Drug-dependent immortalized cell capable of inducing cell death and method for producing same

Abstract

A purpose of the present invention is to provide an immortalized stem cell that can be maintained in a body for a long period of time and can be removed when a desired therapeutic effect is obtained. Another purpose of the present invention is to provide a method for producing the stem cell.　Provided is a stem cell into which a gene for Caspase-9 that is activated in a drug-dependent manner has been introduced. In the present invention, the stem cell is a mesenchymal stem cell, and is preferably an immortalized stem cell. The immortalized stem cell preferably contains at least one gene selected from the group consisting of genes for TERT, Bmi-1, human papillomavirus E6, and human papillomavirus E7. The drug preferably promotes dimerization of Caspase-9.

Description

Drug-dependent cell death inducible immortalized cells and method for producing same

[0001] The present invention relates to immortalized cells that can be induced to undergo cell death by controlling the expression of caspase-9, and a method for producing the same. More specifically, the present invention relates to immortalized stem cells into which a gene encoding drug-dependently activated caspase-9 has been introduced, and a method for producing the same.

[0002] Organs can be damaged by disease, trauma, etc. In some cases, the damage is sufficient to heal naturally, while in other cases it exceeds the natural healing capacity. If the damage exceeds the natural healing capacity, it is desirable to restore it if possible.

[0003] Methods for restoring such damaged organs can be broadly divided into transplant medicine and regenerative medicine. Transplant medicine involves receiving organs from donors and transplanting them to restore organ function.

[0004] In contrast, regenerative medicine involves culturing cells or tissues from several individuals, including the patient, and using these to repair or regenerate lost tissues or organs. Regenerative medicine using stem cells is currently being developed. Stem cells are defined as undifferentiated cells that possess pluripotency and the ability to self-renew. Pluripotency refers to the ability to differentiate into multiple cells with different functions, and self-renewal refers to the ability to continue self-proliferation.

[0005] Stem cells are divided into totipotent stem cells and pluripotent stem cells based on their differentiation potential. Totipotent stem cells are cells that have the ability to become all the cells that make up the body from a single cell (totipotency) and are capable of individual development, while pluripotent stem cells are defined as cells that cannot undergo individual development on their own. Stem cells are also broadly divided into totipotent stem cells and tissue stem cells. Cells present in the body basically have a finite lifespan, and it is known that stem cells supply new cells to replace old cells.

[0006] Here, tissue stem cells are cells that exist in the body under specific physiological conditions and are committed to differentiate into specific tissues or organs, serving as a source of cells for maintaining homeostasis. Examples include mesenchymal stem cells, which differentiate into muscle cells, bone cells, and cartilage; neural stem cells, which differentiate into neurons; and hematopoietic stem cells, which differentiate into platelets, white blood cells, and red blood cells. In other words, tissue stem cells have the ability to differentiate along a defined lineage, but lack the ability to differentiate undefined. Furthermore, tissue stem cells are characterized by their highly potent division potential, yet most of them are in a state of cell cycle arrest (G0 phase), meaning that stem cells present in normal tissues do not proliferate uncontrollably. This quiescent state is also a characteristic of tissue stem cells.

[0007] Stem cells can also be divided into somatic stem cells and embryonic stem cells (hereinafter sometimes referred to as "ES cells"). Here, somatic stem cells are stem cells specific to each organ, and include hematopoietic stem cells and neural stem cells, which differentiate along a defined lineage. ES cells are pluripotent stem cells derived from early embryos (Non-Patent Document 1).

[0008] Currently, three types of stem cells are known to be actually or potentially applicable to regenerative medicine: human somatic stem cells, human embryonic stem cells (ES cells), and human induced pluripotent stem (iPS) cells. Human somatic stem cells, which have already been used for research purposes, are cells present in adult tissues, as mentioned above. Transplantation using one's own cells (hereinafter referred to as "autologous cells") (hereinafter referred to as "autologous transplantation") is known to be free of immune rejection and to have good engraftment. Furthermore, there have been no reports of tumorigenesis in cultured cells over long periods of time. However, it is known that the extraction of somatic stem cells from human tissue involves invasive procedures, and the number of subcultures of the resulting somatic stem cells is limited to just over 40 times, or 100-200 days (Non-Patent Document 2).

[0009] Human embryonic stem cells (ES cells) are stem cells obtained by extracting and culturing the "inner cell mass" from a blastocyst approximately five days after fertilization. Because they form teratomas, an indicator of pluripotency, they are believed to be capable of differentiating into any of the three germ layers, and there have been reports of them differentiating into cardiac muscle, nerves, and retina. Because ES cells have a high self-renewal capacity, a single cell line can be cultured indefinitely, and it is known that, under appropriate culture conditions, it is possible to mass-produce homogeneous cellular products.

[0010] On the other hand, because ES cell production requires the use of fertilized eggs, strict ethical considerations must be taken into account when using ES cells for transplantation. Furthermore, since autologous cells cannot usually be used as ES cells, allogeneic transplantation is generally performed using cells derived from another person (hereinafter referred to as "allogeneic cells"). Therefore, it is necessary to address the risk of rejection due to immune responses. Furthermore, heterologous cells and serum must be used when culturing ES cells. Furthermore, it is known that even the presence of even a small amount of undifferentiated cells in the regenerated tissue after transplantation is likely to result in the formation of teratomas, i.e., the possibility of cancer (Non-Patent Document 3).

[0011] Human induced pluripotent stem (iPS) cells are artificial stem cells established by introducing a portion of a gene specifically expressed in ES cells into differentiated adult cells (e.g., epithelial cells). Therefore, for example, cells can be obtained from one's own skin and used to create iPS cells, and ES cell differentiation techniques can be applied directly. Furthermore, because iPS cells do not require the use of fertilized eggs as ES cells do, the ethical hurdles for resolving them are relatively low.

[0012] The advent of iPS cells has made it possible to generate cells with nearly identical properties to embryonic stem cells using adult tissues, and has also made it possible to autologously transplant stem cells generated from autologous cells, as described above. This has made it possible to avoid the immune rejection problem that plagues allogeneic transplantation. However, it has been known that the percentage of cells established as transfected iPS cells is low, and that they are prone to form not only benign tumors but also malignant tumors (germ cell carcinoma) (Non-Patent Document 4).

[0013] In recent years, mesenchymal stem cells (MSCs), which are pluripotent stem cells, have attracted attention as a source of stem cells for use in regenerative medicine. As mentioned above, mesenchymal stem cells are somatic stem cells, and it has been reported that they can be used for the regeneration of various organs and the treatment of diseases (see Non-Patent Documents 2 and 3).

[0014] WO 2013 / 147082

[0015] 27 / 29 cycles and 81 cycles of 88 cycles Han, Y.; Li, X.; Zhang, Y.; Han, Y.; Chang, F.; Ding, J. Mesenchymal Stem Cells for Regenerative Medicine. Cells 2019, 8, 886. http: / / doi.org / 10.3390 / cells8080886Huang, Y.; Wu, Q.; Tam, PKH Immunomodulatory Mechanisms of Mesenchymal Stem Cells and Their Potential Clinical Applications. Int. J. Mol. Sci. https: / / doi.org / 10.3390 / ijms231710023Katahira et al. Protective effects of conditioned media of immortalized stem cells from human exfoliated deciduous teeth on pressure ulcer formation. Front Immunol. 13, 1010700, 2023, https: / / doi.org / 10.3389 / fimmu.2022.1010700Carlos Almeida Ramos, Zahra Asgari, Enli Liu, Eric Yvon, Helen E. Heslop, Clio M. Rooney, Malcolm K. Brenner, Gianpietro Dotti, An Inducible Caspase 9 Suicide Gene to Improve the Safety of Mesenchymal Stromal Cell Therapies, Stem Cells, Volume 28, Issue 6, June 2010, Pages 1107-1115;Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy. Cancer Gene Ther 26, 11-16 (2019). https: / / doi.org / 10.1038 / s41417-018-0034-1DeRose, R., Miyamoto, T. & Inoue, T. Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pflugers Arch - Eur J Physiol 465, 409-417 (2013). https: / / doi.org / 10.1007 / s00424-012-1208-6Voβ, Stephanie; Klewer, Laura; Wu, Yao-Wen (2015). Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells. Current Opinion in Chemical Biology, Volume 28, 194-201. doi:10.1016 / j.cbpa.2015.09.003Sakai et al. Clinical development of regenerative medicine targeted for intervertebral disc disease. Medicina 58, 267, 2022.

[0016] Stem cell-based treatment techniques are useful in that transplanting stem cells into a patient's body can regenerate tissues and repair wounds. However, normal cells have a finite number of divisions and then age, and stem cells also share this property. As a result, stem cells deteriorate or age after transplantation, leading to problems such as poor engraftment at the transplant site and inability to fully function due to aging. For this reason, techniques have been developed to introduce immortalization genes into mesenchymal stem cells, thereby suppressing aging and allowing mesenchymal stem cells to survive for long periods of time (Patent Document 1 and Non-Patent Document 3, hereinafter referred to as "Prior Art 1" and "Prior Art 2").

[0017] Prior arts 1 and 2 are excellent in that they enable long-term passage without changing the properties of cells. Meanwhile, immortalized stem cells are cells whose lifespan, which is inherently finite, has been extended using special techniques. On the other hand, the property of continuing to proliferate indefinitely is a characteristic of cancer cells. For this reason, there has been a strong social demand for a technology that can eliminate the possibility of immortalized stem cells becoming cancerous.

[0018] Therefore, in recent years, cell therapy has been performed, in which cells themselves are used as therapeutic materials. Here, "cell therapy" is defined as a treatment method for treating a disease using cells from one's own body or another person. Examples include red blood cell transfusion and hematopoietic stem cell transplantation. Here, red blood cell transfusion is a technique in which red blood cells (cells) obtained from a healthy person (another person) are administered to increase the supply of oxygen carried by the red blood cells. Meanwhile, hematopoietic stem cell transplantation is a technique in which blood containing hematopoietic stem cells is administered, and the hematopoietic stem cells are differentiated into white blood cells, red blood cells, platelets, etc., thereby treating the disease in the process.

[0019] Furthermore, a technique has been reported that can induce apoptosis by introducing only a specific enzyme gene into T lymphocytes (see Non-Patent Document 5). However, it has not been reported that when such a technique is used in regenerative medicine, etc., it can be used to survive in the body for a certain period of time without becoming cancerous and then be killed at a predetermined time point.

[0020] Therefore, there has been a strong social demand for transplant cells that can be maintained in the body for a long period of time and can be removed once the desired therapeutic effect has been achieved, as well as a method for producing the same.

[0021] The present invention was made under the above circumstances. Specifically, a first aspect of the present invention relates to stem cells into which a gene encoding caspase-9 that is activated in a drug-dependent manner has been introduced. Here, the drug for activating caspase-9 is preferably AP20187 or AP1903. Furthermore, the stem cells are preferably mesenchymal stem cells, and more preferably, the mesenchymal stem cells are immortalized stem cells.

[0022] The immortalized stem cells are preferably those into which at least one gene selected from the group consisting of TERT, Bmi-1, human papillomavirus E6, and human papillomavirus E7 has been introduced, and the caspase-9 gene is preferably the gene set forth in SEQ ID NO: 1.

[0023] A second aspect of the present invention is a method for producing immortalized stem cells transfected with the Caspase-9 gene, comprising: a step of producing a DNA fragment containing a drug-dependently activated Caspase-9 gene and multiple genes for immortalizing cells; a vector production step of producing a viral vector incorporating the DNA fragment; a transfected stem cell production step of transfecting mammalian stem cells with the viral vector to introduce the gene into the stem cells and produce transfected cells; and a selection step of selecting transfected stem cells from the stem cells obtained in the transfection step.

[0024] Here, the mammalian stem cells are preferably mesenchymal stem cells, and more preferably human dental pulp stem cells. The DNA fragment preferably contains, in addition to the caspase-9 gene, any one of a set of genes selected from the group consisting of (a) to (d) below: (a) human papillomavirus E7 and TERT; (b) Bmi-1 and TERT; (c) Bmi-1, human papillomavirus E6, and TERT; and (d) Bmi-1, human papillomavirus E6, human papillomavirus E7, and TERT.

[0025] The introduction of the Caspase-9 gene is preferably carried out using a virus, which is preferably any one selected from the group consisting of lentivirus, adenovirus, and retrovirus, and the mammalian stem stem cells are preferably any one selected from the group consisting of human dental pulp stem cells, porcine dental pulp stem cells, and porcine adipose stem cells.

