WO2014133090A1

WO2014133090A1 - Pluripotent stem cell for treating brain tumor

Info

Publication number: WO2014133090A1
Application number: PCT/JP2014/054910
Authority: WO
Inventors: 宏樹難波; 真理出澤; 正順吉田
Original assignee: 国立大学法人浜松医科大学; 国立大学法人東北大学; 株式会社Ｃｌｉｏ
Priority date: 2013-02-28
Filing date: 2014-02-27
Publication date: 2014-09-04
Also published as: JPWO2014133090A1

Abstract

The purpose of the present invention is to provide a novel medical use using a pluripotent stem cell (Muse cell). Provided is a cell preparation for treating a brain tumor, said cell preparation comprising SSEA-3 positive pluripotent stem cells that are separated from a mesenchymal tissue in an organism or cultured mesenchymal cells. The cell preparation according to the present invention is based on a mechanism in which Muse cells carrying a suicide gene transferred thereinto is intravenously administered to a subject having a brain tumor, and a prodrug corresponding to the suicide gene is further administered so that the Muse cells are allowed to selectively accumulate around the brain tumor and thus cells constituting the brain tumor are exterminated or the growth of the cells is reduced through the activation of the prodrug accompanied with the expression of the suicide gene.

Description

Pluripotent stem cells for brain tumor treatment

The present invention relates to a cell preparation containing pluripotent stem cells into which a suicide gene effective for treatment of brain tumors is introduced.

Glioma (glioma), which accounts for about ¼ of brain tumors, grows infiltrating into the surrounding normal brain tissue, so that if the brain function is to be preserved, it is difficult to remove it by surgery. Furthermore, radiation therapy and anticancer drug treatment are often used after surgery, but the cure rate is low, and the average malignant glioblastoma (glioblastoma) has an average survival time of only about 1 year. Moreover, despite the development of various treatment strategies, this result has hardly changed for the past 30 years and is said to be one of the last malignant tumors left in the 21st century, and the development of new treatment strategies is urgently needed. It is.

The gene therapy called “suicide gene therapy” introduced in the 1990s is a therapy that uses genes such as viruses to change harmful drugs in mammals into toxic substances (anticancer drugs) in transgenic cells and kill cancer cells. is there. In particular, in the suicide gene therapy using the herpes simplex virus thymidine kinase gene (HSVtk gene) and the antiviral agent ganciclovir (GCV), the bystander effect that all tumors disappear even if the transgenic cells are about 10% of the whole. (Non-Patent Document 1). However, although this treatment showed remarkable effects in animal experiments such as rats, the clinical results were not always satisfactory. One reason for this is that the retrovirus-producing fibroblasts used for gene transfer have a low migration ability and could not cover glioma cells that grow invasively.

On the other hand, neural stem cells that have been discovered recently and are actively researched for nerve regeneration have extremely active migration ability in the brain, and in rat experiments, when brain tumors and neural stem cells are transplanted to the left and right brains separately and observed, It is known that it moves to the side brain tumor and accumulates around the tumor (Non-Patent Document 2). Attempts have been made to treat brain tumors by incorporating suicide genes into such migratory neural stem cells and expressing cytotoxic substances in the tumor site (Patent Document 1). The inventors of the present invention transplanted a neural stem cell into which HSVtk / GCV gene has been introduced around a brain tumor, found an antitumor effect due to the bystander effect, and found that the neural stem cell is a cell suitable for the treatment of malignant glioma. (Non-patent Document 3). However, neural stem cells are extremely effective as a vector for carrying a suicide gene to a tumor site, but when considering clinical application, it is invasive and extremely difficult to remove neural stem cells from a patient. So far, the present inventors have used a mesenchymal stem cell collected from the bone marrow as a vector carrying a suicide gene to the tumor site as a more general-purpose vector having the same mobility as that of a neural stem cell. A treatment test was attempted (Patent Document 2). However, the mesenchymal stem cells used in this treatment test were heterogeneous and the effects were not constant.

