JP7630132B2 - MEGAKARYOCYTIC PROGENITOR CELLS AND MEGAKARYOCYTIC CELLS METHOD, AND MEGAKARYOCYTIC PROGENITOR CELLS AND MEGAKARYOCYTIC CELLS OBTAINED - Google Patents

Description

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 who are bleeding due to surgery, injury, etc., or who have a decreased platelet count. Platelet preparations are currently manufactured from donated blood. However, due to changes in the population structure, the amount of donated blood is decreasing, and there are concerns that there will be a shortage of platelet preparations.
In addition, if a donor of blood has an infectious disease such as a bacterial infection, the blood may be contaminated with bacteria, and there is a risk of infection due to administration of a bacterially contaminated platelet preparation. For this reason, a method for producing platelets in vitro has been developed (Non-Patent Document 1).

It is now possible to produce megakaryocytes from pluripotent stem cells such as ES cells and iPS cells, that is, to produce mature megakaryocytes (multinucleated megakaryocytes) through in vitro differentiation of pluripotent stem cells into blood stem cells and further into megakaryocyte progenitor 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 and various types of growth factors are required, which requires high costs. In addition, in the early stages after differentiation induction, blood system cells other than megakaryocytes are also included, so a long culture period and amount are required for selection.

WO2012/157586

Takayama N et. al, “Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted ”, 2008, Blood, Vol. 111, No. 11, pages 5298-5306

The present invention provides a method for producing a megakaryocyte progenitor cell line capable of efficiently and stably producing mature megakaryocytes, and a method for producing megakaryocytes and platelets from the megakaryocyte progenitor cell line obtained by the production method.

The inventors have succeeded in establishing megakaryocyte precursor cells and megakaryocytes, as well as immortalized cell lines thereof, by expressing the E6 gene and/or E7 gene of human papillomavirus in human bone marrow-derived hematopoietic stem cells and culturing them.

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

That is, the present invention provides the following.
(1) A megakaryocyte progenitor cell or megakaryocyte cell 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 E7 gene on its genome.
(3) The megakaryocyte progenitor cell according to (1) or (2) above, which is mononuclear.
(4) The megakaryocyte progenitor cells or megakaryocyte cells according to (1) above, which are established megakaryocyte progenitor cells or megakaryocyte cells.
(5) The megakaryocyte progenitor cells or megakaryocyte cells according to (3) above, which are established megakaryocyte progenitor cells or megakaryocyte cells.
(6) A frozen product of the megakaryocyte progenitor cells or megakaryocyte cells described in (4) above, or the megakaryocyte progenitor cells or megakaryocyte cells described in (5) above.
(7) A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising:
expressing either or both of the E6 and E7 genes in cells of the megakaryocyte lineage (e.g., hematopoietic stem cells, hematopoietic progenitor cells, or CD34-positive cells);
and culturing the resulting cells in the presence of thrombopoietin (TPO).
(7A) A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising culturing megakaryocyte lineage cells (e.g., hematopoietic stem cells, hematopoietic progenitor cells, or CD34-positive cells) having the E6 gene and/or E7 gene under culture conditions suitable for megakaryocyte differentiation.
(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, comprising the steps of:
expressing either or both of the E6 and E7 genes in a cell of the megakaryocyte lineage (e.g., a hematopoietic stem cell or a hematopoietic progenitor cell);
culturing the resulting cells in the presence of thrombopoietin (TPO) to obtain megakaryocytes and recovering platelets from the culture of megakaryocytes;
A method comprising:
(9A) A method for producing a platelet preparation, comprising: 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 cells are cells in which gene expression of the E6 gene and the E7 gene is suppressed.

According to the present invention, it is possible to provide a method for producing a megakaryocyte progenitor cell line capable of efficiently and stably producing megakaryocyte progenitor cells, and a method for producing megakaryocytes and platelets from the megakaryocyte progenitor cell line obtained by the production method.

