Method for producing reversibly immortalized cells

Abstract

To provide a method for producing a reversibly immortalized cell, by which the cell can be proliferated over a long period without damaging the chromosome of the cell into which an immortalizing gene is transferred and the immortalizing gene can be removed, and to provide a method for obtaining the reversibly immortalized cell capable of being cloned and having stable quality in a large amount.SOLUTION: The present invention provides a method for producing reversibly immortalized cells, comprising the steps of: introducing a non-chromosomal RNA viral vector carrying one or more immortalizing genes selected from the group consisting of Bmi-1 genes, TERT genes, and SV40T genes into mammalian cells to express the immortalizing genes in the cells; and culturing and growing the obtained cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing reversibly immortalized cells, and more particularly to a method for producing reversibly immortalized cells using a chromosomally non-integrating RNA viral vector carrying a specific immortalization gene. [Background technology]

[0002] The main cell sources used in regenerative medicine and cell therapy are somatic stem cells such as mesenchymal stem cells (MSCs) and pluripotent stem cells such as embryonic stem cells (ES cells) and iPS cells. Among stem cells, mesenchymal stem cells (MSCs) are naturally present in the body, so they have a low risk of rejection or tumorigenesis and are therefore safe. However, because there is a limit to the number of cell divisions, securing a sufficient number of cells needed to supply them to medical settings is a challenge.

[0003] Therefore, attempts have been made to introduce immortalizing genes into cells to allow them to proliferate indefinitely. Known immortalizing genes include the telomerase reverse transcriptase (TERT) gene, genes that regulate telomerase expression or activity (e.g., Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.). These immortalizing genes are introduced into cells using vectors such as plasmid DNA, lentiviral vectors, retroviral vectors, and adenoviral vectors (see, e.g., Patent Documents 1, 2, and 3). Viral vectors are preferred because they allow efficient gene transfer by simply filtering the culture supernatant of recombinant viral vector-producing cells and adding it to the target cells. Among the above vectors, plasmid DNA and adenoviral vectors have DNA vector genomes that are integrated into the host chromosomes. Furthermore, lentiviral and retroviral vectors have RNA vector genomes that are converted into DNA by reverse transcriptase within the cell and integrated into the host chromosomes, thereby expressing the harbored gene. As described above, vectors commonly used for immortalization carry the risk of damaging the host cell's genes by integrating foreign DNA into the host cell's chromosome, potentially leading to tumorigenesis, posing a major safety issue in regenerative medicine. On the other hand, Sendai virus vectors (SeV vectors) are non-chromosomally integrated RNA viral vectors that do not take the form of DNA. SeV vectors are capable of transducing genes into a wide range of cell types and are easily able to induce high levels of protein expression, making them widely used for iPS cell induction and other purposes (Patent Document 4). 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 was recognized that SeV vectors would be difficult to achieve long-term expression of transgenes. Therefore, SeV vectors have been limited to transient high expression and have not been used to transduce immortalizing genes into cells.

[0004] Meanwhile, reversibly (conditionally) immortalized cells have also been studied, in which an immortalizing gene is introduced to immortalize cells, and the immortalizing gene can be excised using a site-specific recombinase after proliferation (Non-Patent Document 1, etc.). However, even if the immortalizing gene is excised using a site-specific recombinase, the resulting cells are genetically engineered cells, and there are concerns that such cells may have adverse effects, such as forming tumors in the host, so safety cannot be said to be ensured.

[0005] Furthermore, stem cells obtained from human tissues are a mixed population of young and aged cells, with diverse properties, and age as they divide repeatedly, making it impossible to maintain cells of a consistent quality. This presents another challenge: it is difficult to set quality standards, which are essential for automation and mechanization.

[0006] Discussions are also underway to try to isolate only cells with desired properties from these different cell populations. Methods for assessing the quality of iPS cells include miRNA analysis, surface marker detection, image analysis of cell morphology, and epigenome analysis, and the use of these techniques for MSCs has also been proposed. However, unlike iPS cells, MSCs do not proliferate indefinitely and cannot be cloned, so these methods have not yet been put to practical use.

[0007] Furthermore, when isolating MSCs with a low passage number without repeated division, a large amount of tissue containing MSCs is required, and it is difficult to secure the necessary number of cells, which is a factor in the soaring production costs. As a result, treatments that require a large number of cells are difficult, and most treatments are performed using autologous cells, making it difficult to implement inexpensive regenerative medicine using allogeneic cells. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 3953399 [Patent Document 2] WO2017 / 078176 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-221219 [Patent Document 4] Patent No. 5763340 [Non-patent literature]

[0009] [Non-Patent Document 1] Fang-Ying Meng, et al., Reversible immortalizationof human hepatocytes mediated by retroviral transfer and site-specific recombination. World Journal of Gastroenterology, 01 Sep 2014, 20(36):13119-13126 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention aims to provide a method for producing reversibly immortalized cells, which allows cells into which an immortalizing gene has been introduced to grow for a long period of time without damaging the chromosomes of the cells, and which also allows the immortalizing gene to be removed, and to provide a method for obtaining large quantities of reversibly immortalized cells that can be cloned and have stable quality. [Means for solving the problem]

[0011] The present inventors conducted extensive research to solve the above-mentioned problems. They found that when mesenchymal stem cells (MSCs) were transfected with one or more immortalization genes selected from the group consisting of Bmi-1, TERT, and SV40T, carried by a Sendai virus vector, a non-integrating RNA vector not previously used for somatic cell immortalization, they surprisingly demonstrated that the cells remained mitotically stable for over 80 days, enabling indefinite proliferation (immortalization). Furthermore, they confirmed that the immortalized cells produced were free of chromosomal abnormalities, pluripotent, and easily removed by temperature control. Furthermore, they successfully cloned immortalized cells from the resulting immortalized cell population. Eight of the ten cloned immortalized cells also showed normal karyotypes and were pluripotent. Furthermore, they confirmed that immortalization could be induced in cells at any stage, from young cells to senescent cells in which division had ceased, and that cloning was possible immediately after immortalization. The present invention was based on these findings.

[0012] That is, the present invention includes the following inventions. [1] A method for producing reversibly immortalized cells, comprising the steps of: (1) introducing a chromosomally non-integrating RNA viral vector carrying an immortalization gene into mammalian cells and expressing the immortalization gene in the cells; and (2) Cultivating and growing the cells obtained in step (1) [2] The method according to [1], wherein the immortalization gene is one or more immortalization genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene. [3] The method according to [1] or [2], wherein the immortalizing gene is any one of the following (a) to (d): (a) Combination of the Bmi-1 gene, the TERT gene, and the SV40T gene (b) Combination of Bmi-1 gene and TERT gene (c) Combination of the TERT gene and the SV40T gene (d) TERT gene [4] The method according to any one of [1] to [3], wherein the cells are somatic cells. [5] The method according to [4], wherein the somatic cells are somatic stem cells. [6] The method according to [5], wherein the somatic stem cells are mesenchymal stem cells. [7] The method according to any one of [1] to [6], wherein the chromosomally non-integrating RNA viral vector is a minus-strand RNA viral vector. [8] The method according to [7], wherein the minus-strand RNA viral vector is a paramyxovirus vector. [9] The method according to [8], wherein the paramyxovirus vector is a Sendai virus vector.

[10] The method according to [9], wherein the Sendai virus vector is a temperature-sensitive Sendai virus vector.

[11] The method according to [1], wherein the chromosomally non-integrating RNA viral vector is a Sendai virus vector, and further comprises the step of removing the Sendai virus vector after the culture in step (2).

[12] The method according to

[11] , wherein the Sendai virus vector is removed by changing the culture temperature from 35°C to 37°C.

[13] The method according to any one of [1] to

[12] , further comprising the step of cloning the immortalized cells after the culture in the step (2).

[14] An immortalized cell obtained by the method described in any one of [1] to

[13] .

[15] An immortalized cell comprising, in a removable state, a Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.

[16] A regenerative medicine product comprising the immortalized cells according to

[14] or

[15] .

[17] A temperature-sensitive Sendai virus vector carrying one or more immortalization genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.

[18] A kit for producing reversibly immortalized cells, comprising the temperature-sensitive Sendai virus vector described in

[17] . This application claims priority to Japanese Patent Application No. 2020-186146, filed on November 6, 2020, and includes the contents described in the specification of that patent application. [Effects of the Invention]

[0013] According to the present invention, by introducing an immortalizing gene into cells using a non-chromosomally integrated RNA vector, it is possible to immortalize (infinitely proliferate) cells that are difficult to mass-culture, such as mesenchymal stem cells (MSCs) used in regenerative medicine, without damaging their chromosomes. This immortalization is possible regardless of the number of cell divisions of the cells to be immortalized or the number of cell divisions during immortalization. Furthermore, by using a temperature-sensitive Sendai virus vector (SeV vector) as the non-chromosomally integrated RNA vector, the vector can be easily removed from cells by simply changing the culture temperature, without requiring complicated removal procedures. This allows for the mass supply of highly safe cells. Furthermore, the immortalized cells obtained by the present invention are free of chromosomal abnormalities and retain the same pluripotency as before gene introduction, making them suitable for use in regenerative medicine. Furthermore, while non-immortalized cells are difficult to clone due to their different lifespans and properties, the immortalized cells produced by the present invention are easy to clone and allow for the stable production of high-quality cells. This facilitates quality control through mechanized cell production, significantly reducing production costs, which have been a challenge for industrialization, and will contribute to the advancement of cell therapy and regenerative medicine. [Brief explanation of the drawings]