According to research by Dezawa, one of the present inventors, SSEA-3 (Stage-Specific Embryonic Antigen-3), which is present in the mesenchymal cell fraction and obtained without induction, is expressed as a surface antigen. It has been found that pluripotent stem cells (Multilineage-differentiating Stress Enduring cells; Muse cells) are responsible for the pluripotency of the mesenchymal cell fraction and can be applied to disease treatment aimed at tissue regeneration. (Patent Document 3; Non-Patent Document 4; Non-Patent Document 5). However, there are no examples that show that the expected therapeutic effect can be obtained by using Muse cells for the prevention and / or treatment of brain tumors.

Special Table 2002-526104 JP 2006-345726 A International Publication No. WO2011 / 007900

An object of the present invention is to provide a new medical use using pluripotent stem cells (Muse cells) in brain tumor treatment. More specifically, an object of the present invention is to provide a cell preparation for the prevention and / or treatment of brain tumor, comprising Muse cells into which a suicide gene has been introduced and a prodrug corresponding to the suicide gene.

By adding human Muse cells into which the herpes simplex virus thymidine kinase (HSVtk) gene has been introduced together with ganciclovir (GCV) to the culture system of glioma cells, the bystander effect of Muse cells on the glioma cells And the migration ability was confirmed, it was found that the proliferation of glioma cells was remarkably suppressed, and the present invention was completed.

That is, the present invention is as follows.
[1] A cell preparation for treating a brain tumor comprising a pluripotent stem cell into which a suicide gene has been introduced, wherein the pluripotent stem cell is derived from a mesenchymal tissue or a cultured mesenchymal cell in a living body. Isolated SSEA-3-positive cells, wherein the cell preparation is used with a prodrug of a drug that kills or inhibits the growth of a brain tumor, the prodrug being a substrate for an enzyme produced by expression of the suicide gene A cell preparation.
[2] The cell preparation described in [1] above, wherein glioma cells constituting a brain tumor are treated.
[3] The cell preparation according to [1] and [2] above, wherein the pluripotent stem cell is CD105 positive.
[4] The cell preparation described in [1] to [3] above, wherein the pluripotent stem cells are CD117 negative and CD146 negative.
[5] The cell preparation described in [1] to [4] above, wherein the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative, and CD271 negative.
[6] The above [1], wherein the pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative. The cell preparation according to [5].
[7] The cell preparation described in [1] to [6] above, wherein the pluripotent stem cell is a pluripotent stem cell having all of the following properties:
(I) low or no telomerase activity;
(Ii) has the ability to differentiate into cells of any germ layer of the three germ layers;
(Iii) no neoplastic growth; and (iv) self-renewal ability.
[8] The cell preparation described in [1] to [7] above, wherein the pluripotent stem cell has an ability to accumulate at a brain tumor site.
[9] The above [1] to [8], wherein the suicide gene is herpes simplex virus thymidine kinase (HSVtk) gene, cytosine deaminase gene, uracil phosphoribosyltransferase gene, guanine phosphoribosyltransferase (gpt) gene or nitroreductase gene. ] The cell preparation as described in.
[10] The prodrug is gancyclovir (GCV), acyclovir, penciclovir, PMEA adefovir or PMPA tenofovir when the suicide gene is an HSVtk gene, and 5-fluorocytosine when the suicide gene is a cytosine deaminase gene, 5-fluorouracil when the suicide gene is a uracil phosphoribosyltransferase gene, 6-thioxanthine or 6-thioguanine when the suicide gene is a gpt gene, and a prodrug when the suicide gene is a nitroreductase (ntr) gene The cell preparation according to [1] to [8] above, wherein is CB1954.

The present invention relates to the expression of a suicide gene by administering a suicide gene-introduced Muse cell and a prodrug for the suicide gene from a vein or the like to a subject suffering from a brain tumor, thereby accumulating the Muse cell in the brain tumor. Based on the activation of the prodrug, tumor cells can be killed or their growth can be suppressed.

