Method for producing cell population containing genetically modified mesenchymal stem cells

Abstract

The present invention addresses the problem of providing a method for producing genetically modified amnion-derived mesenchymal stem cells by introducing a suicide gene into amnion-derived mesenchymal stem cells.　Provided is a method for producing a cell population including genetically modified amnion-derived mesenchymal stem cells, the method comprising a step for genetically introducing a suicide gene or a suicide gene expression cassette into a cell population containing amnion-derived mesenchymal stem cells through a method in which a plasmid vector is used, such as the transposon method.

Description

Method for producing a cell population containing genetically modified mesenchymal stem cells

[0001] The present invention relates to a method for producing a cell population comprising genetically modified mesenchymal stem cells.

[0002] Cancer is a disease with a large number of patients and deaths worldwide, with millions of deaths due to cancer each year. Cancer is divided into various types depending on the organ in which it develops and the cells from which it originates. Some cancers are still poorly treated with existing therapies, and research into new treatments is actively underway.

[0003] Malignant brain tumors are one of the cancers with a particularly poor prognosis. The standard existing treatment for malignant brain tumors is surgical removal of the tumor followed by radiation therapy and / or drug therapy. However, depending on the type and location of the brain tumor, surgical removal is often not possible, and even after radiation therapy or drug therapy, cancer cells often remain and recur. Therefore, new therapies for malignant brain tumors that offer improved targeting of cancer cells are needed.

[0004] Mesenchymal stem cells are somatic stem cells that have been reported to exist in bone marrow, adipose tissue, dental pulp, amniotic membrane, etc. Mesenchymal stem cells have immunosuppressive properties and can be transplanted allogeneically, so they have attracted attention as a promising cell source in cell therapy, and in fact, their practical application for acute graft-versus-host disease (GVHD), spinal cord injury, etc. Furthermore, mesenchymal stem cells are known to have the property of specifically migrating and accumulating in cancer lesions in the body, and research is underway to utilize mesenchymal stem cells as a more targeted cancer therapy.

[0005] One cancer treatment method using mesenchymal stem cells is to use mesenchymal stem cells that have been conferred anticancer function by gene transfer with a suicide gene such as herpes simplex virus thymidine kinase (HSV-TK). A typical gene transfer method for this purpose is to use a viral vector, from the viewpoints of gene transfer efficiency and stability of gene expression. For example, Non-Patent Document 1 describes a method for producing target cells by gene transfer of HSV-TK into adipose tissue-derived mesenchymal stem cells using a lentiviral vector. On the other hand, it has been reported that gene transfer of a suicide gene into induced pluripotent stem cells or dental pulp stem cells using a viral vector can sometimes result in cytotoxicity and reduced proliferation ability of the suicide gene-transferred cells (Non-Patent Documents 2 and 3).

[0006] Amniotic membrane-derived mesenchymal stem cells are characterized by the following: they are derived from amniotic membranes, which are treated as medical waste after cesarean section delivery, so cell collection does not require the invasiveness of a new donor; because they are derived from amniotic membranes, which are fetal tissues, there is little variation in cell characteristics between donors; and a large number of mesenchymal stem cells can be obtained from the amniotic membrane of a single donor. These characteristics make amniotic membrane-derived mesenchymal stem cells particularly excellent as a cell therapy material among various tissue-derived mesenchymal stem cells, and their use in the field of cancer treatment is expected to provide low-cost, stable-quality cell therapy.

[0007] de Melo SM, et al. The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor. PLoS One. 2015 Jun 12;10(6): e0128922.Iwasawa C, et al. Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells. Int J Mol Sci. 2019 Feb 14;20(4):810.Oishi T, et al. Efficacy of HSV-TK / GCV system suicide gene therapy using SHED expressing modified HSV-TK against lung cancer brain metastases. Mol Ther Methods Clin Dev. 2022 Jul 6:26:253-265.

[0008] The present inventors first introduced a suicide gene into amnion-derived mesenchymal stem cells using a lentiviral vector, a technique known for its high gene transfer efficiency. As a result, they found that the proliferation ability of the transfected cells was significantly reduced due to cytotoxicity, making it impossible to produce the desired cells. Therefore, an object of the present invention is to provide a method for producing genetically modified amnion-derived mesenchymal stem cells without significantly reducing the proliferation ability of the suicide gene-transfected amnion-derived mesenchymal stem cells.

[0009] The present inventors conducted extensive research to solve the above-mentioned problems. Surprisingly, they found that when a plasmid vector, such as a transposon vector, was used to introduce a suicide gene into the transfected cells, the transfected cells did not experience a significant decrease in proliferation ability due to cytotoxicity. This finding made it possible to produce amnion-derived mesenchymal stem cells that stably express a suicide gene, leading to the completion of the present invention.

[0010] That is, the present invention relates to the following [1] to

[10] . [1] A method for producing a cell population containing genetically modified amnion-derived mesenchymal stem cells, comprising the step of transfecting amnion-derived mesenchymal stem cells with a suicide gene or a suicide gene expression cassette using a plasmid vector. [2] The production method according to [1], wherein the plasmid vector is introduced into cells by electroporation. [3] The production method according to [1] or [2], wherein the suicide gene or suicide gene expression cassette is integrated into a chromosome in the gene transfer step. [4] The production method according to any of [1] to [3], wherein the gene transfer step uses two types of plasmid vectors: a plasmid vector containing a construct in which the suicide gene or suicide gene expression cassette is located adjacent to a repeat sequence recognized by transposase, and a plasmid vector containing a transposase expression cassette. [5] The production method according to any of [1] to [3], wherein the gene transfer step uses a plasmid vector containing a construct in which the suicide gene or suicide gene expression cassette is located adjacent to a repeat sequence recognized by transposase, and a transposase expression cassette. [6] The manufacturing method according to any one of [1] to [5], wherein the suicide gene is a herpes simplex virus thymidine kinase gene or a cytosine deaminase gene. [7] The manufacturing method according to any one of [1] to [6], wherein the suicide gene is a herpes simplex virus thymidine kinase gene. [8] A cell preparation comprising genetically modified amnion-derived mesenchymal stem cells manufactured by the manufacturing method according to any one of [1] to [7]. [9] A cell preparation comprising amnion-derived mesenchymal stem cells, wherein the amnion-derived mesenchymal stem cells have (i) a suicide gene or an expression cassette for the suicide gene, and (ii) a repeat sequence recognized by a transposase integrated into their chromosomes.

