WO2022097716A1 - 可逆的不死化細胞の製造方法 - Google Patents

可逆的不死化細胞の製造方法 Download PDF

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WO2022097716A1
WO2022097716A1 PCT/JP2021/040757 JP2021040757W WO2022097716A1 WO 2022097716 A1 WO2022097716 A1 WO 2022097716A1 JP 2021040757 W JP2021040757 W JP 2021040757W WO 2022097716 A1 WO2022097716 A1 WO 2022097716A1
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cells
gene
cell
vector
immortalized
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French (fr)
Japanese (ja)
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光雄 押村
寿晃 田畑
康宏 香月
愛海 宇野
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Tottori University NUC
Trans Chromosomics Inc
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Tottori University NUC
Trans Chromosomics Inc
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Priority to JP2022560822A priority Critical patent/JP7805590B2/ja
Priority to EP21889268.5A priority patent/EP4242317A4/en
Priority to US18/251,805 priority patent/US20230407268A1/en
Priority to CN202180075178.0A priority patent/CN116419968A/zh
Publication of WO2022097716A1 publication Critical patent/WO2022097716A1/ja
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18841—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18843—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a method for producing a reversible immortalized cell, and more particularly to a method for producing a reversible immortalized cell using a chromosomal non-integrating RNA viral vector carrying a predetermined immortalizing gene.
  • the main cell sources used for regenerative medicine and cell therapy are somatic stem cells such as mesenchymal stem cells (MSC) and pluripotent stem cells such as ES cells and iPS cells.
  • MSC mesenchymal stem cells
  • ES cells pluripotent stem cells
  • iPS cells pluripotent stem cells
  • stem cells mesenchymal stem cells (MSCs) are cells that originally exist in the body, so there is little risk of rejection and tumorigenesis, and they are excellent in terms of safety, but because the number of cell divisions is limited. The challenge is to secure a sufficient number of cells to supply to the medical field.
  • telomerase reverse transcriptase genes that regulate the expression or activity of telomerase (for example, Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.) are known.
  • vectors such as plasmid DNA, lentiviral vector, retroviral vector, and adenoviral vector are used for introduction of these immortalizing genes into cells (Patent Document 1, Patent Document 2, Patent Document 3, etc.).
  • the virus vector is preferable in that the gene can be efficiently introduced by simply filtering the culture supernatant of the recombinant virus vector-producing cell and adding it to the target cell.
  • the plasmid DNA and the adenovirus vector have a vector genome of DNA and are integrated into the host chromosome.
  • the vector genome is RNA, but in the cell, the DNA is taken in the form of DNA by a reverse transcription enzyme and integrated into the chromosome of the host to express the loaded gene.
  • the vector generally used for immortalization has a risk of damaging the gene of the host cell by incorporating foreign DNA into the chromosome of the host cell and, in some cases, forming a tumor, and thus the safety of regenerative medicine.
  • the Sendai virus vector (SeV vector) is a non-chromosomally integrated RNA virus vector that does not take the form of DNA.
  • the SeV vector can be introduced into a wide range of cell types and easily expresses proteins, so that it is widely used for inducing iPS cells (Patent Document 4).
  • stem cells obtained from human tissues are a group of young cells and aged cells, and have various properties. They divide and age, so cells can be maintained at a constant quality. not. Therefore, there is a different problem that it is difficult to set quality standards, which are necessary elements for automation and mechanization.
  • iPS cells Attempts to isolate only cells with the desired properties from those different cell populations are beginning to be discussed.
  • Known methods for evaluating the quality of iPS cells include miRNA analysis, surface marker detection, cell morphology image analysis, and epigenome analysis, and MSC has also proposed the use of these techniques, but unlike iPS cells. Since MSCs do not grow indefinitely and cannot be cloned, they have not been put into practical use.
  • the present invention is a method for producing a reversible immortalized cell, which can proliferate the cell for a long period of time without damaging the chromosome of the cell into which the immortalizing gene has been introduced, and can remove the immortalizing gene. It is an object of the present invention to provide a method for obtaining a large amount of reversible immortalized cells that can be cloned and have stable quality.
  • the present inventors have obtained one or more immortalizing genes selected from the group consisting of Bmi-1 gene, TERT gene, and SV40T gene.
  • MSC mesenchymal stem cells
  • Sendai virus vector which is a non-chromosomal RNA vector that has not been used for cell immortalization
  • it has been used for more than 80 days. It was found that cell proliferation does not occur and that infinite cell proliferation (immortalization) is possible, and that the rate of cell proliferation increases at an early stage after gene transfer. It was also confirmed that the produced immortalized cells had no chromosomal abnormality, had pluripotency, and could easily remove the immortalized gene by temperature control.
  • the immortalized cells were successfully cloned from the obtained immortalized cell population, and 8 out of 10 clones of the cloned immortalized cells also showed normal nuclear type and were pluripotent. It was confirmed that immortalization can be induced in cells at any stage from young cells to senescent cells in which division has stopped, and that cloning is possible immediately after immortalization. The present invention has been completed based on such findings.
  • the present invention includes the following inventions.
  • a method for producing a reversible immortalized cell which comprises the following steps. (1) A step of introducing a chromosome-non-integrating RNA viral vector carrying an immortalizing gene into mammalian cells and expressing the immortalizing gene in the cells, and (2) culturing the cells obtained in step (1). And the process of growing [2] The method according to [1], wherein the immortalizing gene is one or more immortalizing 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 of the following (a) to (d).
  • RNA virus vector is a paramyxovirus vector.
  • paramyxovirus vector is a Sendai virus vector.
  • Sendai virus vector is a temperature sensitive Sendai virus vector.
  • chromosomal non-integrating RNA virus vector is a Sendai virus vector, further comprising a step of removing the Sendai virus vector after culturing in the step (2).
  • removal of the Sendai virus vector is performed by changing the culture temperature from 35 ° C to 37 ° C.
  • a cell that is difficult to mass-culture for example, a mesenchymal stem cell (MSC) used for regenerative medicine
  • MSC mesenchymal stem cell
  • a temperature-sensitive Sendai virus vector (SeV vector) as a non-chromosomal integrated RNA vector, the vector can be easily removed from cells only by changing the culture temperature without performing a complicated removal operation. Therefore, it is possible to supply a large amount of cells having excellent safety.
  • the immortalized cells obtained by the present invention can be used for regenerative medicine because they have no chromosomal abnormality and have pluripotency as before gene transfer.