1 shows the results of gene expression analysis of cultures 21 days after virus removal from hematopoietic stem cells expressing E6 and E7 proteins (Panel A) and the change in CD41 cell count after the initiation of the culture (Panel B). Hereinafter, "E6-2A-E7" will be referred to simply as "E6-E7" in the figure. FIG. 2 shows the percentage of CD41 positive and CD42b positive cells in the cultures 70 days after viral clearance. FIG. 3 shows the percentage of CD41-positive and CD42b-positive cells in cultures 3 weeks after viral introduction of hematopoietic stem cells expressing either the E6 gene or the E7 gene alone (Panel A) and the change (weeks) in the percentage (%) of CD41-positive and CD42b-positive cells in cultures after viral removal of hematopoietic stem cells expressing either the E6 protein or the E7 protein, or both. FIG. 4 shows the change in cell number during culture of hematopoietic stem cells expressing either or both of the E6 and E7 genes. FIG. 5 shows the percentage of PI-positive cells generated in hematopoietic stem cells that have been made to express either or both of the E6 and E7 genes. FIG. 6 shows the production of platelet precursors from immortalized megakaryocyte progenitor cells.

Detailed Description of the Invention

In this specification, "hematopoietic stem cells" refers to stem cells that have the ability to differentiate into blood system cells. Hematopoietic stem cells are known not to have the ability to differentiate into cells other than blood system cells. They are also known to have the ability to differentiate into all kinds of blood system cells. "Hematopoietic stem cells" are also called "blood stem cells". Such "hematopoietic stem cells" are known to be abundantly contained in cell populations separated and collected from tissues such as umbilical cord blood, peripheral blood, bone marrow, and fetal liver by flow cytometry or the like using antibodies that specifically bind to surface antigens of hematopoietic stem cells (such as CD34). In addition, in the present invention, "hematopoietic stem cells" can also be prepared by inducing differentiation from human pluripotent stem cells.

In this specification, the term "megakaryocyte" is used to include mononuclear megakaryocytes and mature megakaryocytes that produce platelets. Among megakaryocytes, mature megakaryocytes have 4N to 128N chromosomes. Mature megakaryocytes are derived from hematopoietic stem cells, and become mature megakaryocytes through CD34-positive hematopoietic stem cells, hematopoietic progenitor cells, megakaryoblasts, and megakaryocyte progenitor cells (megakaryocyte lineage cells). In this specification, megakaryocytes with 2N chromosomes before polyploidization are called mononuclear megakaryocytes. When creating a megakaryocyte cell line, it is created from mononuclear megakaryocytes. Mature megakaryocytes have the ability to produce platelets.

In the present invention, "megakaryocyte progenitor cells" refers to cells that have the ability to differentiate into mature megakaryocyte cells (i.e., multinucleated megakaryocyte cells). Megakaryocyte progenitor cells may be cells that have lost the ability to differentiate into blood cells other than megakaryocytes. Furthermore, "megakaryocyte progenitor cell line" refers to an established megakaryocyte progenitor cell line, specifically, an immortalized megakaryocyte progenitor cell.

As used herein, "human papillomavirus" (HPV) refers to a virus belonging to the Papillomaviridae family that causes warts called papillomas (or papillomas). HPV has early genes E1 to E7 and late genes L1 and L2. E6 and E7 are known to be proteins involved in carcinogenesis (particularly cervical cancer). As used herein, E6 protein refers to the E6 protein of HPV type 16 and an E6 protein having an amino acid sequence corresponding to the amino acid sequence of said protein. As used herein, E7 protein refers to the E7 protein of HPV type 16 and an E7 protein having an amino acid sequence corresponding to the amino acid sequence of said protein.

As used herein, a "cell" refers to a mammalian cell, and may be, for example, a human cell. It is known that once a cell is established, it will undergo cell division indefinitely.