[10] The cell preparation according to [8] or [9], wherein the cell preparation is used in combination with a prodrug.

[11] The cell preparation according to

[10] , wherein the prodrug is ganciclovir or 5-fluorocytosine.

[12] The cell preparation according to

[11] , wherein the prodrug is ganciclovir.

[13] The cell preparation according to any one of [8] to

[12] , wherein the cell preparation is for cancer treatment.

[14] The cell preparation according to

[13] , wherein the cell preparation is for treating a brain tumor.

[15] A cancer treatment kit comprising (i) a cell population comprising genetically modified amnion-derived mesenchymal stem cells produced by the production method according to any one of [1] to [7], and (ii) a prodrug.

[16] A cancer treatment kit comprising the cell preparation according to [9] and a prodrug.

[17] The cancer treatment kit according to

[15] , wherein the prodrug is ganciclovir or 5-fluorocytosine.

[18] The cancer treatment kit according to

[16] , wherein the prodrug is ganciclovir.

[19] The kit according to any one of

[15] to

[18] , wherein the cancer is a brain tumor. This specification incorporates the disclosures of Japanese Patent Application No. 2024-037914, from which the present application claims priority.

[0011] According to the present invention, a cell population containing amnion-derived mesenchymal stem cells that express a suicide gene can be produced without reducing the cell proliferation ability.

[0012] 1 shows bright-field and fluorescent observation results under a microscope of Venus-expressing amnion-derived mesenchymal stem cells produced using a lentiviral vector in Comparative Example 1. (A) Day 3 of culture, (B) Day 10 of culture. 1 shows bright-field and fluorescent observation results under a microscope of HSV-TK-expressing amnion-derived mesenchymal stem cells produced using a lentiviral vector in Comparative Example 2. (A) Day 3 of culture, (B) Day 10 of culture. 1 shows bright-field and fluorescent observation results under a microscope of HSV-TK-expressing amnion-derived mesenchymal stem cells produced using a transposon method in Example 1. (A) Day 3 of culture, (B) Day 10 of culture. 1 shows the results of evaluating the in vivo anti-cancer effect of HSV-TK-expressing amnion-derived mesenchymal stem cells produced using the transposon method in Example 2, based on the time course of tumor volume.

[0013] An embodiment of the present invention will be described below, but the present invention is not limited thereto.

[0014] [1] Explanation of Terms (Amnion-derived mesenchymal stem cells) As used herein, "amnion-derived mesenchymal stem cells" refer to stem cells that can be collected from the amnion and that meet the following definitions: i) They exhibit adhesiveness to plastic when cultured in a standard medium. ii) They are positive for the surface antigens CD73 and CD90, and negative for CD45 and CD326.

[0015] The term "amniotic membrane" as used herein refers to a transparent, poorly vascularized membrane that is the innermost layer of the fetal membrane, and includes the amniotic epithelial layer (epithelial tissue), basement membrane, and stratum densa (interstitial tissue). As used herein, "mesenchymal stem cells (MSCs)" also encompass "mesenchymal stromal cells." The origin of the amniotic membrane as used herein is not particularly limited, and it can be derived from any organism. Examples of such organisms include mammals such as humans, monkeys, cows, horses, dogs, cats, pigs, sheep, mice, rats, and rabbits, and birds such as parakeets, parrots, and chickens. The amniotic membrane as used herein is preferably derived from humans, monkeys, cows, horses, dogs, cats, pigs, sheep, mice, rats, or rabbits, more preferably from humans, monkeys, dogs, or cats, and even more preferably from humans.

[0016] (Cell population containing mesenchymal stem cells) As used herein, a "cell population containing mesenchymal stem cells" refers to a cell aggregate composed of one or more cells, including one or more mesenchymal stem cells. The form of the cell aggregate is not particularly limited, and examples include tissue, tissue fragment, cell pellet, cell aggregate, cell sheet, cell suspension, cell suspension, and frozen products thereof.

[0017] (Suicide Gene) As used herein, the term "suicide gene" refers to a gene that encodes a protein that converts a prodrug into a cytotoxic substance, and that induces cell death in cells expressing the gene in the presence of a prodrug. Examples of suicide genes include the herpes simplex virus thymidine kinase (HSV-TK) gene, the cytosine deaminase (CD) gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the purine nucleoside phosphorylase gene, the β-galactosidase gene, the cytochrome P450 gene, the Escherichia coli GPT gene, the linamarase gene, the horseradish peroxidase gene, the carboxypeptidase A gene, and the carboxypeptidase G2 gene. As used herein, the suicide gene is preferably the HSV-TK gene or the CD gene, and more preferably HSV-TK.

[0018] The term "prodrug" as used herein is not particularly limited as long as it is a precursor drug that can be converted into a cytotoxic anticancer substance by the suicide gene used, and examples thereof include ganciclovir, 5-fluorocytosine (5-FC), 6-methoxypurine arabinoside, CB1954, 6-methylpurine deoxyriboside, Daun02, cyclophosphamide, ifosfamide, and ZD-2767P. The combination of a prodrug with a suicide gene is important; for example, ganciclovir can be converted into an anticancer substance when combined with the HSV-TK gene, 5-FC when combined with the CD gene, 6-methoxypurine arabinoside when combined with the varicella-zoster virus thymidine kinase gene, CB1954 when combined with the nitroreductase gene, and 6-methylpurine deoxyriboside when combined with the purine nucleoside phosphorylase gene. The prodrug herein is preferably ganciclovir or 5-FC, and more preferably ganciclovir.

[0019] (Expression Cassette) As used herein, the term "expression cassette" refers to a collection of nucleic acid regions that contain one or more types of genes and optionally contain one or more nucleic acid regions that control the expression of the genes, and encompass expression units of the genes. The nucleic acid regions that control the expression of genes are not particularly limited. Examples include promoters, enhancers, terminators, polyA signals, and linkers.

