Method for producing deimmortalized cells from reversibly immortalized cells

Abstract

The problem addressed by the present invention is to provide a method for efficiently producing highly safe cells, in which the Sendai virus vector has been removed and which do not cause adverse effects such as triggering tumor formation, from reversibly immortalized cells containing a Sendai virus vector. To this end, the present invention provides: a method for producing deimmortalized cells from reversibly immortalized cells comprising (1) a removal step for removing the Sendai virus vector from reversibly immortalized cells comprising animal cells that contain a Sendai virus vector containing an immortalization gene and (2) an isolation step for isolating the deimmortalized cells from which the Sendai virus vector has been removed from the cells after the removal step; deimmortalized cells produced by the method; and a regenerative medical product containing the immortalized cells.

Description

Method for producing de-immortalized cells from reversibly immortalized cells

[0001] The present invention relates to a method for producing de-immortalized cells from reversibly immortalized cells, de-immortalized cells produced by the method, and regenerative medicine products containing the de-immortalized cells.The present invention further relates to a method for removing a temperature-sensitive Sendai virus vector from reversibly immortalized cells consisting of animal cells containing a temperature-sensitive Sendai virus vector containing an immortalization gene.The present invention also relates to a method for lengthening telomeres in animal cells.

[0002] Cultured cells with differentiation potential are essential biological materials in biomedical, pharmaceutical, and clinical fields. In particular, extending the lifespan and improving the differentiation potential of human mesenchymal stem cells (hMSCs)—through the stable supply of specific cell resources, and in some cases extracellular vesicles (EVs)—are expected to significantly contribute to basic research, drug discovery, and cell therapy. However, because primary cultured cells continue to divide, their lifespan is limited, making it difficult to maintain sufficient cell numbers while maintaining cellular characteristics. To overcome this problem, attempts have been made to introduce immortalizing genes into cells to extend their lifespan or enable indefinite proliferation. Well-known immortalizing genes include the telomerase reverse transcriptase (TERT) gene, oncogenes (c-myc, Bmi-1), cell cycle regulatory genes (mutant CDK4, CCND1), and viral genes (SV40T, HPV E6 / E7, EBV). Furthermore, Sendai virus vectors are RNA viral vectors that demonstrate high gene expression in various mammalian species, including humans, and have high gene transfer efficiency. Because Sendai virus vectors are non-integrating viral vectors, transgenes are expressed in the cytoplasm of host cells. Furthermore, by changing the insertion site of a gene into the vector, Sendai virus vectors allow for the adjustment of gene expression levels and the simultaneous expression of multiple genes, and are widely used to induce pluripotent stem cells, particularly iPS cells (Patent Document 1). However, in cells such as mesenchymal stem cells (MSCs), which have a limited number of divisions and cease division after two months or more, it has been recognized that Sendai virus vectors are difficult to achieve long-term expression of transgenes, and therefore their use is limited to temporary high expression. On the other hand, because Sendai virus vectors do not pose a risk of chromosomal integration, they are expected to be able to introduce immortalizing genes into cells and express genes without damaging the host chromosome into which the vector has been introduced, while maintaining the properties of the host cell. Furthermore, it has been reported that mutant Sendai virus vectors can induce temperature-sensitive viral replication arrest when gene expression is no longer required by applying temperature-sensitive conditions.However, cells immortalized by introducing an immortalization gene into cells using a Sendai virus vector retain the immortalization gene after proliferation, raising concerns about adverse effects such as tumor formation in the host. Therefore, to avoid such risks and put immortalized cells into practical use, it is necessary to remove as much of the Sendai virus vector containing the immortalization gene as possible. Patent Document 2 discloses the immortalization of MSCs using a temperature-sensitive Sendai virus vector and the cancellation of immortalization by temperature change. However, Patent Document 2 does not disclose a method for removing the Sendai virus vector to the extent that it is undetectable by a highly sensitive method (e.g., RT-qPCR). Conventional Sendai virus vectors can also be removed by inhibiting the expression of proteins involved in the replication of the Sendai virus vector. One example of such a method is a method using siRNA. siRNAs capable of inhibiting the expression of proteins involved in the replication of Sendai virus vectors are described, for example, in Patent Document 3 and Non-Patent Document 1.

[0003] Patent No. 5763340 International Patent Publication No. 2022 / 097716 Pamphlet International Patent Publication No. 2010 / 134526 Pamphlet

[0004] Nishimura et al. J. Biol. Chem. 2011. Vol. 286, No. 6, pp. 4760-4771

[0005] When cells such as MSCs are obtained for transplantation in regenerative medicine, they are immortalized by infection with a Sendai virus vector carrying an immortalization gene and then expanded to the required quantity. After expansion, to avoid risks associated with the presence of the immortalization gene, it is necessary to remove as much of the Sendai virus vector carrying the immortalization gene from the cells to be transplanted. Therefore, an object of the present invention is to provide a method for efficiently obtaining safe cells from which as much of the Sendai virus vector carrying the immortalization gene has been removed. As a result of intensive research conducted by the present inventors to achieve this object, they have found that de-immortalized cells can be produced by removing the Sendai virus vector from reversibly immortalized cells containing the Sendai virus vector, thereby isolating de-immortalized cells from which the Sendai virus vector has been removed. Specifically, the present invention encompasses the following: (1) A method for producing de-immortalized cells from reversibly immortalized cells, the method comprising a removal step of removing the Sendai virus vector from reversibly immortalized cells consisting of animal cells containing a Sendai virus vector carrying an immortalization gene. (2) The method according to (1), wherein the Sendai virus vector is a temperature-sensitive Sendai virus vector, and the temperature in the removal step is changed to a temperature that inactivates the temperature-sensitive Sendai virus vector and the temperature-sensitive Sendai virus vector is maintained under the temperature conditions. (3) The method according to (1), wherein the removal step is carried out by suppressing expression of a protein involved in replication of the Sendai virus vector. (4) The method according to (3), wherein expression of a protein involved in replication of the Sendai virus vector is suppressed by RNA interference. (5) The method according to (4), wherein RNA interference is carried out by introducing siRNA. (6) The method according to any one of (1) to (5), further comprising, before the removal step, a culture step of culturing reversibly immortalized cells consisting of animal cells containing the Sendai virus vector containing the immortalization gene. (7) The process according to any one of (1) to (5), further comprising a separation step of separating de-immortalized cells from which the Sendai virus vector has been removed from the cells after the removal step.(8) The method according to (7), further comprising a harvesting step of harvesting the de-immortalized cells after the separation step. (9) The method according to (6), further comprising a measuring step of harvesting a portion of the reversibly immortalized cells after the culture step and measuring the telomere length of the cells. (10) The method according to (9), wherein the removal step is carried out after measuring the telomere length of the reversibly immortalized cells after the culture step to confirm that the telomere length of the reversibly immortalized cells is significantly longer than that of cells not treated with the Sendai virus vector containing the immortalizing gene for the same number of culture days. (11) The method according to any one of (1) to (10), wherein the immortalizing gene comprises one or more genes selected from the group consisting of a Bmi-1 gene, a TERT gene, and an SV40T gene. (12) The method according to any one of (1) to (11), wherein the Sendai virus vector further comprises a gene encoding a marker protein. (13) The method according to (12), wherein the labeling protein is a fluorescent protein. (14) The method according to (7), wherein the de-immortalized cells are separated using the labeling protein as an indicator in the separation step. (15) The method according to any one of (2), (6) to (14), wherein the temperature-sensitive Sendai virus vector is maintained at a temperature of 38°C to 40°C in the removal step. (16) The method according to (15), wherein the reversibly immortalized cells are maintained at a temperature of 38°C to 40°C for 0.5 to 2 days in the removal step. (17) The method according to (15) or (16), wherein the reversibly immortalized cells are maintained at a temperature of 38°C to 40°C in the removal step, and then maintained at a temperature of 36°C to 37.5°C in the removal step. (18) The method according to (17), wherein the reversibly immortalized cells are maintained at a temperature of 36°C to 37.5°C for 6 to 10 days. (19) The method according to any one of (1) to (18), wherein the animal cell is a somatic cell. (20) The method according to (19), wherein the somatic cell is a somatic stem cell. (21) The method according to (20), wherein the somatic stem cell is a mesenchymal stem cell. (22) A de-immortalized cell produced by the method according to any one of (1) to (21).(23) The cells according to (22), in which telomere length is significantly extended over the same number of culture days compared to cells not treated with the Sendai virus vector containing the immortalization gene. (24) A regenerative medicine product comprising the de-immortalized cells according to (22) or (23). (25) A method for removing a temperature-sensitive Sendai virus vector from reversibly immortalized cells consisting of animal cells containing a temperature-sensitive Sendai virus vector containing an immortalization gene, the method comprising a removal step of removing the temperature-sensitive Sendai virus vector from the reversibly immortalized cells. (26) The method according to (25), further comprising a separation step of separating de-immortalized cells from which the Sendai virus vector has been removed from the cells after the removal step. (27) The method according to (25) or (26), in which the Sendai virus is a temperature-sensitive Sendai virus vector, and in the removal step, the temperature is changed to a temperature that inactivates the temperature-sensitive Sendai virus vector and the temperature-sensitive Sendai virus vector is maintained under that temperature condition. (28) A method for extending telomere length in animal cells, comprising: [1] an infection step of infecting animal cells with a Sendai virus vector containing an immortalization gene; and [2] a culture step of culturing the animal cells infected with the Sendai virus vector. The present invention makes it possible to efficiently remove the Sendai virus vector from reversibly immortalized cells containing a Sendai virus vector containing an immortalization gene. De-immortalized cells from which the Sendai virus vector has been removed by the method of the present invention have the Sendai virus vector removed to below the detection limit of a highly sensitive detection method, so there is no concern that the immortalization gene carried by the Sendai virus vector will adversely affect a host receiving the cells. The present invention makes it possible to obtain such safe cells. Furthermore, it has been found that such de-immortalized cells have elongated telomeres, which are responsible for the cell's lifespan, and are capable of proliferating over a long period of time. Therefore, the method of the present invention makes it possible to efficiently obtain large quantities of cells required for regenerative medicine. This specification includes the disclosure of Japanese Patent Application No. 2024-101097, from which the present application claims priority.