  • it is difficult to clone non-immortalized cells because they have different lifespans and properties, but the immortalized cells produced by the present invention are easy to clone and can stably obtain high-quality cells. Therefore, the mechanization of cell manufacturing facilitates quality control, makes it possible to significantly reduce the manufacturing cost, which is an issue for industrialization, and contributes to the development of cell medicine and regenerative medicine.
  • the genomic structure of the SeV vector carrying the immortalizing factor gene is shown [A. SeV (18+) Bmi1 (HNL) OFP / TS15d ⁇ F, B. SeV (PM) hTERT (HNL) EGFP / TS15 ⁇ F, C. SeV (18+). ) SV40T / TS15 ⁇ F, D. SeV (HNL) E6-E7, BFP / TS15 ⁇ F].
  • Transmitted light and fluorescence (OFP) images of Bmi-1 solitary infected cells (MOI: 1,5,20), transmitted light and fluorescence (GFP) images of hTERT solitary infected cells (MOI: 1,5,20) show.
  • the transmitted light image and the fluorescence (OFP, GFP) image of the two-factor (Bmi-1 / hTERT) co-infected cell (MOI: 1,5,20) are shown.
  • the transmitted light image and the fluorescence (OFP, GFP) image of the three-factor (Bmi-1 / hTERT / SV40T) co-infected cell (MOI: 1,5,20) are shown.
  • the cell morphology and cell condition determination 59 days after the introduction of immortalizing factors (No. 1 to No. 16) are shown. Shows the cell proliferation curve of immortalizing factors (No. 1, No. 2, No. 4, No. 12, No. 16) introduced cells (up arrow: morphological (transmitted light) image and fluorescent image point (Fig. 5)).
  • the transmitted light image and the fluorescence (OFP, GFP) image of the three-factor (Bmi-1 / hTERT / SV40T) -introduced cell are shown.
  • the morphology (transmitted light) image of the three-factor (Bmi-1 / hTERT / SV40T) -introduced cell is shown (maintained at 35 ° C, temperature changed from 35 ° C to 37 ° C).
  • the chromosomal analysis results (normal karyotype) after 90 days of subculture of the three-factor (Bmi-1 / hTERT / SV40T) -introduced cells are shown (chromosome analysis point ( ⁇ ) in FIG. 7).
  • telomere analysis points (A) The results of quantitative analysis of telomeres in long-term culture samples of immortalizing factors (No.1, No.2, No.4, No.12, No.16) are shown (A. Effects of immortalizing factor combinations on telomeres). (Day0, Day40: Telomere analysis point in Fig. 6 (*)), B. After introduction of factor 3 (Bmi-1 / hTERT / SV40T), change in telomere amount due to factor non-removal / removal (Day0, Day40: Fig. 6) Telomere analysis points (*), Day85, Day98: Telomere analysis points in Fig. 7 (*)). Pictures of adipocytes differentiated from early cultured MSCs and immortalized MSCs are shown.
  • clone A10 The results of chromosomal analysis of cloned immortalized cells (clone A10) are shown (Day 80: Chromosome analysis point ( ⁇ ) in FIG. 18). It shows the differentiation potential of cloned immortalized cells (fat cell differentiation, neuronal cell differentiation, osteoblast differentiation).
  • Cryopreservation points of MSCs used in single-cell cloning studies (early MSCs: Day9, mid-term MSCs: Day24, late MSCs: Day49) and immortalization induction points by SeV vector infection (early MSCs: Day21, mid-term MSCs: Day36, late MSCs) : Day61) is shown. Photographs of SeV vector infected cells (early MSCs, middle MSCs, late MSCs) and SeV vector non-infected MSCs (controls) before single-cell cloning (immediately before 96-well plate seeding) are shown.
  • results of a single cell cloning test of cell populations of SeV vector non-infected MSC (control) and SeV vector infected cells (early MSC) are shown (numbers in wells: number of clones formed, culture days: 2 weeks).
  • results of a single cell cloning test of a cell population of SeV vector-infected cells are shown (numbers in wells: number of clones formed, number of culture days: 2 weeks).
  • Photographs of SeV vector infection test results for cells in which cell division has stopped Day 90 (cells immediately before SeV vector infection, SeV vector non-infected cells (control cells), and SeV vector infected cells (2 weeks after infection)) ).
  • the test results of single-cell cloning of arrested cells (Day 90) that started reproliferation by SeV vector infection are shown (numbers in wells: number of clones formed, number of culture days: 2 weeks).
  • the cell proliferation curve of adipose tissue-derived human MSC (non-infected cell line, SeV vector infected cell line) in serum-free culture is shown.
  • the cell proliferation curve of bone marrow-derived human MSC (non-infected cell line, SeV vector infected cell line) in serum-free culture is shown.
  • a transmitted light image and a fluorescence (OFP, GFP) image of a SeV vector-infected cell line of a serum-free cultured adipose tissue-derived human MSC (hMSC-AT) and a SeV vector-infected cell line of a bone marrow-derived human MSC (hMSC-BM) are shown.
  • hMSC-AT 16th and 58th days from the date of infection
  • hMSC-BM 24th and 50th days from the date of infection
  • the cell proliferation curve of rat MSC non-infected cell line, SeV vector infected cell line # 1, # 2 is shown.
  • Transmission light images and fluorescence (OFP, GFP) images of SeV vector-infected cell lines of immortalized rat MSC are shown (2nd, 9th, and 58th days from the day of infection).
  • the cell proliferation curve of HFL1 non-infected cell line # 1, # 2, SeV vector infected cell line # 1, # 2, 37 ° C cultured SeV vector infected cell line # 1, # 2) is shown.
  • HUVEC non-infected cell line # 1, SeV vector infected cell line # 1, # 2 cultured at 35 ° C, SeV vector infected cell line # 1, # 2 cultured at 37 ° C
  • Immortalized HUVEC [35 ° C culture (7 days, 36, 72 days from infection date), 35 ° C culture + 37 ° C culture (35 days + 37 days from infection date)] transmitted light images and fluorescence (OFP, GFP) images are shown. .. It is a photograph showing the result of FISH analysis of immortalized MSC by introduction of a multiproliferative factor-inserted human artificial chromosome vector (in the lower right frame: DAPI-stained image of arrow cells). The results of a healing effect test of an enteritis model by transplantation of immortalized MSCs are shown.
  • the present invention is a method for producing reversible immortalized cells, wherein the method (1) introduces a chromosomal non-integrating RNA virus vector carrying an immortalizing gene into mammalian cells. Then, the step of expressing the immortalizing gene in the cell and the step of (2) culturing and proliferating the cell obtained in the step (1) are included.
  • the "cell” refers to all somatic cells other than germline cells (egg and sperm, oocyte, ES cell, etc.) and totipotent cells (iPS cells).
