WO2021060467A1 - Method for producing megakaryocyte progenitor cell and megakaryocytic cell, and megakaryocyte progenitor cell and megakaryocytic cell obtained thereby - Google Patents

Abstract

The present invention provides a megakaryocyte progenitor cell and a megakaryocytic cell, and a method for producing the same. Specifically, the present invention provides: a megakaryocyte progenitor cell or a megakaryocytic cell containing papillomavirus E6 genes and/or E7 genes; and a method for producing the same.

Description

Method for producing megakaryocyte progenitor cells and megakaryocyte cells and the obtained megakaryocyte progenitor cells and megakaryocyte cells

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.

WO2012 / 157586

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.

Next, we attempted to induce the differentiation of the obtained cells into mature megakaryocyte cells. As a result, it was found that the obtained megakaryocyte cells contained multinucleated megakaryocyte cells, and that the megakaryocyte cells formed platelet precursors. The present application is based on these findings.

That is, 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.
(5) The megakaryocyte progenitor cell or megakaryocyte cell according to (3) above, which is an established megakaryocyte progenitor cell or megakaryocyte cell.
(6) A frozen product of the megakaryocyte progenitor cell or megakaryocyte cell according to (4) above or the megakaryocyte progenitor cell or megakaryocyte cell according to (5) above.
(7) A method for producing megakaryocyte progenitor cells or megakaryocyte cells.
Expressing either or both of the E6 and E7 genes in 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).
(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.
(8) The method according to (7) or (7A) above, wherein the culture is carried out in the presence of TPO, stem cell factor (SCF) and fms-related tyrosine kinase 3 (FLT3).
(9) A method for producing a platelet preparation, which is a method for producing a platelet preparation.
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.

According to the present invention, 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. Hereinafter, in the figure, "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.

Specific description of the invention

In the present specification, "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.

In the present specification, "megakaryocytes" are used in the sense of including mononuclear megakaryocytes and mature megakaryocytes that produce platelets. Of the megakaryocytes, 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). In the present specification, a megakaryocyte having a 2N chromosome before becoming a polyploid is referred to as a mononuclear megakaryocyte. When creating a megakaryocyte cell line, it will be created from mononuclear megakaryocytes. Mature megakaryocytes have the ability to produce platelets.

In the present invention, 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. In addition, the "megakaryocyte progenitor cell line" means an established megakaryocyte progenitor cell, and specifically, an immortalized megakaryocyte progenitor cell.

In the present specification, "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). As used herein, 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. As used herein, 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.

In the present specification, the "cell" is a mammalian cell, for example, a human cell. It is known that when cells are established, they repeat cell division indefinitely.

In the present specification, "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. The sequence identity can mean, for example, the value Identity obtained by using the parameters prepared by default by the NEEDLE program (Journal of Molecular Biology, 1970, Vol. 48, p. 443-453). The above parameters can be: Gap penalty = 10, Extended penalty = 0.5, and Matrix = EBLOSUM62.

<Megakaryocyte progenitor cells and megakaryocyte cells>
In the present invention, a cell having at least one selected from the group consisting of a gene encoding the E6 protein of human papillomavirus (HPV) (also referred to as the E6 gene) and a gene encoding the E7 protein (also referred to as the E7 gene) (also referred to as an E7 gene). For example, 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).

The "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). It is known that 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. Has been done. 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. In the case of 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. As the inducible promoter, for example, a promoter that activates transcription in response to an external stimulus can be used. For example, when the external stimulus is a tetracycline antibiotic (tetracycline, doxycycline, etc.), the tetracycline response. 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. In addition, 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. In addition, when the external stimulus is the presence of 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. Furthermore, when the external stimulus is the presence of FKCsA, 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.

Examples of megakaryocyte lineage cells 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.

According to the present invention
Frozen megakaryocyte progenitor cells or megakaryocyte cells containing either or both of the E6 and E7 genes; and megakaryocyte progenitor cells or megakaryocyte cells having either or both of the E6 and E7 genes on their genome. Frozen material is provided. In the frozen product, the megakaryocyte progenitor cells or megakaryocyte cells may be mononuclear. Mononuclear megakaryocyte progenitor cells or mononuclear megakaryocyte cells or compositions containing them (eg, frozen compositions or frozen products) 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. Thus, in some embodiments of the invention, 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. Also, in some embodiments of the invention, 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. , A master cell bank or a working cell bank for use in producing platelets, comprising a frozen product selected from the group consisting of megakaryocyte precursor cells or frozen products of megakaryocyte cells, or the frozen product.