In this specification, "corresponding to a certain amino acid sequence" refers to a protein that is an orthologue of the amino acid sequence and has the same functionality as a protein having the amino acid sequence. The E6 protein and E7 protein may include, for example, E6 protein and E7 protein that have sequence identity of 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 with the above amino acid sequence. The sequence identity may mean, for example, the value Identity obtained by using the parameters provided as default by a NEEDLE program (Journal of Molecular Biology, 1970, Vol. 48, p. 443-453) search. The parameters may be as follows. Gap penalty = 10, Extend penalty = 0.5, and Matrix = EBLOSUM62.

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

The HPV E6 gene is not particularly limited, but can be, for example, the E6 gene of HPV type 16 (also referred to as the "HPV-E6 gene"). The HPV E7 gene is not particularly limited, but can be, for example, the E7 gene of HPV type 16 (also referred to as the "HPV-E7 gene"; the E6 gene and E7 gene of HPV type 16 are collectively referred to as the "HPV-E6/E7 genes").

"HPV-E6/E7 genes" may typically be nucleic acids encoding 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 protein, e.g., SEQ ID NO: 3).

The E6 gene and the E7 gene may be contained in separate nucleic acids (e.g., DNA and RNA), but preferably may be contained in one nucleic acid. When the E6 gene and the E7 gene are contained in one nucleic acid, they may be contained in the nucleic acid polycistronically or monocistronically. 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 (e.g., a fusion protein linked by the 2A peptide). The 2A peptide has a sequence having a length of about 20 amino acids derived from a virus, and is known to be recognized by a protease (2A peptidase) present in a cell and cleaved at one residue from its C-terminus. The 2A peptide may be, for example, a 2A peptide having an amino acid sequence corresponding to the amino acid sequence described in SEQ ID NO: 4, and can be used in the present invention. The 2A peptide can be linked to a protein by interposing a spacer (e.g., an amino acid sequence of about 3 amino acids) before and after it.

The E6 gene and/or the E7 gene may be contained in a mammalian gene expression vector or may be contained on the genome of a cell. Examples of gene expression vectors include virus vectors (e.g., lentivirus, retrovirus, herpes virus, adenovirus, adeno-associated virus, and Sendai virus) and plasmid vectors. In a cell, the gene expression vector may be integrated into the genome or may exist episomally. In the case of an RNA virus vector such as Sendai virus, a microRNA target sequence can be introduced into the genome and the microRNA can be introduced into a cell infected with the virus to remove the vector.

The E6 gene and/or the E7 gene may be operably linked to an expression control sequence (promoter) capable of expressing these genes in mammalian cells. Examples of promoters include promoters that bring about constitutive expression and inducible promoters. As an 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.), a promoter including a tetracycline response element (TRE) and a minimal promoter can be used. A promoter including a tetracycline response element (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 is activated when the tetracycline antibiotic (tetracycline, doxycycline, etc.) is released. In addition, a promoter including a tetracycline response element (TRE) and a minimal promoter is activated when it binds to a tetracycline antibiotic (tetracycline, doxycycline, etc.) in the presence of a reverse tetracycline-regulated transactivator (rtTA), and is inactivated when the tetracycline antibiotic (tetracycline, doxycycline, etc.) is removed. In addition, when the external stimulus is the presence of an ecdysteroid (ecdysone, muristerone A, ponasterone A, etc.), a promoter capable of inducing expression of a downstream gene by binding of the ecdysteroid with an ecdysone receptor-retinoid receptor complex is exemplified. Furthermore, when the external stimulus is the presence of FKCsA, a promoter capable of inducing expression of a downstream gene by binding of FKCsA with a Gal4 DNA binding domain fused to FKBP12-VP16 activator domain complex fused to cyclophilin is exemplified.

Megakaryocyte lineage cells include CD34-positive hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte progenitor cells derived from hematopoietic stem cells, as well as megakaryocytes. Megakaryocyte lineage cells may be cells selected from the group consisting of CD41-positive cells, von Willebrand factor (VMF)-positive cells, and CD41-positive and CD42b-positive cells (e.g., megakaryocyte progenitor cells, and megakaryocytes).