[0014] [Figure 1] The genomic structures of the SeV vectors carrying the immortalization factor gene are shown [A. SeV(18+)Bmi1(HNL)OFP / TS15dΔF, B. SeV(PM)hTERT(HNL)EGFP / TS15ΔF, C. SeV(18+)SV40T / TS15ΔF, D. SeV(HNL)E6-E7,BFP / TS15ΔF]. [Figure 2]Transmitted light and fluorescence (OFP) images of cells infected with Bmi-1 alone (MOI: 1, 5, 20) and transmitted light and fluorescence (GFP) images of cells infected with hTERT alone (MOI: 1, 5, 20) are shown. [Figure 3] Transmitted light and fluorescence (OFP, GFP) images of cells co-infected with two factors (Bmi-1 / hTERT) (MOI: 1, 5, 20) are shown. [Figure 4] Transmitted light and fluorescent (OFP, GFP) images of cells co-infected with three factors (Bmi-1 / hTERT / SV40T) (MOI: 1, 5, 20) are shown. [Figure 5] The morphology and state of the cells 59 days after the introduction of immortalization factors (No. 1 to No. 16) are shown. [Figure 6] The cell growth curves of cells transfected with immortalization factors (No. 1, No. 2, No. 4, No. 12, No. 16) are shown (upper arrows: morphological (transmitted light) image and fluorescent image points (Figure 5); *: telomere analysis point (Figure 11A, B); ▽: chromosome analysis point). [Figure 7] The cell growth curve of the three-factor (Bmi-1 / hTERT / SV40T) transfected cells is shown (NC: no immortalization gene transfected; up arrow: morphological (transmitted light) image and fluorescent image point (Figure 8); double arrow: morphological (transmitted light) image point (Figure 9); *: telomere analysis point (Figure 11B); ▽: chromosome analysis point (Figure 10)). [Figure 8] Transmitted light images and fluorescent (OFP, GFP) images of cells transfected with three factors (Bmi-1 / hTERT / SV40T) are shown. [Figure 9] The morphology (transmitted light) images of the cells transfected with the three factors (Bmi-1 / hTERT / SV40T) are shown (maintained at 35°C, temperature changed from 35°C to 37°C). [Figure 10] The results of chromosome analysis (normal karyotype) of the three-factor (Bmi-1 / hTERT / SV40T)-transfected cells 90 days after subculture are shown (chromosome analysis point (▽) in Figure 7). [Figure 11]The figures show the results of telomere quantitative analysis of long-term culture samples of cells transfected with immortalization factors (No. 1, No. 2, No. 4, No. 12, No. 16). (A. Effect of immortalization factor combinations on telomeres (Day 0, Day 40: telomere analysis points (*) in Figure 6); B. Changes in telomere quantity after transfection with three factors (Bmi-1 / hTERT / SV40T) with and without factor removal (Day 0, Day 40: telomere analysis points (*) in Figure 6; Day 85, Day 98: telomere analysis points (*) in Figure 7). [Figure 12] Photographs of adipocytes differentiated from cultured primary MSCs and immortalized MSCs are shown. [Figure 13] Photographs of osteoblasts differentiated from cultured primary MSCs and immortalized MSCs are shown. [Figure 14] Photographs of neurons differentiated from cultured primary MSCs and immortalized MSCs are shown. [Figure 15] Photographs of chondrocytes differentiated from cultured primary MSCs and immortalized MSCs are shown. [Figure 16] 1 shows the results of a cloning test 1 of an immortalized cell population (2 weeks after transfection of the immortalization gene) (numbers in the wells: number of clones formed). [Figure 17] 1 shows the results of cloning test 2 of the immortalized cell population (4 months after transfection of the immortalization gene) (numbers in the wells: number of clones formed). [Figure 18] The cell growth curve of the cloned immortalized cell (clone A10) is shown (▽: chromosome analysis point). [Figure 19] The results of chromosome analysis of the cloned immortalized cell (clone A10) are shown (Day 80: chromosome analysis point (▽) in Figure 18). [Figure 20] This shows the differentiation potential of cloned immortalized cells (adipocyte differentiation, neuronal differentiation, osteoblast differentiation).

[0015] [Figure 21]The cryopreservation points of the MSCs used in the single-cell cloning test (early MSCs: Day 9, intermediate MSCs: Day 24, late MSCs: Day 49) and the points of immortalization induction by SeV vector infection (early MSCs: Day 21, intermediate MSCs: Day 36, late MSCs: Day 61) are shown. [Figure 22] Photographs of SeV vector-infected cells (early MSCs, intermediate MSCs, and late MSCs) and SeV vector-uninfected MSCs (control) before single-cell cloning (immediately before seeding on a 96-well plate) are shown. [Figure 23-1] The figures show the results of a single-cell cloning test of cell populations of SeV vector-uninfected MSCs (control) and SeV vector-infected cells (primary MSCs) (numbers in wells: number of clones formed, culture days: 2 weeks). [Figure 23-2] The results of a single-cell cloning test of cell populations (mid-stage MSCs, late-stage MSCs) infected with SeV vectors are shown (numbers in wells: number of clones formed; number of days in culture: 2 weeks). [Figure 24] The results of SeV vector infection tests on cells in which cell division had stopped (Day 90) are shown (photographs of cells immediately before SeV vector infection, SeV vector-uninfected cells (control cells), and SeV vector-infected cells (2 weeks after infection)). [Figure 25] The results of a single-cell cloning test of division-arrested cells (Day 90) that had begun to regrow after SeV vector infection are shown (numbers in the wells: number of clones formed; number of days in culture: 2 weeks). [Figure 26] 1 shows cell growth curves of adipose tissue-derived human MSCs (uninfected cell line, SeV vector-infected cell line) in serum-free culture. [Figure 27] 1 shows cell growth curves of bone marrow-derived human MSCs (uninfected cell line, SeV vector-infected cell line) in serum-free culture. [Figure 28]The figures show transmitted light and fluorescence (OFP, GFP) images of adipose tissue-derived human MSC (hMSC-AT) cell lines infected with SeV vectors and bone marrow-derived human MSC (hMSC-BM) cell lines infected with SeV vectors, both cultured in serum-free media (hMSC-AT: days 16 and 58 after infection; hMSC-BM: days 24 and 50 after infection). [Figure 29] 1 shows cell proliferation curves of rat MSCs (uninfected cell line, SeV vector-infected cell lines #1 and #2). [Figure 30] The figures show transmitted light and fluorescence (OFP, GFP) images of an immortalized rat MSC cell line infected with an SeV vector (days 2, 9, and 58 after infection). [Figure 31] The graph shows cell growth curves of HFL1 (uninfected cell lines #1 and #2, SeV vector-infected cell lines #1 and #2 cultured at 35°C, and SeV vector-infected cell lines #1 and #2 cultured at 37°C). [Figure 32] Transmitted light images and fluorescence (OFP, GFP) images of immortalized HFL1 [cultured at 35°C (10, 34, and 255 days after infection), cultured at 35°C + cultured at 37°C (34 days after infection + 221 days after infection)] are shown. [Figure 33] The graph shows cell growth curves of HUVEC (uninfected cell line #1, SeV vector-infected cell lines #1 and #2 cultured at 35°C, and SeV vector-infected cell lines #1 and #2 cultured at 37°C). [Figure 34] Transmitted light and fluorescence (OFP, GFP) images of immortalized HUVECs [cultured at 35°C (7, 36, and 72 days after infection), cultured at 35°C + 37°C (35 + 37 days after infection)] are shown. [Figure 35] Photographs showing the results of FISH analysis of immortalized MSCs introduced with a multiple growth factor-inserted human artificial chromosome vector (bottom right box: DAPI-stained image of the cells indicated by the arrow). [Figure 36] 1 shows the results of a test of the healing effect of transplantation of immortalized MSCs on an enteritis model. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1. Method for producing reversibly immortalized cells The present invention relates to a method for producing reversibly immortalized cells, which comprises the steps of (1) introducing a non-chromosomally integrated RNA viral vector carrying an immortalization gene into mammalian cells and expressing the immortalization gene in the cells, and (2) culturing and growing the cells obtained in step (1).

[0017] (cell) In the present invention, the term "cell" refers to all somatic cells other than germline cells (eggs and sperm, oocytes, ES cells, etc.) and totipotent cells (iPS cells). Furthermore, "somatic cells" may be primary cultured cells, passaged cells, or established cell lines. Furthermore, somatic cells may be naturally occurring or artificially produced by differentiation of iPS cells or the like. 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, monocytic cells, etc.), and 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.). The origin of the cells is not particularly limited as long as they are mammalian, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses.

[0018] (Reversible immortalized cells) "Immortalized cells" refer to cells that do not cease proliferation even after repeated cell division, i.e., cells capable of indefinite proliferation, unlike primary culture cells or cells cultured under normal culture conditions. In the present invention, "immortalized cells" refer to cells that have been made capable of indefinite proliferation by the introduction of a specific immortalization gene, and whose indefinite proliferation ability does not decrease even when cultured and subcultured repeatedly. The "immortalized cells" of the present invention vary in proliferation rate and duration depending on the cell origin or culture conditions. However, as a result of subculture, under the same culture conditions, they can continue exponential proliferation for 20 days or more, preferably 60 days or more, and more preferably 80 days or more, even after the time when untreated cells' proliferation decreases or stops. Furthermore, the immortalized cells of the present invention encompass both the above-mentioned cell populations capable of indefinite proliferation and immortalized cell lines cloned from such cell populations. "Reversible immortalization" refers to the process of introducing an immortalization gene into cells to render them indefinitely proliferative, and then removing the immortalization gene to stop or attenuate cell proliferation.

