060467 A1 WO 2021060467A1
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megakaryocyte
cells
cell
gene
megakaryocyte progenitor
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Japanese (ja)
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啓光 中内
山崎　聡
清純 越智
素生 渡部
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国立大学法人東京大学
株式会社メガカリオン
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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
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to a method for producing immortalized megakaryocyte progenitor cells and platelets from CD34-positive cells.
  • Platelet preparations are administered to patients with decreased platelets during bleeding such as surgery or injury. Platelet preparations are currently produced from blood obtained from blood donations. However, due to changes in the population composition, there is concern that the amount of blood donated will decrease and that platelet preparations will be in short supply. In addition, if the blood donor is infected with an infectious disease such as bacteria, the blood may be contaminated with bacteria, and therefore there is a risk of infectious disease due to administration of the platelet preparation contaminated with bacteria. Therefore, a method for producing platelets in vitro has been developed (Non-Patent Document 1).
  • Producing megakaryocyte cells from pluripotent stem cells such as ES cells and iPS cells that is, through differentiation from pluripotent stem cells to blood stem cells and further to megakaryocyte progenitor cells in vitro, mature megakaryocytes. It is possible to produce sphere cells (pluripotent megakaryocyte cells) (Patent Document 1). However, in these methods, in order to induce differentiation from pluripotent stem cells to megakaryocyte progenitor cells, selection of pluripotent stem cells, various growth factors, and the like are required, so that a large cost is required. In addition, since blood cells other than megakaryocytic cells are also contained in the early stage after induction of differentiation, a large culture period and amount are required for selection.
  • the present invention is a method for producing a megakaryocyte progenitor cell line capable of efficiently and stably producing mature megakaryocyte cells, and a method for producing megakaryocyte cells and platelets from the megakaryocyte progenitor cell line obtained by the production method. I will provide a.
  • the present inventors have established megakaryocyte progenitor cells and megakaryocytes and their immortalized cell lines by expressing and culturing the E6 gene and / or E7 gene of human papillomavirus in human bone marrow-derived hematopoietic stem cells. Successful.
  • the obtained megakaryocyte cells contained multinucleated megakaryocyte cells, and that the megakaryocyte cells formed platelet precursors.
  • the present application is based on these findings.
  • the present invention provides the following. (1) Megakaryocyte progenitor cells or megakaryocyte cells containing either or both of the E6 gene and the E7 gene. (2) The megakaryocyte progenitor cell or megakaryocyte cell according to (1) above, which has either or both of the E6 gene and the E7 gene on the genome. (3) The megakaryocyte progenitor cell according to (1) or (2) above, which is a mononuclear cell. (4) The megakaryocyte progenitor cell or megakaryocyte cell according to (1) above, which is an established megakaryocyte progenitor cell or megakaryocyte cell.
  • the megakaryocyte progenitor cell or megakaryocyte cell according to (3) above which is an established megakaryocyte progenitor cell or megakaryocyte cell.
  • megakaryocyte lineage cells eg, hematopoietic stem cells, hematopoietic progenitor cells or CD34 positive cells
  • a method comprising culturing the resulting cells in the presence of thrombopoietin (TPO).
  • TPO thrombopoietin
  • 7A A method for producing megakaryocyte precursor cells or megakaryocyte cells, in which megakaryocyte lineage cells having the E6 gene and / or the E7 gene (for example, hematopoietic stem cells, hematopoietic precursor cells or CD34-positive cells) are differentiated into megakaryocytes.
  • a method comprising culturing under suitable culturing conditions.
  • a method for producing a platelet preparation which is a method for producing a platelet preparation.
  • E6 and E7 genes Expressing one or both of the E6 and E7 genes in megakaryocyte lineage cells (eg, hematopoietic stem cells or hematopoietic progenitor cells)
  • the obtained cells were cultured in the presence of thrombopoietin (TPO) to obtain megakaryocyte cells, and platelets were recovered from the culture of megakaryocyte cells. Including methods.
  • (9A) A method for producing a platelet preparation, which comprises preparing a culture of megakaryocyte cells having an E6 gene and / or an E7 gene, and recovering platelets from the culture.
  • Method. (10) The method according to (9) or (9A) above, wherein the megakaryocyte cell is a cell in which the gene expression of the E6 gene and the E7 gene is suppressed.