Cells having a 2N karyotype are suitable for proliferation. Therefore, cells having a 2N karyotype can be preferably established as a cell line. The establishment can be performed by immortalizing the cells. The immortalization can be performed using a conventional method, for example, by introducing an immortalization factor (e.g., T antigen of SV40 virus or telomere reverse transcription protein (TERT)) into the cells.

According to the present invention,
Frozen megakaryocyte progenitor cells or megakaryocyte cells containing either or both of the E6 gene and the E7 gene; and frozen megakaryocyte progenitor cells or megakaryocyte cells having either or both of the E6 gene and the E7 gene on their genome are 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 the same (e.g., compositions in a frozen state or frozen products) can be used, for example, to prepare a working cell bank, i.e., can be used as a master cell bank for preparing a working cell bank. Thus, in one aspect of the present invention, frozen megakaryocyte progenitor cells or megakaryocyte cells containing either or both of the E6 gene and the E7 gene; and frozen megakaryocyte progenitor cells or megakaryocyte cells having either or both of the E6 gene and the E7 gene on their genome are used as a master cell bank. In addition, in one aspect of the present invention, there is provided a master cell bank or working cell bank for use in producing platelets, which comprises a frozen material selected from the group consisting of frozen megakaryocyte progenitor cells or megakaryocyte cells containing either or both of the E6 gene and the E7 gene; and frozen megakaryocyte progenitor cells or megakaryocyte cells having either or both of the E6 gene and the E7 gene on their genome, or the frozen material.

Whether a cell has the E6 gene and/or the E7 gene can be detected by conventional methods (e.g., PCR, Southern blotting, and Northern blotting).

<Mechanism of producing megakaryocyte progenitor cell line>
In the method for producing a megakaryocytic progenitor cell line of the present invention, a nucleic acid (e.g., DNA or mRNA) comprising the E6 gene and/or E7 gene of human papilloma virus (HPV) can be introduced into a cell (e.g., a cell at any differentiation stage from hematopoietic stem cell to megakaryocytic progenitor cell (e.g., hematopoietic stem cell and hematopoietic progenitor cell). Thus, according to the present invention, there can be provided a cell at any differentiation stage from hematopoietic stem cell to megakaryocytic progenitor cell (e.g., hematopoietic stem cell and hematopoietic progenitor cell), which comprises a nucleic acid (e.g., DNA or mRNA) comprising the E6 gene and/or E7 gene of human papilloma virus (HPV). In one aspect of the present invention, the hematopoietic stem cell may be a CD34-positive hematopoietic stem cell.
When DNA containing the E6 gene and/or the E7 gene is introduced into a cell, the DNA may contain the E6 gene and/or the E7 gene operably linked to an expression control sequence (promoter).
In one aspect of the present invention, a gene expression vector comprising the E6 and/or E7 genes of human papillomavirus (HPV) operably linked to an inducible promoter is introduced into a cell. An external stimulus that activates the inducible promoter can be added to the cell at any time (e.g., before, during, or after or immediately after the introduction of the vector). In one aspect, an external stimulus that activates the inducible promoter is not added before the introduction of the vector. In one aspect, an external stimulus that activates the inducible promoter may be added to the cell for a certain period of time after the introduction of the vector, but then removed. The removal can be performed before or after the desired cells (e.g., megakaryocyte progenitor cells or megakaryocyte cells) are generated.

An "expression cassette capable of inducing expression of the E6 and E7 genes (HPV-E6/E7 genes) of human papillomavirus (HPV) type 16 in response to an external stimulus" that is introduced into human hematopoietic stem cells is a nucleic acid construct that includes an inducible promoter, for example, a promoter that can induce expression of a downstream gene in response to an external stimulus, the HPV-E6/E7 genes whose expression is controlled by the promoter, and, if necessary, a terminator.