[0019] "Immortalization" refers to the process of overcoming the limitations on the number of cell divisions and cell senescence of initial cells, thereby conferring the ability to continuously divide and proliferate. Specifically, it refers to a state in which cells can be passaged under standard cell culture conditions, typically 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 techniques known to those skilled in the art. Cells obtained after 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)" depending on the number of passaging operations. Furthermore, a step of freezing the cells may be included between each passaging operation.

[0020] (immortalization gene) The immortalized cells of the present invention are prepared by introducing a specific immortalization gene into cells using a non-chromosomally integrated RNA viral vector. Here, "immortalization gene" refers to a gene that immortalizes cells, granting them the ability to proliferate indefinitely, without inducing cell death. The immortalization gene is an exogenous gene, meaning an immortalization gene newly introduced from outside the cell. Furthermore, the immortalization gene may be derived from a non-human source or may be an immortalization gene modified to be expressible in target cells. In the present invention, one or more genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene can be used as the immortalization gene. A combination of two genes is preferred, and a combination of three genes is more preferred. When using one gene, the TERT gene is preferred. When using a combination of two or more genes, preferred combinations include the Bmi-1 gene and the TERT gene, and the TERT gene and the SV40T gene.

[0021] In the present invention, unless otherwise specified, the term "gene" includes not only structural genes that define the primary structure of a protein, but also regions on nucleic acids that have control functions such as promoters and operators. Therefore, in the present invention, the term "gene" refers to regulatory regions, coding regions, exons, and introns without distinction, unless otherwise specified.

[0022] The Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog) gene encodes a protein that has two nuclear localization signals and functions as a member of the polycomb repressive complex 1 (PRC1), which is localized in the cytoplasm or nucleus. As a PRC1, Bmi-1 is involved in the regulation of the expression of various genes, including Hox genes, by regulating chromatin remodeling and histone modification. Bmi-1 is known to regulate cell proliferation by suppressing the expression of cell cycle-related proteins p16 and p19Arf, and to play an important role in maintaining self-renewal of hematopoietic and neural stem cells by participating in their cell division.

[0023] The "TERT (telomerase reverse transcriptase) gene" is a gene that encodes telomere reverse transcriptase (TERT). TERT comprises telomerase, an enzyme that elongates specific repeat sequences at the ends of eukaryotic chromosomes (telomeres), from the telomerase RNA component (TR or TERC) and other regulatory subunits. Cells have a telomere length monitoring mechanism, and cellular senescence is caused by telomere shortening. TERT is known to play a role in maintaining telomere length.

[0024] The SV40T (simian virus 40 large T antigen) gene encodes the simian virus 40 large T antigen. The SV40 (simian vacuolating 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 contains transcriptional control and replication origins. The early region encodes the large T antigen, which is involved in the initiation of viral genome replication and the inactivation of the tumor suppressor gene products p53 and pRB, and the small T antigen, which binds to and inhibits the protein phosphatase PP2A.

[0025] 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, the TERT gene, and the SV40T gene may also be transcriptional variants, splicing variants, or orthologs thereof.

[0026] The Bmi-1, TERT, and SV40T genes may be genes consisting of a nucleotide sequence that shares at least 80%, preferably at least 90%, and more preferably at least 95% sequence identity with the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively, as long as they have equivalent functions and activities. They may also 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 immortalizing genes of the present invention. Furthermore, the Bmi-1, TERT, and SV40T genes 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.

[0027] (chromosomally non-integrating RNA viral vector) In the present invention, a "chromosomally non-integrating RNA viral vector" is used as a vector for introducing and expressing the immortalizing gene into cells. In the present invention, a viral vector refers to a vector that has genomic nucleic acid derived from the virus and can express a transgene by incorporating the transgene into the nucleic acid. Furthermore, a "chromosomally non-integrating RNA viral vector" refers to a viral vector derived from a virus that can introduce a gene into target cells, and is a carrier that does not pose a risk of the introduced gene being integrated into the host's chromosome (nuclear-derived chromosome).

[0028] Examples of chromosomally non-integrating RNA viral vectors used in the present invention include minus-strand RNA viral vectors. A "minus-strand RNA viral vector" refers to a vector consisting of a virus containing minus-strand RNA (an antisense strand complementary to the sense strand encoding the viral protein) as its genome, and minus-strand RNA is also called negative-strand RNA. As the minus-strand RNA virus used in the present invention, single-stranded minus-strand RNA viruses (also called non-segmented minus-strand RNA viruses) are particularly preferred. A "single-stranded negative-strand RNA virus" refers to a virus having a single-stranded negative-strand (minus-strand) RNA in its genome, and includes viruses belonging to the Paramyxoviridae (including the genera Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus), Rhabdoviridae (including the genera Vesiculovirus, Lyssavirus, and Ephemerovirus), and Filoviridae.

[0029] Examples of negative-strand RNA viruses that can be used in the present invention include Sendai virus, Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus, Rinderpest virus, distemper virus, simian parainfluenza virus (SV5), human parainfluenza virus types 1, 2, and 3, influenza virus, Orthomyxoviridae, vesicular stomatitis virus, and rabies virus, all of which belong to the Paramyxoviridae family, with Sendai virus being preferred.

[0030] (Sendai virus vector: SeV vector) In a preferred embodiment of the present invention, a Sendai virus (SeV) vector is used as the chromosomally non-integrating RNA vector. The immortalization factor genes, Bmi-1 gene, TERT gene, and SV40T gene, may be inserted into the SeV vector separately or together into a single SeV vector.

[0031] Sendai virus is a type of virus in the genus Respirovirus in the family Paramyxoviridae, and contains a single negative-strand RNA (the antisense strand to the sense strand that encodes the viral proteins) as its genome.

[0032] SeV vectors have the following characteristics: (i) extremely high efficiency of gene transfer and expression in various mammalian cells, including human; (ii) they are non-chromosomally integrated viral vectors, and because the vector is expressed in the cytoplasm, the introduced gene is not integrated into the host chromosome and there is no risk of chromosomal structural changes; (iii) they are not human pathogenic viruses; (iv) by changing the insertion site in the vector, it is possible to adjust the gene expression level and simultaneously express multiple genes; and (v) the vector can be removed from the introduced cells after the purpose has been achieved.

[0033] The Sendai virus genome contains, from the 3' to the 5' end, the NP (nucleocapsid) gene, P (phospho) gene, M (matrix) gene, F (fusion) gene, HN (hemagglutinin / neuraminidase) gene, and L (large) gene. Of these, the Sendai virus can function sufficiently as a vector if it has the NP, P, and L genes, replicating its genome in cells and expressing the genes it carries. Because the Sendai virus has a negative-strand RNA genome, the 3' end of the genome is upstream and the 5' end is downstream, which is the opposite of the usual genome.

[0034] In the present invention, any of naturally occurring strains, wild-type strains, mutant strains, and commercially available SeV vectors can be used. As long as the desired function can be achieved, the virus may have a structure similar to that of a virus isolated from nature, or may be artificially modified by genetic recombination. For example, the virus may have a mutation or deletion in any of the genes of a wild-type virus. Specifically, a non-transmissible vector (ΔF) in which the F gene is deleted from the genome and infectious particles are not formed in transfected cells, as well as a vector in which the F gene is deleted and the M and / or HN genes are further deleted, or which further has mutations (e.g., temperature-sensitive mutations) in the M and / or HN genes, are preferably used in the present invention. Furthermore, for example, a vector in which the F gene is deleted and the M or HN gene is further deleted and further has mutations (e.g., temperature-sensitive mutations) in the remaining M and / or HN genes is also preferably used in the present invention (see, for example, Japanese Patent No. 5763340).

[0035] Furthermore, the SeV vector used in the method of the present invention is preferably temperature-sensitive. "Temperature-sensitive" means that the activity is significantly reduced at normal cell culture temperatures (e.g., 37-38°C) compared to low temperatures (e.g., 30-36°C). Examples of such vectors include 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 (P protein). Mutations such as the D433A / R434A / K437A mutation in the protein and the L1361C / L1558I mutation in the L protein are temperature-sensitive mutations and can be suitably used in the present invention. These mutations are preferably further introduced into the F gene-deficient SeV vector. For details of these SeV vectors, see Japanese Patent No. 5763340, WO2015 / 046229, etc.

[0036] The SeV vectors of the present invention include not only infectious virus particles but also 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 a gene carried by the vector when introduced into a cell. For example, a ribonucleoprotein (a viral core portion) consisting of the Sendai virus genome and the Sendai virus proteins (NP, P, and L proteins) that bind to it can express a transgene in a cell when introduced into the cell. Introduction into a cell can be carried out using an appropriate transfection reagent or the like. Therefore, such ribonucleoproteins (RNPs) are also encompassed in the SeV vectors of the present invention.

[0037] (Construction of SeV vector carrying immortalizing gene) The positions at which the immortalizing genes (Bmi-1 gene, TERT gene, and SV40T gene) are integrated are not particularly limited, but when each immortalizing gene is inserted into a separate vector, it is preferable that the Bmi-1 gene be inserted upstream of the NP gene, the TERT gene be inserted between the P gene and the M gene, and the SV40T gene be inserted upstream of the NP gene. Alternatively, two or more genes (Bmi-1 and TERT, TERT and SV40T, or Bmi-1, TERT, and SV40T) may be inserted into a single vector.

[0038] The immortalizing gene-carrying SeV vector may be packaged as a kit, which may include, for example, a medium or container for cell culture, instructions for using the kit, and the like.