  • the "somatic cell” may be any of a primary cultured cell, a subcultured cell, and an established cell line.
  • the somatic cells may be naturally derived or artificially produced by differentiating from iPS cells or the like. Specific examples of somatic cells include cells that form tissues (fat cells, fibroblasts, nerve cells, skin cells, blood cells, muscle cells, osteoblasts, cartilage cells, hepatocytes, pancreatic cells, and renal cells.
  • Myocardial cells brain cells, lung cells, splenocytes, adrenal cells, gingival cells, root membrane cells) or their precursor cells, immune system cells (B cells, T cells, monocytic cells) (Cells, etc.), somatic stem cells [mesophageal 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, nerve stem cells, epidermal stem cells, intestinal epithelial stem cells, dental pulp stem cells, tooth roots, etc. Membrane stem cells, etc.] and the like.
  • the origin of the cells is not particularly limited as long as it is a mammal, and examples thereof include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses.
  • the "immortalized cell” refers to a cell whose proliferation does not stop even if cell division is repeated, that is, a cell having an infinite proliferation ability, unlike a primary cultured cell or a cell cultured under normal culture conditions.
  • the "immortalized cell” in the present invention means a cell capable of infinite proliferation by introducing a predetermined immortalizing gene, and a cell whose infinite proliferation ability does not decrease even if it is repeatedly subcultured and cultured. ..
  • the “immortalized cells” according to the present invention have different growth rates and growth periods depending on the origin of the cells or the culture conditions, but as a result of subculture, untreated cells proliferate or cease under the same culture conditions.
  • the immortalized cells of the present invention include both the cell population capable of infinite proliferation as described above and the immortalized cell line cloned from the cell population.
  • reversible immortalization means that a cell proliferation is stopped or attenuated by introducing an immortalizing gene into a cell, making the cell proliferative indefinitely, and then removing the immortalizing gene. It means to let.
  • “Immortalization” means to release the limit of the number of cell divisions and cell aging of early cells, and to impart continuous cell division ability and proliferation ability. Specifically, under standard cell culture conditions, usually 5 or more passages, preferably 7 or more passages, 8 or more passages, 9 or more passages, 10 or more passages, 12 or more passages, 15 passages or more. As mentioned above, it means that more than 20 passages have become possible.
  • Cells having a confluent number of passages of 0 can be amplified and cultured by subculturing using a method known to those skilled in the art. The cells obtained by one passage operation are called “passage number 1 (or second generation)" cells, and “passage number 2, 3, 4 " corresponding to the number of passage operations. It can be expressed as "n (n (integer) is the number of passages) (n + 1th generation)".
  • a step of freezing the cells may be included between each passage operation.
  • the immortalized cell of the present invention is produced by introducing a predetermined immortalizing gene into a cell using a chromosome non-integrating RNA viral vector.
  • the "immortalizing gene” refers to a gene that immortalizes a cell to acquire infinite proliferation ability and does not induce cell death.
  • the immortalizing gene is an extrinsic gene and means an immortalizing gene newly introduced from the outside of the cell.
  • the immortalizing gene may be an immortalizing gene derived from other than human, or may be an immortalizing gene modified into a form expressible in a target cell.
  • the immortalizing gene one or more genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene can be used, but a combination of the two genes can be used. Preferably, a combination of three genes is more preferred.
  • the TERT gene is preferable, and examples of a preferable combination when two or more kinds of genes are combined include a combination of Bmi-1 gene and TERT gene, and a combination of TERT gene and SV40T gene.
  • the "gene” includes not only a structural gene that defines the primary structure of a protein but also a region on a nucleic acid having a control function such as a promoter or an operator. Therefore, in the present invention, the term “gene” is used without distinguishing between a regulatory region, a coding region, an exon, and an intron, unless otherwise specified.
  • Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog) gene
  • PRC1 polycomb repressive complex 1
  • Bmi-1 is involved in the regulation of expression of various genes including Hox gene by controlling chromatin remodeling and histone modification.
  • Bmi-1 has the effect of controlling cell proliferation by suppressing the expression of p16 and p19Arf involved in the cell cycle, and plays an important role in maintaining self-renewal by being involved in cell division of hematopoietic stem cells and nerve stem cells. It is known to play.
  • TERT telomerase reverse transcriptase gene
  • TERT Telomerase reverse transcriptase gene
  • Telomerase RNA component extends specific repeat sequences of eukaryotic chromosome ends (telomeres) from telomere RNA components (Telomerase RNA: TR or Telomerase RNA component: TERC) and other regulatory subunits. It constitutes the enzyme telomerase. It is known that TERT has a role in maintaining telomere length, whereas cell aging has a telomere length monitoring mechanism and cell senescence is caused by telomere shortening.
  • the "SV40T (simianvirus 40large Tantigen) gene” is a gene encoding the Simian virus 40 large T antigen.
  • the genome of SV40 (simianvacuolating virus40) is divided into an early region that is expressed immediately after infection, a late region that is expressed during replication of the viral genome after infection, and a regulatory region that includes transcriptional regulation and the origin of replication.
  • the early region encodes a large T antigen involved in the initiation of viral genome replication and inactivation of p53 and pRB, which are tumor suppressor gene products, and a small t antigen that binds to and inhibits the protein dephosphorylation enzyme PP2A.
  • a specific example of the Bmi-1 gene used in the present invention is the mouse BMI1 gene (SEQ ID NO: 1)
  • a specific example of the TERT gene is the human TERT gene (SEQ ID NO: 2)
  • a specific example of the SV40T gene is SV40 large.
  • the T antigen gene (SEQ ID NO: 3) can be mentioned.
  • the Bmi-1 gene, TERT gene, and SV40T gene may also be these transcriptional variants, splicing variants and their orthologs.
  • the above Bmi-1 gene, TERT gene, and SV40T gene are 80% or more, preferably 90% or more, relative to each of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3 as long as they have the same functions and activities.
  • the Bmi-1 gene, TERT gene, and SV40T gene are artificially modified so that the product of the gene is expressed as a fusion protein with other proteins or peptides as long as it has the same function and activity. It may be a gene.
  • a "chromosome non-integrating RNA virus vector” is used as a vector for introducing and expressing the immortalization gene in cells.
  • the viral vector means a vector having a genomic nucleic acid derived from the virus and capable of expressing the gene by incorporating a transgene into the nucleic acid.
  • the "chromosome non-integrated RNA virus vector” is a virus vector derived from a virus and capable of introducing a gene into a target cell, and the introduced gene is integrated into a host chromosome (nuclear-derived chromosome). A non-hazardous carrier.