[0026] According to the present invention, stem cells incorporating the caspase-9 gene and a specific gene can be produced, which can be used for transplantation or regenerative medicine. The immortalized stem cells incorporating the caspase-9 gene obtained here can be administered with a drug at a desired timing to express caspase-9, thereby inducing cell death in the immortalized cells. Furthermore, according to the present invention, immortalized stem cells incorporating the caspase-9 gene and a specific gene can be efficiently produced.

[0027] Figure 1 shows an experiment to examine the drug activity of iCas9 using iCas9-GFP-transfected SHEDs. (A) A schematic diagram of the experiment. (B) The state of iCas9-GFP-transfected SHEDs after addition of AP20187 at concentrations of 0, 10, 100, or 1000 nM. Figure 2 shows the results of FACS analysis of iCas9-GFP-transfected SHEDs 24 hours after addition of AP20187, showing forward scatter (FSC) and side scatter (SSC) in FACS.

[0028] Figure 3 shows the results of FACS analysis of iCas9-GFP-introduced SHED 24 hours after the addition of AP20187. The upper histogram shows the number of 7ADD-positive cells, and the lower histogram shows the number of GFP-positive cells. Figure 4 shows the results of FACS analysis of iCas9-GFP-introduced SHED 24 hours after the addition of AP20187. (A) shows the percentage of GFP-expressing cells (remaining iCas9-GFP-introduced SHED). (B) shows the percentage of 7ADD-negative live cells among all cells.

[0029] The present invention is described in more detail below. The stem cells of the present invention are obtained by introducing the gene for Caspase-9 (hereinafter sometimes referred to as "iCas9"), which can induce drug-dependent activation, into any stem cell. The stem cells are not particularly limited as long as they are somatic stem cells.

[0030] Examples of the drug include AP20187 and AP1903. AP20187 is a cell membrane-permeable low-molecular-weight compound known as a B / B homodimerizer, which induces dimerization of fusion proteins containing AP20187-binding domains, such as the FK506-binding protein 12 (FKBP) domain. To prevent the FKBP domain from reacting with endogenous ligands, it is preferable to fuse an FKBP mutant (e.g., a mutant with the F36V mutation) to the target protein. AP20187 is nontoxic in vitro and in mice. The recommended dosage is 0.01-100 nM in vitro and 0.005-10 mg / kg in mice. The LNCaP cells used in the in vitro evaluation are an androgen-sensitive human prostate adenocarcinoma cell line derived from left supraclavicular lymph node metastasis in a 50-year-old Caucasian man in 1977, and are a commonly used cell line in the field of oncology.

[0031] AP1903 is a dimerizing agent that acts by cross-linking FKBP domains, and it can be used to rapidly induce apoptosis by dimerizing FKBP-containing caspase-9. FKBP is not only important for its biological function, but is also widely used as a protein domain to initiate artificial molecular interactions.

[0032] Caspases are enzymes that selectively cleave substrates, inducing protein maturation, activation, or inactivation. Over 1,000 substrates have been identified. Caspases are unique cysteine ​​proteases that require an aspartic acid residue at the P1 cleavage site of their substrates, hence the name caspase. Their substrates include ICAD (inhibitor of caspase-activated DNase), which promotes nuclear condensation and DNA fragmentation, characteristic of apoptosis; the scramblase Xkr8, which is involved in the exposure of phosphatidylserine (PS) on the apoptotic cell membrane; the flippase ATP11C, which controls PS asymmetry; and the kinase ROCK-1, which is involved in the cell morphological changes characteristic of apoptosis.

[0033] Caspases are a group of intracellular proteases that share homology with the gene encoding interleukin 1β-converting enzyme (ICE), which matures and secretes interleukin 1 (IL-1)β, cloned from human monocytes, and the gene encoding ced-3, which executes all cell death in C. elegans.

[0034] Caspases form a family of proteases that play a central role in cell death, inflammation, and many other processes. Cysteine ​​aspartate-specific proteases, caspases, and other enzymes are all synthesized in an inactive form called procaspase, which undergoes dimerization / oligomerization followed by cleavage and activation. Activation of caspases is followed by the morphological changes and molecular movements characteristic of apoptosis.

[0035] Specifically, during apoptosis, a group of caspases, caspase-2, -8, -9, -10, -11, and -12, activates another group of downstream effector caspases, caspase-3, -6, and -7, which cleave other proteins to complete the apoptotic program. Caspases-1, -4, -5, -11, and -12 are involved in inflammation. Caspase-14 is not involved in apoptosis or inflammation, but it plays an important role in skin cell development.

[0036] As mentioned above, caspase-9 is one of the proteins involved in the caspase cascade that induces apoptosis. Because caspase-9 activates the apoptosis effector factors caspase 3, 6, and 7, artificial induction of activated caspase-9 means artificial induction of apoptosis.

[0037] Dimerization is required for caspase-9 activation. When using caspase-9 in transplantation or regenerative medicine, it is preferable that it be derived from the same animal species as the target animal. Furthermore, to prevent spontaneous activation of caspase-9 in vivo, it is preferable to insert a domain that can induce dimerization in a drug-dependent manner (drug-dependent dimerization inducing domain, hereinafter referred to as "DDID") in place of the endogenous caspase activation and recruit domain (hereinafter referred to as "CARD").

[0038] Many types of DDIDs are known (see Non-Patent Documents 6 and 7), and an appropriate one can be selected depending on the application. iCas9 can be created by replacing the CARD in human caspase-9 with human FK506 binding protein (hereinafter referred to as "FKBP") and adding an SGGGS linker. The CARD domain is a module consisting of 90-100 amino acids and is involved in apoptotic signal transduction. The association of an adaptor protein, a procaspase, and their respective CARDs forms an adaptor / procaspase heterodimer.

[0039] Because FKBP forms dimers in a small molecule-dependent manner, iCas9 dimerization is induced by small molecules that bind to FKBP. Known examples of such small molecules include AP1903 and AP20187. The iCas9 gene can be, for example, the gene set forth in SEQ ID NO: 1.

[0040] The stem cells of the present invention can be produced by introducing the iCas9 gene into stem cells. Known gene introduction methods include electroporation, drug-dependent methods, and methods using viral vectors. To ensure stable long-term expression of the introduced gene, it is preferable to create a viral vector for gene introduction and use it for the introduction. Examples of viruses used to produce such viral vectors include retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, Sindbis viruses, herpes simplex viruses, rabies viruses, and Sendai viruses.

[0041] The method for producing stem cells of the present invention is described below. Here, we will explain the method using stem cells derived from human deciduous teeth (SHED), but the stem cells of the present invention are not limited to SHED.

[0042] To obtain the immortalized stem cells of the present invention, stem cells are first isolated from mesenchymal cells of a mammal, preferably selected from the group consisting of humans, pigs, horses, and monkeys, due to their high genetic similarity to human cells and low risk of infection.

[0043] As used herein, "mesenchymal cells" refer to cells that are believed to have the ability to differentiate into cells belonging to the mesenchymal system, such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Specific examples of mesenchymal cells include dental pulp cells, bone marrow cells, umbilical cord cells, and adipocytes of the above-mentioned animals. Furthermore, "dental pulp cells" refer to a type of stem cell contained in the nerves of teeth that has the ability to regenerate. Protected by the hard material of teeth, they have the properties of being impermeable to ultraviolet rays and radiation, and of being resistant to genetic damage.

[0044] The above gene set consists of bmi-1, HPV-E6, HPV-E7, and TERT. hTERT is a gene encoding a telomere repair enzyme, and bmi-1 is a gene encoding Bmi-1, a protein that constitutes the polycomb complex. Bmi-1 is necessary for the maintenance of hematopoietic stem cells and has the effect of increasing hematopoietic stem cells by enhancing their activity. E6 and E7 are early genes of HPV-16 or HPV-18 DNA.

[0045] The following describes the preparation of immortalized stem cells from dental pulp cells from exfoliated human deciduous teeth. First, the exfoliated deciduous teeth are disinfected with disinfectants such as chlorhexidine, isodine solution, or other disinfectants, and then the crown is divided and the dental pulp tissue is collected using a dental reamer.

[0046] The collected dental pulp tissue is suspended in a basal medium, such as Dulbecco's Modified Eagle's Medium (DMEM), containing 5-15% calf serum (CS) and 50-150 units / mL of antibiotics, and then treated with 1-5 mg / mL of collagenase and 1-5 mg / mL of dispase at 37°C for 0.5-2 hours.

[0047] As the basal medium, in addition to DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), RPMI1640 medium, etc. can be used. A mixed medium containing two or more types of basal medium may also be used. An example of a mixed medium is a medium containing equal amounts of IMDM and HamF12 (for example, product name: IMDM / HamF12 (GIBCO)). The above basal medium can also be used for culturing for cell selection, as described below, and for culturing the cells after selection.

[0048] In addition, substances that can be added to the basal medium include fetal calf serum (hereinafter referred to as "FCS"), human serum, sheep serum, and other sera, serum substitutes (such as knockout serum replacement (KSR)), bovine serum albumin (hereinafter sometimes referred to as "BSA"), penicillin, streptomycin, and other antibiotics, various vitamins, and various minerals.

[0049] After enzyme treatment, the dental pulp stem cells are centrifuged (3,000-7,000 rpm) for 3-10 minutes to recover the dental pulp cells. If necessary, the cells are sorted using a cell strainer. The sorted cells are resuspended in, for example, 3-6 mL of the above-mentioned basal medium and seeded onto a 4-8 cm diameter dish for adherent cell culture.

[0050] Next, a culture medium, such as DMEM containing 10% FCS, is added, and the cells are cultured at 37°C for approximately two weeks in a 5% CO2 incubator. After removing the culture medium, the cells are washed one to several times with PBS or the like. Instead of removing the culture medium and washing the cells, the adherent dental pulp stem cells that have formed colonies can be collected. The adherent dental pulp stem cells can be detached from the dish and collected by treating them with, for example, 0.025-0.1% trypsin and 0.3-1 mM EDTA for several minutes at 37°C.

[0051] Next, the adhesive cells selected as described above are cultured. For example, the dental pulp stem cells obtained as described above are seeded onto an adherent cell culture dish and cultured in an incubator under conditions of 5% CO2 and 37°C. For example, when the cells reach subconfluency or confluency as observed with the naked eye, they are detached from the culture vessel using trypsin and EDTA as described above, recovered, and seeded again into a culture vessel containing fresh culture medium for subculture.

[0052] Here, "subconfluent" refers to a state in which cells are attached to about 70% of the cell attachment surface in the culture vessel. For example, subculture is performed 1 to 8 times, and the selected cells are cultured to a required number of cells, for example, about 1 x 10 7 The cells are then grown to a density of 100 cells / mL. The grown cells are collected as described above and stored in liquid nitrogen. Cells collected from various donors may be stored in the form of a dental pulp stem cell bank.

[0053] Next, the stem cells are initially cultured, and four types of genes are introduced into the resulting primary cultured cells to produce transgenic cells. The genes to be introduced are preferably hTERT, bmi-1, E6, and E7, as described above, because this allows for the production of immortalized stem cells with a higher population doubling number.

[0054] Such gene introduction can be carried out as follows: A plasmid for incorporating the gene of interest is prepared, and then this is incorporated into a shuttle vector, such as pSuttle2, to clone the gene. E. coli is transformed with this shuttle vector, and kanamycin-resistant transformants are selected. Plasmid DNA from the selected kanamycin-resistant transformants is purified, and the recombinants are identified by analyzing the restriction enzyme sites.

[0055] 1. Preparation of DNA fragments and viral vectors 1.1 Preparation of DNA fragments for viral integration First, obtain the sequence information of the vector used to integrate the DNA fragment into the virus. In the method of the present invention, the virus used as the gene transfer vector is preferably any one selected from the group consisting of lentivirus, adenovirus, and retrovirus, because the introduced gene is expressed non-transiently.

[0056] The following explains the use of a lentiplasmid vector (pLVSIN), which has a high efficiency for producing stable expression strains of introduced genes. Download the sequence information for pLVSIN from, for example, the Takara Bio web catalog (pLVSIN-CMV / EF1α Vector | Takara Bio Inc.) and confirm the multicloning site.

[0057] Next, a DNA fragment is prepared as follows. In the present invention, two to four genes are incorporated into the above DNA fragment to prepare immortalized stem cells. The cells used are not particularly limited as long as they are stem cells obtained from mammals, but it is preferable to use porcine dental pulp tissue, porcine adipose tissue, or human dental pulp tissue, as these are easily available and can yield immortalized stem cells with stable traits.