  • a method for producing a megakaryocyte progenitor cell line capable of efficiently and stably producing megakaryocyte progenitor cells and a method for producing megakaryocyte cells and platelets from the megakaryocyte progenitor cell line obtained by the production method. It becomes possible to provide a method of doing so.
  • FIG. 1 shows the results of gene expression analysis of the culture of hematopoietic stem cells expressing the E6 protein and the E7 protein on the 21st day after virus removal (panel A) and the change in the number of CD41 cells after the start of the culture (panel B). ) Is shown.
  • E6-2A-E7 is simply referred to as “E6-E7”.
  • FIG. 2 shows the proportion of CD41-positive and CD42b-positive cells in the culture 70 days after virus removal.
  • FIG. 3 shows the proportion of CD41-positive and CD42b-positive cells in the culture 3 weeks after the introduction of the virus of hematopoietic stem cells expressing the E6 gene or the E7 gene alone (panel A) and either the E6 protein or the E7 protein.
  • the change (week) in the proportion (%) of the CD41-positive and CD42b-positive cells in the culture of the hematopoietic stem cells expressing both or both after the virus removal is shown.
  • FIG. 4 shows changes in the number of hematopoietic stem cells expressing either or both of the E6 gene and the E7 gene during culture.
  • FIG. 5 shows the proportion of PI-positive cells generated in hematopoietic stem cells expressing either or both of the E6 and E7 genes.
  • FIG. 6 shows the production of platelet precursors from immortalized megakaryocyte progenitor cells.
  • hematopoietic stem cell means a stem cell capable of differentiating into a blood lineage cell. It is known that hematopoietic stem cells usually do not have the ability to differentiate into cells other than the blood system. It is also known to have the ability to differentiate into all kinds of blood cells. "Hematopoietic stem cells” are also referred to as “blood stem cells”. Such “hematopoietic stem cells” are separated and recovered from tissues such as cord blood, peripheral blood, bone marrow, and fetal liver by a flow cytometry method or the like using an antibody that specifically binds to a surface antigen (CD34 or the like) of the hematopoietic stem cells. It is known that it is abundantly contained in the cell population. Further, in the present invention, “hematopoietic stem cells” can also be prepared by inducing differentiation from human pluripotent stem cells.
  • megakaryocytes are used in the sense of including mononuclear megakaryocytes and mature megakaryocytes that produce platelets.
  • mature megakaryocytes have chromosomes of 4N to 128N.
  • Mature megakaryocytes are derived from hematopoietic stem cells and become mature megakaryocytes via CD34-positive hematopoietic stem cells, hematopoietic progenitor cells, megakaryocyte blasts, and megakaryocyte progenitor cells (megakaryocyte lineage cells).
  • a megakaryocyte having a 2N chromosome before becoming a polyploid is referred to as a mononuclear megakaryocyte.
  • a megakaryocyte cell line it will be created from mononuclear megakaryocytes.
  • Mature megakaryocytes have the ability to produce platelets.
  • the "megakaryocyte progenitor cell” means a cell capable of differentiating into a mature megakaryocyte cell (that is, a multinucleated megakaryocyte cell). Megakaryocyte progenitor cells can be cells that have lost the ability to differentiate into blood lineage cells other than megakaryocytes.
  • the "megakaryocyte progenitor cell line” means an established megakaryocyte progenitor cell, and specifically, an immortalized megakaryocyte progenitor cell.
  • HPV human papillomavirus
  • HPV is one of the viruses belonging to the papillomavirus family, and is a virus that forms warts called papilloma (or papilloma).
  • HPV has E1 to E7 as early genes and L1 and L2 as late genes.
  • E6 and E7 are known as proteins involved in carcinogenesis (particularly cervical cancer carcinogenesis).
  • the E6 protein means an E6 protein of type 16 of HPV and an E6 protein having an amino acid sequence corresponding to the amino acid sequence of the protein.
  • the E7 protein means an E7 protein of type 16 of HPV and an E7 protein having an amino acid sequence corresponding to the amino acid sequence of the protein.
  • the "cell” is a mammalian cell, for example, a human cell. It is known that when cells are established, they repeat cell division indefinitely.
  • corresponding to a certain amino acid sequence means an ortholog of the amino acid sequence and a protein having the same functionality as the protein having the amino acid sequence.
  • the E6 protein and E7 protein have the same sequence as the above amino acid sequence, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, respectively.
  • E6 and E7 proteins of sex may be included.