[0039] (Introduction of immortalization genes into cells) The SeV vector carrying the immortalizing gene obtained as described above is introduced into somatic cells by adding the vector (Sendai virus particles) to the culture medium of the cells and infecting the cells with the virus. Since the vector dose varies depending on the type of cell, cell density, and volume of culture medium, an MOI that provides an infection efficiency close to 100% can be determined in advance for each cell type to be used.

[0040] Alternatively, when the SeV vector is in the form of RNP, it can be introduced into cells by techniques such as electroporation, lipofection, and microinjection.

[0041] (Culture of immortalized transfected cells) In the present invention, the cells into which an immortalizing gene has been introduced can be cultured according to the method and conditions used for culturing conventional mammalian somatic cells. The medium used for culture is not particularly limited, and any medium generally used for maintenance or expansion of cells 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) include Dulbecco's Modified Eagle's Medium (D-MEM) medium, Dulbecco's Modified Eagle's Medium:Nutient 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 (EMEM) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, RPMI 1640 medium, Medium 199 medium, αMEM medium, Ham's medium, Fischer's medium, and mixtures thereof. The medium may also contain growth factors (FGF, EGF, etc.), interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplements, N2 supplements, antibiotics (penicillin, streptomycin, etc.), etc., as needed. The medium may be serum-containing or serum-free. From the viewpoint of preventing contamination with components derived from different animal species, it is preferable to use serum-free medium or serum derived from the same animal species as the cells to be cultured. Serum substitutes such as albumin may also be used.

[0042] 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. Cell-adhesive culture vessels may be treated with cell support substrates such as extracellular matrices to improve cell adhesion. Examples of cell support substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

[0043] The culture temperature 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 in a CO2-containing air atmosphere, for example, at a CO2 concentration of 2% to 5%. The culture temperature for removing the immortalization gene is 37°C to 38°C, and preferably 37°C to 37.5°C.

[0044] 2. Differentiation induction According to the present invention, the immortalized cells prepared as described above can be induced to differentiate into specific tissue cells by culturing them in a differentiation-inducing medium. For example, when the immortalized cells are mesenchymal stem cells, they can be differentiated into adipocytes, osteoblasts, neurons, chondrocytes, etc.

[0045] The composition of the medium, differentiation-inducing factors, culture method, passaging method, etc. for inducing differentiation of the immortalized cells according to the present invention into target cells can be appropriately determined from well-known and commonly used techniques.

[0046] The differentiation-inducing medium can be selected appropriately depending on the type of cells to be differentiated. Differentiation-inducing media for various tissues (media containing at least one differentiation-inducing or -promoting factor appropriate for the cells to be differentiated) are commercially available, and these commercially available media may be used. For example, a medium for inducing the differentiation of the immortalized cells of the present invention into adipocytes may be a commercially available adipocyte-inducing medium or a commercially available animal cell medium supplemented with insulin, dexamethasone, indomethacin, 3-isobutyl-1-methylxanthine, troglitazone, biotin, or the like. Examples of commercially available media include Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (PromoCell) and Human Mesenchymal Stem Cell Adipogenic Differentiation Medium BulletKit 8 (Lonza). The medium for inducing the differentiation of the cells of the present invention into osteoblasts may be a commercially available osteoblast induction medium or a commercially available animal cell medium containing dexamethasone, ascorbic acid, β-glycerophosphate, hydrocortisone, BMP4, BMP2, or the like. Examples of commercially available media include Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell) and Human Mesenchymal Stem Cell Osteogenic Differentiation Medium Bullet Kit (Lonza). The medium for inducing the differentiation of the immortalized cells of the present invention into neurons may be a commercially available neuronal culture medium or neuronal differentiation medium (e.g., Mesenchymal Stem Cell Neurogenic Differentiation Medium (PromoCell)). Furthermore, the neuronal culture medium or neuronal differentiation induction medium preferably contains a neuronal induction factor (e.g., brain-derived nerve growth factor (BDNF) or fibroblast growth factor (FGF)).Furthermore, a commercially available chondrocyte induction medium or a commercially available animal cell medium containing dexamethasone, ascorbic acid, and TGF-β3 can be used as a medium for inducing differentiation of the immortalized cells of the present invention into chondrocytes. Examples of commercially available media include Mesenchymal Stem Cell Chondrogenic Differentiation Medium (PromoCell) and Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium Bullet Kit (Lonza).

[0047] The culture conditions for differentiation induction are the same as those for culturing normal stem cells. The culture period for differentiation induction is not particularly limited, but is generally 5 to 20 days, and preferably 7 to 18 days.

[0048] Whether or not stem cells have been induced to differentiate into the target cells can be confirmed by examining the expression of markers specific to each differentiated cell type. For example, differentiation into adipocytes can be confirmed by Oil Red 0 staining, differentiation into osteoblasts by alkaline phosphatase staining, differentiation into neurons by NeuroFluor NeuO staining, and differentiation into chondrocytes by Alcian Blue staining.

[0049] The immortalized cells obtained by the present invention and tissue cells induced to differentiate from the immortalized (stem) cells can be used, for example, by cell transplantation into diseased or damaged sites, and can be provided as regenerative medicine products. Examples of regenerative medicine products include cultured skin, cultured cartilage, cultured corneal epithelium, and various cell sheets (e.g., epidermal cell sheets, fibroblast sheets, corneal endothelial cell sheets, cardiac muscle cell sheets, osteoblast sheets, myoblast sheets, nerve cell sheets, chondrocyte sheets, hepatic cell sheets, pancreatic islet cell sheets, and periodontal ligament cell sheets). [Example]

[0050] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. In the following examples, the serum-containing medium used to culture human mesenchymal stem cells (MSCs) was prepared with a composition of D-MEM (Low Glucose), 20% FBS, 0.01 mol / L Hepes, 100 units / ml penicillin, 100 μg / ml streptomycin, and 20 ng / ml bFGF. Because bFGF has a very short half-life, Gibco Heat Stable Recombinant Human bFGF (Thermo Fisher Scientific), which has excellent stability, was used. MSCs were cultured in the above medium in a 5% CO2 incubator. The human MSCs used in the experiments were derived from bone marrow, adipose tissue, umbilical cord blood, or umbilical cord matrix.

[0051] Example 1: Construction of SeV vector carrying immortalizing gene and examination of infection conditions (1) Construction of SeV vector carrying an immortalizing gene We selected the commonly used immortalization factors Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog), hTERT (human telomerase reverse transcriptase), SV40T (simian virus 40 large T antigen), and E6 / E7 (human papillomavirus 16 E6 and E7 proteins) and incorporated the Bmi-1 gene (SEQ ID NO: 1), hTERT gene (SEQ ID NO: 2), SV40T gene (SEQ ID NO: 3), and E6 / E7 gene (SEQ ID NO: 4) into the SeV vector, a non-integrating RNA viral vector. ID Pharma Co., Ltd. (Headquarters: Tsukuba City) was commissioned to incorporate the immortalization genes into the SeV vectors and manufacture the vectors. Three of the four vectors also contained the ORFs (red), GFP (green), and BFP (blue) of fluorescent dye genes, allowing for easy microscopic detection of gene expression (vector genome). Figure 1 shows the structure of each vector. Regarding the location of the immortalization genes to be loaded into the SeV vector, Bmi-1 and SV40T were loaded at the most upstream position of the vector genome because high expression results in long-term expression, and loading them upstream of the vector genome in the SeV vector results in high expression. The loading positions of hTERT and E6 / E7 were changed to downstream positions due to reduced vector production efficiency, which was thought to be caused by high expression.

[0052] The SeV vector used was the TS15ΔF type vector, which has been improved so that the viral vector disappears from the cells when the culture temperature is changed from 35°C to 37°C (Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, Takada N, Inoue M, Hasegawa M, Kawamata S, Nishikawa S. Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14234-9.).

[0053] (2) Examination of conditions for SeV vector infection When transducing genes with SeV vectors, the infection efficiency varies greatly depending on the cell type. Furthermore, excessive infection can cause damage to some cell types. Therefore, we first examined the infection MOI (multiplicity of infection: the number of vector particles per cell) for each cell type to be used.

[0054] Commercially available bone marrow-derived mesenchymal stem cells (product name: Ultra-Pure Human Mesenchymal Stem Cells (REC), PuREC Co., Ltd.) were cultured at 5 × 10 cells per well in a 48-well plate (FALCON 353230) containing 200 μl of medium. 4 The cells were seeded at 70-80% confluence and infected with four types of SeV vectors carrying immortalization factor genes, either singly or in combination, at an MOI of 1, 5, or 20 for each vector, and then incubated overnight. The medium was replaced with fresh medium the next day, and thereafter every two days. Five days after vector infection, the cells were expanded onto 12-well plates (FALCON 353043), and cells were observed 9 days after infection.

[0055] The results are shown in Figures 2–4. Nine days after SeV vector infection, no cell damage was observed in any wells, and no abnormalities were observed even at an MOI of 20 (MOI of the SeV vector itself: 80). The infection efficiency of the SeV vector was observed using an inverted fluorescence microscope (Nikon) based on the expression of fluorescent dye. In single-vector infection, fluorescent dye expression was observed in most cells at an MOI of 20 (Figure 2). Similar results were obtained with coinfection with two factors, Bmi-1 and hTERT (Figure 3). Similar results were also obtained with coinfection with three factors, Bmi-1, hTERT, and SV40T (Figure 4). These results suggest that infection at an MOI of 20 enables gene transfer to nearly 100% of cells. Therefore, we decided to use an MOI of 20 for subsequent SeV vector infection of MSCs.