[0058] Furthermore, the genes introduced here are preferably a set of at least two genes selected from the group consisting of two papillomavirus early genes, telomerase reverse transcriptase (hereinafter sometimes referred to as "TERT"), and bmi, as this allows for the production of immortalized stem cells with stable traits. Furthermore, the papillomavirus early genes are preferably human papillomavirus E6 (hereinafter sometimes referred to as "E6") or human papillomavirus E7 (hereinafter sometimes referred to as "E7") in terms of expression efficiency.

[0059] It is more preferable to prepare and use a DNA fragment containing any of the following gene sets: (a) E7 and TERT; (b) bmi and TERT; (c) bmi, E6, and TERT; (d) E6, E7, and TERT; or (e) bmi, E6, E7, and TERT. It is preferable to use human TERT (hereinafter sometimes referred to as "hTERT") as TERT in terms of gene expression efficiency.

[0060] Among the above genes, TERT encodes the telomerase reverse transcriptase, which elongates telomere sequences that shorten with age. E6 and E7 are genes present in the open reading frame encoding the early genes used by human papillomaviruses for self-replication. E6 is known to reactivate TERT and degrade proteins with PDZ domains. bmi-1 is a polycomb group gene known to be involved in stem cell self-renewal and differentiation control.

[0061] By incorporating the above-mentioned gene set, it is possible to confirm in what combination these genes should be introduced so that they can be efficiently expressed and the cells can be immortalized.

[0062] The following describes the construction of a lentiviral vector and the introduction of two genes. To incorporate the human papillomavirus E7 gene and telomerase reverse transcriptase (TERT) gene set (a) above, a double-stranded DNA of EcoRI / KoZal / E7 / T2A4 / hTERT / BamHI (SEQ ID NO: 3 in the Sequence Listing) is synthesized according to the sequence information using standard procedures. The resulting DNA fragment is then cloned into the multicloning site of the lentivector (pLVSIN-CMV Neo) using standard procedures to obtain a lentivector (E7T) incorporating the two genes.

[0063] Similarly, when introducing two different genes, for example, when introducing the set (b) above, first, a double-stranded DNA containing Bmi-1, EcoRI / KoZal / Bmi-1 / T2A4 / hTERT / BamHI (SEQ ID NO: 4 in the Sequence Listing), is synthesized using standard procedures. The resulting DNA fragment is cloned into the multicloning site of the lentivector (pLVSIN-CMV Neo) using standard procedures to obtain a lentivector (BT) incorporating the two genes.

[0064] Similarly, to introduce three genes, double-stranded DNA of T2A3E6 is synthesized according to the sequence information and inserted between Bmi-1 and T2A4 of the lentiviral vector prepared above to generate EcoRI / KoZal / Bmi-1 / T2A3 / E6 / T2A4 / hTERT / BamHI double-stranded DNA (SEQ ID NO: 5 in the Sequence Listing). The resulting DNA fragment can be cloned into the multicloning site of the lentivector (pLVSIN-CMV Neo) using standard procedures to obtain a lentivector (BE6T) incorporating the three genes.

[0065] Similarly, to introduce four genes, double-stranded DNA of E6T2A3 is synthesized and inserted between the Kozak sequence and E7 of the lentiviral vector E7T prepared above, and EcoRI / KoZal / E6 / T2A3 / E7 / T2A4 / hTERT / BamHI (SEQ ID NO: 6 in the Sequence Listing) is synthesized using standard procedures. The resulting DNA fragment is cloned into the multicloning site of the above-mentioned lentiplasmid vector (pLVSIN-CMV Neo) using standard procedures to obtain a lentivector (E6E7T) incorporating the above three genes. Synthesis of such DNA fragments can also be outsourced to a company that specializes in DNA synthesis.

[0066] Unlike lentiviral vectors, retroviruses are introduced by co-infection with vectors incorporating individual genes. For example, five immortalization genes (hTERT (SEQ ID NO: 7), human papillomavirus E6 (SEQ ID NO: 8), human papillomavirus E7 (SEQ ID NO: 9), pigTERT (SEQ ID NO: 10), and hBmi-1 (SEQ ID NO: 11)) with a Kozak sequence (gccacc) added upstream of the initiation codon can be prepared according to standard methods and cloned into the PmaC I-Hpa I site of pDON-5 NEO DNA (TaKaRa Code 3657) according to standard methods to prepare retroviral plasmid vectors (pDON-5 Neo hTERT vector, pDON-5 Neo HPV16 E6 vector, pDON-5 Neo HPV16 E7 vector, pDON-5 Neo pHTERT vector, and pDON-5 Neo hBmi1 vector).

[0067] Then, the five types of plasmid DNA prepared as described above are used to transform E. coli according to standard methods, and the resulting transformants are cultured in a CO2 incubator to obtain transfection-grade plasmid DNA.

[0068] Next, 5.5 to 6.5x10 6 G3T-hi cells are seeded onto the desired plate at 1 / dish and cultured in a 5% CO2 incubator at approximately 37°C for approximately 20 to 28 hours. A desired amount of transfection reagent (e.g., 0.3 to 0.5 mL of TransIT-293) is then added, and three of the five plasmid vectors listed above are selected and co-transfected with pGP and pE-Ampho (both vectors included in the Retrovirus Packaging Kit Ampho). The cells are then cultured under the same conditions for a further 40 to 56 hours to transfect these genes.

[0069] 1.2 Preparation of Vector-Producer Cells In parallel with the above steps, recombinant lentiviral vector-producer cells are prepared. Examples of such cell lines include Lenti-X 293T (Clontech, code number 632180) and other commercially available cell lines. Lenti-X 293T can be cultured in a medium containing fetal bovine serum and antibiotics. Examples of such media include minimal medium (MEM) and Dulbecco's modified Eagle's medium (DMEM). For example, DMEM (Sigma, St. Louis, MO) containing 5-15% fetal bovine serum (FBS, HyClone) and 0.5-2% antibiotics (penicillin / streptomycin, Gibco) is preferred in terms of cell growth efficiency.

[0070] For example, DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin can be prepared and used as the basal medium. Hereinafter, this medium will be referred to as "293T basal medium."

[0071] For example, when using Lenti-X 293T as the cell line, 1 to 5 x 10 5 A cell suspension of 100 cells / mL is prepared, and 10 mL of this suspension is placed in a 10 cm dish and cultured in a 5% CO2 incubator for approximately 24 hours. After that, the cells are passaged according to their condition until use. Before use, first remove the culture supernatant from the passaged cells, wash with PBS, and then add a commercially available detachment agent to detach the cells from the dish and collect them.

[0072] Next, a desired amount of the cell suspension diluted approximately 3 to 5 times is taken, trypan blue staining solution is added, and the number of viable cells is counted using a hemocytometer. The above-mentioned basal medium is added to the cell suspension, and the number of viable cells is counted to approximately 5 x 10 5 A cell suspension of 100 cells / mL is prepared and seeded in a dish at a desired concentration, for example, and cultured under desired conditions. For example, 1 to 4 × 10 cells are seeded in a collagen-coated dish with a diameter of 6 cm. 6 The cells are seeded at 400 cells / 4 mL, and then cultured at about 37° C. in a 5% CO 2 incubator for about 24 hours. The medium is then replaced and the culture is continued.

[0073] Furthermore, it is preferable to use G3T-hi cells (manufactured by Takara Bio Inc.), a cell line that highly expresses human N-acetylglucosaminyltransferase III (GnT-III), which was created by introducing human N-acetylglucosaminyltransferase III (GnT-III) into the human kidney-derived cell line 293T (G418-resistant) using a hygromycin resistance gene, as retrovirus-preparing cells.

[0074] The G3T-hi cells are designed to rapidly and transiently produce high-titer recombinant viruses by co-transfecting a recombinant retrovirus vector plasmid incorporating the target gene with an expression vector plasmid for the gag-pol and env genes using the Retrovirus Packaging Kit Eco or Ampho (Takara Bio Inc., product codes 6160 and 6161). Because the G3T-hi cells are derived from 293T, the SV40 T antigen gene has been introduced into them, and the action of this gene amplifies retroviral RNA, resulting in the production of high-titer virus solutions. Transient expression using the Retrovirus Packaging Kit typically requires 10 5 ~10 7 A solution containing cfu / mL of virus is obtained.

[0075] In the G3T-hi cells, the cell membrane glycans are modified by GnT-III. Because budding retroviruses wrap around the host cell membrane, it is presumed that the glycans of the membrane proteins of the recombinant retroviruses obtained from the G3T-hi cells are modified by GnT-III. Due to this glycan modification, retroviruses prepared using the G3T-hi cells have a high affinity for RetroNectin (a recombinant human fibronectin fragment). Therefore, using RetroNectin as a coating agent for cultureware significantly improves the efficiency of gene transfer into target cells. RetroNectin is particularly effective for gene transfer targeting hematopoietic cells.

[0076] When G3T-hi cells are used, it is preferable to use a medium prepared by adding 10% FBS and 1% penicillin / streptomycin to DMEM containing glucose (4.5 g / L) and L-glutamine (584 mg / L) instead of the above-mentioned 293T basal medium, in terms of retrovirus production efficiency.

[0077] 1.3 Preparation of Viral Vectors Below, a method for preparing a viral vector will be explained using a lentiviral vector as an example. Cotransfection is performed by adding the lentiviral vector plasmid prepared as described above to each of multiple dishes containing cells cultured as described above. For such cotransfection, a commercially available packaging system, such as the Lenti-X HTX packaging system (Clontech), can be used. Specific procedures can be performed by following the manual provided with the system.

[0078] After cotransfection, the medium is replaced with fresh complete medium and cultured at approximately 37°C for 24 to 48 hours. The timing for harvesting the viral vector is determined by titering the viral vector, and the culture supernatant containing the viral vector is harvested when the viral titer reaches its maximum. A commercially available simple titer measurement kit can be used to measure the viral vector titer, such as Lenti-X GoStix (Clontech).

[0079] For example, the day after changing the medium, the culture supernatant in the Petri dish is aspirated and collected using a syringe of the desired size, and the collected culture supernatant is filtered to obtain the viral vector. For example, the culture supernatant is aspirated using a 10 mL disposable syringe (Terumo Corporation), and then a 0.45 μm filter (MILEX-HV, Millipore) is attached to the syringe, and the culture supernatant in the syringe is filtered while collecting the viral vector solution in, for example, a 15 mL tube.

[0080] Then, the presence of recombinant lentiviral vector is confirmed using the reagent contained in the commercially available simple titer measurement kit. For example, a desired amount of viral vector solution, for example, 20 μL, is added to the above-mentioned Lenti-X GoStix S, and Chase Buffer 1 is further added, and the reaction is carried out at room temperature for a desired time, for example, 10 minutes. The presence or absence of a band can be used to confirm the presence of recombinant lentiviral vector.

[0081] Cells containing the four plasmids (E7T, BT, BE6T, and E6E7T) obtained as described above are streaked on an agar medium, and the formed colonies are picked and inoculated into a desired amount of medium, for example, about 2 mL of LB medium containing an antibiotic, and cultured with vigorous shaking at about 37° C. for about 8 hours. Next, a desired amount, for example, 100 μL, of the culture medium of the cells cultured as described above is transferred to, for example, about 50 mL of LB medium containing a different antibiotic, for example, ampicillin, and cultured with vigorous shaking at about 37° C. for about 14 to 18 hours.

[0082] The amount of plasmid eluted with water is measured using a commercially available kit, such as the MN NucleoBond Xtra Mid Kit (Clontech). Analysis is also performed on a desired gel, such as an approximately 1% agarose gel, to confirm that the integrated DNA is unique. Examples of markers that can be used for gel electrophoresis analysis include supercoiled DNA ladders and λ-Hind III-digested DNA. Viral vectors can be constructed in this manner.

[0083] 2. Preparation of mammalian stem cells 2.1 Preparation of porcine dental pulp stem cells The jawbone (mandible with mandibular teeth) and mesentery of a 5-6 month old pig are obtained. Porcine dental pulp stem cells (hereinafter sometimes referred to as "SHED") are obtained from the above pig teeth and mandible according to the following procedure.

[0084] First, the teeth and mandible of the pig are disinfected with an appropriate disinfectant, such as isodine. Then, for example, using a dental diamond point, the crowns of the mandibular teeth are cut horizontally, and then the caps are removed by cutting vertically. The dental pulp is then extracted from the crowns and roots using, for example, a dental surgical scaler.

[0085] The obtained dental pulp is chopped using, for example, an under-eye scalpel, suspended in a collagenase solution of a desired concentration, for example, 1 to 3 mg / mL, and placed in an incubator at about 37°C for a desired time, for example, 1 hour, to isolate cells. The isolated cells are pre-cultured in a desired medium, for example, DMEM containing 10% FBS and 1% Anti-Anti (Antibiotic, Anti-myotic, Invitrogen, Carlsbad, CA), at about 37°C in a 5% CO incubator to obtain cells for passage.