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).

<Manufacturing method of megakaryocyte progenitor cell line>
In the method for producing a macronuclear progenitor cell line of the present invention, a nucleic acid (eg, DNA or mRNA) containing the E6 gene and / or the E7 gene of human papillomavirus (HPV) is applied to cells (eg, from hematopoietic stem cells to macronuclear progenitor cells). It can be introduced into cells at any stage of differentiation (eg, hematopoietic stem cells and hematopoietic progenitor cells). Therefore, according to the present invention, it is at any stage of differentiation from hematopoietic stem cells to macronuclear progenitor cells. Cells (eg, hematopoietic stem cells and hematopoietic progenitor cells), comprising nucleic acids (eg, DNA or mRNA) containing the E6 and / or E7 genes of human papillomavirus (HPV), can be provided. In some embodiments, the hematopoietic stem cell can be a CD34 positive hematopoietic stem cell.
When introducing a DNA containing an E6 gene and / or an E7 gene into a cell, the DNA may contain an E6 gene and / or an E7 gene operably linked to an expression regulatory sequence (promoter).
In one aspect of the invention, 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.

"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.

In addition, if necessary, in the human blood stem cells according to the present invention, 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.

In the present invention, 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. For example, 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.

Examples of such expression vectors 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. A lentivirus can be preferably used from the viewpoint of extremely high efficiency of introduction into genomic DNA.

In the method for producing a megakaryocyte progenitor cell line of the present invention, 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". Is a megakaryocyte precursor cell line capable of stably expressing the HPV-E6 / E7 gene in the presence of an external stimulus as a result of being introduced into the cell. From the viewpoint that the expression cassette according to the present invention can be removed together with the nucleus at the stage of differentiation into cells, a megakaryocyte precursor cell line in which the expression cassette according to the present invention is incorporated into genomic DNA is preferable.

In the present invention, "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. Examples of such "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.

Further, regarding 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. For example, when the external stimulus is the presence of doxycycline (DOX), the preferred concentration of DOX added is 1-2 μg / 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, α-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. In addition, if necessary, inorganic salts (ferrous sulfate, etc.), antibiotics (for example, streptomycin, penicillin, etc.) and the like may be added.

In the presence of the external stimulus and blood growth factor, 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.

Furthermore, from the viewpoint of ensuring a period until the expression cassette is stably introduced into the genomic DNA of the blood stem cells, 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.

In some aspects of the invention, 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. ..

<Manufacturing method of mature megakaryocyte cells>
As shown in Examples below, expression of the E6 and / or E7 genes is important in increasing the amount of established megakaryocyte progenitor cell lines. On the other hand, in the process of differentiation and maturation of megakaryocyte cells, the cell division ability gradually decreases from the megakaryocyte progenitor cells. Therefore, the human megakaryocyte progenitor cell line produced by the above method is used as an inducible promoter. May be cultured in the absence of an external stimulus (eg, DOX) that activates.

That is, the method for producing mature macronuclear cells of the present invention is used for inducing differentiation into mature macronuclear cells, and examples of the medium include IMDM solution, α-MEM solution, and DMEM solution. Fetal bovine serum (FBS), bovine serum albumin (BSA), human insulin, human transferase, 2-mercaptoethanol, sodium selenate, ascorbic acid, alpha monothioglycerol, L-glutamine, SCF, TPO, FLT3, etc. May be included. Inorganic salts or antibiotics may be added as needed.

As for such a production method, those skilled in the art can refer to the above-mentioned description of the present invention and the specific description of the examples, and refer to the configuration of the expression cassette according to the present invention, the method of introducing the expression cassette, and the like. It can be carried out by appropriately selecting the culture conditions (for example, the composition of the medium, the culture period) and the like at the differentiation stage, and appropriately modifying or modifying these methods as necessary.

In one aspect of the invention, 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". Is a megakaryocyte precursor cell line capable of stably expressing the HPV-E6 / E7 gene in the presence of an external stimulus as a result of being introduced into the cell. From the viewpoint that the expression cassette according to the present invention can be removed together with the nucleus at the stage of differentiation into cells, a megakaryocyte precursor cell line in which the expression cassette according to the present invention is incorporated into genomic DNA is preferable.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples. The cells, culture medium and vector used for transformation in this example are as follows.