[0056] Furthermore, among the SeV vector combinations tested, co-infection with Bmi-1, hTERT, and SV40T vectors at a high MOI of 20 clearly increased cell numbers compared with other combinations, including the uninfected control (Fig. 4). Actively dividing cells at MOI 20 were smaller than non-fluorescent cells at MOI 1, and both ORF and GFP fluorescence were positive. This suggests that the promotion of cell proliferation is due to the action of the three immortalizing factors: Bmi-1, hTERT, and SV40T.

[0057] (Example 2) Selection of immortalization factors MSCs were infected with SeV vectors carrying the genes for four immortalization factors (Bmi-1, hTERT, SV40T, E6 / E7) and cultured for a long period of time to identify the factors required for MSC immortalization.

[0058] For the study, umbilical cord blood-derived MSCs (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)) were used, and the MSCs were infected with one or a combination of two or more SeV vectors carrying immortalization factor genes (15 combinations in total) (48-well plate, 3 × 104 MSCs were cultured in 48 wells, 12 wells, and 6 wells, with an MOI of 20. MSCs not infected with SeV vector served as a negative control. As the MSCs proliferated, they were expanded from 48 wells to 12 wells, and then to 6 wells. After that, when the cells reached confluence (the adhesive surface of the plate was 100% covered by cells), the cells were counted and passaged. The proliferation rates of a total of 16 types of cells were measured, and the cell morphology was observed under a microscope.

[0059] The cell proliferation rate was measured by immortalization gene transfection (cell number at the time of transfection: 3 × 10 4 The cells were measured over a period of 80 days from the day they were taken. The results (No. 1 to No. 16) sorted in descending order of cell count at 80 days are shown in Table 1 below, along with the cell counts.

[0060] [Table 1]

[0061] The condition of the cells was observed approximately two months (day 59), when cell proliferation in the negative control had stopped, and the uniformity of cell size and the degree of dead cells detached from the culture plate were graded on a two-point scale (Figure 5). The results are shown in Table 1 (uniform cell size and little cell detachment: ◯, heterogeneous cell size and heavy cell detachment: ×, and the presence or absence of the immortalization factor is indicated by + or -).

[0062] As shown in Table 1 and Figure 5, cell morphological abnormalities were observed in all combinations lacking the hTERT factor (including the negative control), suggesting that hTERT is essential for cell immortalization using SeV vectors. Furthermore, when the cell number and immortalization factor combinations were compared, of the four immortalization factors, E6 / E7 had almost no effect on cell proliferation.

[0063] From the above results, combinations of immortalization factors suitable for immortalization using SeV vectors were extracted from Table 1 and summarized in Table 2 (No. 16 is shown as a negative control for comparison).

[0064] [Table 2]

[0065] Figure 6 shows the cell growth curves. Growth of the negative control (No. 16) stopped after approximately two months (66 days), while growth was prolonged for immortalization factor-transfected cells (Nos. 1, 2, 4, and 12). Although growth was prolonged for cells transfected with the hTERT gene alone (No. 12), the growth rate gradually decreased after two months, and further growth was not expected. In contrast, cells transfected with a combination of Bmi-1 and hTERT, hTERT and SV40T, or Bmi-1, hTERT, and SV40T (Nos. 1, 2, and 4) showed stable growth without any decline in growth rate even at 80 days. Among these, the growth rate of the triple factor combination of Bmi-1, hTERT, and SV40T was the fastest.

[0066] Instead of the above-mentioned umbilical cord blood-derived MSCs, combinations of immortalization factors were examined using mesenchymal stem cells derived from human umbilical cord matrix (product name: Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), Promo Cell, product code C-12971). Similarly, the combination of Bmi-1, hTERT, and SV40T resulted in the best proliferation of immortalized cells, and microscopic observation revealed the most uniform and healthy cells.

[0067] Example 3: Analysis of immortalized cultured cells using SeV vector carrying the three-factor (Bmi-1 / hTERT / SV40T) genes (1) Preparation of immortalized cells transfected with three factors (Bmi-1 / hTERT / SV40T) After examining MSCs derived from three different tissues, we determined that the SeV vector carrying the three-factor (Bmi-1 / hTERT / SV40T) gene was most suitable for immortalization, and detailed characterization of MSCs immortalized with this vector was performed. The cells used were the same umbilical cord blood-derived MSCs (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)) used in the selection of the immortalization factor, but these cells had been cultured for a longer period (slightly less than one month) than the time of selection. Gene transduction was performed using the SeV vector carrying the three-factor (Bmi-1 / hTERT / SV40T) gene at an MOI of 20, as in Example 2. MSCs not transfected with the SeV vector were cultured simultaneously as a negative control.

[0068] (2) Comparison of growth rates due to changes in culture temperature The three-factor (Bmi-1 / hTERT / SV40T)-transfected immortalized cell line was cultured in a 6-cm dish in a CO2 incubator at 35°C. When the cells had proliferated to approximately 80% of the adhesive surface of the plate, 1 / 5 of the cells were subcultured onto a new 6-cm dish, and the culture was continued for 75 days.

[0069] The SeV vector carrying the immortalization gene is a temperature-sensitive vector, and the SeV vector genome can be rapidly eliminated from cells by raising the culture temperature from 35°C to 37°C (Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, Takada N, Inoue M, Hasegawa M, Kawamata S, Nishikawa S. Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14234-9). To confirm whether the SeV vector genome can be eliminated from MSCs, we cultured immortalized cells transfected with the three factors mentioned above (Bmi-1 / hTERT / SV40T) at different temperatures and examined the change in cell number.

[0070] To examine SeV vector elimination by temperature change, we used immortalized cells (day 75) at a stage when the proliferation ability of untreated MSCs had decreased. When subculturing the cells on day 78, two petri dishes containing the same number of cells were prepared. One dish was maintained at 35°C, while the other was cultured at 37°C. The proliferation patterns of the two dishes were compared. The results are shown in Figure 7. The SeV vector was eliminated (fluorescence disappeared) from the cells cultured at 37°C, and proliferation rapidly decreased. However, there was no particular change in the cells cultured at 35°C, and they continued to proliferate vigorously even more than 130 days after gene transfer (Figure 7).

[0071] (3) Comparison of fluorescent protein expression and cell morphology with changes in culture temperature When the expression of fluorescent proteins was confirmed using a fluorescence microscope 10 days after the temperature change, fluorescence of OFP (co-transfected with Bmi-1) and GFP (co-transfected with hTERT) was confirmed in cells maintained at 35°C, but expression was hardly detectable in cells whose culture temperature was raised to 37°C (Figure 8).

[0072] When the cell morphology was checked two weeks after subculture (92 days after the start of culture), no change in the cell state was observed at 35°C and the cells were dividing vigorously. However, at 37°C, the cell division rate visibly decreased and cell size varied, significantly different from the uniformity at 35°C (Figure 9).

[0073] These results suggest that the temperature sensitivity of SeV vectors, which has already been demonstrated in iPS cells, was confirmed in MSCs by changing the temperature from 35°C to 37°C. At the same time, removal of the three immortalizing factors reset the immortalization of the cells, returning them to a state of finite proliferation.

[0074] (4)Chromosome analysis Chromosome analysis of the cells was performed using quinacrine-Hoechst banding, as indicated by the triangle in Figure 7. For the quinacrine-Hoechst banding, the chromosome slide was first immersed in 50 ml of McIlvaine's solution (280 ml of 0.1 M citric acid solution and 220 ml of 0.2 M disodium hydrogen phosphate solution, mixed and autoclaved). It was then immersed in 50 ml of McIlvaine's solution containing 10 ng / ml Hoechst 33258 (cat: B-2883-25MG, Sigma) for 30 minutes. After gently running tap water over the back of the chromosome slide, it was again immersed in McIlvaine's solution for 5 minutes and then mounted with a coverslip in McIlvaine's mounting medium (a 1:1 mixture of McIlvaine and glycerol). Analysis was performed using a chromosome analysis microscope (model: AxioImager Z2, ZEISS) and chromosome analysis software (model: Ikaros V5.7.4 CM / V5.4.12, Metasystems). As a result, the immortalized cell lines had a normal karyotype at all points, just like the parental lines (Figure 10).

[0075] (5) Evaluation of telomere length Telomere length was assessed at the points marked with an asterisk (*) in Figures 6 and 7. Telomere length was assessed by real-time PCR using genomic DNA as a template and relative quantification of telomere sequences. Genomic DNA was extracted from cells using the Gentra® Puregene® Kit (Qiagen) according to the manufacturer's instructions. For real-time PCR, the Telomere primer set and Single Copy Reference primer set included with the Relative Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories) were used, and the PCR reagents were FastStart Essential DNA Green Master (Roche). PCR reaction conditions followed the manufacturer's recommendations. PCR reactions and data acquisition were performed using StepOnePlus (Life Technologies Japan), and data analysis was performed using StepOne Software v2.3.

[0076] The results are shown in Figure 11. Telomere elongation was confirmed with the combination of three factors (Bmi-1, hTERT, and SV40T) and the combination of two factors (Bmi-1 and hTERT), but no elongation was observed with hTERT alone (Figure 11A). Furthermore, significant telomere shortening was observed with long-term culture after removal of the immortalization factor (Figure 11B). Although shortening was observed even without removal of the immortalization factor (35°C), the telomere length of the final sample was similar to that of the initial hMSC. Based on these results and comparison with proliferation capacity, it was suggested that hTERT is necessary but not sufficient for immortalization.