[0086] First, the cells are cultured until they become subconfluent, with the medium being changed two to three times a week. The subconfluent cells are detached using a detaching agent, for example, Hepes solution containing 0.05% trypsin, and collected under desired conditions, for example, by centrifugation at 1,500 rpm at room temperature for about 3 minutes. The obtained cells are transferred to fresh medium and subcultured in a desired amount, for example, the entire amount, under the same conditions as above.

[0087] 2.2 Preparation of Porcine Adipose Stem Cells. Adipose tissue is excised from the mesentery of a pig using, for example, dissecting scissors and a scalpel. Excess tissue is removed and blood is flushed out with saline. The resulting adipocytes are transferred to a desired medium, such as DMEM containing 5-15% bovine serum and a desired concentration of antibiotics, and pre-cultured under the same conditions as above. They are then cultured in the DMEM at 37°C and 5% CO2 until subconfluent. All of the above amounts are final concentrations. For example, DMEM containing approximately 10% bovine serum, approximately 100 U / mL penicillin, and approximately 100 μg / mL streptomycin can be used as such a medium. As with porcine SHED, the cells are detached using a detachment agent, centrifuged, and subcultured to obtain adipose stem cells.

[0088] 2.3 Preparation of Human Deciduous Dental Pulp Stem Cells Deciduous teeth obtained from healthy children or extracted teeth are obtained. These teeth are disinfected with an appropriate disinfectant, such as isodine solution, and dental pulp tissue is collected in a manner similar to that used to obtain porcine dental pulp. The obtained dental pulp tissue is digested in a solution of collagenase and dispase at the desired concentrations, e.g., about 3 mg / mL type I collagenase and about 4 mg / mL dispase, at the desired temperature for the desired time, e.g., about 37°C for about 1 hour. The solution is then filtered, e.g., using a 70 μm cell strainer (Falcon), to separate the cells.

[0089] The filtered cells are resuspended in a desired volume (e.g., approximately 4 mL) of the above medium and seeded onto an adherent cell culture dish with a desired diameter (e.g., 6 cm). The desired medium, e.g., DMEM containing approximately 10% FCS, is added, and the cells are cultured for a desired period (e.g., approximately 2 weeks) in an incubator adjusted to 5% CO2 and 37°C. The colonized adherent cells (dental pulp stem cells) are treated with a desired detachment agent, e.g., approximately 0.2 mM EDTA containing approximately 0.05% trypsin, for a desired time (e.g., 5 minutes at approximately 37°C), and the detached cells are collected from the dish.

[0090] Next, the adherent cells recovered as described above are seeded, for example, on an adherent cell culture dish (collagen-coated dish) and cultured, for example, in an incubator adjusted to 5% CO and 37°C to obtain primary cultured cells. When the cells become subconfluent or confluent as observed with the naked eye, they are treated in the same manner as above with the same detachment agent, and the cells contained in the dish are recovered.

[0091] Thereafter, the primary cultured cells (hereinafter sometimes referred to as "primary cultured cells") are cultured in the above medium at a desired concentration, for example, about 1 × 10 4 cells / cm 2 The cells are subcultured at a concentration of 1000 kJ / ml, and the cells are used for experiments after 1 to 3 passages. In this way, human deciduous dental pulp stem cells (SHED) can be obtained.

[0092] If necessary, the porcine deciduous tooth dental pulp stem cells, porcine adipose stem cells, and human deciduous tooth dental pulp stem cells obtained as described above may be placed in vials and cryopreserved at -80°C.

[0093] 3. Preparation of Gene-Transduced Cells 3.1 Gene Transfer Using Lentiviral Vectors The porcine dental pulp stem cells, porcine adipose stem cells, and human dental pulp stem cells obtained as described above were cultured in the same manner as described above. When they reached approximately 70-90% confluence, a mycoplasma check was performed using a commercially available kit. Examples of such kits include the MycoAlert Mycoplasma Detection Kit (Lonza, LT07-318). Next, the lentiviral vectors prepared as described above were infected into each of the stem cells using polybrene reagent, and drug selection was performed to select strains stably expressing the target gene.

[0094] After removing the culture supernatant from each stem cell culture, the cells are washed with, for example, phosphate-buffered saline (hereinafter, sometimes referred to as "PBS" (pH 7.4)), and then detached using a desired detachment agent and collected individually. Examples of such detachment agents include StemPro (registered trademark: GIBCO) for porcine deciduous dental pulp stem cells and porcine adipose tissue stem cells, and Trypsin-EDTA for human deciduous dental pulp stem cells.

[0095] The obtained cells were counted according to a standard method, and approximately 1 × 10 cells were placed in each well of a tissue culture-treated (TC) 6-well plate (manufactured by Corning). 5 The cells are seeded at 1000 cells / well and cultured in a CO2 incubator at approximately 37°C for approximately 24 hours, after which it is confirmed that the cells are uniformly distributed throughout the plate.

[0096] The culture supernatant is then removed, and a desired amount (e.g., 750 μL / well) of a desired medium containing a desired concentration of polybrene (e.g., DMEM medium containing approximately 8 μg / mL of polybrene and approximately 10% FBS) is added to each well for the porcine deciduous dental pulp stem cells. Next, the lentiviral vector solution obtained as described above is added to each well at a rate of, for example, approximately 250 μL / well.

[0097] In the case of human deciduous dental pulp stem cells, a desired amount of DMEM containing polybrene and FBS as described above is added, for example, about 1.2 mL / well, and then a desired amount of the lentiviral vector solution described above is added to each well, for example, about 400 μL / well.

[0098] Each plate containing the viral vector is then centrifuged under desired conditions, for example, at about 1,000 × g for about 30 minutes at about 32°C, to infect the cells with the virus. The cells are then cultured under desired conditions, for example, in a CO2 incubator at about 37°C, for about 4 to about 6 hours. A desired amount of culture medium, for example, about 1 mL / well, is then added to each well, and the cells are further cultured under desired conditions, for example, in a CO2 incubator at 37°C, for about 24 hours to allow gene transfer.

[0099] 3.2 Drug Selection The culture supernatant is removed from each well of the culture plate for the stem cells (porcine dental pulp stem cells, porcine adipose stem cells, or human dental pulp stem cells) cultured as described above, and a desired amount, for example, about 2 mL, of selective medium containing a desired concentration of selective agent, for example, about 0.4 mg / mL or about 0.8 mg / mL GENETICIN (G418, GIBCO) is added to each well, and the selective medium is replaced. Thereafter, the cells are cultured for a desired period, for example, about 3 to about 5 days, with medium changes, to select for transgenic cells.

[0100] A portion of each of the cultured cells is subjected to cloning, and the remaining cells are pooled and continue to be cultured in a selection medium. Cloning can be performed as follows.

[0101] For example, the cells can be diluted using antibiotic-free selection medium, e.g., selection medium that does not contain penicillin, streptomycin, or G418, to a desired concentration, e.g., about 1 x 10 3 cells / 4mL or approximately 5 x 10 3 Cells / 4 mL are seeded into each dish and then cultured for approximately 24 hours in a CO2 incubator at approximately 37°C. The formed colonies are marked on the back of the dish, and when the desired number of colonies, for example, approximately 100, is reached, the culture supernatant is removed and the dish is washed with PBS. A cloning ring is placed on the dish, and the cells from the colonies are detached using a detachment agent and individually placed into each well of a 48-well plate containing the desired amount of medium, for example, approximately 1 mL.

[0102] 3.3 Preparation of total RNA For confirmation of gene expression, total RNA is prepared using, for example, NucleoSpin RNA II (a kit manufactured by MACHEREY-NAAGEL). The various buffers, ring filters, etc. used below are those included in the kit, and it is preferable to perform the procedure according to the manual that comes with the kit, as this will result in obtaining highly pure total RNA.

[0103] A desired number, e.g., about 5 x 10 5 The cell pellet is lysed to prepare a lysate, which is then placed in a purple ring filter and centrifuged at approximately 11,000 × g for approximately 1 minute. After centrifugation, the filter is discarded, and a desired amount of ethanol, e.g., approximately 350 μL of approximately 70% ethanol, is added to the collection tube and pipetted up and down as desired, e.g., approximately 5 times.

[0104] Next, a desired amount of the resulting solution, e.g., about 700 μL, is loaded onto a light blue ring column placed in a collection tube and centrifuged under desired conditions, e.g., about 11,000 × g for about 30 seconds to bind the RNA. After centrifugation, the column is placed in a new collection tube, and a desired amount of reagent, e.g., about 350 μL of Membrane Desalting Buffer (MDB), is added. The column is then desalted by centrifugation under desired conditions, e.g., about 11,000 × g for about 1 minute.

[0105] After centrifugation, a desired amount of rDNase (e.g., about 10 μL) and about 90 μL of DNase reaction buffer are gently mixed to prepare a DNA reaction mixture. A desired amount (e.g., about 95 μL) is added to the column and incubated under desired conditions, for example, at room temperature for about 15 minutes to digest the DNA. Subsequently, a desired amount (e.g., 200 μL) of Buffer RA2 is added to the column and washed by centrifugation under desired conditions, for example, at about 11,000 × g for about 30 seconds. Subsequently, the column is placed in a new collection tube, and a desired amount (e.g., about 700 μL) of Buffer RA3 is added to the column and washed by centrifugation under desired conditions, for example, at about 11,000 × g for another 30 seconds.

[0106] The solution in the collection tube is then discarded, and a desired amount of Buffer RA3, for example, about 250 μL, is added to the column. The column is then centrifuged under desired conditions, for example, at about 11,000 × g for about 2 minutes, and the silica membrane of the column is air-dried. The column is then placed in a collection tube of desired size, for example, about 1.5 mL, and a desired amount of RNAase-free water, for example, about 60 μL, is added to the column. The column is then centrifuged under desired conditions, for example, at about 11,000 × g for about 1 minute, to obtain a total RNA sample.

[0107] 3.4 Reverse transcription reaction The total RNA sample obtained as described above is reverse-transcribed, followed by real-time PCR to obtain a PCR product. For reverse transcription, a commercially available product such as PrimeScript RT reagent Kit (Perfect Real Time, manufactured by TaKaRa Bio) can be used, and reverse transcription can be performed according to the manual provided with the kit.

[0108] Real-time PCR can be performed using a commercially available kit such as SYBR Premix Ex Taq. Examples of the standard gene used here include porcine β-actin and human β-actin, and primers such as those shown in SEQ ID NOs: 12 to 17 in the Sequence Listing can be prepared and used.

[0109] First, prepare a real-time PCR premix by mixing the desired amount, e.g., approximately 350 μL of SYBR Premix Ex Taq II (x2), approximately 28 μL of primer mix (approximately 10 μL), and approximately 266 μL of double-distilled water. Next, dispense the desired amount, e.g., approximately 23 μL of the premix, into real-time PCR tubes, and add approximately 2 μL of template cDNA to each tube. Real-time PCR is then performed under the desired conditions, e.g., approximately 95°C for approximately 30 seconds (approximately 95°C for approximately 5 seconds, followed by approximately 60°C for approximately 30 seconds), for approximately 40 cycles. The melting curve is then determined by dissociating the cells at approximately 95°C for approximately 15 seconds, approximately 60°C for approximately 30 seconds, and approximately 95°C for approximately 15 seconds. The same procedure is performed for human dental pulp stem cells.

[0110] From the calibration curves created as described above, the expression level of each gene in cells transfected with each viral vector can be determined. Accurate results can be obtained by correcting for the expression level of an internal control gene.

[0111] Expression of the introduced gene is confirmed in each of the gene-transfected stem cells. This confirmation allows the gene transfer efficiency of each viral vector prepared as described above to be determined. Next, single-cell cloning of cells is performed from the gene-transfected cells obtained in this manner (hereinafter sometimes referred to as a "population of cells stably expressing the introduced gene" or "pooled cells").

[0112] First, the pooled cells are plated in a dish of a desired size at a desired number, for example, about 1 × 10 cells in a 60 mm dish. 3 The cells are seeded at a cell density of 1000 cells / dish and cultured in a CO2 incubator at a desired temperature for a desired time, for example, 24 hours at 37°C. The growth medium is then replaced with a new one containing the selective agent described above, and the culture is continued to allow colonies to form. The formed colonies are detached individually using a cloning ring and a detachment agent, and each is seeded onto a desired plate, for example, a 24-well plate.

[0113] The seeded cells are then grown and seeded on a culture dish or other medium for expansion, yielding single clones of transgene-stable cells. The resulting clones are then cultured and expanded as described above to obtain total RNA. The resulting total RNA is used as a template for reverse transcription using a desired kit, such as the PrimeScript RT reagent kit, to obtain template cDNA.