The expression cassette may contain an element selected from the group consisting of an enhancer, a silencer, a selection marker gene (e.g., a drug resistance gene such as a neomycin resistance gene), and an SV40 replication origin, as necessary. In addition, a person skilled in the art can construct an expression cassette capable of inducing expression of the HPV-E6/E7 genes at a desired expression level by appropriately selecting and combining enhancers, silencers, selection marker genes, terminators, etc. from known elements, taking into consideration the type of promoter to be used, etc.

In addition, if necessary, the human blood stem cells of the present invention may also be introduced with an expression cassette capable of constitutively expressing in the nucleus a factor that induces the expression of HPV-E6/E7 genes in response to external stimuli (e.g., tetracycline transactivator, tetracycline repressor, ecdysone receptor-retinoid receptor complex, Gal4 DNA binding domain fused to FKBP12-VP16 activator domain complex fused to cyclophilin).

In the present invention, the method for introducing the expression cassette into hematopoietic stem cells is not particularly limited, and any known method can be appropriately selected and used. For example, the expression cassette can be inserted into an appropriate expression vector, and the expression vector can be introduced into cells by infection, lipofection, liposome method, electroporation, calcium phosphate coprecipitation, DEAE-dextran method, or microinjection.

Examples of such expression vectors include viral vectors such as lentivirus, retrovirus, herpes virus, adenovirus, adeno-associated virus, and Sendai virus, as well as animal cell expression plasmids. However, lentiviruses are preferably used because of their extremely high efficiency of introduction into the genomic DNA of blood stem cells, which do not have very high proliferation activity.

In the method for producing a megakaryocyte progenitor cell line of the present invention, the human blood stem cells into which the expression cassette has been introduced can then be cultured in the presence of the external stimuli and blood system growth factors.

Note that "a megakaryocytic progenitor cell line into which an expression cassette capable of inducing expression of the human papillomavirus type 16 E6 gene and E7 gene in response to an external stimulus has been introduced" refers to a megakaryocytic progenitor cell line that can stably express the HPV-E6/E7 genes in the presence of an external stimulus as a result of the expression cassette of the present invention being introduced into the cells. From the viewpoint that the expression cassette of the present invention can be removed together with the nucleus during the establishment of the cell line and further during the differentiation stage into megakaryocytic cells described below, a megakaryocytic progenitor cell line in which the expression cassette of the present invention has been incorporated into the genomic DNA is preferred.

In the present invention, "blood lineage growth factor" refers to 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 lineage growth factors" include SCF and TPO.

The preferred concentration of SCF to be added in the culture medium described below is 50 to 100 ng/ml, and the preferred concentration of TPO to be added is 50 to 100 ng/ml.

Furthermore, regarding the external stimulus, a person skilled in the art can appropriately adjust the amount of the external stimulus to be added to the medium, which will be described later, taking into consideration the type of the promoter to be used, etc. For example, when the external stimulus is the presence of doxycycline (DOX), the preferred concentration of DOX to be added is 1 to 2 μg/ml.

The culture medium used for inducing differentiation into megakaryocyte progenitor cells and to which the external stimuli and blood-based growth factors are added includes, for example, IMDM solution, α-MEM solution, or DMEM solution, and may further contain 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 an external stimulus that activates an inducible promoter (e.g., DOX). In addition, inorganic salts (e.g., ferrous sulfate, etc.) or antibiotics (e.g., streptomycin, penicillin, etc.) may be added as necessary.

The megakaryocyte progenitor cells are continued to be cultured in the presence of the above-mentioned external stimuli and blood-based growth factors while changing the culture medium, and the culture period during which an immortalized cell line is considered to have been established is preferably three months, and more preferably, when the cell line has been cultured for six months or more, it is judged to be an immortalized cell line.

Furthermore, from the standpoint of ensuring a period of time until the expression cassette is stably introduced into the genomic DNA of the blood stem cells, the expression cassette may be introduced into the blood stem cells, and then the cells may be cultured in the absence of the external stimuli and in the presence of blood system growth factors for 1 to 7 days before culturing in the presence of the external stimuli and blood system growth factors.