  • RNA virus vector refers to a vector consisting of a virus containing RNA of minus strand (antisense strand complementary to the sense strand encoding the viral protein) as a genome, and minus strand RNA is also called negative strand RNA. ..
  • a single-stranded negative-strand RNA virus also referred to as a non-segmented negative-strand RNA virus is particularly preferable.
  • Single-stranded negative-strand RNA virus refers to a virus having a single-stranded negative-strand (minus-strand) RNA in its genome, for example, Paramyxoviridae; Paramyxovirus, Morbillivirus, Rubulavirus, Pneumovirus, etc. Includes), Rhabdoviridae (including Rhabdoviridae; Vesculovirus, Lyssavirus, and Ephemerovirus genus), and viruses belonging to the family Filoviridae.
  • minus-strand RNA viruses examples include Paramyxoviridae virus Sendai virus, Newcastle disease virus, Mumps virus, and measles virus.
  • RS virus Respiratory syncytial virus
  • cattle epidemic virus Rinderpest virus
  • distemper virus distemper virus
  • salparainfluenza virus SV5
  • human parainfluenza virus type 1,2,3, orthomixovirus family Orthomyxoviridae influenza virus (Influenza virus), Rhabdoviridae (Rhabdoviridae) vesicular stomatitis virus (Vesicular stomatitis virus), mad dog disease virus (Rabies virus) and the like can be mentioned, but Sendai virus (Sendai virus) is preferable.
  • Sendai virus vector SeV vector
  • a Sendai virus (SeV) vector is used as the non-chromosomal integrated RNA vector.
  • the immortalizing factor genes of the Bmi-1 gene, TERT gene, and SV40T gene may be inserted separately into the SeV vector, or may be inserted together into a single SeV vector.
  • Sendai virus is a type of virus belonging to the genus Respirovirus of the Paramyxoviridae family, and contains a single minus strand (antisense strand against the sense strand encoding the viral protein) RNA as a genome.
  • the Sendai virus genome consists of NP (nucleocapsid) gene, P (phospho) gene, M (matrix) gene, F (fusion) gene, and HN (hemogglutinin / neuraminidase) in order from the 3'end to the 5'end. ) Gene and L (large) gene are included. Of these, Sendai virus can sufficiently function as a vector if it has the NP gene, P gene, and L gene, and can replicate the genome in cells and express the loaded gene. Since the Sendai virus has minus-strand RNA in its genome, the 3'side of the genome is upstream and the 5'side is downstream, which is the opposite of the normal case.
  • the SeV vector can be used as a natural strain, a wild strain, a mutant strain, or a commercially available product.
  • the virus may be a virus having a structure similar to that of a virus isolated from nature, or a virus artificially modified by genetic recombination, as long as the desired function can be achieved.
  • any gene possessed by the wild-type virus may be mutated or deleted.
  • a non-propagating vector ( ⁇ F) in which the F gene is deleted from the genome and no infectious particles are formed from the gene-introduced cells, and the F gene is deleted, and the M and / or HN gene is deleted.
  • a vector that further deletes or has a mutation in the M and / or HN gene is preferably used in the present invention.
  • a vector in which the F gene is deleted, the M or HN gene is further deleted, and the remaining M and / or HN gene is further mutated is also preferably used in the present invention (patented). See No. 5763340, etc.).
  • the SeV vector used in the method of the present invention is preferably temperature sensitive.
  • “Temperature sensitivity” means that the activity is significantly reduced at a normal cell culture temperature (for example, 37 to 38 ° C) as compared with a low temperature (for example, 30 to 36 ° C).
  • Sendai virus TS7 L protein Y942H / L1361C / L1558I mutation
  • TS12 P protein D433A / R434A / K437A mutation
  • TS13 P protein D433A / R434A / K437A mutation and L protein L1558I mutation
  • TS14 P.
  • Mutations such as protein D433A / R434A / K437A mutations and L protein L1361C) and TS15 are temperature-sensitive mutations and are preferred in the present invention. These mutations can be utilized. It is preferable to further introduce these mutations into the above-mentioned F gene-deficient SeV vector.
  • SeV vectors refer to Japanese Patent No. 5763340, WO2015 / 046229, etc. be able to.
  • the SeV vector in the present invention is a complex consisting of an infectious virus particle, a virus core, a complex of a virus genome and a virus protein, a non-infectious virus particle, and the like, and is loaded by introduction into cells. Includes a complex capable of expressing a gene that does.
  • a ribonucleoprotein core part of a virus consisting of a Sendai virus genome and Sendai virus proteins (NP, P, and L proteins) that bind to it expresses the introduced gene in the cell by introducing it into the cell.
  • the introduction into cells may be carried out by appropriately using a transfection reagent or the like. Therefore, such a ribonucleoprotein (RNP) is also included in the SeV vector in the present invention.
  • the position where the immortalizing gene (Bmi-1 gene, TERT gene, and SV40T gene) is integrated is not particularly limited, but when each immortalizing gene is inserted into multiple separate vectors, the Bmi-1 gene is the NP gene. Upstream, the TERT gene is preferably inserted between the P and M genes, and the SV40T gene is preferably inserted upstream of the NP gene. Also, two or more genes (Bmi-1 and TERT, TERT and SV40T, or Bmi-1 and TERT and SV40T) may be inserted into a single vector.
  • the SeV vector carrying the immortalization gene obtained as described above is introduced into the cells by adding the vector (Sendai virus particles) to the medium of the somatic cells and infecting the cells with the virus. .. Since the dose of the vector varies depending on the cell type, cell density, and amount of medium, the MOI whose infection efficiency is close to 100% may be investigated and determined in advance for each cell to be used.
  • the SeV vector when in the form of RNP, it can be introduced into cells by a method such as an electroporation method, a lipofection method, or a microinjection method.
  • the method for culturing cells into which an immortalizing gene has been introduced can be carried out according to the method and conditions for culturing normal mammalian somatic cells.
  • the medium used for culturing is not particularly limited, and a medium generally used for maintenance culture or expansion culture of cells and suitable for virus infection may be used, and either a commercially available medium or a self-made medium may be used. good.
  • a basal medium containing components necessary for cell survival and proliferation inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids
  • D-MEM Dulbecco's Modified Eagle's Medium
  • Dulbecco's Modified Eagle's Medium Natural Mixture F-12 (D-MEM / F-12) Medium, Glassgow MEM (G-MEM) Medium, Basal Medium Eagle (BME) Medium, Minimum Essential Medium (MEM) Medium, Eagle's minimal essential Examples include medium (EMEM) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, RPMI 1640 medium, Medium 199 medium, ⁇ MEM medium, ham medium, Fischer medium, and a mixed medium thereof.