The accumulation of Muse cells in human glioblastoma (glioblastoma cell type) is shown. The bystander effect in Muse cells (Muse-TK) and human glioma (A172) introduced with a suicide gene is shown. FIG. 2 shows the results of observing the culture state of glioma cells over time when glioma cells and Muse-TK are co-cultured at different cell ratios. FIG. 3 is a diagram showing the decrease in glioma cells by counting the number of cells based on the results obtained in FIG. The result of having measured the migration ability of the Muse cell with respect to a glioma cell using the Boyden chamber is shown. The result of having verified the bystander effect by Muse-TK in vivo is shown.

The present invention relates to a cell preparation for treating a brain tumor, comprising pluripotent stem cells (Muse cells) into which a suicide gene has been introduced. The present invention is described in detail below.

1. Applicable Diseases The present invention aims at treatment of brain tumors, in particular, treatment of killing glioma cells or inhibiting their growth, using cell preparations containing pluripotent stem cells (Muse cells) into which a suicide gene has been introduced. Here, “brain tumor” refers to a state in which any type of nerve cell proliferates abnormally. Examples of brain tumors include, but are not limited to, glioma, medulloblastoma, neuroblastoma, meningioma, pituitary adenoma, schwannoma, primary central nervous system lymphoma, sarcoma, and spinal cord tumor Can be mentioned. The cell preparation of the present invention can target the above-mentioned various brain tumors, but it is preferable to treat gliomas that account for about 1/4 of the brain tumors as treatment targets.

2. Cell preparation (1) Pluripotent stem cells (Muse cells)
The pluripotent stem cell used in the cell preparation of the present invention was found by Dezawa, one of the present inventors, in the human body and named “Muse (Multilineage-differentiating Stress Ending) cell”. It is. Muse cells can be obtained from skin tissues such as bone marrow fluid and dermal connective tissue, and are also scattered in connective tissues of each organ. In addition, this cell is a cell having the properties of both pluripotent stem cells and mesenchymal stem cells. For example, the cell surface markers “SSEA-3 (Stage-specific embryonic antigen-3)” and “ Identified as "CD105" double positive. Accordingly, Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, these antigen markers as indicators. Details such as a method for separating Muse cells, an identification method, and characteristics are disclosed in International Publication No. WO2011 / 007900. Also, as reported by Wakao et al. (2011, supra), when mesenchymal cells are cultured from bone marrow, skin, etc. and used as the population of Muse cells, all SSEA-3 positive cells are CD105 It is known to be a positive cell. Accordingly, in the cell preparation of the present invention, when separating Muse cells from living mesenchymal tissue or cultured mesenchymal stem cells, the Muse cells can be purified and used simply with SSEA-3 as an antigen marker. . In the present specification, pluripotent stem cells isolated from living mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker, which can be used in cell preparations for treating brain tumors ( Muse cells) or a cell population containing Muse cells may be simply referred to as “SSEA-3 positive cells”. In the present specification, “non-Muse cells” refer to cells other than “SSEA-3-positive cells”, which are cells contained in a mesenchymal tissue or cultured mesenchymal tissue in a living body.

Briefly, Muse cells or cell populations containing Muse cells can be obtained from living tissue (eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively) , Mesenchymal tissue). Here, “living body” means a living body of a mammal. In the present invention, the living body does not include embryos whose developmental stage is earlier than the fertilized egg or blastocyst stage, but includes embryos in the developmental stage after the blastocyst stage including the fetus and blastocyst. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. It is done. Muse cells used in the cell preparation of the present invention are clearly distinguished from embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) in that they are derived from living tissues. “Mesenchymal tissue” refers to tissues existing in various organs such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, and umbilical cord. For example, Muse cells can be obtained from bone marrow or skin. For example, it is preferable to collect a mesenchymal tissue of a living body and separate and use Muse cells from this tissue. Moreover, you may isolate | separate a Muse cell from a cultured mesenchymal cell using the said isolation | separation means. In the cell preparation of the present invention, the Muse cell used may be autologous to the recipient who receives the cell transplant, or may be another family.