[0006] Figure 1 is a photograph showing the results of measuring the expression of green fluorescent protein (GFP), red (orange) fluorescent protein (OFP), and blue fluorescent protein (BFP) in mesenchymal stem cells transfected with a Sendai virus (SeV) vector carrying a fluorescent gene together with an immortalization gene. Figure 2 is a diagram showing the schedule (condition 1 and condition 2) for removing the Sendai virus vector by raising the temperature of mesenchymal stem cells (hMSCs) transfected with a temperature-sensitive SeV vector to 37°C or 39°C, sorting of fluorescent-negative cells by FACS, and expansion of the sorted cells. Figure 3 is a photograph showing the expression of fluorescent proteins (GFP, OFP, and BFP) in mesenchymal stem cells and bright-field images after removing the temperature-sensitive SeV vector under the two conditions (condition 1 and condition 2) shown in Figure 2. The photograph on the left is of cells cultured at 35°C for 9 days, the photograph in the center is of cells cultured at 37°C for 9 days, and the photograph on the right is of cells cultured at 39°C for 1 day and then at 37°C for 8 days. Figure 4 shows the results of RT-qPCR measurements of the amount of SeV vector in hMSCs treated with each of the following methods: the leftmost column in Figure 4 shows the results of measurements in parent hMSCs not transfected with the SeV vector (hMSCs); the second column from the left shows the results of measurements in hMSCs transfected with the SeV vector and cultured at 35°C (hMSCs GOB 35°C); the second column from the right shows the results of measurements in hMSCs transfected with the SeV vector and cultured at 37°C followed by FACS sorting of fluorescence-negative cells (hMSCs GOB 37°C after sorting); and the rightmost column shows the results of measurements in hMSCs transfected with the SeV vector and cultured at 39°C and 37°C followed by FACS sorting of fluorescence-negative cells (hMSCs GOB 39-37°C after sorting). The vertical axis shows the relative amount of SeV vector in each treatment group, with the amount of SeV vector measured in hMSC GOB at 35°C set to 1. Figure 5 shows the results of infecting hMSCs with SeV vector, changing the temperature according to condition 2 in Figure 2 on day 22 after infection, sorting by FACS on day 32, and culturing hMSCs from which it was confirmed that the SeV vector had been removed, and assessing their proliferation ability.The open circles represent hMSCs infected with the SeV vector, while the crosses represent control cells that were not infected with the SeV vector. The horizontal axis represents the number of days since infection, and the vertical axis represents the number of cells. Figure 6 is a photograph showing the results of karyotype analysis of hMSCs that were removed after SeV vector introduction. The numbers below the photographs of each chromosome represent the human chromosome number. Figure 7 shows the results of relative quantitative analysis of telomeres in hMSCs that had undergone various treatments. In Figure 7 , the leftmost column shows the measurement results for hMSCs before treatment with SeV vector (hMSC Day 0), the second column from the left shows the measurement results for hMSCs cultured for 30 days without SeV vector treatment (hMSC Day 30), the second column from the right shows the measurement results for cells on Day 22 after SeV vector treatment (SeV-hMSC Day 22), and the rightmost column shows the measurement results for cells infected with SeV vector and cultured after vector removal (cells on Day 59 after SeV vector treatment) (rej-hMSC SeV-removed Day 59). The vertical axis shows the relative telomere amount, where the telomere amount measured on hMSC Day 0 is set to 1. Figure 8 shows the experimental schedule for infecting hMSCs with a conventional SeV vector carrying an immortalizing gene and then removing the SeV vector with siRNA. 9 is a photograph showing the results of investigating the reduction of SeV vectors when hMSCs were infected with a conventional SeV vector carrying an immortalization gene and then removed with siRNA, using the fluorescent protein OFP as an indicator. As shown in FIG. 8, OFP fluorescence was observed on days 1 (Day 1), 4 (Day 4), 8 (Day 8), 11 (Day 11), and 17 (Day 17).In Figure 9, hMSC (untreated) represents the results for cells not infected with the SeV vector, hMSCsiL527 represents the results for cells infected with the SeV vector and then removed with the target siRNA siL527, hMSCsiL1913 represents the results for cells infected with the SeV vector and then removed with the target siRNA siL1913, and hMSCsiLuc represents the results for cells infected with the SeV vector and then treated with the negative control siRNA siGL3Luc. Figure 10 is a graph showing the amount of SeV vector in hMSCs that had undergone various treatments, as determined by RT-qPCR. The leftmost column shows the results for cells transfected with control siRNA (siControl), the second column from the left shows the results for cells in which the SeV vector was removed with siL527 (siL527), the second column from the right shows the results for cells in which the SeV vector was removed with siL1913 (siL1913), and the rightmost column shows the results for untreated cells in which the SeV vector was not removed with siRNA. The vertical axis shows the amount of SeV vector in cells that underwent various treatments, with the amount of SeV vector measured in the siControl set at 1.