[0020] (Gene introduction) As used herein, "gene introduction" refers to the artificial introduction of nucleic acids into a target object such as a cell. Gene introduction methods generally include methods using viral vectors and methods using non-viral vectors. Examples of viral vectors include adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, vaccinia viral vectors, and herpes simplex viral vectors. Examples of non-viral vectors include plasmid vectors. Examples of gene introduction methods using plasmid vectors include the transposon method, a method using cationic lipids, calcium phosphate coprecipitation, diethylaminoethyl (DEAE)-dextran, a method using cationic polymers, electroporation, microinjection, a method using a gene gun, a method using a laser, and a method using magnetic nanoparticles.

[0021] The term "electroporation" as used herein refers to a gene transfer method in which cells are exposed to an electric pulse to temporarily form pores in the cell membrane, and nucleic acids such as plasmid vectors are delivered into the cells through the pores. In this specification, the term "electroporation" also includes "electroporation."

[0022] As used herein, the term "transposon method" refers to a gene transfer method that utilizes a mechanism of gene transfer involving a combination of a transposase and a specific repeat sequence recognized by the transposase. The transposase is not particularly limited. Examples include Sleeping Beauty, piggyBac, Tol2, Frog Prince, and HimarI. As used herein, "transposon" also includes "DNA transposons" and "retrotransposons."

[0023] Genetically Modified As used herein, a "genetically modified" cell refers to a cell that has an artificially introduced nucleic acid.

[0024] (Cell Preparation Comprising Amnion-Derived Mesenchymal Stem Cells) As used herein, the term "cell preparation comprising amnion-derived mesenchymal stem cells" refers to a composition prepared ex vivo and containing one or more genetically modified amnion-derived mesenchymal cells. The form of the cell preparation is not particularly limited, and examples thereof include a cell suspension, a cell suspension, a cell pellet, a cell aggregate, a sheet, a sealant, and frozen products thereof. The term "cancer" as used herein is not particularly limited. Examples include squamous cell carcinoma, lung cancer, peritoneal cancer, mesothelioma, colon cancer, bile duct tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral cancer, osteosarcoma, soft tissue sarcoma, gallbladder cancer, kidney cancer, leukemia, bladder cancer, melanoma, brain cancer, glioma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, esophageal cancer, colon cancer, gastric cancer, cervical cancer, endometrial cancer, prostate cancer, ovarian cancer, head and neck cancer, rectal cancer, and testicular cancer. As used herein, "cancer" includes "carcinoma," "tumor," and "malignant tumor."

[0025] [2] Method for producing a cell population comprising genetically modified amnion-derived mesenchymal stem cells One or more embodiments of the present invention provide a method for producing a cell population comprising genetically modified amnion-derived mesenchymal stem cells. The method for producing a cell population comprising genetically modified amnion-derived mesenchymal stem cells of the present invention comprises the step of transfecting amnion-derived mesenchymal stem cells with a suicide gene or a suicide gene expression cassette.

[0026] The cell population containing amnion-derived mesenchymal stem cells used as a starting material for producing a cell population containing genetically modified amnion-derived mesenchymal stem cells of the present invention is not particularly limited as long as it contains one or more amnion-derived mesenchymal stem cells, and may also contain other cells. The percentage of amnion-derived mesenchymal stem cells in the cell population containing amnion-derived mesenchymal stem cells is, for example, 0.1% or more, 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0027] The cell population containing amnion-derived mesenchymal stem cells of the present invention may be pre-cultured or frozen, and may be, for example, tissue or cells collected from an individual, or a frozen product thereof, or a culture of tissue or cells collected from an individual, or a frozen product thereof.

[0028] The present invention may include a cell population obtaining step of enzymatically treating an amniotic membrane to obtain a cell population containing amniotic membrane-derived mesenchymal stem cells as a raw material. The cell population obtaining step may further include a step of obtaining the amniotic membrane by Caesarean section.

[0029] According to one or more embodiments of the present invention, the method for producing a cell population containing amnion-derived mesenchymal stem cells preferably comprises enzymatically treating a sample containing amniotic membrane. The enzymatic treatment is preferably a treatment with an enzyme capable of liberating mesenchymal stem cells contained in the extracellular matrix layer of the amniotic membrane without degrading the epithelial cell layer. Examples of enzymes having this activity include collagenase and metalloproteinase. Metalloproteinases include, but are not limited to, thermolysin and dispase, which are metalloproteinases that cleave the N-terminal side of nonpolar amino acids. Furthermore, two or more enzymes having this activity may be used in combination.

[0030] The present invention may further comprise the step of culturing a cell population comprising amnion-derived mesenchymal stem cells. In this case, the method for culturing the cell population comprising amnion-derived mesenchymal stem cells may be the same as the method for culturing a cell population comprising genetically modified amnion-derived mesenchymal stem cells described below.

[0031] The present invention comprises a step of transfecting a suicide gene or a suicide gene expression cassette into amnion-derived mesenchymal stem cells contained in a cell population containing amnion-derived mesenchymal stem cells. From the viewpoint of the efficiency of producing suicide gene-transfected cells, it is preferable that the suicide gene or the suicide gene expression cassette be integrated into the chromosome of the genetically modified cells. This enables more stable production of genetically modified cells compared to genetically modified cells having an extrachromosomal vector.

[0032] A commonly used gene transfer method for integrating a transgene into the chromosome of a target cell is the use of a viral vector. However, the present inventors have first confirmed that when amnion-derived mesenchymal stem cells are subjected to gene transfer of a suicide gene or a suicide gene expression cassette using a viral vector, the cell proliferation ability is reduced and the efficiency of producing genetically modified cells is significantly reduced. Therefore, the present invention is characterized by using a plasmid vector for gene transfer.

[0033] The plasmid vector used in the present invention may be at least one type of plasmid vector, or two or more types of plasmid vectors. There are no particular limitations on the types. For example, a plasmid vector having a suicide gene or a suicide gene expression cassette in its structure may be used in combination with a plasmid vector that assists or controls the integration of the suicide gene into the genome of a target cell or the expression of the suicide gene.

[0034] In the present invention, methods for introducing a plasmid vector into cells include, for example, a method using a cationic lipid, calcium phosphate coprecipitation, a diethylaminoethyl (DEAE)-dextran method, a method using a cationic polymer, electroporation, a microinjection method, a method using a gene gun, a method using a laser, and a method using magnetic nanoparticles. From the viewpoints of introduction efficiency and ease of operation, the method for introducing a plasmid vector into cells in the present invention is preferably a method using a cationic lipid, calcium phosphate coprecipitation, a diethylaminoethyl (DEAE)-dextran method, a method using a cationic polymer, or electroporation, more preferably a method using a cationic lipid, a method using a cationic polymer, or electroporation, and most preferably electroporation.