[0077] Example 4: Differentiation Potential of Immortalized Population Cells The three-factor (Bmi-1 / hTERT / SV40T)-transfected immortalized cells (MSCs) prepared in Example 3 were examined for their ability to differentiate into adipocytes, osteoblasts, neurons, and chondrocytes. For the differentiation test, cord blood-derived MSCs (ATCC PCS-500-010) were used, which were immortalized with the three factors Bmi-1, hTERT, and SV40T and cultured for a long period (four months after gene transfection). Four months is a difficult period for general non-immortalized MSCs to culture.

[0078] Commercially available differentiation kits (Promo Cell) were used for MSC differentiation. Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (product code C-28016) was used for adipocyte differentiation, Mesenchymal Stem Cell Osteogenic Differentiation Medium (product code C-28013) for osteoblast differentiation, Mesenchymal Stem Cell Neurogenic Differentiation Medium (product code C-28015) for neuronal differentiation, and Mesenchymal Stem Cell Chondrogenic Differentiation Medium (product code C-28012) for chondrocyte differentiation were used. The procedures were carried out according to the attached protocols.

[0079] Adipocyte differentiation was confirmed using the fluorescent dye Lipi-Green (Dojin Chemical: product code LD02), which specifically stains lipid droplets, an indicator of adipocyte differentiation. Osteoblast differentiation was confirmed by alkaline phosphatase staining. Neuronal differentiation was confirmed using the fluorescent dye NeuroFluor NeuO (VERITAS: product code ST-01801), which specifically stains neurons. Chondrocyte differentiation was confirmed by Alcian blue staining.

[0080] We compared the differentiation potential of immortalized MSCs cultured for approximately two weeks after purchase with that of untransfected MSCs in the logarithmic growth phase, which retained differentiation potential, as a positive control. Lipid droplets, an indicator of differentiation, were observed in both immortalized MSCs and the positive control during adipocyte differentiation. The immortalized MSCs showed a lower degree of differentiation than the positive control (Figure 12). For osteoblast differentiation, alkaline phosphatase staining was observed in both immortalized MSCs and the positive control, indicating osteoblast differentiation (Figure 13). The immortalized MSCs showed a higher differentiation rate than the positive control. For neuronal differentiation, fluorescence was observed in almost all cells in both immortalized MSCs and the positive control, indicating neuronal differentiation (Figure 14). Although we were unable to compare the degree of differentiation between immortalized MSCs and the positive control, the induced MSCs stained significantly more intensely with Alcian blue than the negative control (not induced), confirming their differentiation into chondrocytes (Figure 15). These results confirmed that immortalized cells (MSCs) transfected with three factors (Bmi-1 / hTERT / SV40T) maintained pluripotency, similar to that of the positive control MSCs, even four months after immortalization.

[0081] (Example 5) Cloning test of immortalized MSCs Experiments were conducted to confirm whether the three-factor (Bmi-1 / TERT / SV40T)-transfected immortalized cells (MSCs) prepared in Example 3 could be cloned, and to what extent it would be difficult to clone non-immortalized MSCs.

[0082] Cloning was performed using three types of MSCs: non-transfected umbilical cord blood-derived MSCs (ATCC PCS-500-010) that were initially cultured for approximately two weeks after purchase and retained their differentiation potential and were in the logarithmic growth phase; immortalized MSCs that had been recently transfected with genes (two weeks had passed); and immortalized MSCs that had been cultured for a long period of time (four months had passed) after transfection with genes.

[0083] The three types of MSCs were cultured and maintained, then completely dissociated into single cells, the cell count was measured, and the cells were seeded into four 24-well sections of a 96-well plate at varying cell numbers (4 cells, 8 cells, 16 cells, and 32 cells / 100μl per well). They were cultured in a 35℃, 5% CO2 incubator until the single cells formed colonies. Half of the medium was replaced every three days to avoid disrupting colony formation.

[0084] After culturing for 10 days and observing under a microscope, it was found that the single cells had proliferated to colonies of sufficient size. The number of colonies formed per well was then counted and the results were summarized in diagrams (Figures 16 and 17). The numbers in the 96 wells in the diagrams indicate the number of colonies formed.

[0085] These results make it possible to determine at a glance how easily colonies could be formed. Non-immortalized MSCs in the early stage of proliferation correspond to typical MSCs, but colonies were formed in only two wells of the 32 cells / well compartment. In contrast, MSCs transfected with the immortalization gene using the SeV vector formed colonies at a high frequency of 9 wells / 24 wells, even at a low density of 4 cells / well, just two weeks after transfection (Figure 16). After four months, colonies formed in 16 wells / 24 wells, more than half of the wells, even at a low density of 4 cells / well (Figure 17).

[0086] These results demonstrate that even MSCs, which are difficult to clone, can be easily cloned by introducing three immortalizing genes using an SeV vector, regardless of the time that has passed since then. The ability to clone at any stage is extremely useful from the perspective of quality control in regenerative medicine.

[0087] (Example 6) Various analyses of clonal immortalized MSCs (1) Proliferation potential of clonal cells Two weeks after SeV vector infection, five MSC clones were cloned as single cells. After 60 days of culture, they were divided into two groups and cultured at 35°C and 37°C. Three of the five clones showed a decrease in growth at 37°C upon removal of the SeV vector. The growth curve for clone A10 is shown (Fig. 18).

[0088] (2) Confirmation of SeV vector elimination by temperature change At 35°C, the presence of SeV vectors could be confirmed by fluorescence observation, as in the mass culture experiment, but the presence of SeV vectors could not be confirmed in the 37°C group.

[0089] (3) Chromosome analysis of cloned cells Chromosome analysis of the 10 clones revealed that 8 clones had a normal karyotype. The results of karyotype analysis of clone A10, which exhibited cell proliferation as shown in Figure 18, are shown in Figure 19.

[0090] (4) Differentiation potential of clonal immortalized MSCs Of the cloned immortalized MSCs, five clones were examined for their differentiation potential into adipocytes, osteoblasts, and neurons using the same method as in Example 4. As a result, differences in morphology and the degree of differentiation were observed among the clones. However, differentiation into adipocytes, osteoblasts, and neurons was observed in all clones, and multipotency was maintained (Figure 20). Some clones showed a much better differentiation state than the cells before cloning, and in the neuronal differentiation, not only was neural marker expression observed, but also the formation of a neural network with neurites spreading out in a mesh-like pattern was observed.

[0091] (Example 7) Immortalization and single-cell cloning of MSCs with different cell ages (degrees of senescence) Single-cell cloning is important in regenerative medicine because cell populations expanded from a single MSC have the same genetic properties, resulting in consistent quality and facilitating quality control. Single-cell cloning is also essential for selecting only the target cells when genes are introduced from outside.

[0092] MSCs isolated from the human body contain a mixture of cells of various stages, from young cells to senescent cells. The ratio of these cells varies from person to person, making it difficult to extract and proliferate only cells of a specific stage (youngness). Conversely, if MSCs of any stage, from young cells to senescent cells, could be immortalized and further cloned, the range of applications in regenerative medicine would be greatly expanded.

[0093] Therefore, we investigated whether single-cell cloning was possible by immortalizing three types of cells, ranging from young cells to aged cells, or cells in between.

[0094] The cells used were umbilical cord blood-derived MSCs (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)). The purchased cells were cultured at 37°C for approximately 80 days until proliferation ceased. During this time, a portion of the cells was cryopreserved after passage approximately every week (Figure 21).

[0095] Three types of cells, early, middle, and late (Day 9, 24, and 49) from the cryopreserved cells, were thawed simultaneously, cultured for 11 days to minimize the effects of freezing and thawing, and then seeded onto a 24-well plate (24 wells, 1 × 10 5 The next day, the cells were infected with an SeV vector carrying the three factors (Bmi-1 / hTERT / SV40T) genes at an MOI of 20 for immortalization (Days 21, 36, and 61).

[0096] After immortalization induction, the cells were transferred from 37°C to a 35°C CO2 incubator and expanded for 8 days (24 wells → 6 wells). The immortalized cells were then seeded into 96-well plates at 1, 2, or 4 cells per well, while the non-immortalized control cells were seeded at 5, 10, 15, or 20 cells per well. These were cultured for approximately 2 weeks until a single cell divided and proliferated to form a cell cluster (colony), and the number of colonies that emerged was counted under a microscope.

[0097] The morphology of SeV vector-infected and non-SeV vector-infected MSCs of three different cell ages (early, middle, and late) was observed immediately before seeding on a 96-well plate (8 days after SeV vector infection), and the differences in appearance were striking (Fig. 22).

[0098] As shown in Figure 22, the cell density, which reflects cell number, is higher for younger cells, indicating active cell proliferation. Although there was no significant difference in cell number between SeV vector-infected and uninfected cells, a clear increase in cell number was observed in SeV vector-infected cells at the late stage. Changes in cell morphology and size were particularly pronounced. In SeV vector-uninfected control cells, cells enlarged over time and their division rate decreased, whereas in SeV vector-infected cells, cell division became more active and cell size became similar, making it impossible to distinguish between early, middle, and late stages based on size.

[0099] The results of cloning using 96-well plates confirmed that no colonies appeared in non-SeV vector-infected cells at 5 cells per well, making cloning difficult, as in Example 5. In the case of SeV vector-infected cells, many colonies were observed even in single-cell cloning plates seeded at 1 cell per well (Figures 23-1 and 23-2).

[0100] In the early stage, colonies were observed in 30 wells per 96 wells, in the intermediate stage in 47 wells, and in the late stage in 27 wells. These results demonstrated that it is possible to easily obtain cell populations derived from single cells from MSCs immortalized with SeV vectors, regardless of their degree of senescence.