In one aspect of the present invention, culture conditions suitable for megakaryocyte differentiation may be conditions in the presence of a factor selected from the group consisting of TPO, SCF and FLT3, for example, conditions in the presence of TPO.

<Method of producing mature megakaryocyte cells>
As shown in the Examples below, the expression of the E6 gene and/or the E7 gene is important in increasing the yield of the established megakaryocyte progenitor cell line. On the other hand, since the cell division ability of megakaryocyte progenitor cells is gradually reduced during the differentiation and maturation process of megakaryocyte cells, the human megakaryocyte progenitor cell line produced by the above-mentioned method may be cultured in the absence of an external stimulus (e.g., DOX) that activates the inducible promoter.

That is, the method for producing mature megakaryocytes of the present invention is used for inducing differentiation into mature megakaryocytes, and examples of the culture medium include IMDM solution, α-MEM solution, and DMEM solution, and the culture medium may contain fetal bovine serum (FBS), bovine serum albumin (BSA), human insulin, human transferrin, 2-mercaptoethanol, sodium selenate, ascorbic acid, alpha monothioglycerol, L-glutamine, SCF, TPO, or FLT3, etc. Furthermore, inorganic salts or antibiotics may be added as necessary.

Furthermore, those skilled in the art can carry out such a manufacturing method by referring to the above-mentioned explanation of the present invention and the specific explanations of the examples, appropriately selecting the configuration of the expression cassette of the present invention, the method of introducing the expression cassette, the culture conditions at each differentiation stage (e.g., medium composition, culture period), etc., and by appropriately modifying or altering these methods as necessary.

In one aspect of the present invention, an expression cassette capable of inducing expression of the human papillomavirus type 16 E6 and E7 genes in response to an external stimulus is introduced, and a human megakaryocyte progenitor cell line is provided which proliferates in the presence of external stimuli and blood growth factors and has the ability to produce megakaryocytes and platelets when cultured in the absence of the external stimuli.

Note that "a megakaryocytic progenitor cell line into which an expression cassette capable of inducing expression of the human papillomavirus type 16 E6 gene and E7 gene in response to an external stimulus has been introduced" refers to a megakaryocytic progenitor cell line that can stably express the HPV-E6/E7 genes in the presence of an external stimulus as a result of the expression cassette of the present invention being introduced into the cells. From the viewpoint that the expression cassette of the present invention can be removed together with the nucleus during the establishment of the cell line and further during the differentiation stage into megakaryocytic cells described below, a megakaryocytic progenitor cell line in which the expression cassette of the present invention has been incorporated into the genomic DNA is preferred.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The cells, culture medium, and vectors used for transformation in the examples are as follows.

<Cells>
Human bone marrow-derived hematopoietic stem cells (CD34-positive cells) were purchased from Lonza.

<Vector>
In the process of establishing megakaryocytic progenitor cell lines, CSIV-TRE-RfA-UbC-KT lentiviral vectors were used to introduce the human papillomavirus-E6/E7 (HPV-E6/E7) gene and a gene encoding tetracycline transactivator (rtTA). Furthermore, lentiviruses using these lentiviral vectors were prepared using standard techniques.

Example 1: Induction of differentiation from hematopoietic stem cells into CD41-positive CD42b-positive megakaryocytes Hematopoietic stem cells differentiate into CD41-positive and CD42b-positive cells (megakaryocytes) in the presence of TPO via CD41-positive cells (megakaryocyte precursor cells). In this example, induction of hematopoietic stem cells into megakaryocytes was attempted.