[0035] The gene transfer method using a plasmid vector in the present invention is preferably the transposon method. The transposon method involves introducing a construct containing a transgene or a transgene expression cassette adjacent to a repeat sequence recognized by a transposase into a target cell, and then expressing the transposase in the target cell, thereby stably integrating the transgene into the chromosome of the target cell. This method therefore has a relatively high efficiency of genomic integration of the transgene. Surprisingly, when the transposon method is used to introduce a suicide gene or a suicide gene expression cassette into amniotic membrane-derived mesenchymal stem cells, the reduction in cell proliferation ability that occurs when using a viral vector is not observed, making it possible to efficiently produce genetically modified cells. Therefore, the present invention preferably uses two types of plasmid vectors: a plasmid vector containing a construct containing a suicide gene or a suicide gene expression cassette adjacent to a repeat sequence recognized by a transposase, and a plasmid vector containing a transposase expression cassette. Alternatively, a plasmid vector having both a construct in which a suicide gene or a suicide gene expression cassette is placed adjacent to a repeat sequence recognized by the transposase, and a transposase expression cassette can also be used.

[0036] The transgene of the present invention may contain one or more suicide genes, or may contain two or more suicide genes, and may also contain one or more other genes as needed.

[0037] The other gene or its expression cassette may be introduced before, after, or simultaneously with the introduction of the suicide gene or suicide gene expression cassette. The other gene may also be included as a component of the suicide gene expression cassette. In this case, the other gene may be introduced simultaneously with the introduction of the suicide gene or suicide gene expression cassette.

[0038] The other gene is not particularly limited as long as it is a gene other than a suicide gene. Examples include antibiotic resistance genes, reporter genes, tag peptides, genes involved in undifferentiation, and genes involved in cell differentiation. Examples of antibiotic resistance genes include genes that confer resistance to puromycin, neomycin, hygromycin, blasticidin, and the like. Examples of reporter genes include GFP, EGFP, RFP, Venus, DsRed, β-galactosidase, β-glucuronidase, and luciferase. The tag peptide is not particularly limited as long as it is a short peptide consisting of a dozen to several tens of amino acids that can label a protein, and examples include FLAG, HA, His, and myc. Examples of genes involved in undifferentiation include Nanog, Oct4, Sox2, and Klf4. Examples of the genes involved in cell differentiation include Runx2, CBF-1α, Sox9, PPARγ, GATA4, GATA6, and MyoD.

[0039] The method for introducing a gene other than the suicide gene or its expression cassette may be a method using a viral vector or a method using a non-viral vector, regardless of whether it is integrated into a chromosome. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, vaccinia viral vectors, and herpes simplex viral vectors. Examples of methods using non-viral vectors include, but are not limited to, the transposon method, a method using cationic lipids, calcium phosphate coprecipitation, diethylaminoethyl (DEAE)-dextran, a method using cationic polymers, electroporation, microinjection, a method using a gene gun, a laser, and a method using magnetic nanoparticles. When the introduction of the other gene or its expression cassette is carried out simultaneously with the introduction of the suicide gene or its expression cassette, the introduction of the other gene or its expression cassette is carried out using a method using a non-viral vector, preferably the transposon method.

[0040] The transgene of the present invention may be operably linked to a promoter, an enhancer, or a combination thereof. The promoter is not particularly limited, but examples thereof include a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a simian virus 40 (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, and a CAG promoter.

[0041] The transgene of the present invention may be formed by linking two or more genes with a linker. The linker is not particularly limited as long as it is a nucleic acid or peptide that links two or more genes, and examples thereof include an IRES (internal ribosome entry site), Ubx-IRES, 2A peptides (P2A, T2A, E2A, F2A, etc.), and a GS linker.

[0042] The present invention may further include a step of selecting genetically modified amnion-derived mesenchymal stem cells. Methods for selecting genetically modified amnion-derived mesenchymal stem cells include, for example, methods that utilize other genes (antibiotic resistance genes, reporter genes, tag peptides, etc.) as described above. More specific examples include, but are not limited to, a method in which a gene-transfected cell population is cultured in a medium containing an antibiotic to select cells that express an antibiotic resistance gene, a method in which cells that express a reporter gene are selected using a cell sorter, and a method in which cells that express a tag peptide are separated by chromatography using an antibody that binds to the tag peptide. Furthermore, two or more selection methods can be combined.

[0043] The cell population containing genetically modified amnion-derived mesenchymal stem cells of the present invention is not particularly limited as long as it contains one or more genetically modified amnion-derived mesenchymal stem cells, and may also contain genetically unmodified amnion-derived mesenchymal stem cells or other cells. The proportion of genetically modified amnion-derived mesenchymal stem cells in the cell population containing genetically modified amnion-derived mesenchymal stem cells is, but is not limited to, for example, 0.01% or more, 0.1% or more, 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0044] The present invention may further include a step of culturing a cell population containing genetically modified amnion-derived mesenchymal stem cells. The medium used in the step of culturing a cell population containing amnion-derived mesenchymal stem cells can be prepared by using any liquid medium for animal cell culture as a basal medium and appropriately adding other components as needed. Examples of the other components include, but are not limited to, albumin, serum, serum replacement reagents, platelet lysates, cytokines, and antibiotics. Alternatively, two or more other components may be added in combination.

[0045] Examples of the basal medium include BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, and RPMI. Media that can be used include, but are not limited to, 1640 medium, Fischer's medium, and mixed media thereof (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)). Various commercially available serum-free media can also be used.

[0046] The seeding density in the step of culturing a cell population containing genetically modified amnion-derived mesenchymal stem cells is not particularly limited. For example, 500 to 100,000 cells / cm ２ The lower limit of the seeding density is, for example, 500 cells / cm. ２ More than 1,000 cells / cm ２ More than 2,000 cells / cm ２ More than 3,000 cells / cm ２ More than 4,000 cells / cm ２ or more than 5,000 cells / cm ２From the viewpoint of cell culture efficiency, the upper limit of the seeding density is preferably 100,000 cells / cm. ２ It is common to use: Others, e.g., 50,000 cells / cm ２ Below, 30,000 cells / cm ２ Below, 20,000 cells / cm ２ or less than 15,000 cells / cm ２ The following can be used: The step of culturing the cell population containing the genetically modified amnion-derived mesenchymal stem cells may include a passaging step, or may include a step of repeating the culture multiple times under different culture conditions.