  • HPV human papillomavirus
  • E7 gene also referred to as an E7 gene
  • cells of the megakaryocyte lineage such as megakaryocyte progenitor cells and megakaryocyte cells are provided.
  • the HPV E6 gene is not particularly limited, but can be, for example, the 16-type HPV E6 gene (also referred to as “HPV-E6 gene”).
  • the E7 gene of HPV is not particularly limited, but for example, the E7 gene of type 16 HPV (also referred to as "HPV-E7 gene”; the E6 gene and E7 gene of type 16 HPV are collectively referred to as "HPV-E6 / E7 gene”. It can be said).
  • HPV-E6 / E7 gene is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (HPV-E6 protein) and a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (HPV-E7). It can be a protein, eg, a nucleic acid encoding SEQ ID NO: 3).
  • the E6 gene and the E7 gene may be contained in separate nucleic acids (for example, DNA and RNA), but may be preferably contained in one nucleic acid. When the E6 gene and the E7 gene are contained in one nucleic acid, they may be polycistronicly contained in the nucleic acid or monocistronicly contained in the nucleic acid.
  • the nucleic acid containing the E6 gene and the E7 gene may contain a gene encoding a fusion protein of the E6 protein and the E7 protein (for example, a fusion protein linked with a 2A peptide).
  • the 2A peptide has a sequence having a length of about 20 amino acids derived from a virus, is recognized by a protease (2A peptidase) inherent in the cell, and is cleaved at one residue from the C-terminal thereof.
  • Examples of the 2A peptide include a 2A peptide having an amino acid sequence corresponding to the amino acid sequence shown in SEQ ID NO: 4, which can be used in the present invention.
  • the 2A peptide can be linked to a protein by interposing a spacer (for example, an amino acid sequence having a length of about 3 amino acids) before and after the spacer.
  • the E6 gene and / or the E7 gene may be contained in a gene expression vector for mammals, or may be contained on the genome of a cell.
  • Gene expression vectors include viral vectors (eg, lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, and Sendaivirus) and plasmid vectors.
  • the gene expression vector may be integrated into the genome or present episomally in the cell.
  • an RNA viral vector such as Sendai virus, it can be removed by introducing a microRNA target sequence into the genome and introducing microRNA into infected cells of the virus.
  • the E6 gene and / or the E7 gene may be operably linked to an expression regulatory sequence (promoter) capable of expressing these genes in mammalian cells.
  • Promoters include promoters that result in constitutive expression and inducible promoters.
  • the inducible promoter for example, a promoter that activates transcription in response to an external stimulus can be used.
  • the external stimulus is a tetracycline antibiotic (tetracycline, doxycycline, etc.)
  • the tetracycline response can be used.
  • a promoter containing a factor (TRE) and a minimal promoter can be used.
  • a promoter containing a tetracycline response factor (TRE) and a minimal promoter is inactivated when a tetracycline antibiotic (tetracycline, doxycycline, etc.) binds in the presence of a tetracycline-regulated transactivator (tTA), and the tetracycline antibiotic It is activated when (tetracycline, doxycycline, etc.) comes off.
  • TRE tetracycline response factor
  • tTA tetracycline-regulated transactivator
  • a promoter containing a tetracycline response factor (TRE) and a minimal promoter is activated by binding to a tetracycline antibiotic (tetracycline, doxycycline, etc.) in the presence of a rivers tetracycline-regulated transactivator (rtTA), and is a tetracycline antibiotic. It is inactivated when antibiotics (tetracycline, doxycycline, etc.) are removed.
  • a tetracycline response factor tetracycline, doxycycline, etc.
  • the binding of the ecdysteroid with the ecdysone receptor-retinoid receptor complex causes the downstream gene.
  • ecdysteroids ecdysone, mristolone A, ponasterone A, etc.
  • the binding of the ecdysteroid with the ecdysone receptor-retinoid receptor complex causes the downstream gene. Examples include promoters capable of inducing expression.
  • the expression of downstream genes can be induced by binding FKCsA to the Gal4 DNA binding domain fused to FKBP12 and the VP16 activator domain complex fused to cyclophilin. Promoters can be mentioned.
  • megakaryocyte lineage cells examples include CD34-positive hematopoietic stem cells, hematopoietic progenitor cells, megakaryocyte progenitor cells, and megakaryocytes derived from hematopoietic stem cells.