[0007] The present invention will be described in further detail. 1. Method for Producing De-immortalized Cells from Reversibly Immortalized Cells 1-1. Overview of the Method for Producing De-immortalized Cells The present invention provides a method for producing de-immortalized cells, which includes a removal step of removing the Sendai virus vector from reversibly immortalized cells consisting of animal cells containing a Sendai virus vector containing an immortalization gene. According to the method of the present invention, the Sendai virus vector can be inactivated and removed from reversibly immortalized cells containing a temperature-sensitive Sendai virus vector by maintaining the reversibly immortalized cells at a temperature at which the temperature-sensitive Sendai virus vector is inactivated. If necessary, the de-immortalized cells from which the Sendai virus vector has been inactivated and removed can be sorted using a marker protein expressed from a marker protein gene incorporated into the Sendai virus vector as an indicator, thereby reducing the amount of Sendai virus vector in the de-immortalized cells to below the detection limit of highly sensitive RT-qPCR. Furthermore, such de-immortalized cells have elongated telomeres, which are responsible for cell lifespan, and are capable of proliferating over a long period of time. Furthermore, even in the case of non-temperature-sensitive Sendai virus vectors, it is possible to eliminate the Sendai virus vector by reducing proteins involved in the replication of the Sendai virus vector. In the Examples herein, the time course of the cell number of human mesenchymal stem cells infected with a temperature-sensitive Sendai virus vector carrying an immortalization gene was measured. Specifically, reversibly immortalized cells infected with a temperature-sensitive Sendai virus vector carrying an immortalization gene were de-immortalized by raising the temperature, and then the de-immortalized cells were isolated and their proliferation during this period was examined. As a result, the reversibly immortalized cells infected with the Sendai virus vector had a higher proliferation capacity than uninfected cells, and acquired superior proliferation capacity during the period in which the immortalization gene was expressed. Furthermore, it was possible to de-immortalize the cells by removing the Sendai virus vector after infection with the Sendai virus vector.Therefore, by optimizing the timing of removing the Sendai virus vector after infection with the vector, it is possible to obtain a large amount of de-immortalized cells with high proliferation capacity. 1-2. Definitions (Cells) In the present invention, "cells" refers to all somatic cells other than germline cells (eggs and sperm, oocytes, ES cells, etc.) and totipotent cells (iPS cells). When the "cells" are derived from animals, they are referred to as "animal cells" in the present invention. Cells are preferably derived from mammals, such as humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses. Preferably, the animal cells are somatic cells, more preferably somatic stem cells, and even more preferably mesenchymal stem cells (MSCs). Here, "somatic cells" refer to cells other than germ cells that constitute a multicellular organism. Furthermore, somatic cells in the present invention may be primary cultured cells, passaged cells, or established cell lines. Specific examples of somatic cells include differentiated cells such as tissue-forming cells (adipocytes, fibroblasts, nerve cells, skin cells, blood cells, muscle cells, osteoblasts, chondrocytes, hepatocytes, pancreatic cells, kidney cells, cardiac muscle cells, brain cells, lung cells, spleen cells, adrenal gland cells, gingival cells, and periodontal ligament cells), or their precursor cells, immune system cells (B cells, T cells, monocyte cells, etc.), somatic stem cells [mesenchymal stem cells (adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord blood-derived stem cells, placenta-derived stem cells, etc.), hematopoietic stem cells, neural stem cells, epidermal stem cells, intestinal epithelial stem cells, dental pulp stem cells, and periodontal ligament stem cells, etc.]. (Immortalization) In the present invention, "immortalization" refers to overcoming the limitations on the number of cell divisions of initial cells and cell senescence, and imparting continuous cell division and proliferation capabilities. Specifically, this refers to a state in which passage under standard cell culture conditions is possible, usually for 5 or more passages, preferably 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, or 20 or more passages. Confluent cells at passage number 0 can be expanded and cultured by passaging them using a method known to those skilled in the art. Cells obtained by a single passaging operation are called "passage number 1 (or second generation)" cells, and can be expressed as "passage number 2, 3, 4, ... n (n (an integer) is the number of passages) (n+1 generation)" according to the number of passaging operations.The process may also include a step of freezing the cells between each passaging operation. (Immortalization Gene) In the present invention, the term "immortalization gene" refers to a gene that immortalizes cells, granting them the ability to proliferate indefinitely, without inducing cell death. Furthermore, the immortalization gene is an exogenous gene, meaning an immortalization gene newly introduced from outside the cell. For example, the immortalized cells of the present invention can be produced by introducing a specific immortalization gene into a cell using a Sendai virus vector. The immortalization gene in the present invention is not particularly limited as long as it immortalizes cells, granting them the ability to proliferate indefinitely, without inducing cell death. It may be an immortalization gene of non-human origin, or an immortalization gene modified to a form that can be expressed in target cells. In the present invention, the immortalization gene may be one or more genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene. A combination of two genes is preferred, and a combination of three genes is more preferred. When one type of gene is used, the TERT gene is preferred, and when two or more types of genes are combined, preferred examples of combinations include the combination of the Bmi-1 gene and the TERT gene, and the combination of the TERT gene and the SV40T gene. The "Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog) gene" is a gene that has two nuclear localization signals and encodes a protein that functions as one of the polycomb repressive complex 1 (PRC1) proteins localized in the cytoplasm or nucleus. As a PRC1 protein, Bmi-1 is involved in the regulation of expression of various genes, including Hox genes, by controlling chromatin remodeling and histone modification. Bmi-1 is known to control cell proliferation by suppressing the expression of p16 and p19Arf, which are involved in the cell cycle, and to play an important role in maintaining self-renewal by being involved in cell division of hematopoietic stem cells and neural stem cells.The "TERT (telomerase reverse transcriptase) gene" is a gene encoding telomere reverse transcriptase (TERT). Telomere reverse transcriptase (TERT) comprises telomerase, an enzyme that elongates specific repeat sequences at the ends of eukaryotic chromosomes (telomeres), from the telomere RNA component (TR or TERC) and other regulatory subunits. Cells have a telomere length monitoring mechanism, and while cellular senescence is caused by telomere shortening, TERT is known to play a role in maintaining telomere length. The "SV40T (simian virus 40 large T antigen) gene" is a gene encoding the simian virus 40 large T antigen. The SV40 (simian vaccinating virus 40) genome is divided into an early region that is expressed immediately after infection, a late region that is expressed during viral genome replication after infection, and a regulatory region that includes transcriptional control and replication origins. The early region encodes large T antigen, which is involved in the initiation of viral genome replication and inactivation of tumor suppressor gene products p53 and pRB, and small T antigen, which binds to and inhibits protein phosphatase PP2A. A specific example of the Bmi-1 gene used in the present invention is the mouse BMI1 gene (SEQ ID NO: 1), a specific example of the TERT gene is the human TERT gene (SEQ ID NO: 2), and a specific example of the SV40T gene is the SV40 large T antigen gene (SEQ ID NO: 3). The Bmi-1 gene, TERT gene, and SV40T gene may also be transcriptional variants, splicing variants, or orthologs thereof.The Bmi-1 gene, TERT gene, and SV40T gene may be genes consisting of a nucleotide sequence that shares 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively, as