<Cell>
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. A KT wrench virus vector was used. In addition, the preparation of the wrench virus using these wrench virus vectors was carried out by adopting a standard procedure.

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). In this example, we attempted to induce hematopoietic stem cells to megakaryocyte cells.

2 × 10 ⁵ cells of human bone marrow-derived hematopoietic stem cells (CD34-positive cells) (Lonza) were seeded in a 6-well plate dish, and 10% FBS (Invitrogen), ITS (10 μg / ml human insulin, 5.5 μg / ml human). 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 In 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. Then, 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. -TRE-RfA-UbC-KT wrench virus vector (TAKARA BIO) (CSIV-TRE-HPV16-T2A-E7-UbC-KT) with MOI = 20 (MOI confirmed using 293T cells) every 12 hours After two infections, 100 ng / ml recombinant human SCF, 100 ng / ml recombinant human TPO, and 100 ng / ml recombinant human FLT3 and 1 μg / ml DOX were added to the IMDM culture medium. Expression of E6 and E7 was induced. This system was an inducible system to verify the effects of E6 and E7. Specifically, 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. .. In addition, 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.

After removing the virus, cell growth was observed over time while exchanging the culture medium. After 20 days of culturing, some cultured cells were collected, and the expression of CD41 was fluorescently stained with an anti-human CD41 antibody and confirmed with a flow cytometer. At this time, in order to confirm whether or not the wrench virus infection was established, the expression of GFP was simultaneously confirmed by flow cytometry.

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. As isotype controls, 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.

The results were as shown in Fig. 1. As shown in 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. When the SCF concentration dependence of the cells was confirmed, as shown in 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. In the developmental differentiation of megakaryocyte cells, 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.

In addition, cells cultured for 70 days after virus removal were collected, and the expression of CD41a and CD42b was confirmed by flow cytometry using an anti-human CD41a antibody and an anti-human CD42b antibody. The results were as shown in FIG. As shown in FIG. 2, most of the cells were CD41a positive and CD42b positive. This revealed that CD41a-positive and CD42b-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).

Next, changes in the number of CD34-positive cells infected with wrench virus containing GFP only, GFP and E6, GFP and E7, or GFP and E6-2A-E7 were examined.

CD34-positive cells infected with lentivirus containing GFP only (control), GFP and E6, GFP and E7 or GFP and E6-2A-E7, SCF ( ^{1 × 10 4 cells) in each group after 3 days.} 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. On the other hand, in 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.
These results suggest that co-expression of E6 and E7 suppresses apoptosis, and cell amplification is more efficient than in other 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.

As a differentiation condition, immortalized megakaryocyte progenitor cells cultured in the absence of DOX for 20 days were transferred to a culture dish coated with fibronectin, and after culturing for 3 days, the distribution of CD41 antibody (red) was observed under a microscope. As a result, a probe-like structure was confirmed.

Sequence Listing 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 MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGSGATNFSLLKQAGDVEENPGPLINMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL

Example of amino acid sequence of SEQ ID NO: 4: 2A peptide ATNFSLLKQAGDVEENPGP

Claims (10)

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 claim 1, which has either or both of the E6 gene and the E7 gene on the genome.
3. The megakaryocyte progenitor cell or megakaryocyte cell according to claim 1 or 2, which is a mononuclear cell.
4. The megakaryocyte progenitor cell or megakaryocyte cell according to claim 1, which is an established megakaryocyte progenitor cell or megakaryocyte cell.
5. The megakaryocyte progenitor cell or megakaryocyte cell according to claim 3, which is an established megakaryocyte progenitor cell or megakaryocyte cell.
6. The frozen product of the megakaryocyte progenitor cell or megakaryocyte cell according to claim 4 or the megakaryocyte progenitor cell or megakaryocyte cell according to claim 5.
7. A method for producing megakaryocyte progenitor cells or megakaryocyte cells, which comprises culturing cells of the megakaryocyte lineage having the E6 gene and / or the E7 gene under culture conditions suitable for megakaryocyte differentiation.
8. The method of claim 7, wherein the culture is carried out in the presence of thrombopoietin (TPO), stem cell factor (SCF) and fms-related tyrosine kinase 3 (FLT3).
9. 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.
10. The method according to claim 9, wherein the megakaryocyte cell is a cell in which the gene expression of the E6 gene and the E7 gene is suppressed.
    
    

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