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 cultured for 48 hours in IMDM (Sigma) medium containing 10% FBS (Invitrogen), ITS (10 μg/ml human insulin, 5.5 μg/ml human transferrin, 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 recombinant human SCF, 100 ng/ml recombinant human TPO, and 100 ng/ml recombinant human FLT3. Thereafter, the human bone marrow-derived hematopoietic stem cells (CD34-positive cells) were infected twice every 12 hours with CSIV-TRE-RfA-UbC-KT lentivirus vector (TAKARA BIO) (CSIV-TRE-HPV16-T2A-E7-UbC-KT) at MOI=20 (MOI was confirmed using 293T cells), which contains a gene (E6-2A-E7) encoding a fusion protein in which the E6 and E7 proteins of papillomavirus are linked via the 2A peptide, and then the cells were infected twice every 12 hours with CSIV-TRE-RfA-UbC-KT at MOI=20 (MOI was confirmed using 293T cells). Then, 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 to induce the expression of E6 and E7. This system was an inducible system to verify the effects of E6 and E7. Specifically, the expression of the E6 and E7 genes can be artificially induced 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, GFP was incorporated into the lentivirus in an expressible manner so that GFP is expressed when the lentivirus infects cells.

DOX was added simultaneously with the first lentivirus infection, and the medium was replaced with fresh medium 24 and 48 hours after the second infection. This medium replacement removed the lentivirus from the medium.

After removing the virus, the culture medium was replaced and cell proliferation was observed over time. After 20 days of culture, some of the cultured cells were harvested and the expression of CD41 was fluorescently stained using an anti-human CD41 antibody and confirmed with a flow cytometer. At this time, GFP expression was simultaneously confirmed by flow cytometry to confirm whether lentivirus infection had been established.

Flow cytometry Cells were stained with monoclonal antibodies for 30 minutes on ice. The cell count was ^1x106 or less in 100 μl of staining solution. After staining, cells were washed twice with staining solution without antibodies, and then measured using a FACS analyzer (BD). Antibodies against human antigens were labeled with fluorescein isothiocyanate (FITC), Alexa Fluor 488 (Alexa488), or allophycocyanin (APC). CD34, CD36, CD41, and CD42b were labeled with FITC, and CD11b was labeled with Alexa488. As isotype controls, CD33, CD45, and 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 well even after 20 days of culture after virus removal, GFP-positive cells were present, and half of the GFP-positive cells were CD41-positive. Furthermore, when the culture was continued for several more days, almost all of the GFP-positive cells changed to CD41-positive cells. When the SCF concentration dependency 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, suggesting that the cells obtained above were CD41-positive cells. In the development and differentiation of megakaryocytes, 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 anti-human CD41a and anti-human CD42b antibodies. The results were as shown in Figure 2. As shown in Figure 2, the majority of the cells were CD41a-positive and CD42b-positive. This demonstrated that megakaryocyte progenitor cells that were CD41a-positive and CD42b-positive were obtained.

The E6 and E7 genes were introduced individually 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, CD41-positive and CD42b-positive cells were confirmed 3 weeks after gene transfer, regardless of whether the E6 gene or the E7 gene was expressed alone. As shown in panel B of Fig. 3, the peak of CD41- and CD42b-positive cells occurred 4 weeks after gene transfer (2 weeks after DOX removal).

Next, we examined the change in cell number of CD34 positive cells infected with lentiviruses containing GFP only, GFP and E6, GFP and E7, or GFP and E6-2A-E7.

CD34 positive cells infected with lentivirus containing GFP only (control), GFP and E6, GFP and E7, or GFP and E6-2A-E7 were cultured in the presence of SCF (100 ng/ml) and TPO (100 ^ng /ml) for 3 days, and the percentage of cells in the culture was measured after the indicated number of days. The results are shown in panel B of Figure 3. As shown in panel B of Figure 3, an increase in cell number was observed in the group transfected with E6 alone or E7 alone and the group co-expressing E6 and E7. On the other hand, in the group expressing the E6-2A-E7 gene, the number of co-positive cells for CD42b and CD41 was maintained at nearly 50% even after 10 weeks.

After infection with lentivirus containing GFP, E6, E7, or E6-2A-E7, the cells were cultured with a GFP-positive cell count of 10 ³ on Day 0, and the cell count was measured using a cell counter. The results are shown in FIG. 4. As shown in FIG. 4, in the group transfected with E6 alone or E7 alone, a long-term increase in cell count was observed compared to the control (GFP) group, but it took more than 3 weeks for the cells to grow. On the other hand, in the group in which the E6-2A-E7 gene was expressed, the period until the cell count increased was very short compared to all other groups.