  • the cells of the megakaryocyte lineage are cells selected from the group consisting of CD41-positive cells, von Willebrand factor (VMF) -positive cells, and CD41-positive and CD42b-positive cells (eg, megakaryocyte precursor cells, etc.). And can be a megakaryocyte).
  • Cells with a 2N karyotype are suitable for proliferation. Therefore, cells with a 2N karyotype can be preferably strained. Straining can be done by immortalizing the cells. Immortalization can be performed using conventional methods, for example, by introducing an immortalizing factor (eg, SV40 virus T antigen, telomere reverse transcriptase (TERT), etc.) into cells.
  • an immortalizing factor eg, SV40 virus T antigen, telomere reverse transcriptase (TERT), etc.
  • the megakaryocyte progenitor cells or megakaryocyte cells may be mononuclear.
  • Mononuclear megakaryocyte progenitor cells or mononuclear megakaryocyte cells or compositions containing them can be used, for example, to prepare working cell banks, ie, It can be used as a master cell bank for preparing a working cell bank.
  • a frozen megakaryocyte progenitor cell or megakaryocyte cell containing either or both of the E6 and E7 genes; and either or both of the E6 and E7 genes are present on the genome.
  • Megakaryocyte progenitor cells or frozen megakaryocyte cells are provided for use as a master cell bank.
  • a frozen megakaryocyte precursor cell or megakaryocyte cell containing either or both of the E6 and E7 genes; and either or both of the E6 and E7 genes are present on the genome.
  • Whether or not a cell has the E6 gene and / or the E7 gene can be detected by conventional methods (for example, PCR method, Southern blotting, and Northern blotting).
  • a nucleic acid eg, DNA or mRNA
  • HPV human papillomavirus
  • Cells eg, hematopoietic stem cells and hematopoietic progenitor cells
  • nucleic acids eg, DNA or mRNA
  • the hematopoietic stem cell can be a CD34 positive hematopoietic stem cell.
  • the DNA may contain an E6 gene and / or an E7 gene operably linked to an expression regulatory sequence (promoter).
  • a gene expression vector containing the E6 and / or E7 gene of human papillomavirus (HPV) operably linked to an inducible promoter is introduced into cells.
  • the external stimulus that activates the inducible promoter can be applied to the cells at any time (eg, before, during, or immediately after the introduction of the vector). In some embodiments, no external stimulus that activates the inducible promoter is added prior to vector introduction. In some embodiments, the external stimulus that activates the inducible promoter is added to the cells for a period of time after vector introduction, but may then be removed. Removal can be done before or after the desired cells (eg, megakaryocyte progenitor cells or megakaryocyte cells) are born.
  • desired cells eg, megakaryocyte progenitor cells or megakaryocyte cells
  • An expression cassette capable of inducing the expression of human papillomavirus (HPV) type 16 E6 gene and E7 gene (HPV-E6 / E7 gene) in response to external stimuli introduced into human hematopoietic stem cells ,
  • Inducible promoters eg, nucleic acids comprising a promoter capable of inducing the expression of a downstream gene in response to an external stimulus, the HPV-E6 / E7 gene whose expression is regulated by the promoter, and optionally a terminator. It is a structure.
  • the expression cassette may optionally contain a factor selected from the group consisting of enhancers, silencers, selectable marker genes (eg, drug resistance genes such as neomycin resistance genes), and the SV40 origin of replication. Further, a person skilled in the art can obtain a desired expression level by appropriately selecting and combining enhancers, silencers, selectable marker genes, terminators, etc. from known ones in consideration of the type of the promoter to be used.
  • An expression cassette capable of inducing the expression of the HPV-E6 / E7 gene can be constructed.
  • factors that induce the expression of the HPV-E6 / E7 gene in response to external stimuli for example, tetracycline transactivator, tetracycline repressor, ecdison receptor
  • an expression cassette capable of constitutively expressing the retinoid receptor complex, Gal4 DNA-binding domain fused to FKBP12-VP16 activator domain complex fused to cyclophyllin may also be introduced.
  • the method for introducing the expression cassette into hematopoietic stem cells is not particularly limited, and a known method can be appropriately selected and used.
  • the expression cassette is inserted into an appropriate expression vector, and the expression vector is introduced into cells by infection, lipofection method, liposome method, electroporation method, calcium phosphate co-precipitation method, DEAE dextran method, or microinjection method. Can be done.
  • expression vectors examples include viral vectors such as lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, and Sendai virus, and animal cell expression plasmids, but blood stem cells having not very high proliferative activity.