  • D-MEM / F-12 Natural Mixture F-12
  • G-MEM Basal Medium Eagle
  • MEM Minimum Essential Medium
  • the medium may contain growth factors (FGF, EGF, etc.), interleukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, antibiotics, if necessary. It may contain a substance (penicillin, streptomycin, etc.) and the like. Further, the medium may be a serum-containing medium or a serum-free medium. From the viewpoint of preventing contamination of heterologous animal-derived components, it is preferable to use serum that does not contain serum or that is derived from the same animal as the cells to be cultured. Alternatively, a serum substitute such as albumin may be used.
  • the culture method is not limited, but is limited to three-dimensional culture under non-adhesive conditions, for example, suspension culture (for example, dispersion culture, aggregate suspension culture, etc.), or two-dimensional culture under adhesive conditions, for example, flat plate. Examples thereof include culture and culture in which three-dimensional culture and two-dimensional culture are combined.
  • the incubator used for culturing cells is not particularly limited as long as it can cultivate cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. ..
  • the incubator may be cell non-adhesive or adhesive, and is appropriately selected depending on the intended purpose.
  • cell adhesion incubator one treated with a cell-supporting substrate or the like using an extracellular matrix or the like may be used for the purpose of improving the adhesion to cells.
  • the cell-supporting substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.
  • the culture temperature is 30 ° C to 36 ° C, preferably 32 ° C to 35 ° C, and more preferably 33 to 35 ° C. Culturing is carried out in an atmosphere of CO 2 containing air, for example, at a CO 2 concentration of 2% to 5%.
  • the culture temperature for removing the immortalizing gene is 37 ° C to 38 ° C, preferably 37 ° C to 37.5 ° C.
  • the immortalized cells prepared as described above can be induced to differentiate into specific tissue cells by culturing them in a differentiation-inducing medium.
  • the immortalized cell when it is a mesenchymal stem cell, it can be differentiated into adipocyte, osteoblast, nerve cell, chondrocyte and the like.
  • composition of the medium for inducing the differentiation of the immortalized cells according to the present invention into the target cells, the differentiation-inducing factor, the culture method, the passage method, etc. can be appropriately set from well-known and conventional techniques.
  • the differentiation-inducing medium can be appropriately selected according to the type of cells targeted for differentiation-inducing. Differentiation-inducing media for various tissues (mediums to which at least one type of differentiation-inducing or promoting factor according to the target cell for differentiation-inducing is added) are commercially available, and these commercially available media may be used.
  • the medium for inducing the differentiation of immortalized cells according to the present invention into adipocytes is a commercially available adipocyte-inducing medium or a commercially available animal cell medium containing insulin, dexamesazone, indomethacin, or 3-isobutyl-1-methylxanthin. , A medium containing troglycazone, biotin, etc. can be used.
  • Examples of commercially available media include Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (manufactured by PromoCell), Human Mesenchymal Stem Cell Adipogenic Differentiation Medium BulletKit8 (manufactured by Lonza), and the like.
  • the medium for inducing the differentiation of the cells according to the present invention into osteoblasts includes dexamesazone, ascorbic acid, ⁇ -glycerophosphate, hydrocortisone, BMP4, in a commercially available osteoblast-inducing medium or a commercially available animal cell medium.
  • a medium containing BMP2 etc. can be used.
  • Examples of commercially available media include Mesenchymal Stem Cell Osteogenic Differentiation Medium (manufactured by PromoCell), Human Mesenchymal Stem Cell Osteogenic Differentiation Medium Bullet Kit (manufactured by Lonza), and the like.
  • a medium for inducing differentiation of immortalized cells into nerve cells according to the present invention, a commercially available nerve cell culture medium or nerve differentiation medium (for example, Mesenchymal Stem Cell Neurogenic Differentiation Medium (manufactured by PromoCell), etc.) can be used.
  • the nerve cell culture medium or the nerve differentiation inducing medium preferably contains a nerve cell inducing factor (for example, brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF)).
  • BDNF brain-derived neurotrophic factor
  • FGF fibroblast growth factor
  • chondrocyte-inducing medium a commercially available chondrocyte-inducing medium or a commercially available animal cell medium containing dexamesazone, ascorbic acid, and TGF- ⁇ 3 is used.
  • examples of commercially available media include MesenchymalStemCellChondrogenic DifferentiationMedium (manufactured by PromoCell), HumanMesenchymalStemCellChondrogenicDifferentiationMedium BulletKit (manufactured by Lonza), and the like.
  • the culture conditions for inducing differentiation are the same as the culture conditions for culturing normal stem cells.
  • the culture period for inducing differentiation is also not particularly limited, but is generally 5 to 20 days, preferably 7 to 18 days.
  • 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. For example, differentiation into adipocytes can be confirmed by oil red 0 staining, differentiation into osteoblasts by alkaline phosphatase staining, differentiation into nerve cells by NeuroFluorNeuO staining, and differentiation into chondrocytes by alcian blue staining.
  • the immortalized cells and tissue cells induced to differentiate from immortalized (stem) cells obtained by the present invention can be used, for example, by transplanting cells to a diseased or damaged site, and can be provided as a product for regenerative medicine.
  • products for regenerative medicine include cultured skin, cultured cartilage, cultured corneal epithelium, various cell sheets (for example, epidermal cell sheet, fibroblast sheet, corneal endothelial cell sheet, myocardial cell sheet, osteoblast sheet, myoblast).
  • the serum-containing medium for culturing human mesenchymal stem cells is D-MEM (Low Gulucose), 20% FBS, 0.01 mol / L Hepes, Penicillin 100units / ml, Streptomycin 100 ⁇ g / It was prepared with a composition of ml and bFGF 20 ng / ml. Since 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% CO 2 incubator. The human MSC used in the experiment is either bone marrow-derived, adipose tissue-derived, cord blood-derived, or umbilical cord matrix-derived.
  • Example 1 Preparation of seV vector carrying an immortalizing gene and examination of infection conditions
  • a SeV vector carrying an immortalizing gene Bmi-1 B lymphoma Mo-MLV insertion region 1 homolog
  • Bmi-1 B lymphoma Mo-MLV insertion region 1 homolog
  • HTERT human telomerase reverse transcriptase
  • SV40T simian virus 40 large T antigen
  • E6 / E7 human papillomavirus 16 E6 protein and E7 protein
  • Bmi was used as the SeV vector for the non-chromosomal RNA virus vector.
  • FIG. 1 shows the structure of each vector.
  • Bmi-1, SV40T is the vector genome. It was installed at the most upstream of. The mounting positions of hTERT and E6 / E7 were changed downstream due to the decrease in vector production efficiency, which is thought to be due to high expression.