The mortality rate of CD34 positive cells infected with lentivirus containing GFP alone, GFP and E6, GFP and E7, or GFP and E6-2A-E7 was measured using a flow cytometer after 14 days of culture as a percentage of the number of PI positive cells. In the groups transfected with E6 alone or E7 alone, the same number of PI positive cells was confirmed compared to the control (GFP) group. However, the group in which the E6-2A-E7 gene was expressed had a reduced number of PI positive cells compared to all other groups.
These results suggest that co-expression of E6 and E7 suppresses apoptosis, thereby resulting in more efficient cell proliferation than other groups.

Example 2: Analysis of platelet precursor release CD41+CD42b+ megakaryocyte precursor cells expressing E6 and E7 were subcultured for more than one month to obtain immortalized CD41+CD42b+ megakaryocyte precursor cells. The immortalized megakaryocyte precursor cells (5× ¹⁰⁶ cells) were cultured for 20 days, washed with phosphate buffered saline (PBS), transferred to a fibronectin-coated culture dish, and cultured for 3 days. They were then stained with CD41 antibody and measured under a microscope.

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

Sequence Listing SEQ ID NO: 1: An example of the amino acid sequence of the E6 protein of HPV type 16 MFQDTEEKPRTLHDLCQALETTIHNIELQCVECKKPLQRSEVYDFAFAD LTVVYREGNPFGICKLCLRFLSKISEYRHYNYSVYGNTLEQTVKKPLNEILIRCIICQRPLCPQEKKRHVDLNKRFHNISGRWAGRCAACWRSRRRETAL

SEQ ID NO: 2: An example of the amino acid sequence of the E7 protein of HPV type 16 MRGHKPTLKEYVLDLYPEPTDLYCYEQLSDSSDEDEGLDRPDGQAQPATADYYIVTCCHTCNTTVRLCVNSTASDLRTIQQLLMGTVNIVCPTCAQQ

SEQ ID NO: 3: An example of the amino acid sequence of a fusion protein of E7 protein and E6 protein of HPV type 16 MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGSGATNFSLLKQAGDVEENPGPLI NMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYS KISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL

SEQ ID NO: 4: An example of the amino acid sequence of 2A peptide ATNFSLLKQAGDVEENPGP

Claims (10)

A megakaryocyte progenitor cell or megakaryocyte cell expressing either or both of the E6 and E7 genes. The megakaryocyte progenitor cell or megakaryocyte cell according to claim 1, which has either or both of the E6 gene and E7 gene on its genome. The megakaryocyte precursor cells or megakaryocyte cells according to claim 1 or 2, which are mononuclear. The megakaryocyte progenitor cells or megakaryocyte cells according to claim 1, which are established megakaryocyte progenitor cells or megakaryocyte cells. The megakaryocyte progenitor cells or megakaryocyte cells according to claim 3, which are established megakaryocyte progenitor cells or megakaryocyte cells. A frozen product of megakaryocyte progenitor cells or megakaryocyte cells according to claim 4 or megakaryocyte progenitor cells or megakaryocyte cells according to claim 5. A method for producing megakaryocyte progenitor cells or megakaryocyte cells, comprising obtaining megakaryocyte progenitor cells from megakaryocyte lineage cells that express the E6 gene and/or the E7 gene. The method according to 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). A method for producing a platelet preparation, comprising : obtaining megakaryocyte progenitor cells from megakaryocyte lineage cells expressing the E6 gene and/or E7 gene; culturing the obtained megakaryocyte progenitor cells under conditions suitable for differentiation into megakaryocyte cells to obtain a culture of mature megakaryocyte cells containing platelets; and recovering platelets from the culture. The method according to claim 9, wherein the mature megakaryocytic cells are cells in which gene expression of the E6 gene and the E7 gene is suppressed.

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