As described above, a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using, for example, SSEA-3 positive and SSEA-3 and CD105 double positive as an index. Are known to include various types of stem cells and progenitor cells. However, Muse cells are not the same as these cells. Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblast (MB), perivascular cells (PC), endothelial progenitor cells (EP), adipose-derived stem cells (ADSC). ). Muse cells can be isolated using “non-expression” of a marker unique to these cells as an index. More specifically, Muse cells are CD34 (EP and ADSC markers), CD117 (c-kit) (MB markers), CD146 (PC and ADSC markers), CD271 (NGFR) (NCSC markers), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and At least one of 11 markers selected from the group consisting of Dct (MB marker), for example 2, 3, 4, 5, 6, 7, 8, 9, 10 The non-expression of individual or eleven markers can be separated into indicators. For example, without limitation, non-expression of CD117 and CD146 can be separated as an index, and non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index, and The non-expression of 11 markers can be separated as an index.

Further, the Muse cell having the above characteristics used in the cell preparation of the present invention is as follows:
(I) low or no telomerase activity;
(Ii) has the ability to differentiate into cells of any germ layer of the three germ layers;
It may have at least one property selected from the group consisting of (iii) showing no neoplastic growth; and (iv) having a self-renewal capability. In one aspect of the present invention, the Muse cell used in the cell preparation of the present invention has all the above properties. Here, with regard to (i) above, “telomerase activity is low or absent” means that, for example, when telomerase activity is detected using TRAPEZE XL telomerase detection kit (Millipore), it is low or cannot be detected. Say. “Low” telomerase activity means, for example, telomerase having a telomerase activity comparable to that of somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less compared to Hela cells. It means having activity. Regarding the above (ii), the Muse cell has the ability to differentiate into three germ layers (endoderm, mesodermal, and ectoderm) in vitro and in vivo, for example, induction culture in vitro Can be differentiated into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. In addition, when transplanted to the testis in vivo, it may show the ability to differentiate into three germ layers. Furthermore, it has the ability to migrate, engraft and differentiate into organs (heart, skin, spinal cord, liver, muscle, etc.) that have been damaged by implantation into a living body by intravenous injection. As for (iii) above, Muse cells have the property of proliferating at a growth rate of about 1.3 days in suspension culture, but stop growing in about 10 days. Further, when transplanted to the testis, Muse cells have cancer for at least half a year. It has the property of not becoming Moreover, about said (iv), a Muse cell has self-renewal (self-replication) ability. Here, “self-renewal” refers to culturing cells contained in an embryoid body-like cell mass obtained by suspension culture of one Muse cell, and forming an embryoid body-like cell mass again. . The self-renewal may be repeated once or multiple times.

(2) Preparation and use of cell preparation The present invention provides a cell preparation for treating brain tumors, including Muse cells into which a suicide gene has been introduced, which is used together with a prodrug corresponding to the suicide gene. To do.
(A) Suicide gene As used herein, “suicide gene” means a gene that can kill itself when expressed in a cell, and typically includes a metabolic toxicity gene. Examples of suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (HSVtk) gene (Proc. Natl. Acad. Sci, USA, 78, 1441-1445 (1981)), cytosine deaminase gene (EG11326 codA 355395..356678, E. coli), uracil phosphoribosyltransferase gene (EG11332 upp 2618894..2618268, E. coli), guanine phosphoribosyltransferase (gpt) gene (EG10414 gpt 255597..256435, E. coli), nitroreductase (ntr) gene (EG11261 nfs7.40 891129, E. coli).
(B) Prodrug The cell preparation of the present invention can be used together with a prodrug to treat a brain tumor. Here, the “prodrug” used together with the cell preparation of the present invention is a prodrug of a drug that kills the target tumor cell or inhibits its growth, and itself exhibits such cytotoxicity. It means a drug that you do not have. This prodrug is converted into a drug having pharmacological activity (cytotoxicity) by an enzyme generated by expression of a suicide gene. Examples of the prodrug include ganciclovir (GCV), acyclovir, pencyclovir, PMEA adefovir, PMPA tenofovir, etc., preferably GCV, acyclovir, and pencyclovir when the suicide gene is herpes simplex virus thymidine kinase (HSVtk) gene. it can. These are all analogs of guanine in the purine form of nucleic acid, and when used for DNA synthesis, DNA synthesis stops there and exerts an antiviral effect. When the suicide gene is a cytosine deaminase gene, 5-fluorocytosine can be used as the prodrug (Human Gene Therapy, 7, 713-720 (1996)). When the suicide gene is a uracil phosphoribosyltransferase gene, 5-fluorouracil can be used as a prodrug (Int. J. Oncol, 18, 117-120 (2001)). When the suicide gene is a guanine phosphoribosyltransferase gene, 6-thioxanthine or 6-thioguanine can be used as the prodrug (Human Gene Therapy, 8, 2043-2055 (1997)). When the suicide gene is a nitroreductase gene, CB1954 can be used as a prodrug (Cancer Gene Therapy, 7, 721-731 (2000)).