[0047] The culture step may include a step of changing the medium during the culture period. The medium to be changed may have the same composition as the original medium, or may have a different composition. The frequency of changing the medium is not particularly limited. For example, the frequency may be every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0048] The confluence rate after the step of culturing the cell population containing the genetically modified amnion-derived mesenchymal stem cells is not particularly limited. From the viewpoint of obtaining a larger number of cells, the confluence rate is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more. Other examples include 1% or more, 10% or more, 30% or more, and 100%.

[0049] The present invention may further include a step of recovering the cells cultured in the culturing step. The method for recovering the cultured cells is not particularly limited, but may be performed, for example, as follows. First, cells cultured to a predetermined confluence are treated with a cell detachment means to detach them from the culture vessel. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in medium or any solvent. If necessary, the method may further include a step of centrifuging the cell suspension and washing it with a washing solution.

[0050] As the cell detachment means, for example, a cell detachment agent may be used. The cell detachment agent is not particularly limited as long as it is an agent that has at least the effect of detaching adherent cells from the culture vessel, and examples thereof include trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA), etc. Commercially available cell detachment agents may be used. Examples include, but are not limited to, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technologies), and Accumax (manufactured by Stemcell Technologies). Alternatively, a physical cell detachment means may be used, such as, but not limited to, a cell scraper (manufactured by Corning). The cell detachment means may be used alone or in combination.

[0051] The washing solution is not particularly limited, but from the viewpoint of reducing damage to cells, a buffer solution or an isotonic solution is preferable, and examples thereof include, but are not limited to, PBS, HBSS(-), Ringer's solution, lactated Ringer's solution, glucose solution, various infusion solutions, physiological saline solution, culture solution, basal medium, albumin solution, blood-derived components, and mixtures thereof.

[0052] The present invention may further comprise a step of cryopreserving the cell population containing genetically modified amnion-derived mesenchymal stem cells recovered through the recovery step. The means for cryopreservation is not particularly limited. Examples include cryopreservation using a programmed freezer, a deep freezer, and cryopreservation in liquid nitrogen. When a programmed freezer is used as the cryopreservation means, the freezing temperature is preferably −30°C or lower, −40°C or lower, −50°C or lower, −80°C or lower, −90°C or lower, −100°C or lower, −150°C or lower, −180°C or lower, or −196°C (liquid nitrogen temperature) or lower. When a programmed freezer is used, the freezing rate is preferably 15°C / min or lower, 11°C / min or lower, 10°C / min or lower, 9°C / min or lower, 5°C / min or lower, 2°C / min or lower, or 1°C / min or lower. When a programmable freezer is used as the cryopreservation means, it is preferable to set the freezing rate to be 1°C / min or more and 2°C / min or less, for example, at least between room temperature and -10°C, and then change the cooling rate appropriately outside of that range to ultimately reach the desired freezing temperature (for example, -80°C to -150°C). Furthermore, when liquid nitrogen is used as the freezing means, the temperature can be rapidly lowered to, for example, -196°C to freeze the sample, and then the sample can be stored in liquid nitrogen (gas phase). Alternatively, the sample can be stored in liquid nitrogen (liquid phase).

[0053] When freezing by the above-mentioned freezing means, the cells to be frozen may be frozen in any storage container, such as, but not limited to, a cryotube, a cryovial, a freezing bag, and an infusion bag.

[0054] When freezing cells by the freezing method, the cells may be frozen in any cryopreservation solution. A commercially available cryopreservation solution may be used. Examples of cryopreservation solutions include, but are not limited to, BAMBANKER (manufactured by Lymphotec), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), CP-1 (manufactured by Kyokuto Pharmaceutical Industries Co., Ltd.), ReproCryo RM (manufactured by ReproCell, Inc.), CryoNovo (manufactured by Akron Biotechnology, Inc.), MSC Freezing Solution (manufactured by Biological Industries, Inc.), and CryoStor (manufactured by HemaCare).

[0055] The cryopreservation solution may contain a polysaccharide at a predetermined concentration. A preferred polysaccharide concentration is, for example, 1% by mass or more, 2% by mass or more, 4% by mass or more, or 6% by mass or more. A preferred polysaccharide concentration is, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, or 13% by mass or less. Examples of polysaccharides include, but are not limited to, hydroxyethyl starch (HES) and dextran (Dextran 40, etc.).

[0056] The cryopreservation solution may contain a predetermined concentration of dimethyl sulfoxide (DMSO). A preferred concentration of DMSO is, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, or 5% by mass or more. A preferred concentration of DMSO is, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, 12% by mass or less, or 10% by mass or less.

[0057] The cryopreservation solution may contain albumin at a predetermined concentration greater than 0% by mass. A preferred albumin concentration is, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, or 4% by mass or more. A preferred albumin concentration is, for example, 30% by mass or less, 20% by mass or less, 10% by mass or less, or 9% by mass or less. Examples of albumin include, but are not limited to, bovine serum albumin (BSA), mouse albumin, and human albumin.

[0058] [3] Cell preparation comprising gene-transfected amnion-derived mesenchymal stem cells In another embodiment of the present invention, there is provided a cell preparation comprising amnion-derived mesenchymal stem cells transfected with a suicide gene or a suicide gene expression cassette.

[0059] The cell preparation of the present invention contains, as an active ingredient, amnion-derived mesenchymal stem cells transfected with a suicide gene or a suicide gene expression cassette, and optionally contains pharmaceutically acceptable solvents and / or other additives. The active ingredient may be a component of a cell population containing genetically modified amnion-derived mesenchymal stem cells produced by the production method described in [2] Method for producing a cell population containing genetically modified amnion-derived mesenchymal stem cells.

[0060] The content of the active ingredient is not particularly limited and can be determined appropriately depending on the dosage form of the cell preparation, the type of solvent or carrier, the dosage, the number of administrations, the administration route, the purpose of use, and the age, weight, and symptoms of the patient or subject.