long as they have equivalent functions and activities. Alternatively, they may be genes in which several nucleotides (e.g., 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3) have been substituted, inserted, added, and / or deleted from the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively. Such homologous genes are also encompassed by the immortalized genes of the present invention. Furthermore, the Bmi-1 gene, TERT gene, and SV40T gene may be artificially modified so that their products are expressed as fusion proteins with other proteins or peptides, as long as they have equivalent functions and activities. (Immortalized Cells) In the present invention, "immortalized cells" refers to cells that have been "immortalized" to enable indefinite proliferation. More specifically, in the present invention, cells can be immortalized by introducing an immortalization gene into them, for example, by infecting the cells with a viral vector carrying a specific immortalization gene. Although the growth rate and duration of immortalized cells vary depending on the cell origin or culture conditions, as a result of subculture, under the same culture conditions, they can continue exponential growth for 20 days or more, preferably 60 days or more, and more preferably 80 days or more, even after the growth of untreated cells decreases or stops. (Reversibly Immortalized Cells) In the present invention, "reversibly immortalized cells" refer to cells that have been reversibly immortalized so that the immortalized state can be released after the cells have been immortalized. For example, one embodiment of "reversible immortalization" is to introduce the aforementioned "immortalization gene" into cells, render them capable of indefinite proliferation, and then remove the immortalization gene to stop or attenuate cell growth. (De-immortalized cells) In the present invention, "de-immortalized cells" refer to cells that have been de-immortalized by removing the immortalizing gene from "reversibly immortalized cells" to release them from the state of being capable of infinite proliferation. By removing the immortalizing gene from the above-mentioned "reversibly immortalized cells," it is possible to obtain "de-immortalized cells" in which cell proliferation has been stopped or attenuated.(Sendai virus vector) Sendai virus is a type of virus belonging to the genus Respirovirus in the family Paramyxoviridae, and contains a single minus-strand RNA (antisense strand relative to the sense strand encoding the viral protein) as its genome. Sendai virus can be converted into a vector, and a Sendai virus vector is a chromosomally non-integrating RNA vector. In this specification, Sendai virus vectors are also referred to as SeV vectors. The Sendai virus vector used in the present invention has the following characteristics: (i) extremely high efficiency of gene transfer and expression in various mammalian cells, including human; (ii) being a chromosomally non-integrating viral vector and expressed in the cytoplasm, the introduced gene is not integrated into the host chromosome, eliminating the risk of chromosomal structural changes; (iii) it is not a human pathogenic virus; (iv) it is possible to adjust the gene expression level or simultaneously express multiple genes by changing the insertion site in the vector; and (v) it is possible to remove the vector from the introduced cell after the purpose is achieved. The genome of the Sendai virus contains, in order from the 3' end to the 5' end, the NP (nucleocapsid) gene, the P (phospho) gene, the M (matrix) gene, the F (fusion) gene, the HN (hemagglutinin / neuraminidase) gene, and the L (large) gene. Of these, the Sendai virus can fully function as a vector if it has the NP gene, the P gene, and the L gene, and is able to replicate its genome in cells and express the genes it carries. Because the Sendai virus has minus-strand RNA in its genome, the 3' end of the genome is upstream and the 5' end is downstream, which is the opposite of the usual situation. The Sendai virus vector of the present invention includes infectious virus particles, as well as complexes consisting of a virus core, a complex of a virus genome and a virus protein, or a non-infectious virus particle, which are capable of expressing the genes they carry when introduced into cells.For example, ribonucleoproteins (viral core moieties) consisting of the Sendai virus genome and the Sendai virus proteins (NP, P, and L proteins) bound thereto can be introduced into cells to express transgenes in the cells. Introduction into cells can be carried out using an appropriate transfection reagent or the like. Therefore, such ribonucleoproteins (RNPs) are also encompassed by the Sendai virus vectors of the present invention. In the present invention, any of natural strains, wild-type strains, mutant strains, and commercially available Sendai virus vectors can be used. As long as the intended function can be achieved, the virus may have a structure similar to that of a virus isolated from nature, or may be a virus artificially modified by genetic recombination. 1-3. Removal Step 1-3-1. Overview of the Removal Step In the removal step of the present invention, the Sendai virus vector is removed from reversibly immortalized cells consisting of animal cells containing a Sendai virus vector containing an immortalization gene. As described in the section on the culture step in 1-4-1 below, reversibly immortalized cells can be prepared by culturing and infecting target cells with a Sendai virus vector into which an immortalization gene has been inserted. The reversibly immortalized cells of the present invention are (reversibly) immortalized by the immortalization gene contained in the Sendai virus vector, but the immortalization can be reversed by removing the Sendai virus vector, thereby producing de-immortalized cells. Removal of the Sendai virus vector removes the immortalization gene carried by the Sendai virus vector, thereby reversing the immortalization conferred by the immortalization gene. As described in 1-3-2 below, when a temperature-sensitive Sendai virus vector is used, the Sendai virus vector can be removed by changing the temperature to a temperature that inactivates the temperature-sensitive Sendai virus vector and maintaining the temperature at that temperature. When a non-mutated normal Sendai virus vector is used, removal can be achieved by suppressing the expression of proteins involved in the replication of the Sendai virus vector, as described in 1-3-3 below.In addition, the use of somatic stem cells, particularly mesenchymal stem cells, as cells is a preferred embodiment of the present invention. Here, "somatic stem cells" refer to cells collected from an animal or cells resulting from the division of such cells, which are pluripotent and maintain the ability to self-replicate. Furthermore, "mesenchymal stem cells" refer to a type of somatic stem cell that has the ability to differentiate into mesodermally derived tissues such as bone, cartilage, vascular, and cardiomyocytes. In the present invention, the immortalization gene is preferably one or more genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene, as described in detail above. 1-3-2. Method for Removing Temperature-Sensitive Sendai Virus Vectors Temperature-sensitive Sendai Virus vectors can be used as the Sendai Virus vectors of the present invention. "Temperature sensitivity" refers to a significant decrease in activity at normal cell culture temperatures (e.g., 37-38°C) compared to low temperatures (e.g., 30-36°C). For example, mutations such as Sendai virus TS7 (Y942H / L1361C / L1558I mutations in the L protein), TS12 (D433A / R434A / K437A mutations in the P protein), TS13 (D433A / R434A / K437A mutations in the P protein and L1558I mutation in the L protein), TS14 (D433A / R434A / K437A mutations in the P protein and L1361C mutation in the L protein), and TS15 (D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein) are temperature-sensitive mutations and can be suitably used in the present invention. For details of these Sendai virus vectors, reference can be made to Japanese Patent No. 5763340, WO 2015 / 046229, WO 2023 / 127871, etc. More specifically, the temperature-sensitive Sendai virus vector described above can be inactivated and the removal step can be achieved by maintaining the temperature-sensitive Sendai virus vector at a temperature of 38°C to 40°C. In a more preferred embodiment, the removal step can be achieved by maintaining the reversibly immortalized cells at a temperature of 38°C to 40°C for 0.5 to 2 days.In a more preferred embodiment, the removal step can be achieved by maintaining the reversibly immortalized cells at a temperature of 38°C to 40°C, followed by a temperature of 36°C to 37.5°C. In a more preferred embodiment, the removal step can be achieved by