Also, the reaction mechanism of the enzyme and prodrug produced by the expression of the suicide gene will be described below using, for example, the HSVtk (herpes simplex virus thymidine kinase) gene and GCV (ganciclovir). For example, the HSVtk gene is introduced into cancer cells and expressed, and GCV, which is an antiviral agent, is administered as a prodrug. Cancer cells into which the HSVtk gene has been introduced phosphorylate GCV with HSVtk to form a monophosphorylated GCV (GCV-1P). GCV-1P is then phosphorylated to triphosphate by its own thymidine kinase, and in order to inhibit DNA polymerase, cells that undergo DNA replication fall into apoptosis and die. Furthermore, GCV-1P is also taken into neighboring cells through a gap junction, and neighboring cells without gene transfer are also killed due to inhibition of DNA synthesis (bystander effect).

(C) Introduction of suicide gene into Muse cell and preparation of cell preparation In the cell preparation of the present invention, a Muse cell into which a suicide gene has been introduced is used. As a method of introducing the suicide gene into Muse cells, a method of introducing a vector incorporating the gene is common. Gene transfer by use of a vector includes, but is not limited to, viral or non-viral gene transfer (eg, plasmid transfer, phage integrase, transposon, adenovirus, adeno-associated virus, and lentivirus). More specifically, when the herpes simplex virus thymidine kinase (HSVtk) gene is used as the suicide gene, the virus is infected to the cells with the supernatant of the mouse-derived HSVtk-retrovirus producing cell (PA317) culture. Can be introduced. When the cytosine deaminase gene is used, it is incorporated into an adenovirus vector with cytomegalovirus early gene enhancer / promoter added to the cytosine deaminase gene cDNA of E. coli (Human Gene Therapy, 6, 1055-1063 (1995)). Can be introduced. When using a uracil phosphoribosyltransferase (UPRT) gene, the gene can be introduced using a retroviral vector LXSN incorporating the UPRT gene of E. coli. When the guanine phosphoribosyltransferase (gpt) gene is used, the gene can be introduced by infecting the cells with the virus using the supernatant of the E. coli gpt-retrovirus producing cell (GP / E86 gpt) culture. When using a nitroreductase (ntr) gene, a plasmid in which the cytomegalovirus early gene enhancer / promoter is added to the Escherichia coli ntr gene can be prepared, and the gene can be introduced into the cell by electroporation. In order to secure a predetermined number of cells before gene introduction, the cells may be appropriately proliferated.

The cell preparation of the present invention can be obtained by suspending Muse cells after introduction of a suicide gene in physiological saline or an appropriate buffer (for example, phosphate buffered saline). In this case, when the number of Muse cells isolated from the tissue of the home or other family is small, the cell preparation may be cultured before being administered to the treatment subject and grown until a predetermined cell concentration is obtained. . In addition, as already reported (International Publication No. WO2011 / 007900), Muse cells do not become tumors. Therefore, even if cells collected from living tissue remain undifferentiated, there is a possibility of canceration. Low and safe. The culture of the collected Muse cells is not particularly limited, but can be performed in a normal growth medium (for example, α-minimal essential medium (α-MEM) containing 10% calf serum). More specifically, referring to the above International Publication No. WO2011 / 007900, in the culture and proliferation of Muse cells, a medium, additives (for example, antibiotics, serum) and the like are appropriately selected, and Muse cells at a predetermined concentration are selected. A solution containing can be prepared.