  • SeV vector For the SeV vector, we used a TS15 ⁇ F type vector that was improved so that the viral vector disappeared from the cells by changing 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 . 2011 Aug 23; 108 (34): 14234-9.).
  • bone marrow-derived mesenchymal stem cells product name: ultra-high-purity human mesenchymal stem cells (REC), PuREC Co., Ltd.
  • REC ultra-high-purity human mesenchymal stem cells
  • PuREC Co., Ltd. are placed in a 48-well plate (FALCON 353230) supplemented with a medium of 200 ⁇ l / well in 5 ⁇ 10 4 Sprinkle individual / well (70-80% confluent) and add 4 types of immortalizing factor gene-laden SeV vectors to the cells so that the MOI of each vector becomes 1,5,20 by single or multiple co-infection. , Incubated overnight. The next day, the medium was replaced with fresh medium, and the medium was replaced every 2 days thereafter. The medium was expanded to a 12-well plate (FALCON 353043) on the 5th day after the vector infection, and the cells were observed 9 days after the infection.
  • Example 2 Selection of immortalizing factor MSC by infecting MSC with a SeV vector carrying four kinds of immortalizing factor (Bmi-1, hTERT, SV40T, E6 / E7) genes alone and culturing for a long period of time. The factors necessary for immortalization of the disease were selected.
  • immortalizing factor Bmi-1, hTERT, SV40T, E6 / E7
  • MSC derived from umbilical cord blood (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)) was used for the study, and one or a combination of two or more SeV vectors carrying the immortalizing factor gene was used. (15 ways in total) were infected with the above MSC (48 well plate, 3 ⁇ 10 4 / well, MOI: 20). MSCs not infected with SeV vector were used as negative controls.
  • the cells are expanded and cultured in the order of 48 well ⁇ 12 well ⁇ 6 well, and after that, when the cells become confluent (the adhesive surface of the plate is 100% of the cells), the number of cells is measured and subcultured to obtain a total of 16 types of cells. At the same time as measuring the growth rate of the cells, the morphology of the cells was observed under a microscope.
  • the cell proliferation rate was measured over 80 days from the introduction of the immortalizing gene (number of cells at the time of introduction: 3 ⁇ 10 4 cells).
  • the results (No. 1 to No. 16) arranged in descending order of the number of cells at 80 days are shown in Table 1 below together with the number of cells.
  • the cell condition was observed at about 2 months (59th day) when the cell proliferation of the negative control stopped, and the uniformity of cell size and the degree of dead cells detached from the culture plate were observed in two stages. Judgment was made (Fig. 5). The judgment results are shown in Table 1 (cell size is uniform and cell peeling is small: ⁇ , cell size is non-uniform and cell peeling is large: ⁇ , presence or absence of immortalizing factor is present. Notated by + or-).
  • FIG. 6 shows the cell growth curve.
  • the growth stopped after about 2 months (66 days), but the growth of the immortalizing factor-introduced cells (No. 1, 2, 4, 12) was prolonged.
  • the growth of hTERT gene-introduced cells (No. 12) was prolonged, the growth rate gradually decreased after 2 months, and further growth could not be expected.
  • the combination-introduced cells (No. 1, 2, 4) of Bmi-1 and hTERT, hTERT and SV40T, and Bmi-1 and hTERT and SV40T proliferated stably without slowing down even on the 80th day. was observed.
  • the growth rate of the combination of the three factors Bmi-1, hTERT and SV40T was the fastest.
  • human umbilical cord matrix-derived mesenchymal stem cells product name: HumanMesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), Promo Cell, product code C-12971
  • hMSC-UC HumanMesenchymal Stem Cells from Umbilical Cord Matrix
  • Promo Cell product code C-12971
  • Example 3 Analysis of immortalized cultured cells using a SeV vector carrying a 3-factor (Bmi-1 / hTERT / SV40T) gene (1) Preparation of 3-factor (Bmi-1 / hTERT / SV40T) -introduced immortalized cells In the examination of MSCs derived from different tissues, it was judged that the SeV vector carrying the three-factor (Bmi-1 / hTERT / SV40T) gene was the most suitable for immortalization, and detailed property analysis of the MSCs immortalized with this vector was performed. ..
  • the cells used were MSCs derived from cord blood (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)) used in the selection of immortalizing factors, but the cells were longer than those used in the selection. Cultured (less than 1 month) cells were used. A 3-factor (Bmi-1 / hTERT / SV40T) gene-loaded SeV vector was introduced under the same MOI: 20 conditions as in Example 2. MSCs without gene transfer with SeV vector were simultaneously cultured as a negative control.
  • the immortalized cell line introduced with 3 factors (Bmi-1 / hTERT / SV40T) was cultured in a CO 2 incubator at 35 ° C using a 6 cm dish, and the plates adhered. When the surface was grown to about 80% of the cells, 1/5 was subcultured to a new 6 cm dish, and the culture was continued for 75 days.
  • the SeV vector carrying the immortalization gene is a temperature-sensitive vector, and it is possible to rapidly eliminate the intracellular SeV vector genome 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 Natl Acad Sci U S A. 2011 Aug 23; 108 (34): 14234-9).
  • Chromosome analysis of cells was performed by the quinacrine-Hoechst fractionation method at the point ⁇ in FIG.
  • the chromosome slide is first immersed in 50 ml of Macylben solution (280 ml of 0.1 M citrate solution and 220 ml of 0.2 M disodium hydrogen phosphate solution are mixed and autoclaved), and then 50 ml of Macylben. It was soaked in a solution of Hoechst 33258 (cat: B-2883-25MG, Sigma) at 10 ng / ml for 30 minutes.
  • the immortalized cell line was the parent strain at all points. It was a normal karyotype as well (Fig. 10).
  • telomere length was evaluated at the points marked with * in FIGS. 6 and 7.
  • the telomere length was evaluated by performing real-time PCR using genomic DNA as a template and by relative quantification of telomere sequences. Genomic DNA extraction from cells was performed using Gentra® Puregene® Kit (Qiagen) with reference to the manufacturer's procedure.
  • Gentra® Puregene® Kit Qiagen
  • the Telomere primer set and Single copy reference primer set attached to the Relative Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories) were used as primers, and FastStart Essential DNA Green Master (Roche) was used as the PCR reagent.
  • the PCR reaction conditions were as recommended by the manufacturer. StepOnePlus (Life Technologies Japan) was used for PCR reaction and data acquisition, and data analysis was performed with StepOne Software v2.3.