[0114] Thereafter, real-time PCR is performed in the same manner as described above, using, for example, the cDNA (reverse transcription product) as a template, the above-mentioned SYBR Premix Ex TaqII (Tli RNaseH Plus), and primers (primers specific to the introduced gene and the internal control gene, respectively, for example, the primers shown in SEQ ID NOs: 12 to 17 in the Sequence Listing).

[0115] A calibration curve for measuring the expression level of each gene was created by plotting the Ct value calculated from the second derivative curve of the real-time PCR on the X axis and the relative value of total RNA on the Y axis. By determining the relative expression levels of the introduced genes derived from porcine dental pulp stem cells and human dental pulp stem cells, with the expression level of the pooled cells set at 1, it was possible to determine whether all of the introduced genes were expressed and their expression levels, and to select the desired clones.

[0116] In this way, a population of immortalized stem cells transfected with the desired gene can be obtained, and single clones of immortalized stem cells can be obtained from that population. From the resulting transformants, ampicillin-resistant transformants (hereinafter referred to as "Amp+") are selected and purified using, for example, ampicillin, and the recombinants are identified by analyzing the restriction enzyme sites. The recombinant adenovirus is then digested with, for example, PacI, and the resulting fragments are transfected into HEK293 cells for growth. The cells are then collected and the titer of the virus is measured. The virus is purified according to standard methods and used to infect target cells, SHED-P.

[0117] After viral infection, HEK293 cells were stained with FITC according to standard methods, and STRO-1-positive cells were detected using a flow cytometer. STRO-1 is considered to be one of the markers for multipotent mesenchymal stem cells in bone marrow and serves as an indicator of cell immortalization. By following these procedures, immortalized stem cells derived from dental pulp can be obtained.

[0118] The resulting immortalized stem cells are then cultured in the above-mentioned basal medium, for example, DMEM supplemented with 10% FBS, under conditions of 5% CO2 and 37°C for 24 to 48 hours to obtain a culture supernatant. The culture supernatant can be collected using, for example, a Komagome pipette.

[0119] Furthermore, the immortalized stem cells secrete insulin-like growth factor binding proteins (IGFBPs), vascular endothelial growth factor (VEGF), tissue metalloproteinases (TIMPs), hepatocyte growth factor (HGF), and other growth factors into the culture supernatant. Here, "growth factor" is a general term for polypeptides that promote cell division and induce morphological changes or hypertrophy. Growth factors vary depending on the type of cell that produces them, and are broadly classified into epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), transforming growth factor (TGF), and others.

[0120] Furthermore, receptors on the cell membrane of each cell have tyrosine kinase activity, and when a growth factor binds to them, the tyrosine residues of the protein are phosphorylated, causing cell proliferation and differentiation. There are several known examples of growth factors acting as mesoderm-inducing substances during ontogeny. There are also several known examples of lymphokines, which regulate the immune system, acting as mesoderm-inducing substances during ontogeny. These growth factors can be quantified using known ELISA or microarray techniques.

[0121] The IGFBPs are polypeptides with a sequence highly similar to insulin and, like insulin, induce mitogenesis and other responses in cell cultures. They are also known to affect the growth of nerve cells. VEGF is a group of glycoproteins involved in angiogenesis, the formation of new blood vessels where none exist, and angiogenesis, the formation of new blood vessels by branching and extending from existing blood vessels, during embryonic development. HGF possesses versatile physiological activities, including promoting cell proliferation, promoting cell motility, preventing apoptosis (cell death), inducing morphogenesis, and promoting angiogenesis, as well as regenerating and protecting tissues and organs, in not only hepatocytes but also various other cells.

[0122] The various stem cells described above can be cultured in DMEM supplemented with 15% FCS for a predetermined period at 37°C to obtain a culture supernatant containing the growth factors. Note that the stem cell culture supernatant contains many proteins in addition to IGFBPs, VEGF, and HGF.

[0123] A 15 mL aliquot of the resulting culture supernatant was placed in an Amicon Ultra Centrifugal Filter Unit-10K (Millipore) and centrifuged at 4,000 x g for approximately 60 minutes to concentrate to approximately 200 μL. An equal volume of sterile PBS was then added to the tube, and the mixture was centrifuged again at 4,000 x g for approximately 60 minutes to replace the solvent with PBS. The resulting 200 μL solution was collected in a microtest tube and used as concentrated stem cell culture supernatant.

[0124] Instead of using the Amicon® method, ethanol precipitation can also be used for concentration. For example, 45 mL of 100% ethanol is added to 5 mL of culture supernatant, mixed, and left at -20°C for 60 minutes. The mixture is then centrifuged at 15,000 x g for 15 minutes at 4°C, and the supernatant is removed. Next, for example, 10 mL of 90% ethanol is added, mixed thoroughly, and centrifuged again at 15,000 x g for 5 minutes at 4°C. The supernatant is removed, and the resulting pellet can be dissolved in, for example, 500 μL of sterile water. After dissolution, the entire volume is collected in a microtest tube and used as concentrated stem cell culture supernatant.

[0125] The culture supernatant obtained as described above can be freeze-dried in a conventional manner to obtain a powder that can be prepared immediately before use.

[0126] 4. Generation of iCas9-transduced stem cells First, prepare the iCas9 gene. The iCas9 gene can be obtained from plasmid repositories such as Addgene. For example, pMSCV-F-del-Casp9.IRES.GFP (Plasmid #15567, SEQ ID NO: 2) is available from Addgene. Addgene is a non-profit organization that distributes plasmids to researchers. In collaboration with hundreds of laboratories, it has compiled a library of high-quality plasmids used in published papers and built a plasmid repository. By linking plasmids to literature, researchers can access the plasmids and related data they need at any time.

[0127] The iCas9 gene can also be produced by cloning the caspase-9 gene and the FKBP gene from a cDNA library and fusing them using PCR. Here, the caspase-9 gene of iCas9 is preferably the same as that of the animal species being treated, taking immunogenicity into consideration. Furthermore, it is preferable to delete the endogenous dimerization domain of caspase-9 so that the introduced caspase-9 does not induce cell death in response to endogenous signals. An FKBP domain is then fused in place of the deleted dimerization domain. Preferably, a mutation is introduced into the endogenous ligand-binding region of the FKBP domain to prevent dimerization in response to endogenous ligands, with the F36V mutant of FKBP being more preferred. The plasmid vector carrying iCas9 is appropriately selected depending on the viral vector used. For example, when using a retrovirus as a vector, a plasmid vector such as pMSCV can be used. The iCas9 gene is incorporated into the plasmid vector using restriction enzymes, DNA ligase, or other conventional methods.

[0128] The resulting iCas9 gene-carrying plasmid is then incorporated into a viral vector. While there are no limitations on the type of viral vector, the following describes the use of retrovirus as an example. To prepare retroviral vectors, commercially available kits are preferably used, such as the Platinum series of retroviral packaging cell lines. Approximately 0.25-0.75 μg of the iCas9-carrying plasmid prepared above and approximately 1-2 μL of a transfection reagent, such as Fugene 6 (Promega), are added to the cells, and the cells are cultured, for example, in 10% FBS-containing DMEM at 5% CO2 and approximately 37°C.

[0129] The next day, the cells are washed with PBS and cultured in fresh DMEM containing 10% FBS for 3 days. The culture supernatant is then transferred to a tube fitted with a 0.22 μM filter and centrifuged. The culture supernatant after centrifugation is stored at 4°C or -20°C until use.

[0130] The retrovirus prepared as described above is used to introduce the iCas9 gene into stem cells. While the type of stem cells is not particularly limited, the following describes the use of SHEDs. SHEDs can be obtained, for example, from the Cell Engineering Laboratory at RIKEN BRC. Alternatively, they can be obtained from human deciduous teeth.

[0131] The SHED you obtained can be divided into 2x10 4 The cells were cultured at a density of 100 cells in a 24-well plate containing 10% FBS-containing DMEM medium. After removing the culture medium, 12.5-37.5 μL of polybrene (1 mg / ml, Sigma) was added to approximately 0.25-0.75 ml of the virus-containing supernatant to a final concentration of 5 μg / ml, and the culture was continued. The following day, the cultured cells were washed with PBS, and fresh 10% FBS-containing DMEM was added and the cells were cultured for 10 days. After culture, SHEDs were collected according to standard procedures and stored at -80°C until use.

[0132] The iCas9-transfected SHEDs created here can be used for cell transplantation therapy. When transfecting with iCas9, it is preferable to introduce a separate gene that allows for the selection of iCas9-transfected cells from a mixture of iCas9-transfected and non-transfected cells. Drug resistance genes such as ampicillin resistance or fluorescent proteins such as GFP can be used as genes for transfected cell selection. For example, when using pMSCV-F-del-Casp9.IRES.GFP, GFP-positive cells can be sorted and purified before use in cell transplantation therapy.

[0133] (Example 1) Preparation of a viral vector for cell immortalization 1. Preparation of an adenovirus introduction vector (1) Reagents, etc. (1-1) Reagents for plasmid extraction Kanamycin (Kan), ampicillin (Amp), LB liquid medium and LB agar medium, glycogen, agarose, sterilized water, ammonium acetate, sodium acetate, sodium dodecyl sulfate, and RNase A were used. 50 mg / mL Kan and Amp were prepared and stored at -20°C as stock solutions. Glycogen was adjusted to 20 mg / mL. 10 mg / mL RNase A was prepared and stored at -20°C. 10 M (saturated) ammonium acetate (NH4OAc) and 3 M sodium acetate (NaOAc; pH 5.2) were prepared.

[0134] (1-2) Restriction enzymes, etc. E. coli competent cells (Supercharge EZ10 Electrocompetent Cells, product code 636756), SwaI (product code 1111A, equivalent to SmiI), XhoI (product code 1094A), T4 DNA Ligase (product code 2011A), NucleoBond Xtra Midi (product code 740410.10 / .50 / .100), and NucleoSpin Plasmid (product code 740588 10 / 50 / 250) were all purchased from Takara Bio Inc. PacI was purchased from New England Biolabs.

[0135] (1-3) Buffers, etc. 1xTE Buffer (10 mM Tris-HCl [pH 8.0] containing 1 mM EDTA) and phenol:chloroform:isoamyl alcohol (25:24:1, hereafter referred to as "PCI mixture") saturated with 100 mM Tris-HCl (pH 8.0) were prepared. Ethanol was used at 100% and 70% concentrations. The following buffers 1 to 4 were prepared for purifying pAdeno-X plasmid DNA to be used in mini-scale recombination.

[0136] Buffer 1: 25 mM Tris-HCl (pH 8.0) containing 10 mM EDTA and 50 mM glucose (store at 4°C after autoclaving) Buffer 2: 0.2 M NaOH containing 1% SDS (prepared immediately before use, sealed, and stored at room temperature) Buffer 3: 5 M KOAc (store at 4°C after autoclaving) Buffer 4: 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA and 20 μg / mL RNase (add RNase immediately before use. Store at -20°C)

[0137] (2) Reagents for Adenovirus Purification and β-gal Assay. Human HEK293 cells (ATCC #CRL1573) transformed with human adenovirus type 5 were used. HEK293 cells were cultured in complete medium. The complete medium consisted of DMEM (basal medium) supplemented with 100 units / mL of penicillin G sodium, 100 μg / mL of streptomycin, 4 mM glutamine, and 10% FBS. Penicillin G sodium solution was prepared at 10,000 units / mL, and streptomycin sulfate solution was prepared at 10,000 μg / mL and stored as stock solutions. 60 mm plates, 100 mm plates, 6-well plates, T75, and T175 flasks were used for culture.

[0138] Trypsin-EDTA (product code CC-5012) was purchased from Takara Bio Inc. PBS (Ca 2＋ and Mg 2＋ (Ca-free) and Dulbecco's phosphate buffered saline (DPBS, 2＋ and Mg 2＋ A 0.33% neutral red staining solution and a 0.4% trypan blue staining solution were also used. For the β-gal assay, a solution of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (25 mg / mL)) in dimethylformamide (DMF) was stored at -20°C, protected from light. The Luminescent β-gal Detection Kit II (product code 631712, Takara Bio Inc.) was used.

[0139] (3) Preliminary Test (3-1) Construction of Recombinant Adenovirus Containing lacZ (pAdeno-X-lacZ) HEK293 cells were thawed and DMSO was removed from them and resuspended in 10 mL of the complete medium described above, and the entire volume was transferred to a 100 mm diameter culture plate. After the HEK293 cells had attached, the medium was removed, the cells were washed once with sterile PBS, and 1 mL of trypsin-EDTA solution was added and treated for approximately 2 minutes. Next, 10 mL of complete medium was added to stop the trypsin reaction and the cells were gently suspended. Viable counts were performed, and 10 cells were transferred to a 100 mm diameter plate containing 10 mL of culture medium. 5 Cells were transferred and spread evenly.