(D) Use of cell preparations When using Muse cells in cell preparations, dimethyl sulfoxide (DMSO) or serum albumin is used to protect the cells, and antibiotics are used to prevent bacterial contamination and growth. You may make it contain in a cell formulation. Furthermore, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) Cells or components other than Muse cells contained in mesenchymal stem cells may be contained in the cell preparation. One skilled in the art can add these factors and agents to the cell preparation at appropriate concentrations.

The number of Muse cells into which the suicide gene contained in the cell preparation prepared above is introduced depends on the sex of the subject so that a desired effect (eg, disappearance of tumor, reduction of tumor size) can be obtained in the treatment of brain tumors. In consideration of the age, weight, state of the affected area, state of cells to be used, etc., it can be appropriately adjusted. In addition, the cell preparation of the present invention can be used a plurality of times (for example, 2 to 10 times) at appropriate intervals (for example, twice a day, once a day, once a week) until a desired therapeutic effect is obtained. 2 times, once a week, once every two weeks). Therefore, although depending on the condition of the subject, the therapeutically effective dose is preferably, for example, 1 × 10 ³ cells to 1 × 10 ⁶ cells per individual and 1 to 10 doses. The total dose in one individual includes, but is not limited to, 1 × 10 ³ cells to 1 × 10 ⁷ , 1 × 10 ⁴ cells to 5 × 10 ⁶ cells, and the like.

As described above, treatment of brain tumors with the cell preparation of the present invention is based on the expression of a suicide gene introduced into Muse cells, and the subsequent activation of the prodrug by the enzyme that is the gene product. Therefore, in the use of the cell preparation of the present invention, the administration site and administration method (local tumor administration, intracarotid artery administration, intravenous administration) of the cell preparation are not limited. The prodrug used together with the cell preparation of the present invention may be intravenous administration as described above, or may be intraperitoneal administration. For example, according to the present invention, but not limited to, by using the migration ability of Muse cells (accumulated brain around the tumor), for example, to a subject having a brain tumor, after the intravenous administration of the cell preparation, a prodrug Can be administered intravenously or intraperitoneally to obtain the desired therapeutic effect. The administration time of the prodrug is not limited, but may be any time after administration of the cell preparation of the present invention, at the same time as administration, and before administration as long as it has the above effect. In one aspect of the present invention, there is provided a pharmaceutical composition comprising a Muse cell into which a suicide gene has been introduced, a prodrug, and optionally a carrier or excipient.

The following examples further illustrate the present invention, but the present invention is not limited to these examples.

Example 1: Preparation of Muse cells into which herpes simplex virus thymidine kinase (HSVtk) gene was introduced (1) Preparation of human Muse cells Human Muse cells were prepared according to the method described in International Publication No. WO2011 / 007900. . More specifically, mesenchymal cells having adhesiveness were cultured from human bone marrow fluid, and after proliferation, Muse cells or a cell population containing Muse cells were separated as SSEA-3 positive cells by FACS. Non-Muse cells are a group of SSEA-3 negative cells among the above mesenchymal cells and used as controls. Then, it adjusted to the predetermined density | concentration using the phosphate buffered saline or a culture solution, and used for the evaluation of the following bystander effects and cell migration ability. When cells obtained by culturing mesenchymal cells such as bone marrow mesenchymal cells are used as the population of Muse cells, SSEA-3-positive cells are reported as reported by Wakao et al. (2011, supra). Are all known to be CD105 positive cells.
(2) HSVtk gene introduction HSVtk retrovirus-producing cells (PA317, mouse fibroblasts, Genetic Therapy Inc.) (provided by Gaithersburg, MD) were cultured in MSC medium for 48 hours, and then HSVtk retrovirus was transferred from the supernatant. Obtained. This was added to Muse cells in culture together with 8 μg / ml polybrene (Aldrich Chemical Company Inc., Milwaukee, Wis.), Further cultured for 5 hours, and then replaced with fresh medium after washing. By culturing with 150 μg / ml G418 (Sigma-Aldrich Japan KK, Tokyo, Japan) for 1 week and selecting drug-resistant cells, only transgenic cells were obtained. Among these cell lines, a ganciclovir GCV-sensitive line was selected and further expanded to obtain a sufficient amount of HSVtk gene-introduced cells (Muse-TK cells).