maintaining the reversibly immortalized cells at a temperature of 36°C to 37.5°C for 6 to 10 days. 1-3-3. Conventional Sendai virus vector removal method In an embodiment using a conventional Sendai virus vector, the removal step can be achieved by inhibiting the proliferation of the Sendai virus vector by suppressing the expression of a protein involved in the replication of the Sendai virus vector. Specific examples of proteins involved in the replication of the Sendai virus vector include the NP protein, P protein, and L protein. However, the proteins whose expression can be suppressed are not limited to these. Examples of methods for suppressing the expression of proteins involved in the replication of the Sendai virus vector include RNA interference (RNAi), nucleic acid aptamers, antisense nucleic acids, ribozymes, and aptamers, but an embodiment using RNAi is the most preferred. RNAi is a phenomenon in which mRNA with a sequence complementary to double-stranded RNA is specifically degraded. This phenomenon can be utilized to artificially introduce double-stranded RNA to suppress the expression of only the target gene. More specifically, means for achieving RNAi include siRNA (small interfering RNA), shRNA (short hairpin RNA), or miRNA (micro RNA) (including pri-miRNA and pre-miRNA). Among these, the use of siRNA is preferred. siRNA is a double-stranded RNA of about 20 base pairs, which is incorporated into Argonaute 2 protein, which forms the core of the protein complex RNA-induced silencing complex (RISC), to become single-stranded, and the target mRNA is cleaved by the RISC.For RNAi, refer to, for example, Bass B.L., 2000, Cell, 101, 235-238; Sharp P.A., 2001, Genes Dev., 15, 485-490; Zamore P.D., 2002, Science, 296, 1265-1269; Dernburg, A.F. & Karpen, G.H., 2002, Cell, 111, 159-162.The RNAi functional nucleic acid of these targets can be designed by using the techniques known in the art. Specific examples of siRNAs used in the present invention include, but are not limited to, siL527, siL913, N121, N335, P234, and P1021. 1-4. Other Optional Steps In addition to the removal step described in 1-3, the method of the present invention may include the following optional steps. 1-4-1. Culturing Step Before the removal step, a culturing step may be further included in which reversibly immortalized cells consisting of animal cells containing a Sendai virus vector containing an immortalization gene are cultured. This culturing step allows the reversibly immortalized cells to be maintained or expanded. This culturing step can be carried out according to the methods and conditions used for culturing conventional mammalian somatic cells. The medium used for culturing is not particularly limited; any medium commonly used for cell maintenance or expansion and suitable for viral infection may be used, and may be either a commercially available medium or a homemade medium.For example, basal media containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), specifically, Dulbecco's Modified Eagle's Medium (D-MEM) medium, Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (D-MEM / F-12) medium, Glasgow MEM (G-MEM) medium, Basal Medium Eagle (BME) medium, Minimum Essential Medium (MEM) medium, Eagle's minimal essential medium Examples of suitable medium include EMEM (Eutrex®) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, RPMI 1640 medium, Medium 199 medium, αMEM medium, Ham's medium, Fischer medium, and mixtures thereof. The medium may also contain growth factors (e.g., FGF, EGF), interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, antibiotics (e.g., penicillin, streptomycin), etc., as needed. The medium may be serum-containing or serum-free. To prevent contamination with components derived from different animal species, it is preferable to use serum-free or serum derived from the same animal species as the cells to be cultured. Serum substitutes, such as albumin, may also be used. Culture methods include, but are not limited to, three-dimensional culture under non-adhesive conditions, such as suspension culture (e.g., dispersed culture, aggregated suspension culture, etc.), two-dimensional culture under adhesive conditions, such as plate culture, or a combination of three-dimensional and two-dimensional culture. The culture vessel used for cell culture is not particularly limited as long as it is capable of culturing cells, and examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, etc. The culture vessel may be either non-adhesive or adhesive, and is selected appropriately depending on the purpose.The cell adhesive culture vessel may be treated with a cell support substrate such as an extracellular matrix to improve cell adhesion. Examples of cell support substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. The culture temperature is the temperature commonly used in this technical field, and is 30°C to 36°C, preferably 32°C to 35°C, and more preferably 33°C to 35°C. The culture is carried out under CO2. ２ In an atmosphere containing air, e.g., CO ２The method is carried out at a concentration of 2% to 5%. 1-4-2. Separation Step After the removal step described in 1-3, a separation step may be included in which de-immortalized cells from which the Sendai virus vector has been removed are separated from the cells that have been subjected to the removal step. The separation means used in this separation step is not particularly limited as long as it can separate de-immortalized cells from reversibly immortalized cells. Suitable separation methods include flow cytometry, which separates cells based on the fluorescence emitted by the target cells, and magnetic cell separation using magnetic beads. Furthermore, cells that do not express the HN protein on the Sendai virus vector on their cell surface can also be separated as de-immortalized cells using flow cytometry or magnetic cell separation using magnetic beads. In the following examples, a gene encoding a fluorescent protein, together with an immortalizing gene, is carried out in the Sendai virus vector, and de-immortalized cells are separated by a flow cytometry step using a FACS Asia Fusion (BD Biosciences) using the fluorescence derived from the expressed fluorescent protein as an indicator. In a preferred embodiment of the present invention, the Sendai virus vector may contain a gene encoding a marker protein together with an immortalization gene. In the method of the present invention, by incorporating a gene encoding a marker protein into the Sendai virus vector together with an immortalization gene, a separation step can be performed using the expressed marker protein as a marker, and removal of the Sendai virus vector containing the immortalization gene can be confirmed until the level of the marker protein falls below the detection limit of a highly sensitive detection method such as RT-qPCR. The term "marker protein" refers to a polypeptide whose activity can be used to determine the presence or absence and expression level of a marker gene or its fusion gene. Here, "based on activity" means based on the results of activity detection. The type of marker protein is not particularly limited, but is preferably one that is less invasive to animal cells into which the Sendai virus vector is introduced. Examples include fluorescent proteins and luminescent proteins. As used herein, "fluorescent protein" refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light. It may be either a natural or non-natural type.The excitation wavelength and fluorescence wavelength are not particularly limited. Specific examples of fluorescent proteins include green fluorescent protein, red fluorescent protein, blue fluorescent protein, and yellow fluorescent protein. As used herein, the term "luminescent protein" refers to a substrate protein that can emit light without the need for excitation light, or an enzyme that catalyzes the luminescence of the substrate protein. Examples include luciferin or aequorin as the substrate protein, and luciferase as the enzyme. 