[0140] Using pShuttle2-lacZ (a positive control vector included in the Adeno-X Expression System 1) and the Adeno-X Viral DNA (PI-SceI and I-CeuI digested) included in the kit, a recombinant adenovirus containing lacZ was constructed according to the protocol provided with the kit. Target cells (SHED) were infected with the vector, and β-galactosidase expression was assayed to confirm the construction of the vector.

[0141] (3-2) Construction of Recombinant pShuttle 2 Plasmid Before constructing the recombinant pShuttle 2 Vector (hereinafter referred to as "rpShuttle 2 Vector"), DH5α E. coli was transformed with the pShuttle 2 Vector and pShuttle 2-lacZ Vector included in the kit. Transformants were selected on LB agar plates containing 50 μg / mL Kan (hereinafter referred to as "LB / Kan"), and bacterial cells from single colonies were streaked onto fresh LB / Kan plates and incubated overnight at 37°C.

[0142] Next, hTERT, bmi-1, HPV-E6, and HPV-E7 were cloned into pShuttle 2 using the following procedure. The pShuttle 2 Vector was cleaved with restriction enzymes appropriate for these genes. The pShuttle 2 Vector Information Packet (PT3416-5) included with the kit was then used to determine the multicloning site that matched the DNA to be inserted. The restriction enzyme-treated plasmid was then purified with alkaline phosphatase.

[0143] The target DNA fragment was prepared and purified according to standard methods. The vector digested with the above restriction enzymes was ligated to the above gene fragment, and DH5α cells (competent cells) were transformed with the ligation product. A portion of the above competent cells was transformed with the control vector pShuttle2-lacZ Vector included in the kit as a positive control.

[0144] The mixture containing the transformed E. coli was inoculated onto an LB / Kan agar plate, and kanamycin-resistant (Kanr) transformants (colonies) were selected. Five to ten Kan-resistant clones were selected and inoculated into a small volume of liquid medium for amplification. After confirming that these clones contained the rpShuttle 2 Vector, the culture was incubated overnight. The constructed plasmid DNA was then purified using a commercially available silica adsorption column according to standard procedures.

[0145] The plasmid DNA was digested with restriction enzymes and subjected to 1% agarose gel electrophoresis to identify the recombinant plasmid of interest. The orientation and insertion site of the inserted fragment were confirmed by sequencing, and positive clones were identified. The recombinant pShuttle 2 plasmid DNA (hereinafter referred to as "rpShuttle2 plasmid DNA") was directly transfected into target cells, and Western blot analysis was performed to preliminary check for expression of the target protein.

[0146] (3-3) PI-Sce I / I-Ceu I double digestion of rpShuttle 2 plasmid DNA. The expression cassette of the introduced gene was excised from the rpShuttle2 plasmid DNA prepared as described above with PI-Sce I and I-Ceu I. The excised expression cassette was integrated into Adeno-X Viral DNA according to the in vitro ligation method described in the protocol attached to the kit. 30 μl of PI-Sce I / I-Ceu I double digestion solution of rpShuttle 2 plasmid DNA was prepared, and the reagents listed in Table 1 below were added to a 1.5 mL sterile microcentrifuge tube and mixed.

[0147]

[0148] After thorough mixing, the mixture was placed in a microcentrifuge tube and centrifuged briefly, followed by incubation at 37°C for 3 hours. The reaction mixture (5 µL) after the double digestion was electrophoresed on a 1% agarose / EtBr gel together with a 1 kb ladder (DNA size marker).

[0149] (3-4) Phenol:chloroform:isoamyl alcohol extraction: To the remaining double digestion solution (25 μl), 70 μL of 1x TE buffer (pH 8.0) and 100 μL of PCI mixture were added to a centrifuge tube and thoroughly mixed by vortexing. The mixture was then centrifuged at 14,000 rpm for 5 minutes at 4°C using a microcentrifuge, and the aqueous layer was transferred to a clean 1.5 mL microcentrifuge tube. 400 μL of 95% ethanol, 25 μL of 10 M ammonium acetate, and 1 μL of glycogen (20 mg / mL) were added and thoroughly mixed by vortexing.

[0150] The mixture was then centrifuged at 14,000 rpm for 5 minutes at 4°C, and the supernatant was removed by aspiration to obtain a pellet. 300 μL of 70% ethanol was added to the pellet, and the mixture was centrifuged at 14,000 rpm for 2 minutes at room temperature. The supernatant was carefully removed by aspiration, and the pellet was air-dried at room temperature for approximately 15 minutes. After the pellet was dried, it was dissolved in 10 μL of sterile 1x TE buffer (pH 8.0) and stored at -20°C until use.

[0151] (4) Construction of recombinant Adeno-X plasmid DNA (4-1) Subcloning of expression cassette into Adeno-X viral genome The reagents shown in Table 2 below were placed in the specified order into a 1.5 mL sterilized microcentrifuge tube, gently mixed, briefly centrifuged, and then incubated overnight at 16°C.

[0152]

[0153] To each sample, 90 μL of 1×TE buffer (pH 8.0) and 100 μL of PCI mixture were added and gently mixed by vortexing. After centrifugation at 14,000 rpm for 5 minutes at 4°C, the aqueous layer was transferred to a clean 1.5 mL microcentrifuge tube, to which 400 μL of 95% ethanol, 25 μL of 10 M ammonium acetate, and 1 μL of glycogen (20 mg / mL) were added and gently mixed by vortexing.

[0154] The mixture was centrifuged at 14,000 rpm for 5 minutes at 4°C, and the supernatant was removed by aspiration to obtain a pellet. The following ethanol precipitation procedure was carried out in the same manner as in (3-4) above. After the pellet was dried, it was dissolved in 15 μL of sterile deionized water.

[0155] (4-2) SwaI Digestion of Recombinant Adeno-X Plasmid DNA The digestion solutions shown in Table 3 below were prepared and added to each sample placed in a centrifuge tube, followed by incubation at 25°C for 2 hours.

[0156]

[0157] To each sample, 80 μL of 1x TE Buffer (pH 8.0) and 100 μL of PCI mixture were added and gently mixed by vortexing. The mixture was then centrifuged at 14,000 rpm for 5 minutes at 4°C in a microcentrifuge tube. The following ethanol precipitation procedure was performed as described above in (3-4), and the pellet solution was stored at -20°C until use.

[0158] (4-3) Confirmation of transformation of E. coli with recombinant Adeno-X plasmid DNA Competent cells for electroporation (E. coli) were transformed with the SwaI digestion product obtained in (4-2) above using Supercharge EZ10 Electrocompetent Cells (product code 636756). The transformation mixture was inoculated onto an agar plate containing LB medium supplemented with Amp (final concentration 100 μg / mL) (hereinafter referred to as "LB / Amp agar plate") and incubated overnight at 37°C. Approximately 10 ampicillin-resistant (Ampr) transformants were obtained. 6 Colonies were obtained, which were checked using the Adeno-X System PCR Screening Primer Set provided with the product.

[0159] Five milliliters of fresh LB / Amp liquid medium was inoculated with cells from a single colony and cultured overnight. The next day, Adeno-X plasmid DNA was purified according to the mini-scale method described below.

[0160] (5) Mini-scale preparation of recombinant Adeno-X plasmid DNA. Five mL of logarithmically growing culture medium was centrifuged at 14,000 rpm for 30 seconds, and the supernatant was removed. The pellet was centrifuged again at 10,000 rpm for 1 minute, and the supernatant was removed using a micropipette. 150 μL of Buffer 1 was added and gently pipetted to resuspend the cells. 150 μL of Buffer 2 was added to this cell suspension, mixed gently by inversion, and left on ice for 5 minutes. 150 μL of Buffer 3 was added to the cooled cell suspension, mixed again by inversion, and left on ice for 5 minutes.

[0161] The cell suspension was centrifuged at 14,000 rpm for 5 minutes at 4°C, and the clear supernatant was transferred to a clean 1.5 mL centrifuge tube. 450 μL of PCI mixture was added to the supernatant and mixed by inversion. The mixture was then centrifuged at 14,000 rpm for 5 minutes at 4°C, and the aqueous layer was transferred to a clean 1.5 mL microcentrifuge tube.

[0162] The following ethanol precipitation procedure was carried out in the same manner as in (4-1) above, and the pellet solution was stored at −20° C. until use. The target rDNA was identified by restriction enzyme analysis and PCR, as described below.

[0163] (6) Restriction enzyme site analysis of the obtained rAdeno-X plasmid DNA Analysis was performed using PI-Sce I and I-Ceu I. The reagents shown in Table 4 below were placed in a 1.5 mL sterilized microcentrifuge tube, and 30 μL of the PI-Sce I / I-Ceu I double digestion reaction solution was added. The tube was thoroughly stirred and gently spun to collect the contents.

[0164]

[0165] The mixture was incubated at 37°C for 3 hours and digested with a restriction enzyme. The reaction mixture after this digestion was subjected to electrophoresis on a 1% agarose / EtBr gel.

[0166] (7) Production of recombinant adenovirus (7-1) Preparation of rAdeno-X plasmid DNA for transfection of HEK293 cells The reagents listed in Table 5 below were mixed in a 1.5 mL sterilized centrifuge tube and briefly centrifuged in a microcentrifuge. The mixture was then incubated at 37°C for 2 hours, and the rAdeno-X plasmid DNA was digested with Pac I restriction enzyme.

[0167]

[0168] 60 μL of 1×TE Buffer (pH 8.0) and 100 μL of PCI mixture were added, gently mixed by vortexing, and centrifuged at 14,000 rpm in a microcentrifuge for 5 minutes at 4° C. The aqueous layer was carefully transferred to a clean 1.5 mL sterile centrifuge tube.

[0169] The following ethanol precipitation procedure was performed in the same manner as in (3-4) above, and the pellet solution was stored at -20°C until use. (7-2) Transfection of PacI-digested Adeno-X plasmid DNA into HEK293 cells. 24 hours before transfection with the above plasmid DNA, cells were cultured at 1-2 x 10 cells per 60 mm diameter culture plate. 6 (approximately 100 cells / mm2 HEK293 cells were inoculated onto the plate so that the total number of cells was 100,000, and the plate was incubated at 37°C in the presence of 5% CO2.

[0170] Each culture plate was transfected with 10 μL of PacI-digested Adeno-X plasmid DNA, and Adeno-X DNA was introduced into HEK293 cells using standard transfection methods (CalPhos Mammalian Transfection Kit, product number 631312, Takara Bio Inc.). The day after transfection, cells were monitored for the occurrence of cytopathic effect (CPE). After one week, cells adhering to the bottom and sides of the culture plate were gently agitated to release them. The resulting cell suspension was transferred to a 15 mL sterile conical centrifuge tube and centrifuged at 1,500 × g for 5 minutes at room temperature.

[0171] The resulting precipitate was suspended in 500 μL of sterile PBS. The cells were frozen in dry ice / ethanol and thawed in a 37°C incubator three times to obtain a fully thawed cell lysate. The supernatant was then removed by brief centrifugation, and the supernatant was transferred to a sterile tube for immediate use. Any unused portion was stored at −20°C. 250 μL of the lysate was added to the cultured cells in a 60 mm plate, and the culture was continued. Adenovirus titers were measured using the anti-Hexon antibody included in the Adeno-X Rapid Titer Kit (product code 631028, Takara Bio Inc.) according to the kit's instruction manual (PT3651-1).

[0172] (7-3) Virus amplification for high-titer virus preparation Approximately 24 hours before starting the titer measurement, HEK293 cells were inoculated into a T75 flask and cultured overnight at 37°C in the presence of 5% CO2 until they reached 50-70% confluence. The next day, the medium was replaced with fresh medium containing the virus, and the cells were infected at an MOI of 10. After 90 minutes of culture at 37°C in the presence of 5% CO2, the flask was removed and 10 mL of medium was added.

[0173] The cells were cultured at 37°C in the presence of 5% CO2 for 3-4 days, and CPE was confirmed. When 50% of the cells had detached, they were prepared as a free cell suspension in the same manner as above and transferred to a 15 mL sterilized conical centrifuge tube. The cells were thawed using the same freeze-thaw procedure as above. The Adeno-X Rapid Titer Kit (product number 631028) was used to thaw 10 7 The titer was measured in PFU / mL, and Western blotting was performed to confirm whether the packaged adenovirus genome contained functional copies of the transcription unit specific to the gene of interest.