Example 2: Detection of Muse cells around glioblastoma (glioblastoma) It was examined whether or not Muse cells were present in human glioblastoma collected from human subjects. A tissue section of the collected glioblastoma was prepared and immunostained using an antibody against the cell surface antigen (SSEA-3 ) of Muse cells according to a conventional method. As shown in FIG. 1, it was found that Muse cells exist around glioblastoma.

Example 3: Confirmation of bystander effect in human glioma cells by Muse-TK cells To confirm the bystander effect by Muse-TK prepared in Example 1, co-culture of Muse-TK cells and glioma cells in vitro Was examined. 96-well human glioma cells (A172) 1.5 × 10 ⁴ cells / well, Muse-TK cells (1/1), 1/4, 1/8, 1/16, 1/32 cells The cells were mixed as a number, cultured in the presence of 2 μg / ml ganciclovir (GCV) for a predetermined number of days, observed with a phase contrast microscope, and imaged. As shown in FIG. 2, on the ninth day after the addition of GCV, the death of glioma cells was observed in the culture systems in which the ratio of Muse-TK cells to glioma cells was 1: 1, 1: 4, and 1: 8. It was done. Further, FIG. 3 shows the results of actually counting glioma cells in culture on the 5th and 9th days after GCV addition based on the results of FIG. As can be seen from this result, cell proliferation of glioma cells was reduced to a cell ratio of 1: 1 to 1: 8. Thus, a bystander effect on glioma cells was observed in Muse-TK cells.

Example 4: Confirmation of migration ability to glioma cells by Muse-TK cells As shown in Example 2, Muse cells have the property of accumulating around tumors. Thus, the in vitro migration ability of Muse cells was examined using culture supernatants of glioma cells (A172 and YKG1). Muse cell migration was quantitatively measured using the Boyden chamber method (Boyden, S., J. Exp. Med., Vol. 115, p. 453-466 (1962)). The Boyden chamber used was a QCM Chemotaxis Cell Migration Assay Kit (QCM 24 Well Colorimetric Cell Migration Assay) commercially available from Millipore. The Boyden chamber includes an insert having a filter having uniform fine pores of 8 μm at the bottom inside the chamber. The culture solution containing Muse cells or non-Muse cells was added to the upper part of the filter of the insert, the culture supernatant of glioma cells was added to the lower part of the insert, and after culturing for 18 hours, the number of cells that passed through the micropores of the filter was counted. . The result is shown in FIG. "Muse 　 A172 "and" Muse " 　 In the “YKG-1” system, Muse cells passed through the micropores and moved significantly to the lower part of the insert. On the other hand, when the culture supernatant of glioma cells was used for non-Muse cells, and when DMEM was used instead of this culture supernatant, the ability to migrate Muse cells was not exhibited. In this way, it is suggested that some factor that activates the migration ability of Muse cells is present in the culture supernatant of glioma cells, and this factor promotes the accumulation of Muse cells in brain tumors in vivo. It is thought that there is.