1-4-3. Harvesting Step The present invention may further include a harvesting step of harvesting de-immortalized cells after the separation step. This is a step of harvesting the de-immortalized cells obtained in the separation step for purposes such as basic research, drug discovery, and cell therapy. Specifically, the de-immortalized cells obtained in the separation step can be harvested in an appropriate container such as a sterilized bottle or a sterilized petri dish. In the method of the present invention, the separation step may also serve as the harvesting step. 1-4-4. Measuring Step of Telomere Length Measurement The method of the present invention may further include a measuring step of harvesting a portion of the reversibly immortalized cells in the culture step and measuring the telomere length of the cells. As shown in the examples herein, treating animal cells with a Sendai virus vector carrying an immortalization gene and culturing the cells (i.e., reversibly immortalized cells) can extend the telomere length. Therefore, by performing such a measurement process, reversibly immortalized cells with telomere lengths reaching the desired length can be obtained. Furthermore, when a removal process for removing the Sendai virus vector from the reversibly immortalized cells is performed, telomere length gradually shortens. For many applications, such as regenerative medicine products, it is preferable that the cells obtained by the method of the present invention have long telomere lengths, or in other words, are "young." Furthermore, cells with long telomere lengths can be cultured for long periods of time, making it easy to obtain large quantities of cells.In a preferred embodiment of the present invention, the telomere length of reversibly immortalized cells that have been cultured is measured in a measurement step to confirm that the telomere length of the reversibly immortalized cells is significantly longer than that of cells not treated with a Sendai virus vector containing an immortalizing gene for the same number of culture days, and then the Sendai virus vector removal step is carried out. Additionally, measuring the telomere length of reversibly immortalized cells is advantageous in maintaining the quality of the de-immortalized cells obtained by the method of the present invention and the cells derived from the de-immortalized cells. The point in the culture step at which the telomere length of the reversibly immortalized cells becomes significantly longer than that of cells not treated with a Sendai virus vector containing an immortalizing gene depends on many factors, including the type of immortalizing gene introduced, and therefore cannot be generally stated. In the Examples herein, telomere length was measured on day 22 after infection in human mesenchymal stem cells (MSCs) infected with a Sendai virus vector carrying the immortalization genes Bmi-1, hTERT, and SV40T. The results confirmed that telomere length was significantly longer than that before infection (Figure 7). It is believed that telomere length shortens with increasing culture time in cells not treated with a Sendai virus vector containing an immortalization gene. Therefore, it is believed that after approximately 20 days of culture after infection, the telomere length of reversibly immortalized cells is significantly longer than that of cells not treated with a Sendai virus vector containing an immortalization gene. 2. Immortalized Cells Produced by the Method of the Present Invention and Regenerative Medicine Products Comprising the Immortalized Cells. As described above in relation to the method for producing immortalized cells, the immortalized cells obtained by the method of the present invention have the immortalized Sendai virus vector removed to a level below the detection limit of RT-qPCR, a highly sensitive assay. In addition, it has been confirmed in the Examples that the de-immortalized cells obtained by the method of the present invention have no karyotypic abnormalities and do not affect chromosomes.Additionally, the de-immortalized cells obtained by the method of the present invention have extended telomere length over the same number of culture days compared to cells not treated with a Sendai virus vector containing an immortalization gene. Therefore, the de-immortalized cells obtained by the method of the present invention are safe cells in which the Sendai virus vector containing the immortalization gene has been highly removed and no abnormalities in the karyotype have been observed. Therefore, the de-immortalized cells produced by the method of the present invention are excellent materials for regenerative medicine products. In the present invention, "regenerative medicine products" are products prepared by processing living cells or tissues through culture, activation, differentiation induction, etc., and include products that reconstruct, repair, or form damaged bodily structures and functions, as well as cell therapy products administered for the treatment or prevention of disease. The regenerative medicine products of the present invention are cell-processed products derived from the de-immortalized cells obtained by the method of the present invention and used for regenerative medicine purposes. While the scope of the products is not particularly limited, examples include cell sheets, cellular cartilage, cell therapy drugs, and secreted extracellular vesicles. Method for Removing a Temperature-Sensitive Viral Vector from Reversibly Immortalized Cells The method for removing a temperature-sensitive Sendai virus vector from reversibly immortalized cells, which are animal cells containing a temperature-sensitive Sendai virus vector carrying an immortalization gene of the present invention, includes a removal step of removing the temperature-sensitive Sendai virus vector from the reversibly immortalized cells. The method may also include an optional separation step of separating de-immortalized cells from which the Sendai virus vector has been removed from the cells after the removal step. The removal and separation steps here are equivalent to those described in 1-3 and 1-4-2 above. In a preferred embodiment of the present invention, the removal step can be carried out by changing the temperature of cells containing a temperature-sensitive Sendai virus vector carrying an immortalization gene to a temperature that inactivates the viral vector and maintaining the cells under that temperature condition. Subsequently, de-immortalized cells from which the Sendai virus vector has been removed can be isolated, for example, using as an indicator a marker derived from a gene for a marker protein carried by the viral vector together with the immortalization gene.4. Method for extending telomere length in animal cells The method for extending telomere length in animal cells of the present invention comprises: (1) an infection step of infecting animal cells with a Sendai virus vector containing an immortalization gene; and (2) a culture step of culturing the animal cells infected with the Sendai virus vector. The infection step of the present invention is carried out by adding a Sendai virus vector containing an immortalization gene to a culture medium for animal cells, preferably mesenchymal stem cells, and infecting and introducing the cells with the Sendai virus vector. The immortalization gene here is as described in Section 1-2 "Immortalization Gene." Furthermore, a "Sendai virus vector containing an immortalization gene" refers to a Sendai virus vector into which such an immortalization gene has been incorporated. Then, by culturing animal cells infected with the Sendai virus vector into which the immortalization gene has been incorporated, the telomere length of the animal cells can be extended. The culture medium used in the culture step is the same as that described in Section 1-4-1. By culturing animal cells infected with a Sendai virus vector, the telomere length of the animal cells is extended by the immortalization gene carried by the Sendai virus vector. Cells with such extended telomere length can be cultured for long periods of time, which is advantageous for obtaining large quantities of target cells. The method can further include a step of removing the Sendai virus vector after infecting and culturing cells with a Sendai virus vector carrying an immortalization gene. This step is similar to the removal step described in 1-3. By performing this removal step, risks associated with the immortalization gene carried by the Sendai virus vector can be avoided. When a temperature-sensitively mutated Sendai virus vector is used as the Sendai virus vector, this removal step can be carried out by changing the temperature to a temperature that inactivates the temperature-sensitive Sendai virus vector and maintaining the temperature under these conditions. When a normal Sendai virus vector is used, the Sendai virus vector can be removed by suppressing the expression of proteins involved in the replication of the Sendai virus vector.Suppression of the expression of proteins involved in the replication of Sendai virus vectors can be achieved, for example, by RNA interference. While the means of RNA interference are not particularly limited, it can be preferably achieved by introducing siRNA. In the examples below, experimental data was obtained showing that when mesenchymal stem cells were infected with a Sendai virus vector containing an immortalization gene and cultured, telomere length was significantly increased on day 22 after infection compared to day 0 after infection. Furthermore, when the Sendai virus vector was subsequently removed and the cells were continued to be cultured, telomere length was shortened.