[0101] (Example 8) Confirmation of effect on proliferation-arrested MSCs The effect of SeV vectors carrying immortalizing genes on senescent MSCs in which cell division had completely stopped was confirmed.

[0102] Cryopreserved cells whose cell proliferation had almost stopped (Day 72) were frozen and thawed and seeded on a 24-well plate to suppress the effects of freezing and thawing. After confirming that the cells were not proliferating (Day 90), the cells were infected with the SeV vector (24 wells, 1 × 10 5 (cells / well, MOI: 20) Then, changes in the cells were observed under a microscope.

[0103] In cells not infected with SeV vector (control cells), no significant changes were observed, and all cells were observed to become round and die. In contrast, in SeV vector-infected cells, no changes were observed for several days, but after 1 week, many cells became round and appeared to die. However, small, adherent cells were observed among them, and after 2 weeks, they transformed into a cell population that proliferated vigorously, similar to the immortalized cells (Fig. 24).

[0104] The cells that had begun to regrow were subjected to single-cell cloning in the same manner as in Example 7. As a result, although the percentage was lower than the results for the single cells described above in Figures 23-1 and 23-2, proliferated cell populations were observed in 17 of the 96 wells, demonstrating that even cells that had completely stopped dividing could resume division by infection with the SeV vector, enabling single-cell cloning (Figure 25).

[0105] This change differs from the cell proliferation that accompanies cell immortalization and is thought to involve the removal of cytoplasm that is not necessary for cell proliferation. Although the detailed mechanism is unknown, since cells that have completely stopped proliferating due to aging are repopulated, in this case it is more appropriate to describe it as "cell rejuvenation" rather than "immortalization."

[0106] (Example 9) Examination of cell proliferation of human mesenchymal stem cells in serum-free medium Adipose tissue-derived human mesenchymal stem cells (hMSC-AT) (PromoCell; C-12977) and bone marrow-derived human mesenchymal stem cells (hMSC-BM) (PromoCell; C-12974) were used in this experiment. The day of SeV vector infection of hMSC-AT and hMSC-BM was defined as the "infection date." Uninfected and infected hMSC-AT cells were cultured in serum-containing medium until days 13 and 11, respectively, from the day of infection. Uninfected and infected hMSC-BM cells were cultured until days 11 and 17, respectively, from the day of infection. The serum-containing medium consisted of D-MEM (low glucose), 20% FBS, 0.01 mol / L Hepes, penicillin 100 units / ml, streptomycin 100 μg / ml, and bFGF 20 ng / ml. The medium was then replaced with serum-free medium for continued culture. The serum-free medium used was Stem Fit For Mesenchymal Stem Cells (AJINOMOTO; A3) medium, and the culture dishes used were 6 cm dishes (CORNING; 353004) coated with iMatrix-511 silk (Matrixome; 892 091).

[0107] SeV vector infection was 2 × 10 5 hMSC-AT cells or hMSC-BM cells were infected with SeV vectors at an MOI of 40. Non-infected and infected cells were each infected once. The medium was replaced 24 hours after infection, and maintenance culture was continued. Cell culture was performed in a 35°C, 5% CO2 incubator.

[0108] In the experiment with hMSC-AT cells, a decrease in cell number was observed in the uninfected cell line, so culture was terminated on day 52 after infection. On the other hand, the SeV vector-infected cell line continued to grow even after day 52 (Fig. 26).

[0109] In the hMSC-BM cell experiment, the uninfected cell line also stopped growing after 40 days from the day of infection, and cell number was measured on day 72. As the cell number was below the detection limit, cell culture was terminated at this point. On the other hand, the SeV vector-infected cell line continued to grow even on day 40 (Figure 27).

[0110] These results confirmed that SeV vector infection can extend the cell division capacity of adipose tissue- and bone marrow-derived MSC cells, similar to that of cord blood-derived MSC cells, and that infected cells can continue to grow in serum-free medium as well as in serum-containing medium.

[0111] After SeV vector infection, GFP (hTERT) and OFP (Bmi-1) fluorescence was observed. Comparison of hMSC-AT and hMSC-BM cells at the initial stage of serum-free culture (days 16 and 24 after infection) with those at the time points after continued culture in serum-free medium (days 58 and 50 after infection) revealed that GFP and OFP expression was maintained (Figure 28). These results confirmed that SeV expression was maintained during continued culture in serum-free medium, similar to that observed in culture in serum-containing medium.

[0112] (Example 10) Examination of cell proliferation of immortalized rat MSCs Rat subcutaneous adipose tissue-derived mesenchymal stem cells (rMSCs) (Cosmo Bio Co., Ltd., MSA01C) were cultured in a growth medium for rat subcutaneous adipose tissue-derived mesenchymal stem cells (Cosmo Bio Co., Ltd., MSA-GM).

[0113] SeV vector infection was 2 × 10 5The SeV vector was injected into rMSC cells at an MOI of 40. The uninfected cells were infected once, and the infected cells were infected twice. The medium was changed 24 hours after infection, and maintenance culture was continued. Culture dishes were placed on collagen-coated dishes (IWAKI, 4810-010) and maintained at 35°C in a 5% CO2 incubator.

[0114] During long-term culture, we observed that rMSC cell lines, which normally lose their proliferation potential after several passages, continued to proliferate in uninfected cells (Figure 29). This is thought to be due to accidental immortalization of the rMSC cell line. However, after 75 days of culture, we observed that the proliferation rate of infected cells was more than twice as fast as that of uninfected cells.

[0115] After SeV vector infection, GFP (hTERT) and OFP (Bmi-1) were observed for fluorescence. Fluorescent images were taken using an all-in-one microscope (KEYENCE; BZ-X800). Photographs were taken at 20x magnification with exposure times of 1 / 5 and 1 / 20 seconds for GFP and OFP, respectively. Fluorescent image observations revealed that expression of OFP (Bmi-1) and GFP (hTERT) was observed at 35°C even after 58 days of culture after infection (Figure 30).

[0116] (Example 11) Examination of cell proliferation of immortalized HFL1 Human fibroblasts (HFL1 cells, RIKEN: RCB0521) were cultured in Ham's-F12 medium (Nakalai; 17458-65) supplemented with 15% (v / v) heat-inactivated fetal bovine serum (NICHIREI; 175012) and 100 units / mL of penicillin-streptomycin solution (Fujifilm Wako; 168-23191).

[0117] SeV vector infection was 2 × 10 5HFL1 cells were infected with SeV vectors at an MOI of 40. Non-infected and infected cells were each tested twice. The medium was replaced 24 hours after infection, and maintenance culture was continued. On day 34 after infection, the infected cells were divided into two groups: one cultured at 35°C and the other at 37°C (Figure 31, arrow). Cell culture was performed at each temperature in a 5% CO2 incubator.

[0118] The culture of uninfected cells was terminated on day 148 after infection because a decrease in cell number was observed. On the other hand, the culture of infected cell lines was terminated on day 259 or 273 after infection because cells cultured at 37°C no longer grew (Fig. 31). This demonstrates that SeV vector infection can extend the cell division ability even in HFL1 cells.

[0119] We calculated how much the cells proliferated after the start of culture. The results showed that proliferation stopped at an order of 17 for uninfected cells, whereas proliferation stopped at an order of 31 for cells infected with the SeV vector and cultured at 37°C. Cells infected with the SeV vector and cultured at 35°C continued to proliferate to approximately an order of 43 without cessation of growth even during the culture period, resulting in a difference of 26 orders of magnitude in cell number compared to cells uninfected with the SeV vector over 260 days. These results confirmed that SeV vector infection can increase cell proliferation in human fibroblast HFL1 cells.

[0120] After SeV vector infection, GFP (hTERT) and OFP (Bmi-1) fluorescence were observed. The results are shown in Figure 32. On day 34 after infection, the cells showed GFP and OFP fluorescence signals similar to those observed on day 10 after infection. The cells were then cultured for an additional 221 days at 35°C and 37°C. Cells cultured at 35°C showed a significant decrease in GFP expression compared with cells on days 34 and 255, whereas OFP expression remained similar to that on days 10 and 34. On the other hand, cells cultured at 37°C showed a significant decrease in both GFP and OFP positivity compared with cells on days 10 and 34. These results confirmed that SeV was eliminated in a temperature-sensitive manner.

[0121] In addition, cells undergoing cellular senescence generally exhibit characteristics such as an increase in cell size, a flattened shape, and the formation of vacuoles. Infected cells cultured at 35°C did not exhibit the characteristics of cellular senescence compared to infected cells cultured at 37°C. These results confirmed that cells cultured at 35°C did not undergo cellular senescence in terms of cell morphology.

[0122] (Example 12) Examination of cell proliferation of immortalized HUVEC Human umbilical vein endothelial cells (HUVEC cells, Promocell, C-12205) were cultured in endothelial cell medium (ScienCell; 1001).

[0123] SeV vector infection was 2 × 10 5 HUVEC cells were infected at an MOI of 40. The non-infected condition was tested once, and the infected condition was tested twice. 24 hours after infection, the medium was replaced and maintenance culture continued. 35 days after infection, the infected cells were divided into two conditions: one cultured at 35°C and the other at 37°C (Figure 33, arrow). Cell culture was performed at each temperature in a 5% CO2 incubator.

[0124] The culture of uninfected cells was terminated on day 39 after infection due to a decrease in cell number. On the other hand, infected cells continued to proliferate even on day 74 after infection (Fig. 33). This confirmed that SeV vector infection can prolong cell proliferation even in HUVEC cells.