Example 5: Verification of tumor formation inhibitory effect in vivo The bystander effect by Muse-TK prepared in Example 1 was confirmed in vivo. U87-luc2 cells (glioma cell line) into which Muse-TK cells and luciferase gene were introduced were co-transplanted into the right brain of nude mice (male 8 weeks old). More specifically, the numbers of Muse-TK cells and U87-luc2 cells to be transplanted were 2.5 × 10 ⁴ and 10 × 10 ⁴ (1: 4), respectively. After transplanting the cells into the brain (day 0), the next day (day 1), ganciclovir (GCV) (Wako) is diluted with PBS to 5 mg / ml, and 1 mg / 200 μl PBS once per mouse. It was administered intraperitoneally twice a day for 10 days. As a control, a group administered with PBS instead of GCV and a non-transplanted group of Muse-TK cells (U87-luc2 cells only) were used. On the 1st day after cell transplantation and every 7th day after transplantation, the process of tumor formation (increase or decrease) was observed with Bioluminescence imaging (BLI) using IVIS200. The result is shown in FIG. Tumor formation was observed up to day 35, but in the control group PBS administration group (2nd and 4th stage) and the non-transplanted group of Muse-TK cells (3rd stage), brain tumors were observed over time. An increase was seen. On the other hand, in the Muse-TK cell + GCV group (first stage), the formation of brain tumor was suppressed from the 7th day after transplantation, and the brain tumor almost disappeared on the 35th day. When the fluorescence intensity by luciferase was measured over time for each of the four groups tested, a marked decrease in fluorescence intensity was observed in the Muse-TK cell + GCV group as described above (data not shown). Furthermore, a Kaplan-Meier curve was prepared to examine the survival rate of the mice. As a result, on the 54th day after transplantation, in the Muse-TK cell + GCV group, all the mice used in the experiment were alive, but in the other 3 groups, all the mice were observed on the 31st to 37th days. The tumor died (data not shown). From these results, a bystander effect on glioma cells by Muse-TK cells was confirmed also in vivo.

When the cell preparation of the present invention is administered to a brain tumor subject, Muse cells into which the suicide gene has been introduced accumulate around the brain tumor, and further, the cells constituting the brain tumor are killed by administration of a prodrug corresponding to the suicide gene. Can be applied to the treatment of brain tumors.

All publications and patent literature cited herein are hereby incorporated by reference in their entirety. While specific embodiments of the invention have been described herein for purposes of illustration, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims

A cell preparation for treating a brain tumor comprising pluripotent stem cells introduced with a suicide gene, wherein the pluripotent stem cells are isolated from mesenchymal tissue or cultured mesenchymal cells in a living body SSEA-3-positive cells, wherein the cell preparation is used with a prodrug of a drug that kills or inhibits the growth of a brain tumor, and the prodrug is a cell that is a substrate for an enzyme produced by expression of the suicide gene Formulation.
The cell preparation according to claim 1, wherein glioma cells constituting a brain tumor are treated.
The cell preparation according to claim 1 or 2, wherein the pluripotent stem cells are CD105 positive.
The cell preparation according to any one of claims 1 to 3, wherein the pluripotent stem cells are CD117 negative and CD146 negative.
The cell preparation according to any one of claims 1 to 4, wherein the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative, and CD271 negative.
The pluripotent stem cell is CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative. The cell preparation according to claim 1.
The cell preparation according to any one of claims 1 to 6, wherein the pluripotent stem cell is a pluripotent stem cell having all of the following properties:
(I) low or no telomerase activity;
(Ii) has the ability to differentiate into cells of any germ layer of the three germ layers;
(Iii) no neoplastic growth; and (iv) self-renewal ability.
The cell preparation according to any one of claims 1 to 7, wherein the pluripotent stem cells have an ability to accumulate at a brain tumor site.
9. The suicide gene according to claim 1, wherein the herpes simplex virus thymidine kinase (HSVtk) gene, cytosine deaminase gene, uracil phosphoribosyltransferase gene, guanine phosphoribosyltransferase (gpt) gene, or nitroreductase gene. The cell preparation described in 1.
The prodrug is ganciclovir (GCV), acyclovir, pencyclovir, PMEA adefovir or PMPA tenofovir when the suicide gene is an HSVtk gene, and 5-fluorocytosine when the suicide gene is a cytosine deaminase gene. The uracil phosphoribosyltransferase gene is 5-fluorouracil, the suicide gene is 6-thioxanthine or 6-thioguanine when the suicide gene is gpt gene, and the prodrug is CB1954 when the suicide gene is nitroreductase (ntr) gene The cell preparation according to any one of claims 1 to 8, wherein