[0008] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1: Infection and removal of cells with a Sendai virus (SeV) vector carrying an immortalization gene 1. Infection and culture of human mesenchymal stem cells with a Sendai virus vector carrying an immortalization gene and a fluorescent protein gene The commonly used Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog), hTERT (human telomerase reverse transcriptase), and SV40T (simian virus 40 large T antigen) were selected as immortalization genes. The Bmi-1 gene (SEQ ID NO: 1), hTERT gene (SEQ ID NO: 2), and SV40T gene (SEQ ID NO: 3) were each incorporated into a temperature-sensitive Sendai virus (SeV) vector. To facilitate detection of the presence of the temperature-sensitive SeV vector, the fluorescent pigment genes red (orange) fluorescent protein (ORF), green fluorescent protein (GFP), and blue fluorescent protein (BFP) were also incorporated into the SeV vector and coexpressed with the immortalization gene described above. Specifically, a SeV vector was constructed in which the hTERT gene was co-loaded with a gene encoding GFP, the Bmi-1 gene was co-loaded with a gene encoding OFP, and the SV40T gene was co-loaded with a BFP gene. This resulted in the expression of various fluorescent proteins along with the immortalization genes, making it possible to easily detect the presence of the SeV vector. The temperature-sensitive SeV vector used was the TS15ΔF vector, which has been improved so that the viral vector disappears from cells when the culture temperature is changed from 35° C. to 37° C. These SeV vectors were used to verify the introduction and removal of SeV vectors carrying immortalizing genes using human mesenchymal stem cells (hMSCs) derived from human umbilical cord matrix.The medium used to culture hMSCs was composed of D-MEM (Low glucose) (Wako), 20% FBS (Cytiva), 0.01 mol / L Hepes (Wako), 100 units / ml Penicillin, 100 μg / ml Streptomycin (Wako), and 20 ng / ml bFGF. Because bFGF has a very short half-life, Gibco Heat Stable Recombinant Human bFGF (Thermo Fisher Scientific), which boasts excellent stability, was used. The medium was maintained at 5% CO. ２ hMSCs were cultured in an incubator. SeV vector infection was performed at a concentration of 2 × 10 ５ hMSC cells were infected with each immortalized SeV vector at an MOI of 40. Non-infected and infected conditions were each tested once. 24 hours after infection, the medium was replaced and maintenance culture was continued. Cell culture was performed at 35°C, 5% CO ２ The cells were cultured in an incubator. Figure 1 shows a bright-field image of the cells and photographs of the GFP, OFP, and BFP fluorescence expression measurements. As a result, it was confirmed that most cells expressed the three fluorescent proteins co-loaded with the immortalization gene after infection with the SeV vector. 2. Culture under varying temperature conditions and acquisition of SeV vector-cleared cells Before sorting the cells, culture for SeV vector removal was performed under two conditions (Figure 2). Condition 1 was at 37°C, 5% CO ２ Condition 2 was maintained in an incubator at 39°C and 5% CO for 9 days. ２ After culturing in an incubator for 1 day, the cells were incubated at 37°C and 5% CO ２The cells were cultured in an incubator for 8 days. As a result, under both conditions, fluorescent protein expression was no longer observed in most of the cells (Figure 3). On the other hand, fluorescent protein expression was observed in cells cultured at 35°C for 9 days. The cells were suspended in 1xD-PBS (Nacalai Tesque) supplemented with 1% FBS, and cells that were negative for BFP / OFP / BFP were sorted using a FACS Aria™ Fusion (BD Biosciences). After cell sorting, the cells were incubated at 37°C, 5% CO under both conditions shown in Figure 2. ２The cells were cultured for an additional 11 days in an incubator. RNA was then collected from the cells to confirm SeV vector removal, as described below. 3. Confirmation of SeV Vector Removal Removal of the SeV vector was confirmed using highly sensitive RT-qPCR. RNA was extracted from the cells using the RNeasy® kit (Qiagen). cDNA synthesis was performed using 1 μg of RNA with SuperScript® III reverse transcriptase (Thermo Fisher Scientific). 20 μl of cDNA was diluted with RNase-free water to a total volume of 250 μl, and qPCR reactions were performed using 5 μl per reaction as template and TaqMan™ Fast Advanced Master Mix (Applied Biosystems) on a StepOne™ Real-Time PCR system (Applied Biosystems). TaqMan Assay Mr04269880_mr (Thermo Fisher Scientific) was used to detect the SeV vector, and TaqMan Assay Hs01060665_g1 (Thermo Fisher Scientific) was used to detect β-actin (Actin beta: ACTB) as an endogenous control. PCR was performed under the following reaction conditions: 50°C for 2 minutes, 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. Analysis revealed that compared with the amount of SeV vector present in immortalized cells cultured at 35°C (hMSC GOB 35°C), the amount of SeV vector present in cells cultured and sorted at 37°C (hMSC GOB 37°C sorted) was extremely low. Cells sorted after culture at 39°C and 37°C (hMSC GOB 439-37°C sorted) were below the detection limit, similar to the parent hMSC (Figure 4). 4. Culturing of SeV Vector-Removed hMSCs hMSCs were infected with SeV vector, and then the temperature was changed from 35°C to 39°C for 1 day on day 22 after infection, then shifted to 37°C. Sorting was performed on day 32. hMSCs confirmed to have been removed from the SeV vector were cultured and their proliferation ability was evaluated. The results are shown in Figure 5.Control cells not infected with SeV vector were 10. ６ On the other hand, in cells infected with SeV vectors (extended survival cells), the number of infected cells eventually reached at least 10 １８ The number of cells at the time of SeV vector removal was 10 ６ Since there are only 10 units, even after removing the SeV vector, １２An increase in the number of individual cells was observed. These results suggest that removal of the SeV vector from immortalized cells can reverse immortalization and that the cells acquire greater proliferation ability than the original parent cell line (control cells) during the period when the immortalization gene is expressed. 5. Karyotype Analysis of SeV Vector-Removed hMSCs Karyotype analysis was performed to confirm the absence of chromosomal abnormalities in the SeV vector-removed cells. Samples were prepared by Carnoy fixation of dividing cells treated with 0.25 μg / mL colcemid solution (Nacalai) for 3 hours. Hoechst 33258 (Sigma) was added to McIlvaine's solution to a final concentration of 25 ng / mL, and staining was performed for 30 minutes. Chromosome images were captured using an Axio Imager Z2 (Zeiss) and analyzed using Ikaros software (MetaSystems). The analysis results are shown in Figure 6. As shown in Figure 6, the obtained cells were found to have a normal karyotype. This result suggests that the series of treatments involving SeV vector introduction, maintenance, and removal does not affect the karyotype of hMSCs. 6. Analysis of the effect of SeV vector treatment on telomere length. Relative quantitative analysis of telomeres was performed on cells cultured before SeV vector treatment, after SeV vector treatment, and after SeV vector removal. Genomic DNA was extracted from the cells using the Gentra® Puregene® Kit (Qiagen) according to the manufacturer's instructions. For real-time PCR, the primers used were the Telomere primer set and Single copy reference (SCR) primer set included with the Relative Human Telomere Length Quantification qPCR Assay Kit (ScienceCell Research Laboratories). The PCR reagent used was the 2x GoldNStart TaqGreen qPCR master mix included with the kit. PCR reaction conditions followed the manufacturer's recommended conditions. PCR reactions and data acquisition were performed using StepOnePlus (Life Technologies Japan), and data analysis was performed using StepOne Software v2.3. The results are shown in Figure 7.A statistically significant increase in telomere mass was observed in cells treated with SeV vectors on day 22 (SeV-hMSC Day 22) compared with hMSCs before treatment with SeV vectors (hMSC Day 0). This result suggests that telomere lengthening occurs due to the introduction of the immortalization gene by SeV vectors. Furthermore, telomere shortening was observed in cells cultured after SeV vector removal (cells treated with SeV vectors on day 59: rej-hMSC SeV-removed Day 59) compared with the cells on day 22 described above. This result suggests that telomere lengthening ceases when the SeV vector is removed, and that telomere length shortens with cell division. Furthermore, telomere mass was measured on day 30 (hMSC Day 30) in cells not treated with SeV vectors, and telomere length was shortened compared to day 0. Considering these results, it can be said that treating cells with SeV vectors "extends" the lifespan of those cells. Example 2: Removal of SeV vectors using siRNA We investigated whether the cell lifespan extension procedure using SeV vector treatment shown in Example 1 is also applicable to conventional SeV vectors that are not temperature-sensitive. SeV vectors were removed using siRNA. 1. Introduction of siRNA The medium used to culture hMSCs consisted of D-MEM (low glucose) (Wako), 20% FBS (Cytiva), 0.01 mol / L Hepes (Wako), Penicillin 100 units / ml, Streptomycin 100 μg / ml (Wako), and bFGF 20 ng / ml. Since bFGF has a very short half-life, we used Gibco Heat Stable Recombinant Human bFGF (Thermo Fisher Scientific), which has excellent stability. The cells were cultured in the above medium at 35°C and 5% CO. ２MSCs were cultured in an incubator. The siRNA sequences used were control siRNA (siGL3Luc) and target siRNAs (siL527, siL1913). The siRNAs used were synthesized, purified, and annealed by Nippon Gene Co., Ltd. These siRNAs were reported in Nishimura et al. J. Biol. Chem. 2011. Vol. 286, No. 6, pp. 4760-4771. The base sequences of the sense strand (S) and antisense strand (AS) of these siRNAs are as follows: siRNA transfection was performed using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocol. siRNA was introduced on days 0 (Day 0), 3 (Day 3), and 7 (Day 7) (Figure 8). Fluorescence of hMSCs was observed on days 1 (Day 1), 4 (Day 4), 8 (Day 8), 11 (Day 11), and 17 (Day 17), and the expression of the fluorescent gene OFP, a marker gene carried by the SeV vector, was observed. The results confirmed that OFP expression was significantly reduced in hMSCsiL527 and hMSCsiL1913 transfected with target siRNA compared to untreated cells and cells transfected with the control siRNA siGL3Luc. These results suggested that the SeV vector was eliminated by the two target siRNAs (siL527, siL1913) (Figure 9). 2. Evaluation of SeV Vector Elimination Effect SeV vector elimination was confirmed by highly sensitive RT-qPCR. RNA was extracted from cells on day 11 using NucleoSpin® RNA Plus (MACHEREY-NAGEL). cDNA synthesis was performed using 1 μg of RNA with SuperScript® III reverse transcriptase (Thermo Fisher Scientific). 20 μl of cDNA was diluted with RNase-free water to a total volume of 250 μl, and 5 μl was used as a template per reaction. RT-qPCR reactions were performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems) with the StepOne™ Real-Time PCR System (Applied Biosystems). TaqMan Assay Mr04269880_mr (Thermo Fisher Scientific) was used to detect the SeV vector, and TaqMan Assay Hs01060665_g1 (Thermo Fisher Scientific) was used to detect the endogenous control, ACTB.The RT-qPCR reaction conditions were 50°C for 2 minutes, 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. RT-qPCR analysis confirmed that the amount of SeV vector present in cells transfected with siL527 or siL1913 was significantly reduced compared to untransfected cells (untreated cells) and cells transfected with control siRNA (si control) ( Figure 10 ). These results demonstrate that SeV vectors can be removed from SeV vector-infected hMSCs using siRNA. This suggests that siRNA-mediated removal can be used to extend cell life using a conventional SeV vector.