[0174] 2. Preparation of Retroviral Vectors (1) Reagents: G3T-hi cells (Takara Bio, product code 6163), TransIT-293 (transfection reagent, Takara Bio, product code MIR2704), Retrovirus Packaging Kit Ampho (Takara Bio, product code 6161), Retrovirus Titer Set (for Real Time PCR) (Takara Bio, product code 6166), One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time, Takara Bio, product code RR066A), and the Thermal Cycler Dice Real System (Takara Bio, model TP800) were used as a real-time PCR instrument.

[0175] (2) Plasmid Preparation: To prepare a retroviral plasmid for retroviral vector preparation, pDON-5 Neo (Takara Bio, catalog number 3657) was used. The immortalization genes were TERT (human or porcine, hereafter sometimes abbreviated as "hTERT" or "pTERT"), human papillomavirus E6 and E7, and a Kozak sequence (gccacc) was added upstream of the start codon of hBmi-1 according to standard methods.

[0176] The five immortalization genes with Kozak sequences added were cloned into the PmacI-HpaI site of the pDON-5 Neo DNA to prepare 10 mL of recombinant retroviral vectors (Amphotropic Envelope) carrying the pDON-5 Neo hTERT Vector (SYN5587-1-4), pDON-5 Neo HPV16 E6 Vector (SYN5587-2-10), pDON-5 Neo HPV16 E7 Vector (SYN5587-3-7), pDON-5 Neo pigTERT Vector (SYN5587-4-9), and pDON-5 Neo hBmi1 Vector (SYN5587-5-1).

[0177] The inserted gene sequence of the plasmid DNA obtained as described above was confirmed by single-strand analysis. The plasmid DNA was used to transform Escherichia coli to obtain transformants. After culturing the resulting transformants, transfection-grade plasmid DNA (approximately 50 μg) was purified according to standard methods and dissolved in sterile water to obtain a plasmid DNA solution.

[0178] (3) Production of recombinant retroviral vectors Five tissue culture dishes (100 mm diameter) were used, and 6 × 10 6 G3T-hi cells were seeded onto the dishes. Culture was then performed at 37°C for approximately 24 hours. Three retrovirus production plasmids (pDON-5 Neo hTERT vector, pDON-5 Neo HPV16E6 vector, pDON-5 Neo HPV16E7 vector, pDON-5 Neo pigTERT vector, and pDON-5 Neo hBmi1 vector, as well as pGP and pE-Ampho included in the Retrovirus Packaging Kit Ampho) were co-transfected into each dish using a transfection reagent, and the culture was continued for an additional 48 hours at 37°C. After incubation, the culture supernatant containing the retrovirus vectors was collected and sterilized by filtration using a 0.45 μm filter (Millipore). The filtrate was dispensed in 1 mL aliquots into sterile tubes.

[0179] (4) Calculation of the Titer of Recombinant Retroviral Vectors The titer of recombinant retroviral vectors was quantified using the Retrovirus Titer Set (for Real-Time PCR) and the One Step SYBR PrimeScript RT-PCR Kit (Perfect Real-Time) according to the instructions provided with the kit. Tables 6 and 7 below show the template treatment and real-time PCR reaction conditions used in measuring the titer of retroviral vectors, along with the reaction scale. Table 6 below shows the composition of the reaction solution used in template treatment (treatment of the template with Dnase I) in measuring the titer of retroviral vectors. Table 7 below shows the reaction conditions. Specifically, the solution shown in Table 6 below was incubated at 37°C for 30 minutes, then heated to 70°C, maintained at this temperature for 10 minutes, and rapidly cooled to 4°C.

[0180]

[0181] Real-time PCR was performed using the solutions shown in Table 7 under the following reaction conditions: (s1) 42°C for 5 minutes, (s2) 95°C for 10 seconds, followed by (s3) 95°C for 5 seconds and 60°C for 30 seconds, for a total of 40 cycles, and a dissociation curve was generated.

[0182]

[0183] The copy number of the RNA Control Template included in the kit is plotted on the X-axis, and the second derivative curve (2 nd A standard curve was created by plotting the Ct values ​​calculated from the chromatograms (Ct values ​​calculated from the chromatograms) on the Y-axis. Table 8 below shows the Ct values ​​of the standard curve for real-time PCR. In the table below, for example, "1.00E+08" represents "1.00 x 10 8 ". The same applies below.

[0184]

[0185] The Ct values ​​were calculated using the 2nd1 Derivative Maximum (SDM) method. The calculated Ct values ​​yielded a slope of -3.54, an intercept of 43.88, an amplification efficiency of 91.6, and a coefficient of determination of 0.999. The titers of the test samples were calculated using this calibration curve, and the results are shown in Table 9. In the table, for example, E+04 is multiplied by x10 4 Shows.

[0186]

[0187] In the table above, ED indicates that dilution buffer EAST Dilution (for Real Time PCR) was used. The titer (copy number / mL) was calculated as follows: calculated copy number (copy number / μL) x 2 (dilution factor during Dnase I treatment) x dilution factor during real time PCR reaction (100 or 500) x 1,000 (μL to mL conversion).

[0188] Real-time PCR showed that DON-5 Neo hTERT was 1.46 x 10 10 copies / mL, pDON-5 Neo HPV16E6 was 1.15 x 10 10 copies / mL, pDON-5 Neo HPV16E7 was 4.05 × 10 10 copies / mL, pDON-5 Neo pigTERT was 1.27 × 10 10 copies / mL, or 2.83 x 10 for pDON-5 Neo hBmi1 10 Titers were given in copies / mL.

[0189] Example 2: Preparation of Immortalized SHEDs (1) Preparation of Dental Pulp Stem Cells An exfoliated deciduous tooth obtained from a 10-year-old healthy boy was used. After disinfecting the exfoliated deciduous tooth with isodine solution, the crown was cut horizontally using a dental diamond point, and dental pulp tissue was collected using a dental reamer. The obtained dental pulp tissue was digested in a solution of 3 mg / mL type I collagenase and 4 mg / mL dispase at 37°C for 1 hour. The solution was then filtered using a 70 mm cell strainer (Falcon).

[0190] The filtered cells were resuspended in 4 mL of the above medium and seeded onto a 60 mm diameter dish for adherent cell culture. DMEM containing 10% FCS was added to the dish, and the cells were cultured for approximately two weeks in an incubator adjusted to 5% CO2 and 37°C. The adherent cells (dental pulp stem cells) that formed colonies were treated with 0.05% trypsin-0.2 mM EDTA for 5 minutes at 37°C, and the cells detached from the dish were collected.

[0191] Next, the adherent cells selected as described above were seeded onto a dish for adherent cell culture (collagen-coated dish) and cultured in an incubator adjusted to 5% CO2 and 37°C to obtain primary cultured cells. When the cells reached subconfluence (approximately 70% of the surface of the culture vessel was occupied by cells) or confluence as observed with the naked eye, they were treated with 0.05% trypsin and 0.2 mM EDTA for 5 minutes at 37°C to detach and recover the cells from the culture vessel. The cells thus obtained were seeded again onto a dish containing the above medium and subcultured several times to obtain a cell population of approximately 1 x 10 7 The cells were grown to a density of 100 cells / mL and stored in liquid nitrogen.

[0192] Then, the primary cultured cells were cultured in the above medium until they were approximately 1 x 10 4 cells / cm 2 The cells were subcultured at a concentration of 1 × 10 for each sample. Cells that had been subcultured 1 to 3 times were used in the experiments. Human BMMSCs (Bone Marrow Mesenchymal Stem Cells) were purchased from Lonza and cultured according to the manufacturer's instructions. In this way, human exfoliated deciduous tooth pulp stem cells (SHED) were obtained. The obtained SHEDs were cultured at approximately 1 × 10 for each sample using a FACSTARPLUS (Becton Dickinson). 6 STRO-1 positive cells were sorted as follows.

[0193] The proliferation rate of SHED cells was assessed by incorporation of BrdU for 12 hours according to the manufacturer's instructions for the bromodeoxyuridine (BrdU) staining kit (Invitrogen). Experiments were repeated five times (n = 3 per group). Statistical significance was assessed using one-way analysis of variance followed by the Tukey-Kramer test.

[0194] For immunofluorescence detection of STRO-1, SHEDs were fixed with 3% paraformaldehyde, rinsed twice with PBS, and treated with 100 mM glycine for 20 min. The cells were then permeabilized with 0.2% Triton-X (Sigma-Aldrich) for 30 min and then incubated in a mixture of 5% donkey serum and 0.5% bovine serum albumin for 20 min.

[0195] Next, the cells were incubated with mouse anti-human STRO-1 antibody (1:100, R&D) for 1 hour, followed by goat anti-mouse immunoglobulin M-FITC antibody (1:500, Southern Biotech) for 30 minutes. The cells were then mounted with Vectashield DAPI (Vector Laboratories Inc.). The sorted cells were then seeded into 6-well plates containing α-MEM supplemented with 15% FBS for cloning. Approximately 300 colonies were pooled for testing.

[0196] (2) Gene transfer (2-1) Gene transfer using adenovirus vectors As described above, four genes, bmi-1, E6, E7, and hTERT, were inserted into adenovirus vectors to generate viral vectors expressing these gene products. As a control, a control vector without these genes was also prepared.

[0197] SHED was placed in a 100 mm diameter collagen-coated dish at 1 × 10 6 100 cells were seeded on the cells, and DMEM supplemented with 10% FBS was added and cultured until subconfluent. The medium was removed by aspiration, and 500 μL of virus solution diluted with the above medium was added (MOI = 10). The cells were cultured at 37°C in a 5% CO2 incubator for 1 hour to infect the cells with the above virus vector. 48 hours after infection, the infected cells were transferred to the above medium supplemented with puromycin (1 pg / mL) and cultured for 10 days for selection, and 500-600 resistant clones were pooled. Approximately 0.5 x 10 cells were cultured every 3-4 days. 5The SHEDs were seeded in a 100 mm diameter culture dish and subcultured. The SHEDs into which the gene was introduced were designated SHED-T, and the SHEDs without the gene introduced were designated SHED-C.

[0198] (2-2) Gene transfer using retroviral vectors. The retroviral vectors obtained as described above were infected into human dental pulp-derived cells (KA_01_W14_P5D3) or human dental pulp-derived cells (KA_02_W4-6_P4D3) to obtain drug-resistant pooled cells (hereinafter simply referred to as "pooled cells"). The resulting pooled cells were subjected to real-time PCR analysis to confirm the number of inserted genes. The primers listed in Table 10 below (all manufactured by Takara Bio) were used for real-time PCR.

[0199]

[0200] The KA_01_W14_P5103 cells were cultured in Corning cell culture dishes (6- or 10-cm diameter cell culture dishes, TC-treated surface) at 37°C in a 5% CO2 incubator. The passage number at the time of seeding was designated as [0] (hereafter, "+P0"), with the first passage designated as "+1" and the second passage designated as "+2." When the cells reached approximately 80% confluency, the culture supernatant was removed, washed two to three times with PBS, and then incubated with 0.25% trypsin-EDTA solution (1x) (containing phenol red) (Thermo Fisher Scientific, 25200-056) as a detachment agent for 3 minutes at 37°C in a 5% CO2 incubator to detach the adherent cells from the dish bottom.

[0201] An appropriate amount of medium was added to the mixture and pipetted to prepare a single-cell cell suspension. The cell suspension was then diluted appropriately and subcultured in a new dish. The culture was continued in the same manner, and no abnormalities in cell growth or morphology were confirmed. Furthermore, the culture supernatant collected during subculture was confirmed to be free of mycoplasma contamination using a MycoAlert Mycoplasma Detection Kit (Loza, product code LT07-318).

[0202] (3) Study of drug resistance concentration To select transfected cells using drugs, we studied the G418 resistance concentration of target cells. Corning 6-well plates (6 Well Clear TC-Treated Multiple Well Plate, product code 3516) were used, and 2.0 x 10 cells in the logarithmic growth phase were placed per well. 4 The cells were seeded in a 5% CO2 incubator at 37°C for approximately 24 hours, after which the adhesion of the cells to the bottom of the wells was confirmed. The medium in each well was replaced with medium containing different concentrations of G418. Photographs of the cells were taken 4, 8, and 10 days after the start of drug selection, and the drug resistance concentration was examined.

[0203] Four days after the start of drug selection, cell proliferation activity was lost in wells with drug concentrations of 250 μL / mL or higher, and after 11 days, most cells were confirmed to have died in wells with drug concentrations of 500 μL / mL or higher. Based on these results, the G418 selection drug concentration for target cells was determined to be 500 μL / mL, which shows a sufficient selective effect.