[0061] The solvent is not particularly limited as long as it is pharmaceutically acceptable. Examples include water, physiological saline, an isotonic solution containing glucose or other adjuvants, a phosphate buffer solution, and a sodium acetate buffer solution. Examples of the other adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, and polyoxyethylene sorbitan fatty acid esters.

[0062] The other additives are not particularly limited as long as they are pharmaceutically acceptable additives, and examples thereof include excipients, binders, disintegrants, fillers, emulsifiers, flow regulators, lubricants, human serum albumin, and prodrugs.

[0063] The cell preparation of the present invention may be used in combination with a prodrug. The prodrug may be administered to a subject before, simultaneously with, or after the administration of the cell preparation.

[0064] The cell preparation of the present invention is useful for treating, for example, cancer, immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease (GVHD), inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen diseases, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular plegia, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides (Alibert-Bazin syndrome), and the like. The cell preparation of the present invention can be used as a therapeutic agent for a disease selected from the group consisting of scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus damage, osteochondrosis elastosis, avascular necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial damage, fibrosis, pulmonary diseases, muscular dystrophy, chronic pancreatitis, and chronic nephritis. The cell preparation of the present invention can be preferably used as a therapeutic agent for cancer.

[0065] Examples of the cancer include squamous cell carcinoma, lung cancer, peritoneal cancer, mesothelioma, colon cancer, bile duct tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral cancer, osteosarcoma, soft tissue sarcoma, gallbladder cancer, kidney cancer, leukemia, bladder cancer, melanoma, brain cancer, glioma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, esophageal cancer, colon cancer, gastric cancer, cervical cancer, endometrial cancer, prostate cancer, ovarian cancer, head and neck cancer, rectal cancer, and testicular cancer. The cell preparation of the present invention can be preferably used as a therapeutic agent for brain tumors.

[0066] The method of administering the cell preparation of one or more embodiments of the present invention is not particularly limited, and examples thereof include subcutaneous injection, intralymph node injection, intravenous injection, intraperitoneal injection, intrathoracic injection, direct injection into a local area, and direct transplantation into a local area.

[0067] The cell preparation according to one or more embodiments of the present invention can also be used as an injectable preparation, a preparation for transplantation of a cell mass or a sheet-like structure, or a gel preparation mixed with any gel.

[0068] The patient or subject for which the cell preparation of one or more embodiments of the present invention is intended is typically a human, but may also be other animals, including mammals such as monkeys, cows, horses, dogs, cats, pigs, sheep, mice, rats, and rabbits, and birds such as parakeets, parrots, and chickens.

[0069] [4] Cancer Treatment Kit In another embodiment of the present invention, there is provided a cancer treatment kit comprising: (i) a cell population comprising amnion-derived mesenchymal stem cells transfected with a suicide gene or a suicide gene expression cassette; and (ii) a prodrug.

[0070] The cancer treatment kit of the present invention is manufactured for the purpose of combined treatment with a prodrug and a cell population containing amnion-derived mesenchymal stem cells transfected with a suicide gene or a suicide gene expression cassette. The kit is manufactured so that the suicide gene or the cell population containing amnion-derived mesenchymal stem cells transfected with a suicide gene expression cassette can be administered to a subject requiring treatment before, simultaneously with, or after the administration of the prodrug, or in a combination of two or more of these.

[0071] The cell population may be a cell population containing genetically modified amnion-derived mesenchymal stem cells produced by the production method described in [2] Method for producing a cell population containing genetically modified amnion-derived mesenchymal stem cells.

[0072] The subject is typically a human, but may be another animal. Examples of other animals include mammals such as monkeys, cows, horses, dogs, cats, pigs, sheep, mice, rats, and rabbits, and birds such as parakeets, parrots, and chickens. The subject is preferably a mammal, including a human, and more preferably a patient in need of cancer treatment. Patients in need of cancer treatment include patients currently undergoing cancer treatment, patients who have previously undergone cancer treatment, and patients who need to undergo cancer treatment, and may also include patients who have undergone surgery to remove cancer for cancer treatment.

[0073] The cancer is similar to the cancer described in "[3] Cell preparation comprising gene-transfected amnion-derived mesenchymal stem cells."

[0074] The cancer treatment kit of the present invention may be composed of a first compartment and a second compartment, the first compartment containing a cell population including amnion-derived mesenchymal stem cells transfected with a suicide gene or a suicide gene expression cassette, and the second compartment containing a prodrug. For convenience and portability, the kit may contain each of the components in a single container, each in a single dose, or in several different small containers.

[0075] The kit of the present invention may optionally contain equipment necessary for use and instruction materials describing the method of administration of each component.

[0076] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0077] (Comparative Example 1) Preparation of Venus-expressing amnion-derived mesenchymal stem cells (amnion MSCs) using a lentiviral vector Human amnion MSCs (passage 2) were cultured at 20,000 cells / cm ２ The cells were seeded at a seeding density of 1000 kJ / well onto a 6-well plate (Corning) and cultured for 2 days in αMEM containing a final concentration of 5% human platelet lysate. After culture, the medium was removed, and a lentiviral vector-containing solution carrying a Venus expression cassette was added. The cells were cultured for 0.5 hours, and then an equal volume of medium to the lentiviral vector-containing solution was added and cultured for 18 hours. The virus-containing solution was then removed, medium was added, and the cells were cultured for 24 hours. After that, the cultured cells were detached using TrypLE Select (Thermo Fisher Scientific), diluted with medium, and centrifuged to collect a cell population containing Venus-expressing amniotic membrane MSCs.

[0078] The lentiviral vector-containing solution carrying the Venus expression cassette was prepared as follows. First, a nucleic acid sequence in which the Venus gene was linked under the control of a CMV promoter was introduced into a lentiviral vector construction plasmid to prepare a lentiviral vector construction plasmid carrying a Venus expression cassette. This plasmid and a lentiviral packaging plasmid were transfected into Lenti-X 293T (Takara Bio Inc.), a lentiviral packaging cell line, using Lipofectamine 3000 (Thermo Fisher Scientific), and the cells were cultured for 48 hours. Thereafter, the Lenti-X 293T culture supernatant was collected and filtered through a 0.8 μm filter to prepare a lentiviral vector-containing solution carrying a Venus expression cassette.