  • viral vectors such as lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, and Sendai virus
  • animal cell expression plasmids but blood stem cells having not very high proliferative activity.
  • a lentivirus can be preferably used from the viewpoint of extremely high efficiency of introduction into genomic DNA.
  • human blood stem cells into which the expression cassette has been introduced can then be cultured in the presence of the external stimulus and blood system growth factor.
  • the expression cassette according to the present invention is "a megakaryocyte precursor cell line into which an expression cassette capable of inducing the expression of human papillomavirus type 16 E6 gene and E7 gene in response to an external stimulus is introduced".
  • a megakaryocyte precursor cell line in which the expression cassette according to the present invention is incorporated into genomic DNA is preferable.
  • blood system growth factor means a factor that contributes to the induction of differentiation of blood stem cells into megakaryocyte progenitor cells or the proliferation of megakaryocyte progenitor cells.
  • blood growth factors include SCF and TPO.
  • the suitable addition concentration of SCF in the culture solution described later is 50 to 100 ng / ml, and the suitable addition concentration of TPO is 50 to 100 ng / ml.
  • the external stimulus a person skilled in the art can appropriately adjust the amount to be added to the medium described later in consideration of the type of the promoter to be used and the like.
  • the external stimulus is the presence of doxycycline (DOX)
  • DOX doxycycline
  • the preferred concentration of DOX added is 1-2 ⁇ g / ml.
  • Examples of the culture medium used for inducing differentiation into megakaryocyte progenitor cells and to which the external stimulus and the blood system growth factor are added include IMDM solution, ⁇ -MEM solution or DMEM solution, and further. , Fetal bovine serum (FBS), bovine serum albumin (BSA), human insulin, human transferrin, 2-mercaptoethanol, sodium selenate, ascorbic acid, alpha monothioglycerol, L-glutamine, SCF, TPO, FLT3, or induction External stimuli (eg, DOX) that actuate the sex promoter may be included.
  • FBS Fetal bovine serum
  • BSA bovine serum albumin
  • 2-mercaptoethanol sodium selenate
  • sodium selenate sodium selenate
  • ascorbic acid alpha monothioglycerol
  • SCF alpha monothioglycerol
  • SCF alpha monothioglycerol
  • TPO glutamine
  • FLT3 FLT3
  • the culture of megakaryocyte precursor cells was continued while exchanging the culture medium, and the culture period considered to be able to establish an immortalized cell line is preferably 3 months. It is judged to be an immortalized cell line when it can be cultured for 6 months or more.
  • the expression is performed before culturing in the presence of the external stimulus and the blood system growth factor.
  • the cassette may be cultured in the absence of the external stimulus and in the presence of blood growth factors for 1 to 7 days after introduction into the blood stem cells.
  • the culture conditions suitable for megakaryocyte differentiation can be conditions in the presence of factors selected from the group consisting of TPO, SCF and FLT3, eg, conditions in the presence of TPO. ..
  • DOX an external stimulus
  • the method for producing mature macronuclear cells of the present invention is used for inducing differentiation into mature macronuclear cells
  • examples of the medium include IMDM solution, ⁇ -MEM solution, and DMEM solution.
  • Inorganic salts or antibiotics may be added as needed.
  • an expression cassette capable of inducing the expression of human papillomavirus type 16 E6 and E7 genes in response to external stimuli has been introduced, the presence of external stimuli and blood growth factors.
  • a human megakaryocyte precursor cell line capable of producing megakaryocyte cells and platelets by growing underneath and culturing in the absence of the external stimulus is provided.
  • the expression cassette according to the present invention is "a megakaryocyte precursor cell line into which an expression cassette capable of inducing the expression of human papillomavirus type 16 E6 gene and E7 gene in response to an external stimulus is introduced".
  • a megakaryocyte precursor cell line in which the expression cassette according to the present invention is incorporated into genomic DNA is preferable.
  • the cells, culture medium and vector used for transformation in this example are as follows.
  • Human bone marrow-derived hematopoietic stem cells (CD34-positive cells) were purchased from Lonza.
  • ⁇ Vector> CSIV-TRE-RfA-UbC-to introduce the human papillomavirus-E6 / E7 (HPV-E6 / E7) gene and the gene encoding the tetracycline transactivator (rtTA) in the process of establishing a macronuclear cell precursor cell line.