  • Example 4 Differentiation ability of immortalized population cells Regarding the immortalized cells (MSC) introduced with the three factors (Bmi-1 / hTERT / SV40T) prepared in Example 3, fat cells, osteoblasts, nerve cells, and chondrocytes. The ability to differentiate into cells was investigated.
  • MSC (ATCC PCS-500-010) derived from cord blood that was immortalized with three factors of Bmi-1, hTERT, and SV40T and cultured for a long period of time (cultured for 4 months after gene transfer) was used. Four months is a difficult period for culturing in general MSCs that are not immortalized.
  • a commercially available (PromoCell) differentiation kit was used for MSC differentiation.
  • Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (product code C-28016) for adipose cell differentiation
  • Mesenchymal Stem Cell Osteogenic Differentiation Medium (product code C-28013) for osteoblast differentiation
  • Mesenchymal Stem Cell Neurogenic Differentiation for nerve cell differentiation.
  • Medium (product code C-28015) and Mesenchymal Stem Cell Chondrogenic Differentiation Medium (product code C-28012) were used for cartilage cell differentiation, and the operation method was carried out according to the attached protocol.
  • the confirmation of adipocyte differentiation was performed using the fluorescent dye Lipi-Green (Doujin Kagaku: product code LD02), which specifically stains lipid droplets, which is an index of adipocyte differentiation. Osteoblast differentiation was confirmed by alkaline phosphatase staining. Nerve cell differentiation was confirmed using the fluorescent dye NeuroFluor NeuO (VERITAS: product code ST-01801), which specifically stains nerve cells. Chondrocyte differentiation was confirmed by alcian blue staining.
  • Lipi-Green Doujin Kagaku: product code LD02
  • Osteoblast differentiation was confirmed by alkaline phosphatase staining.
  • Nerve cell differentiation was confirmed using the fluorescent dye NeuroFluor NeuO (VERITAS: product code ST-01801), which specifically stains nerve cells. Chondrocyte differentiation was confirmed by alcian blue staining.
  • MSCs that retain the differentiation potential and have not been gene-introduced in the logarithmic growth phase were used as positive controls, and compared with the differentiation potential of immortalized MSCs that had passed 4 months, fat.
  • lipid droplets which are indicators of differentiation, were observed in both cases.
  • the immortalized MSC had a lower degree of differentiation than the positive control (Fig. 12).
  • osteoblast differentiation staining with alkaline phosphatase was confirmed in both immortalized MSC and positive control, and the cells were differentiated into osteoblasts (Fig. 13).
  • Immortalized MSCs had a higher differentiation rate than positive controls.
  • Example 5 Cloning test of immortalized MSCs For the three-factor (Bmi-1 / TERT / SV40T) -introduced immortalized cells (MSCs) prepared in Example 3, whether cloning is possible or whether the MSCs are not immortalized We conducted a confirmation experiment to see how difficult cloning is.
  • MSC (ATCCPCS-500-010) derived from umbilical cord blood that retains differentiation potential and has not been gene-introduced in the logarithmic growth phase in early cells cultured for about 2 weeks after purchase, has just been gene-introduced ( Cloning was performed using three types of MSCs: an immortalized MSC (2 weeks old) and an immortalized MSC (4 months old) cultured for a long time after gene transfer.
  • the three types of MSCs that had been cultured and maintained were once completely separated into single cells, and the number of cells was measured.
  • the cells were seeded at a variable number such as 32 cells / 100 ⁇ l, and cultured in a 5% CO 2 incubator at 35 ° C until single cells formed colonies. For medium exchange, half the amount was exchanged every 3 days so as not to disturb colonization.
  • Non-immortalized early growth MSCs corresponded to common MSCs, but only 2 wells of 32 cells / well compartments were formed.
  • MSCs into which the immortalizing gene was introduced with the SeV vector colonies were formed at a high frequency of 9 well / 24 well even at a low density of 4 cells / well in the two weeks immediately after the introduction (Fig. 16). After 4 months, colonies were formed in 16 wells / 24 wells and more than half of the wells even at a low density of 4 cells / well (Fig. 17).
  • Example 6 Various analyzes of clone immortalized MSC (1) Proliferative ability of cloned cells Five clones of MSC cloned as a single cell 2 weeks after infection with SeV vector were divided into 35 ° C and 37 ° C after culturing for 60 days. Was cultured. Three of the five clones proliferated and decreased with the removal of the SeV vector at 37 ° C. A growth curve is shown for clone A10 among them (Fig. 18).
  • Example 7 Immortalization and single-cell cloning of MSCs with different cell ages (aging degree) Since the cell populations grown from one MSC have the same genetic properties, the quality is constant and quality control becomes easy. In that respect, single-cell cloning has important significance in regenerative medicine, and single-cell cloning is indispensable in that when a gene is introduced from the outside, only the target cell is selected.
  • MSC isolated from the human body is a mixture of cells of various stages, from young cells to aged cells. The ratio also varies from individual to individual, and it is difficult to extract and proliferate only cells at a specific time (youth). Conversely, if MSCs can be immortalized at any time from young cells to senescent cells and can be further cloned, their application range will be greatly expanded in regenerative medicine.
  • the cells used were MSCs derived from umbilical cord blood (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)).
  • the purchased cells were cultured for a long period of about 80 days at 37 ° C until proliferation stopped. During that time, a part of the cells was cryopreserved at about every other week of passage (Fig. 21).
  • the cells were transferred to a CO 2 incubator at 37 ° C to 35 ° C and expanded and cultured for 8 days (24 well ⁇ 6 well). After that, the immortalized cells were seeded on a 96-well plate so that the number of cells per well was 1, 2, or 4, and the number of non-immortalized control cells was 5, 10 per well. The cells were seeded to 15 or 20 cells, cultured for about 2 weeks until one cell divided and proliferated and a cell population (colony) appeared, and the number of colonies generated by microscopic observation was counted.
  • Example 8 Confirmation of effect on MSCs in which proliferation has stopped The effect of the SeV vector carrying an immortalizing gene on MSCs in which cell division has completely stopped due to aging was confirmed.
  • SeV vector non-infected cells control cells
  • SeV vector-infected cells no change was observed for several days, but after one week, many cells changed roundly and seemed to die as they were, but small adhesive cells were observed. After 2 weeks, the cells changed to a vigorously proliferating cell population similar to immortalized cells (Fig. 24).
  • Infected cells and infected cells were cultured in a medium containing serum from the day of infection to the 11th and 17th days, respectively.
  • a medium having a composition of D-MEM (Low Gulucose), 20% FBS, 0.01 mol / L Hepes, Penicillin 100units / ml, Streptomycin 100 ⁇ g / ml, and bFGF 20 ng / ml was used. Then, it was replaced with a serum-free medium and the culture was continued.