[0079] In order to mass-produce Venus-expressing amniotic MSCs, we attempted to proliferate the Venus-expressing amniotic MSCs contained in the collected cell population containing Venus-expressing amniotic MSCs. Specifically, we cultured the collected cell population containing Venus-expressing amniotic MSCs at a density of 10,000 cells / cm. ２ The cells were seeded at a seeding density of 1000 kJ / ml into T25 flasks (Sumitomo Bakelite Co., Ltd.) and cultured in α-MEM containing human platelet lysate at a final concentration of 5%. On day 3 of culture, the cell culture status was observed under a microscope in bright field (×40 magnification, 1 / 25 second exposure time) and green fluorescence (×40 magnification, 1 second exposure time) (Figure 1A). After a further 7 days of culture, on day 10, the cell culture status was again observed under a microscope in bright field (×40 magnification, 1 / 25 second exposure time) and green fluorescence (×40 magnification, 1 second exposure time) (Figure 1B). Cell proliferation was observed in the bright field image, and an increase in Venus-expressing cells emitting fluorescent signals in the green fluorescence image indicated the proliferation of Venus-expressing amniotic membrane MSCs in the seeded cell population. These results indicate that the proliferation potential of amnion-derived mesenchymal stem cells is not affected when genes other than suicide genes are introduced using lentiviral vectors.

[0080] (Comparative Example 2) Preparation of HSV-TK-expressing amniotic membrane MSCs using lentiviral vector Human amniotic membrane MSCs (passage 2) were cultured at 20,000 cells / cm ２ The cells were seeded at a seeding density of 1000 μg / well onto a 6-well plate (Corning Incorporated) and cultured for 2 days in αMEM containing a final concentration of 5% human platelet lysate. After culture, the medium was removed, and a solution containing a lentiviral vector carrying an HSV-TK expression cassette was added. The cells were cultured for 0.5 hours, and then an equal volume of medium to the lentiviral vector-containing solution was added and cultured for 18 hours. The virus-containing solution was then removed, and medium was added. The cells were cultured for 24 hours, after which a cell population containing HSV-TK-expressing amniotic membrane MSCs was detached and collected using the same method as in Comparative Example 1.

[0081] The lentiviral vector-containing solution carrying the HSV-TK expression cassette was prepared as follows. First, the HSV-TK gene was placed under the control of the EF-1α promoter, and the Venus gene was further linked downstream of the HSV-TK gene via an IRES. This nucleic acid sequence was introduced into a lentiviral vector construction plasmid to prepare a lentiviral vector construction plasmid carrying the HSV-TK expression cassette. Linking the Venus gene downstream of the HSV-TK gene via an IRES made it possible to distinguish HSV-TK-expressing cells by the Venus fluorescent signal. This plasmid and a lentiviral packaging plasmid were transfected into Lenti-X 293T (Takara Bio Inc.), a lentiviral packaging cell line, using Lipofectamine 3000 (Thermo Fisher Scientific), and the cells were cultured for 48 hours. Thereafter, the Lenti-X 293T culture supernatant was collected and filtered through a 0.8 μm filter to prepare a solution containing a lentiviral vector carrying an HSV-TK expression cassette.

[0082] In order to mass-produce HSV-TK-expressing amniotic MSCs, we attempted to proliferate the HSV-TK-expressing amniotic MSCs contained in the collected cell population containing HSV-TK-expressing amniotic MSCs. Specifically, we cultured the collected cell population containing HSV-TK-expressing amniotic MSCs at a density of 10,000 cells / cm. ２The cells were seeded at a seeding density of 1000 kJ / ml into T25 flasks (Sumitomo Bakelite Co., Ltd.) and cultured in α-MEM containing human platelet lysate at a final concentration of 5%. On day 3 of culture, the cell culture status was observed under a microscope in bright field (×40 magnification, 1 / 25 second exposure time) and green fluorescence (×40 magnification, 2.5 second exposure time) (Figure 2A). After a further 7 days of culture, on day 10, the cell culture status was again observed under a microscope in bright field (×40 magnification, 1 / 25 second exposure time) and green fluorescence (×40 magnification, 5 second exposure time) (Figure 2B). While bright field images confirmed cell proliferation, green fluorescence images did not reveal any proliferation of HSV-TK-expressing cells, which emitted fluorescent signals. This indicates that proliferation of HSV-TK-expressing amniotic membrane MSCs was unsuccessful. The proliferation of cells not transfected with HSV-TK was observed in bright field observation images, indicating that the proliferation ability of amniotic MSCs was significantly reduced by the transfection and expression of HSV-TK gene.

[0083] Example 1: Preparation of HSV-TK-expressing amniotic MSCs using the transposon method To prepare HSV-TK-expressing amniotic MSCs using the transposon method, the Venus gene was first introduced into amniotic MSCs using a lentiviral vector in order to improve the visibility of the HSVTK-expressing amniotic MSCs. Specifically, a cell population containing Venus-expressing amniotic MSCs was collected using the same method as in Comparative Example 1, and Venus-expressing amniotic MSCs were selected and collected using a cell sorter FACSAriaIII (BD) using the fluorescent signal as an indicator.

[0084] Venus-expressing amniotic membrane MSCs were cultured at 10,000 cells / cm ２The cells were seeded at a seeding density of 1000 kJ / ml onto 15 cm dishes (Sumitomo Bakelite Co., Ltd.) and cultured for 3 days in α-MEM containing human platelet lysate at a final concentration of 5%. After culture, the cells were detached using TrypLE Select (Thermo Fisher Scientific), diluted with medium, and collected by centrifugation. Using Cell Line Nucleofector Kit L (Ronza) and Nucleofector II / 2b (Ronza), the collected cells were transfected with a plasmid vector containing a construct in which an HSV-TK expression cassette was placed adjacent to a repeat sequence recognized by the transposase, and a plasmid vector containing a transposase expression cassette (a total of two plasmid vectors).

[0085] The construct contained an HSV-TK gene under the control of the EF-1α promoter, and further contained a puromycin resistance gene linked downstream of the HSV-TK gene via T2A, with repeat sequences recognized by transposase at both ends of the expression cassette. Linking the puromycin resistance gene downstream of the HSV-TK gene via T2A enabled the selection of HSV-TK-expressing cells by drug selection.