  • HPV-E6 / E7 human papillomavirus-E6 / E7
  • rtTA tetracycline transactivator
  • Example 1 Induction of differentiation of hematopoietic stem cells into CD41-positive CD42b-positive megakaryocytes
  • the hematopoietic stem cells are CD41-positive and CD42b-positive cells (megakaryocytes) via CD41-positive cells (megakaryocyte progenitor cells) in the presence of TPO. It differentiates into cells).
  • CD41-positive and CD42b-positive cells megakaryocytes
  • CD41-positive cells megakaryocyte progenitor cells
  • Transtransferase 5 ng / ml sodium selenate; Sigma), 50 mg / ml ascorbic acid (Sigma), PSQ (100 units / ml penicillin, 100 mg / ml streptomycin, 2 mM L-glutamine; Invitrogen), 100 ng / ml
  • an IMDM (Sigma) culture solution containing recombinant human SCF, 100 ng / ml recombinant human TPO, and 100 ng / ml recombinant human FLT3, the mixture was cultured for 48 hours.
  • CSIV incorporating a gene (E6-2A-E7) encoding a fusion protein in which the E6 protein and E7 protein of papillomavirus were ligated into the human bone marrow-derived hematopoietic stem cells (CD34-positive cells) via a 2A peptide.
  • the E6 and E7 genes can be artificially induced to be expressed in the presence of doxycycline (DOX), and the expression of the E6 and E7 genes can be suppressed in the absence of DOX. ..
  • DOX doxycycline
  • the wrench virus was incorporated into the lentivirus so that it can express GFP so that it expresses GFP when it infects cells.
  • DOX was added at the same time as the first wrench virus infection, and the medium was replaced with fresh medium 24 hours and 48 hours after the second infection. By this medium exchange, the lentivirus in the medium was removed.
  • Flow cytometry Cells were stained with a monoclonal antibody for 30 minutes on ice. Cell numbers were the 1 ⁇ 10 6 or less in the staining solution of 100 [mu] l. After staining, it was washed twice with a staining solution containing no antibody, and then measured using a FACS analyzer (BD). Antibodies to human antigens were labeled with fluorescein isothiocyanate (FITC), Alexa Fluor 488 (Alexa 488) or allophycocyanin (APC). CD34, CD36, CD41, and CD42b were labeled with FITC, and CD11b was labeled with Alexa488.
  • FITC fluorescein isothiocyanate
  • Alexa 488 Alexa Fluor 488
  • APC allophycocyanin
  • CD33, CD45, c-KIT were labeled with APC. These labeled antibodies were purchased from BD Biosciences. Cell viability was measured by Propidium Iodide (PI) staining. PI-positive cells were excluded as dead cells, and PI-negative cells were analyzed using CellQuest analysis software.
  • PI Propidium Iodide
  • Fig. 1 The results were as shown in Fig. 1.
  • panel A of FIG. 1 the cells cultured under the above conditions continued cell division satisfactorily even after culturing for 20 days after virus removal, GFP-positive cells were present, and the cells were present. Half of the GFP-positive cells were CD41-positive. In addition, when the culture was continued for several more days, almost all GFP-positive cells changed to CD41-positive cells.
  • Panel B of FIG. 1 the efficiency of cell proliferation changed depending on the concentration of SCF in the medium. Therefore, the cells obtained above were CD41 positive. It was suggested that the cells were.
  • CD41-positive cells are the phenotype of megakaryocyte progenitor cells. Therefore, in this example, it was suggested that CD41-positive megakaryocyte progenitor cells were obtained.
  • the E6 and E7 genes were independently introduced into bone marrow-derived CD34-positive cells, and changes in the expression of surface markers CD41 and CD42b-positive cells after long-term culture were examined. As shown in panel A of FIG. 3, even in the single expression of either the E6 gene or the E7 gene, CD41-positive and CD42b-positive cells could be confirmed 3 weeks after the gene transfer. As shown in panel B of FIG. 3, the peak time of CD41 and CD42b-positive cells was 4 weeks after gene transfer (2 weeks after DOX removal).
  • the cells were cultured in the presence of 100 ng / ml) and TPO (100 ng / ml), and the proportion of cells in the culture was measured after the lapse of a specified number of days.
  • the results were as shown in panel B of FIG. As shown in panel B of FIG. 3, an increase in the number of cells was observed in the gene transfer group of E6 alone or E7 alone and the co-expression group of E6 and E7.