  • Stem Fit For Mesenchymal Stem Cell (AJINOMOTO; A3) medium was used as the serum-free medium.
  • As the culture dish a 6 cm dish (CORNING; 353004) coated with iMatrix-511 silk (Matrixome; 892 091) was used.
  • Infection with the SeV vector was performed on 2 ⁇ 10 5 hMSC-AT cells or hMSC-BM cells under the condition of MOI: 40.
  • the non-infection condition and the infection condition were carried out once each.
  • the medium was changed 24 hours after infection and maintenance culture was continued.
  • Cell culture was performed in a 5% CO 2 incubator at 35 ° C.
  • the non-infected cell line was found to have a decrease in the number of cells, so the culture was terminated 52 days after the day of infection.
  • the SeV vector-infected cell line continued to proliferate after the 52nd day (Fig. 26).
  • Example 10 Examination of cell proliferation of immortalized rat MSC Rat subcutaneous fat-derived mesenchymal stem cells rMSC cells (Cosmo Bio Co., Ltd., MSA01C) are media for proliferation of rat subcutaneous fat-derived mesenchymal stem cells (Cosmo Bio Co., Ltd.). Cultivated in MSA-GM).
  • Infection with the SeV vector was performed on 2 ⁇ 10 5 rMSC cells under the condition of MOI: 40.
  • the non-infected condition was once and the infected cell line was twice.
  • the medium was changed 24 hours after infection and maintenance culture was continued.
  • Culture dishes were placed in a collagen coated dish (IWAKI, 4810-010) and maintained at 35 ° C. in a 5% CO 2 incubator.
  • cells with advanced cellular senescence may exhibit characteristics such as an increase in cell size, a flat shape, and the formation of vacuoles.
  • Infected cells cultured at 35 ° C showed no characteristic of cellular senescence compared to infected cells cultured at 37 ° C. From this result, it was confirmed that the cells continued to be cultured at 35 ° C. did not undergo cell senescence in terms of cell morphology.
  • Example 12 Examination of cell proliferation of immortalized HUVEC Human umbilical vein endothelial cells (HUVEC cells, Promocell, C-12205) were cultured in an endothelial cell medium (ScienCell; 1001).
  • Infection with the SeV vector was performed on 2 ⁇ 10 5 HUVEC cells under MOI: 40 conditions. The non-infection condition was once and the infection condition was twice. The medium was changed 24 hours after infection and maintenance culture was continued. Infected cells were divided into two conditions, one was to culture at 35 ° C and the other was to culture at 37 ° C on the 35th day after infection (Fig. 33, arrow). Cell culture was performed at each temperature in a 5% CO 2 incubator.
  • the non-infected cells were found to have a decrease in the number of cells, so the culture was terminated on the 39th day from the day of infection. On the other hand, the infected cells continued to proliferate even 74 days after the infection (Fig. 33). From this, it was confirmed that cell proliferation can be prolonged even in HUVEC cells by SeV vector infection.
  • Example 14 Verification of enteritis healing effect of immortalized MSC in an inflammatory bowel disease model (1) CD4 positive CD45RB high positive CD25 negative (CD4 + CD45RB High + CD25- ) Isolation of T cells 8 weeks old BALB / cAJcl ( After acclimatization of 30 females of Claire Japan) for 1 week, blood was removed by cardiac blood sampling under anesthesia, and the spleen and mesenteric lymph nodes were collected. The spleen was hemolyzed after tissue dispersion using the MACS system (Miltenyi Biotech). The mesenteric lymph nodes were crushed with a 1 mL syringe plunger and filtered through a 40 ⁇ m cell strainer.
  • Antibodies to isolate CD4 + CD45RB High + CD25-T cells after combining spleen and mesenteric lymph node cells and separating CD4 positive cells by CD4 microbeads (Miltenyi Biotech) treatment. Labeled with mouse CD4 antibody (BD), FITC Rat Anti-Mouse CD45RB antibody (BD), PE / Cy7 anti-mouse CD25 antibody (BioLegend)) and fractionated by flow cytometer (MoFlo XDP, BECKMAN).
  • CD4 + CD45RB High + CD25 --T cell transfer 9-week-old female SCID CB-17 / lcr-scid / scidJcl: Japan Marie
  • Cells were transplanted by tail vein injection (cell transplant group: 1 group 8 animals x 5 groups, untreated group (PBS administration): 1 group x 7 animals). After transplantation, body weight was measured three times a week and the coat and stool condition were observed. Twenty-one days after cell administration, randomization was performed using the "multivariable block allocation" system of the statistical analysis software JMP based on body weight changes, and the cell transplantation group was divided into groups. At that time, individuals who deviated from the average relative body weight of ⁇ 2 SD were excluded.
  • hMSC-UC parent MSC
  • immortalized MSC were cultured under each cell culture condition.
  • MSC administration (therapeutic cell transplantation)
  • parental MSC and immortalized MSC were administered 1.0 ⁇ 10 6 per mouse by tail vein injection.
  • Dexamethasone (Sigma): Dex (1 mg / kg, 100 ⁇ L / animal) was subcutaneously administered from the 21st day to the 14th day. Dex solution was prepared at the time of use based on the group average body weight on the 21st and 28th days.
  • the present invention can be used in the fields of cell medicine and regenerative medicine.
  • the cell immortalization technique of the present invention can be used not only for normal cells with slow cell proliferation but also for cancer cells and the like, and can also be used in the field of basic research. Furthermore, single-cell cloning, which is essential for gene transfer and chromosome transfer, is also possible.
  • the cells obtained by the present invention continue to proliferate without aging, which facilitates quality control, promotes mechanization of culture, significantly reduces cell production costs, and handles a large number of cells. Is possible. Therefore, the present invention expands the range of applicable diseases in cell medicine and regenerative medicine, and contributes to the activation of domestic and foreign medical-related industries. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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CN118109517A (zh) * 2024-02-04 2024-05-31 南京市第一医院 一种永生化人结直肠癌成纤维细胞系及其构建方法和应用
WO2026005077A1 (ja) * 2024-06-24 2026-01-02 株式会社Trans Chromosomics 可逆的不死化細胞から不死化解除細胞を製造する方法

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WO2024111576A1 (ja) 2022-11-22 2024-05-30 国立大学法人 鹿児島大学 抗ｇｐａ３３抗体及びその利用
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WO2026005077A1 (ja) * 2024-06-24 2026-01-02 株式会社Trans Chromosomics 可逆的不死化細胞から不死化解除細胞を製造する方法

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