[0086] After transfection, the cells were seeded into a T75 flask and cultured for 24 hours. Puromycin was then added at a final concentration of 1 μg / mL. On day 3 of culture, the cell culture status was observed under a microscope in bright field (×40 magnification, 1 / 25 second exposure time) and in green fluorescence (×40 magnification, 3 second exposure time) (Figure 3A). After a further 7 days of culture, on day 10, the cell culture status was again observed under a microscope in bright field (×40 magnification, 1 / 25 second exposure time) and in green fluorescence (×40 magnification, 1 second exposure time) (Figure 3B). The bright field and green fluorescence images showed an increase in HSV-TK-expressing cells resistant to puromycin, indicating the proliferation of HSV-TK-expressing amniotic membrane MSCs within the seeded cell population.

[0087] The above results demonstrated that amniotic MSCs transduced with HSV-TK using a viral vector exhibited inhibited proliferation, whereas amniotic MSCs transduced with HSV-TK using a plasmid vector by the transposon method could be efficiently produced without a decrease in proliferation ability.

[0088] Example 2 Evaluation of the anticancer effect of HSV-TK-expressing amniotic MSCs produced using the transposon method on brain tumors An experiment was conducted to confirm that HSVTK-expressing amniotic MSCs (TK-AMSCs), produced using the transposon method for gene transfer of an HSV-TK expression cassette, have an anticancer effect on brain tumors. First, TK-AMSCs were produced by the method described in Example 1 and selected using puromycin resistance as an indicator, and then detached and collected in the same manner as in Comparative Example 1. The collected TK-AMSCs were cultured at a density of 1,000 cells / cm. ２ The TK-AMSCs were seeded onto a 15 cm dish (manufactured by Sumitomo Bakelite Co., Ltd.) at a seeding density of 1000 x 1000 and cultured for 7 days in α-MEM containing human platelet lysate at a final concentration of 5%. After culture, the TK-AMSCs were detached and collected in the same manner as in Comparative Example 1, and then collected at a density of 2 x 100 x 1000. ６ TK-AMSC cells were suspended in 50 μL of physiological saline containing 50% Matrigel (manufactured by Corning) to prepare a TK-AMSC administration solution. ６ U-87MG, a human brain tumor-derived cell line from TK-AMSCs, was suspended in 50 μL of physiological saline containing 50% Matrigel (manufactured by Corning Incorporated) to prepare a U-87MG administration solution. The TK-AMSC administration solution and / or the U-87MG administration solution was subcutaneously transplanted into the right flank of 5-week-old immunodeficient nude mice (Balb / c-nu / nu). Starting the day after cell transplantation, ganciclovir (GCV) was administered intraperitoneally once daily at a dose of 50 mg / kg for 7 days. The major and minor axes of the tumor were measured 1, 3, 7, 10, 14, 17, 21, 24, 28, and 31 days after cell transplantation, and the tumor volume was calculated using the formula (tumor volume (mm ３) = major axis (mm) × minor axis (mm) × minor axis (mm) ÷ 2), and the tumor volume was calculated and graphed (Figure 4). As a result, the experimental group that received TK-AMSC transplantation and GCV administration treatment (TK-AMSC(+), GCV(+)) showed significantly suppressed tumor growth compared to the untreated group (TK-AMSC(-), GCV(-)) and the experimental group that received only ganciclovir administration treatment (TK-AMSC(-), GCV(+)). These results demonstrate that tumor growth inhibitory effects were observed in the test group that received HSV-TK-expressing amnion-derived mesenchymal stem cell transplantation and ganciclovir administration.

[0089] These results confirm that HSV-TK-expressing amniotic MSCs prepared by the method described in the Examples have excellent anticancer effects and are highly useful as a cell preparation for cancer treatment. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for producing a cell population containing genetically modified amnion-derived mesenchymal stem cells, comprising the step of introducing a suicide gene or a suicide gene expression cassette into amnion-derived mesenchymal stem cells using a plasmid vector.

2. The method of claim 1, wherein the plasmid vector is introduced into cells by electroporation.

3. The method of claim 1, wherein the suicide gene or a suicide gene expression cassette is integrated into a chromosome in the gene introduction step.

4. The manufacturing method described in claim 1, wherein the gene introduction step uses two types of plasmid vectors: a plasmid vector containing a construct in which the suicide gene or a suicide gene expression cassette is positioned adjacent to a repeat sequence recognized by a transposase, and a plasmid vector containing a transposase expression cassette.

5. The manufacturing method described in claim 1, wherein the gene introduction step uses a construct in which the suicide gene or a suicide gene expression cassette is positioned adjacent to a repeat sequence recognized by a transposase, and a plasmid vector containing a transposase expression cassette.

6. The method of claim 1, wherein the suicide gene is a herpes simplex virus thymidine kinase gene or a cytosine deaminase gene.

7. The method of claim 1, wherein the suicide gene is the herpes simplex virus thymidine kinase gene.

8. A cell preparation comprising genetically modified amnion-derived mesenchymal stem cells, produced by the production method described in any one of claims 1 to 7.

9. A cell preparation comprising amnion-derived mesenchymal stem cells, wherein the amnion-derived mesenchymal stem cells have (i) a suicide gene or an expression cassette for the suicide gene, and (ii) a repetitive sequence recognized by a transposase integrated into their chromosomes.

10. The cell preparation according to claim 8 or 9, wherein the cell preparation is used in combination with a prodrug.

11. The cell preparation of claim 10, wherein the prodrug is ganciclovir or 5-fluorocytosine.

12. The cell preparation of claim 11, wherein the prodrug is ganciclovir.

13. The cell preparation according to claim 8 or 9, wherein the cell preparation is for cancer treatment.

14. The cell preparation according to claim 12, wherein the cell preparation is for treating brain tumors.

15. A cancer treatment kit comprising: (i) a cell population containing genetically modified amnion-derived mesenchymal stem cells produced by the production method described in any one of claims 1 to 7; and (ii) a prodrug.

16. A cancer treatment kit comprising the cell preparation of claim 9 and a prodrug.

17. The cancer treatment kit according to claim 15 or 16, wherein the prodrug is ganciclovir or 5-fluorocytosine.

18. The cancer treatment kit of claim 17, wherein the prodrug is ganciclovir.

19. The kit of claim 15 or 16, wherein the cancer is a brain tumor.

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