[0204] The retroviral vector solution was infected into target cells using the polybrene method, and the immortalized gene stable expression clones were selected using medium B. 1.40 x 10 cells in the logarithmic growth phase were placed in each well of the 6-well plate. 5Cells were seeded and cultured at 37°C in a 5% CO2 incubator for approximately 24 hours. Polybrene (Hexadimethrine bromide, Sigma-Aldrich, product code H9268) was then added to a recombinant retroviral vector solution diluted 4-fold or 10-fold with medium A to a final concentration of 8 μg / mL, and equal volumes were added to each well to infect the cells with the virus. Approximately 4 hours after viral infection, 1.0 mL of medium A was added to each well. The plate was incubated at 37°C in a 5% CO2 incubator, and after 24 hours, medium B was added to begin drug selection.

[0205] After 10 days of drug selection, cells that had become drug-resistant and proliferated were collected, and 1 mL of CELLBANKER 1 Plus (Nihon Zenyaku Kogyo Co., Ltd., product code CB021) was added to the cells, and the cells were diluted to 1x10 6 A suspension containing 100 cells / mL was prepared and stored at -80°C as a cell stock for transplantation.

[0206] (Example 3) Preparation of iCas9 gene-introduced stem cells (1) Preparation of viral vector A plasmid containing the iCas9 and GFP genes (pMSCV-F-del-Casp9.IRES.GFP (Plasmid #15567), SEQ ID NO: 2) was obtained from Addgene (hereinafter referred to as "iCas9 plasmid").

[0207] The iCas9 gene and GFP gene were incorporated into a viral vector using the iCas9 plasmid described above. Platinum-A Retroviral Packaging Cell Line, Amphotropic (Cosmo Bio) was used to generate the viral vector. Cells from the Platinum-A Retroviral Packaging Cell Line, Amphotropic, were quickly thawed in a 37°C water bath. The thawed cells were suspended in a 15 mL tube containing 10 mL of DMEM medium and centrifuged at 1,300-1,500 rpm. The supernatant was discarded, and 2 mL of medium was added to the tube and gently suspended. The cell suspension was added to a 10 cm diameter culture dish containing 8 mL of medium and cultured until 70-90% confluent.

[0208] The cells in the dish were washed with PBS, and 4 mL of 0.05% Tripsin-0.5 mM EDTA was added to the dish and incubated at 37°C for 3 minutes. The dish was washed three times with 4 mL of Tripsin-EDTA, and then medium was added to detach the cells, which were then collected in a 50 mL tube. The collected cells were centrifuged at 1,300-1,500 rpm, the supernatant was discarded, and 5 mL of 10% FBS-containing DMEM was added to the tube and suspended. The cells were seeded in a 24-well plate until the cell count reached 2 x 10 4 The culture was continued until the density reached 0.5 cells / mL.

[0209] 0.5 μg of iCas9 plasmid and 1.5 μL of Fugene6 (Promega) were mixed and added to each well. The next day, the cells were washed with PBS, replaced with fresh medium, and cultured at 37°C for 3 days. The culture supernatant was centrifuged and passed through a 0.22 μM filter (Merck) to prepare a viral vector solution.

[0210] (2) Preparation of iCas9-transduced stem cells The iCas9 gene and GFP gene were introduced into the immortalized SHED prepared in Example 2 using the viral vector prepared in (1) above. First, the immortalized SHED were suspended in 10% FBS-containing DMEM medium and placed in a 24-well plate (Greiner) at 2 × 10 4 Cells were seeded at 1000 cells / well. 0.5 mL of the virus vector solution obtained in (1) above was added to each well, followed by 2.5 μL of polybrene (1 mg / mL, Sigma) (final concentration: 5 μg / mL), and the cells were cultured overnight at 37°C in a 5% CO2 incubator. The next day, the cells in each well were washed with PBS, and fresh 10% FBS-containing DMEM medium was added. The cells were then cultured at 37°C in a 5% CO2 incubator for an additional 10 days.

[0211] After the culture was completed, iCas9-GFP-positive and -negative cells were present, and the iCas9-GFP transfection efficiency was approximately 40%. The transfected cells in this state were used to confirm drug-dependent apoptosis induction as described in Example 4.

[0212] Example 4 Confirmation of Drug-Dependent Apoptosis Induction DMEM medium containing 10% FBS containing 0, 10, 100, or 1000 nM AP20187 (purchased from Sigma) was prepared, and 0.5 mL of each medium was added to each well of a 24-well plate. 5 × 10 cells were added to each of the four types of medium. 5 The cells were added to the wells at a concentration of 1000 cells / well and cultured at 37°C in a 5% CO2 incubator for 24 hours (see Figure 1(A)). An optical microscope image of the culture results after 24 hours is shown in Figure 1(B).

[0213] As shown in Figure 1(B), the number of transfected cells prepared in Example 3 decreased in an AP20187 concentration-dependent manner. The cells cultured as described above were then collected from each well and placed in a 1.5 mL tube. These tubes were centrifuged at 400 x g, and the supernatant was discarded. After centrifugation, 100 μL of PBS was added to each tube to suspend the cells. Then, 0.5 μL of 7-AAD (Biolegend), a dead cell stain, was added to each tube and the tube was left to stand at room temperature for 5 minutes to stain dead cells, resulting in the production of stained cells.

[0214] The stained cells were subjected to FACS. FACS was performed using a FACSCanto II flow cytometer (BD Biosciences, hereafter referred to as the "FACS instrument"). Each tube containing the stained cells was placed in the FACS instrument, and the instrument settings were adjusted according to the manual. GFP and 7AAD were both excited with 488 nm excitation light, and the respective fluorescence was separated using a 502LP or 655LP dichroic mirror. Subsequently, the wavelengths passing through a 530 / 30 or 670LP bandpass filter were detected.

[0215] The results of FACS are shown in Figure 2. Figure 2 shows the width (W) and area (A) of the side scatter (SSC) and forward scatter (FSC) of the cells. SSC indicates the granularity and internal complexity of the cells, while FSC indicates the size of the cells.

[0216] Figure 3 shows the results of the cell death rate obtained in Example 3 in the presence of various concentrations of AP20187. The horizontal axis in the upper row represents the fluorescence intensity of 7AAD staining, and the horizontal axis in the lower row represents the fluorescence intensity of GFP expression. The vertical axes both represent the number of cells. The four graphs show the results when the AP20187 concentration was set to 0, 10, 100, and 1000 nM, from left to right.

[0217] In the figure, the numbers (e.g., 92.7, 84.3, etc.) indicate the percentage of cells present within the range of the black bar (line) below each number as a percentage of the total cell number. The thick downward arrows in the figure indicate a decrease in iCas9-GFP-expressing SHED. The numbers in the upper and lower rows of Figure 3 are shown as graphs in Figures 4(A) and 4(B).

[0218] The bottom panel of Figure 3 shows that at 0 nM AP20187, two populations of iCas9-GFP-transfected SHEDs and non-iCas9-GFP-transfected SHEDs were present, with iCas9-GFP-transfected SHEDs accounting for 38.9% of the total. However, with increasing AP20187 concentrations to 10, 100, and 1000 nM, the proportion of iCas9-GFP-transfected SHEDs significantly decreased to 2.6, 3.3, and 4.3%, respectively. These results indicate that only iCas9-GFP-transfected SHEDs underwent AP20187 concentration-dependent cell death, demonstrating that iCas9-GFP-transfected SHEDs undergo drug-dependent cell death.

[0219] On the other hand, in the upper panel of Figure 3, at 0 nM AP20187, 92.7% of cells were unstained with 7AAD, indicating that most cells survived. In contrast, at 10 nM and 100 nM AP20187 concentrations, the percentage of unstained cells decreased to approximately 84%. At 1000 nM AP20187, the percentage of unstained cells further decreased to 66.9%. This confirms that iCas9-GFP-transfected SHED cells died in an AP20187 concentration-dependent manner.

[0220] Furthermore, when the concentration of AP20187 was 1000 nM, the percentage of unstained cells, i.e., live cells, was 66.9%, which, when taken together with the percentage of iCas9-GFP-introduced SHED of 38.9%, indicates that only iCas9-GFP-introduced SHED selectively underwent cell death, while iCas9-GFP-unintroduced SHED was not affected.

[0221] These results demonstrate that drug-dependent cell death is induced in iCas9-introduced immortalized stem cells without affecting non-iCas9-introduced immortalized stem cells, and that this allows selective elimination of iCas9-introduced immortalized stem cells.

[0222] The present invention is useful in the fields of cell medicine, regenerative medicine, transplant medicine, and the like.

[0223] SEQ ID NO: 1: Nucleotide sequence of the iCas9 gene. SEQ ID NO: 2: Nucleotide sequence of pMSCV-F-del-Casp9.IRES.GFP. SEQ ID NO: 3: Nucleotide sequence (1) of a DNA fragment containing a gene to be introduced into a cell. SEQ ID NO: 4: Nucleotide sequence (2) of a DNA fragment containing a gene to be introduced into a cell. SEQ ID NO: 5: Nucleotide sequence (3) of a DNA fragment containing a gene to be introduced into a cell. SEQ ID NO: 6: Nucleotide sequence (4) of a DNA fragment containing a gene to be introduced into a cell.

[0224] SEQ ID NO: 7: The base sequence (5) of the DNA fragment containing the gene to be introduced into the cell. SEQ ID NO: 8: The base sequence (6) of the DNA fragment containing the gene to be introduced into the cell. SEQ ID NO: 9: The base sequence (7) of the DNA fragment containing the gene to be introduced into the cell. SEQ ID NO: 10: The base sequence (8) of the DNA fragment containing the gene to be introduced into the cell. SEQ ID NO: 11: The base sequence (9) of the DNA fragment containing the gene to be introduced into the cell.

[0225] SEQ ID NO: 12: PCR primer (1). SEQ ID NO: 13: PCR primer (2). SEQ ID NO: 14: PCR primer (3). SEQ ID NO: 15: PCR primer (4). SEQ ID NO: 16: PCR primer (5). SEQ ID NO: 17: PCR primer (6).

[0226] SEQ ID NO: 18: PCR primer (7). SEQ ID NO: 19: PCR primer (8). SEQ ID NO: 20: PCR primer (9). SEQ ID NO: 21: PCR primer (10).

Claims

1. Stem cells transfected with the gene for caspase-9, which is activated in a drug-dependent manner.

2. The stem cells according to claim 1, characterized in that the drug is AP20187 or AP1903.

3. Stem cells according to claim 1 or 2, characterized in that the stem cells are mesenchymal stem cells.

4. The stem cells according to claim 3, characterized in that the mesenchymal stem cells are immortalized stem cells.

5. The stem cells described in claim 4, characterized in that the immortalized stem cells have at least one gene selected from the group consisting of TERT, Bmi-1, human papillomavirus E6, and human papillomavirus E7.

6. The stem cell according to claim 1 or 2, wherein the caspase-9 gene is the gene shown in SEQ ID NO:

1.

7. A method for producing immortalized stem cells carrying the Caspase-9 gene, comprising: a step of producing a DNA fragment containing a drug-dependently activated Caspase-9 gene and multiple genes for immortalizing cells; a vector production step of producing a viral vector incorporating the DNA fragment; a gene transfer step of transfecting mammalian stem cells with the viral vector to introduce the gene into the stem cells and produce gene-transduced cells; and a selection step of selecting gene-transduced stem cells from the stem cells obtained in the gene transfer step.

8. A method for producing immortalized stem cells transfected with the Caspase-9 gene according to claim 7, characterized in that the mammalian stem cells are mesenchymal stem cells.

9. The method for producing immortalized stem cells transfected with the Caspase-9 gene according to claim 7, characterized in that the DNA fragment contains, in addition to the Caspase-9 gene, any one of a set of genes selected from the group consisting of (a) to (d) below: (a) human papillomavirus E7 and TERT; (b) Bmi-1 and TERT; (c) Bmi-1, human papillomavirus E6 and TERT; and (d) Bmi-1, human papillomavirus E6, human papillomavirus E7 and TERT.

10. A method for producing immortalized stem cells carrying the Caspase-9 gene as described in claim 7, characterized in that the viral vector is produced using any virus selected from the group consisting of adenovirus, lentivirus, and retrovirus.

11. A method for producing immortalized stem cells transfected with the Caspase-9 gene according to claim 7, characterized in that the immortalized stem cells are selected using a drug.

12. A method for producing immortalized stem cells transfected with the Caspase-9 gene according to any one of claims 7 to 9, characterized in that the Caspase-9 gene is the gene shown in SEQ ID NO: 1.

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