[0125] We calculated how much the cells proliferated after the start of culture. While proliferation stopped at around 8 orders of magnitude for uninfected cells, SeV vector-infected cells cultured at 37°C proliferated to 18 orders of magnitude. When SeV vector-infected and cultured at 35°C, cells continued to proliferate to around 21 orders of magnitude, resulting in a 13-order difference in cell number compared to uninfected cells over 74 days. These results confirmed that SeV vector infection can increase cell proliferation in human umbilical vein endothelial (HUVEC) cells.

[0126] After SeV vector infection, GFP (hTERT) and OFP (Bmi-1) fluorescence was observed. The results are shown in Figure 34. On day 36 after infection, cells cultured at 35°C showed GFP and OFP fluorescence signals similar to those of cells cultured on day 7 after infection. Cells were then cultured for an additional 37 days at 35°C and 37°C. Although the percentage of fluorescent-positive cells in the cells cultured at 35°C was reduced compared with cells cultured on days 7 and 36 after infection, fluorescent signals were still observed in many cells. On the other hand, the percentage of fluorescent-positive cells in the cells cultured at 37°C was significantly reduced compared with cells cultured on days 7 and 36 after infection. These results confirmed that SeV was eliminated in a temperature-sensitive manner.

[0127] (Example 13) Introduction of a multiple growth factor-inserted human artificial chromosome vector into immortalized MSC cells and stability and differentiation induction after long-term culture

[0128] (1) Chromosome transfer by microcell fusion and isolation of drug-resistant clones CHO cells carrying 21HAC2, which are loaded with HGF (hgf), GDNF (gdnf), IGF-1 (igf-1), and luciferase (e-luc) as described in Watanabe et al. (Mol Ther Nucleic Acids. 2015), were used as chromosome donor cells, and hMSC-UC No. 3 cells, the immortalized MSC cells described in Example 1, were used as chromosome recipient cells. Micronuclear cell fusion and culture were performed as described in Katoh et al. (BMC Biotechnology, 2010, 10:37). After one week of culture under BS selection culture, resistant colonies appeared, and a total of nine colonies obtained from four fusions were isolated, expanded, and further analyzed.

[0129] (2) Confirmation of the transferred chromosome (2-1) Fluorescence microscopy When the nine cloned colonies were observed under a fluorescence microscope, GFP-positive cells were observed in all clones, with the positive rate ranging from 50 to 100%.

[0130] (2-2)FISH analysis Four clones confirmed by PCR analysis (clone names: hMSC-UC No. 3 #1-01, #1-02, #2-01, #3-01) were subjected to FISH analysis using PACs containing HGF (hgf), GDNF (gdnf), IGF-1 (igf-1), and luciferase (e-luc) as probes. Two clones were confirmed to contain human artificial chromosomes without karyotypic abnormalities (Figure 35, hMSC-UC No. 3, #3-01).

[0131] (3) In vitro differentiation induction The above four clones confirmed by PCR analysis can be induced to differentiate into bone, cartilage, and fat cells according to the method of Okamoto et al. (BBRC, 295:354, 2002), and it can be confirmed whether they maintain the same differentiation ability as the parent strain.

[0132] From the above analytical results (1) to (3), it was confirmed that two clones of immortalized MSCs carrying multiple growth factor-inserted human artificial chromosome vectors were obtained by microcell fusion.

[0133] (Example 14) Verification of the effect of immortalized MSCs on healing of enteritis in an inflammatory bowel disease model (1) CD4 positive CD45RB high positive CD25 negative (CD4 + CD45RB High+ CD25 - ) Isolation of T cells Thirty 8-week-old female BALB / cAJcl (Japan CLEA) rats were acclimatized for 1 week, and then bled by cardiac vein sampling under anesthesia. The spleen was hemolyzed after tissue dissociation using a MACS system (Miltenyi Biotech). The mesenteric lymph nodes were crushed using a 1 mL syringe plunger and filtered through a 40 μm cell strainer. Spleen and mesenteric lymph node cells were combined and treated with CD4 microbeads (Miltenyi Biotech) to separate CD4+ cells. CD4+CD45RBHigh+CD25- T cells were then isolated by antibody labeling (APC-H7 rat anti-mouse CD4 antibody (BD), FITC rat anti-mouse CD45RB antibody (BD), and PE / Cy7 anti-mouse CD25 antibody (BioLegend)). The cells were then sorted using a flow cytometer (MoFlo XDP, BECKMAN).

[0134] (2) CD4 + CD45RB High+ CD25 - T cell transfer 4.0 × 10 per 9-week-old female SCID (CB-17 / lcr-scid / scidJcl: CLEA Japan) 5 CD4+CD45RBHigh+CD25- T cells were transplanted via tail vein injection (cell transplant group: 8 mice x 5 groups, untreated group (PBS administered): 1 group x 7 mice). After transplantation, mice were weighed three times a week and their coat and feces were observed. 21 days after cell transplantation, randomization was performed based on weight change using the "multivariate block allocation" system in the statistical analysis software JMP, and the cell transplantation groups were divided into groups. Individuals outside the mean relative body weight ± 2SD were excluded.

[0135] (3) Transplanted cell culture To obtain cells for transplantation, hMSC-UC (parent MSCs) and immortalized MSCs were cultured under various cell culture conditions.

[0136] (4) MSC administration (therapeutic cell transplantation) CD4+CD45RB High+On days 21, 28, and 35 after CD25-T cell transplantation, parental MSCs and immortalized MSCs were added at 1.0 × 10 per mouse. 6 Each mouse was administered a dose of 1 mg / kg of Dexamethasone (Sigma) via tail vein injection. As a positive control, Dex (1 mg / kg, 100 μL / mouse) was administered subcutaneously for 14 days starting from day 21. The Dex solution was prepared freshly based on the group's average body weight on days 21 and 28.

[0137] (5) Collection of materials On day 42 after the start of the experiment, whole blood was collected and serum was prepared (stored at -80°C). Furthermore, the digestive tract from the pylorus to the anus (colon to rectum) was sampled, and after evaluation of the condition, weight and length were measured, the sample was fixed in 10% formalin.

[0138] The results are shown in Figure 36. The efficacy of MSC transplantation in treating enteritis in an adoptive transfer IBD model using SCID mice was examined. Clinical scores (weight loss score, hypertrophy score, stool score, and coat condition score) were significantly reduced in the Dex-treated, parental MSC-treated, and immortalized MSC-treated groups compared to the untreated group. In particular, the parental MSC-treated group showed a nearly half-life reduction, indicating a high efficacy in improving enteritis. The immortalized MSC-treated group showed no significant difference from the parental MSC-treated group, equivalent to Dex administration, demonstrating that the enteritis-healing effect of the MSC parent line was maintained even after immortalization and long-term culture following immortalization. Regarding the culture of cells for transplantation, the immortalized cells proliferated significantly better than the parental cells at the initial stage of culture, and cell preparation was not time-consuming. [Industrial Applicability]

[0139] The present invention can be used in the fields of cell therapy and regenerative medicine. The cell immortalization technology of the present invention can be used not only on normal cells with slow cell proliferation but also on cancer cells, and can be used in the field of basic research. Furthermore, single-cell cloning, which is essential for gene transfer and chromosome transfer, is also possible. In addition, the cells obtained by the present invention continue to proliferate without aging, making quality control easier, facilitating the mechanization of culture, significantly reducing cell production costs, and enabling the handling of large numbers of cells. Therefore, the present invention will expand the range of diseases applicable to cell therapy and regenerative medicine, and contribute to the revitalization of medical-related industries both domestically and internationally. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for producing reversibly immortalized cells, comprising the steps of: (1) introducing a non-chromosomally integrating RNA viral vector carrying an immortalization gene into mammalian cells and expressing the immortalization gene in the cells; and (2) Cultivating and growing the cells obtained in step (1)

2. The method according to claim 1, wherein the immortalization gene is one or more immortalization genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.

3. The method according to claim 1 or 2, wherein the immortalization gene is any one of the following (a) to (d): (a) Combination of the Bmi-1 gene, the TERT gene, and the SV40T gene (b) Combination of Bmi-1 gene and TERT gene (c) Combination of the TERT gene and the SV40T gene (d) TERT gene

4. The method according to any one of claims 1 to 3, wherein the cells are somatic cells.

5. The method of claim 4 , wherein the somatic cells are somatic stem cells.

6. The method of claim 5 , wherein the somatic stem cells are mesenchymal stem cells.

7. The method according to any one of claims 1 to 6, wherein the chromosomally non-integrating RNA viral vector is a minus-strand RNA viral vector.

8. The method according to claim 7, wherein the minus-strand RNA viral vector is a paramyxovirus vector.

9. The method according to claim 8, wherein the paramyxovirus vector is a Sendai virus vector.

10. The method according to claim 9, wherein the Sendai virus vector is a temperature-sensitive Sendai virus vector.

11. The method according to claim 1, wherein the chromosomally non-integrating RNA viral vector is a Sendai virus vector, and further comprises the step of removing the Sendai virus vector after the culture in step (2).

12. The method according to claim 11, wherein the Sendai virus vector is removed by changing the culture temperature from 35°C to 37°C.

13. The method according to any one of claims 1 to 12, further comprising the step of cloning the immortalized cells after the culture in step (2).

14. An immortalized cell obtained by the method according to any one of claims 1 to 13.

15. An immortalized cell comprising, in a removable state, a Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.

16. A regenerative medicine product comprising the immortalized cells of claim 14 or 15.

17. A temperature-sensitive Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.

18. A kit for producing reversibly immortalized cells, comprising the temperature-sensitive Sendai virus vector according to claim 17.

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