  • the group expressing the E6-2A-E7 gene nearly 50% of the co-positive cells of CD42b and CD41 could be maintained even after 10 weeks.
  • GFP, E6, E7, or after infection with lentivirus containing E6-2A-E7 were cultured cells as 10 three the number of GFP-positive cells in Day 0, cell numbers were determined by cell counter. The results were as shown in FIG. As shown in FIG. 4, in the gene transfer group of E6 alone or E7 alone, a long-term increase in the number of cells was observed as compared with the control (GFP) group, but it took more than 3 weeks for the cells to proliferate. did. On the other hand, in the group expressing the E6-2A-E7 gene, the period until the increase in the number of cells was very short as compared with all the groups.
  • the mortality rate of CD34-positive cells infected with lentivirus containing GFP only, GFP and E6, GFP and E7 or GFP and E6-2A-E7 was defined as the ratio of PI-positive cells, and 14 days after culturing, using a flow cytometer. It was measured. In the gene transfer group of E6 alone or E7 alone, similar PI-positive cells could be confirmed as compared with the control (GFP) group. However, in the group in which the E6-2A-E7 gene was expressed, the number of PI-positive cells decreased as compared with all the groups.
  • Example 2 Analysis of platelet precursor release CD41-positive CD42b-positive megakaryocyte progenitor cells expressing E6 and E7 were subcultured, and the subculture was performed for 1 month or longer to immortalize the CD41. Positive CD42b-positive megakaryocyte progenitor cells were obtained. Immortalized macronuclear progenitor cells (5 ⁇ 10 6 cells) were cultured for 20 days, washed with phosphate buffer (PBS), transferred to a culture dish coated with fibronectin, cultured for 3 days, stained with CD41 antibody, and microscopically stained. Measured below.
  • PBS phosphate buffer
  • SEQ ID NO: 1 An example of the amino acid sequence of HPV 16 type E6 protein MFQDTEEKPRTLHDLCQUARETTIHNIELQCVECKPLQRSEVYDFAFADLTVVYREGNPFGICKLCLLRFLSKISEYRHYNYSVYGNTLEQTVKPRIRKPRIrk
  • SEQ ID NO: 2 An example of the amino acid sequence of HPV 16 type E7 protein MRGHKPTLKEYVLDLYPEPTDLYCYEQLSDSDEDEGLDRPDGQAQPATADYYIVTCCHTCNTTVRLLCVNSTASDLRTIQQLLMGTVNIVCPTCAQQ
  • SEQ ID NO: 3 An example of the amino acid sequence of the fusion protein of E7 protein and E6 protein of HPV 16 type MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGSGATNFSL

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Abstract

La présente invention concerne une cellule progénitrice de mégacaryocyte et une cellule mégacaryocyte, et leur procédé de production. Spécifiquement, la présente invention concerne : une cellule progénitrice de mégacaryocyte ou une cellule mégacaryocyte contenant des gènes E6 et/ou des gènes E7 de papillomavirus ; et un procédé de production de celles-ci.
PCT/JP2020/036272 2019-09-25 2020-09-25 Procédé de production de cellule progénitrice de mégacaryocyte et de cellule mégacaryocyte, et cellule progénitrice de mégacaryocyte et cellule mégacaryocyte ainsi obtenue WO2021060467A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005068613A1 (fr) * 2004-01-14 2005-07-28 Renomedix Institute Inc. Differenciation de cellules positives cd34 en megacaryocytes et multiplication
JP2014503190A (ja) * 2010-11-02 2014-02-13 ヘルムホルツ−ツェントルム フュア インフェクツィオンスフォルシュンク ゲーエムベーハー 細胞不死化のための方法及びベクター
JP2014036651A (ja) * 2012-07-20 2014-02-27 Institute Of Physical & Chemical Research ヒト赤血球前駆細胞株及びヒト脱核赤血球の製造方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005068613A1 (fr) * 2004-01-14 2005-07-28 Renomedix Institute Inc. Differenciation de cellules positives cd34 en megacaryocytes et multiplication
JP2014503190A (ja) * 2010-11-02 2014-02-13 ヘルムホルツ−ツェントルム フュア インフェクツィオンスフォルシュンク ゲーエムベーハー 細胞不死化のための方法及びベクター
JP2014036651A (ja) * 2012-07-20 2014-02-27 Institute Of Physical & Chemical Research ヒト赤血球前駆細胞株及びヒト脱核赤血球の製造方法

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