[0009] The present invention provides a method for producing de-immortalized cells from reversibly immortalized cells. The cells obtained by the method of the present invention are highly safe and are therefore useful for obtaining cells useful for transplantation in regenerative medicine, such as MSCs.

[0010] SEQ ID NO: 1: Nucleotide sequence of mouse BMI1 gene SEQ ID NO: 2: Nucleotide sequence of human TERT gene SEQ ID NO: 3: Nucleotide sequence of SV40 large T antigen gene SEQ ID NO: 4: Nucleotide sequence of siGL3Luc-S SEQ ID NO: 5: Nucleotide sequence of siGL3Luc-AS SEQ ID NO: 6: Nucleotide sequence of siL527-S SEQ ID NO: 7: Nucleotide sequence of siL527-AS SEQ ID NO: 8: Nucleotide sequence of siL1913-S SEQ ID NO: 9: Nucleotide sequence of siL1913-AS All publications, patents and patent applications cited in this specification are hereby incorporated by reference in their entirety.

Claims

1. A method for producing de-immortalized cells from reversibly immortalized cells, the method comprising a removal step of removing the Sendai virus vector from reversibly immortalized cells consisting of animal cells containing a Sendai virus vector containing an immortalization gene.

2. The method according to claim 1, wherein the Sendai virus vector is a temperature-sensitive Sendai virus vector, and in the removal step, the temperature is changed to a temperature that inactivates the temperature-sensitive Sendai virus vector, and the temperature-sensitive Sendai virus vector is maintained under that temperature condition.

3. The method according to claim 1, wherein the removal step is carried out by suppressing the expression of a protein involved in the replication of the Sendai virus vector.

4. The method according to claim 3, wherein the expression of a protein involved in the replication of the Sendai virus vector is suppressed by RNA interference.

5. The method according to claim 4, wherein RNA interference is carried out by introducing siRNA.

6. The method according to any one of claims 1 to 5, further comprising a culture step of culturing reversibly immortalized cells consisting of animal cells containing a Sendai virus vector containing the immortalization gene before the removal step.

7. The process according to any one of claims 1 to 5, further comprising a separation step of separating de-immortalized cells from which the Sendai virus vector has been removed from the cells after the removal step.

8. The method of claim 7, further comprising a harvesting step of harvesting the de-immortalized cells after the separating step.

9. The method according to claim 6, further comprising a measuring step of collecting a portion of the reversibly immortalized cells after the culturing step and measuring the telomere length of the cells.

10. The method according to claim 9, wherein the removal step is carried out after measuring the telomere length of the reversibly immortalized cells after the culture step to confirm that the telomere length of the reversibly immortalized cells is significantly longer than that of cells not treated with the Sendai virus vector containing the immortalization gene for the same number of culture days.

11. The method according to any one of claims 1 to 10, wherein the immortalizing gene comprises one or more genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.

12. The method according to any one of claims 1 to 11, wherein the Sendai virus vector further comprises a gene encoding a marker protein.

13. The method of claim 12, wherein the label protein is a fluorescent protein.

14. The method according to claim 7, wherein in the separation step, the de-immortalized cells are separated using a label protein as an indicator.

15. The method according to any one of claims 2, 6 to 14, wherein the temperature-sensitive Sendai virus vector is maintained at a temperature of 38°C to 40°C in the removal step.

16. The method according to claim 15, wherein in the removal step, the reversibly immortalized cells are maintained at a temperature of 38°C to 40°C for 0.5 to 2 days.

17. The method described in claim 15 or 16, wherein in the removal step, the reversibly immortalized cells are maintained at a temperature of 38°C to 40°C, and then maintained at a temperature of 36°C to 37.5°C.

18. The method according to claim 17, wherein the reversibly immortalized cells are maintained at a temperature of 36°C to 37.5°C for 6 to 10 days.

19. The method according to any one of claims 1 to 18, wherein the animal cells are somatic cells.

20. The method of claim 19, wherein the somatic cells are somatic stem cells.

21. The method of claim 20, wherein the somatic stem cells are mesenchymal stem cells.

22. A de-immortalized cell produced by the method of any one of claims 1 to 21.

23. The cells according to claim 22, in which telomere length is significantly extended compared to cells not treated with the Sendai virus vector containing the immortalizing gene over the same number of days of culture.

24. A regenerative medicine product comprising the de-immortalized cells of claim 22 or claim 23.

25. A method for removing a temperature-sensitive Sendai virus vector from a reversibly immortalized cell consisting of an animal cell containing a temperature-sensitive Sendai virus vector that includes an immortalization gene, the method comprising a removal step of removing the temperature-sensitive Sendai virus vector from the reversibly immortalized cell.

26. The method according to claim 25, further comprising a separation step of separating de-immortalized cells from which the Sendai virus vector has been removed from the cells after the removal step.

27. The method according to claim 25 or 26, wherein the Sendai virus is a temperature-sensitive Sendai virus vector, and in the removal step, the temperature is changed to a temperature that inactivates the temperature-sensitive Sendai virus vector, and the temperature-sensitive Sendai virus vector is maintained under that temperature condition.

28. A method for extending telomere length in animal cells, comprising: (1) an infection step of infecting animal cells with a Sendai virus vector containing an immortalization gene; and (2) a culture step of culturing the animal cells infected with the Sendai virus vector.

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