Method for culturing cells into which reprogramming factor is introduced

Abstract

To meet a need for an efficient method for culturing cells into which a reprogramming factor is introduced.SOLUTION: According to the present disclosure, provided is a method for culturing cells into which a reprogramming factor is introduced, the method including: culturing cells into which a reprogramming factor is introduced; and recovering all cells into which the reprogramming factor is introduced and seeding and passaging at least part of the recovered cells in a medium. In addition, provided is a method for culturing cells into which a reprogramming factor is introduced, the method including: culturing cells into which a reprogramming factor is introduced; and inducing the cells into which the reprogramming factor is introduced into somatic cells different from pluripotent stem cells without passaging.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to cell technology and to a method for culturing cells into which reprogramming factors have been introduced. do. [Background technology]

[0002] Induced pluripotent stem (iPS) cells are cells with two distinctive abilities. The ability to transform into any cell that makes up the body. Because iPS cells have these two abilities, they can be used to grow cells from the patient's own somatic cells. By creating iPS cells from the tissue and transforming them into the desired somatic cells, we can achieve transplant treatment without rejection. Therefore, iPS cells are expected to become a powerful technology for regenerative medicine. (See, for example, Patent Documents 1 to 4 and Non-Patent Documents 1 and 2.) When introducing programming factors and inducing iPS cells, the stem cells are observed under a microscope. Cell-like colonies are picked up with a pipette and passaged. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2000 / 70070 [Patent Document 2] International Publication No. 2010 / 008054 [Patent Document 3] International Publication No. 2012 / 029770 [Patent Document 4] International Publication No. 2015 / 046229 [Non-patent literature]

[0004] [Non-Patent Document 1] Nature 448, 313-317 [Non-patent document 2] Nature Biotechnol 26(3): 313-315, 2008. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for an efficient method for culturing cells into which reprogramming factors have been introduced. The present invention aims to provide an efficient method for culturing cells into which reprogramming factors have been introduced. It shall be one. [Means for solving the problem]

[0006] According to an aspect of the present invention, a method for producing a reprogramming cell is provided, comprising culturing a cell into which a reprogramming factor has been introduced, and All cells into which the programming factors have been introduced are collected, and at least a portion of the collected cells are cultured. and seeding and passage the cells into a medium. A method is provided in which all the collected cells may be mixed.

[0007] In the above method, the cells do not have to be cloned when they are passaged.

[0008] In the above method, multiple colonies formed by cells into which reprogramming factors have been introduced are The method may not involve isolating the components from each other.

[0009] In the above method, a single colony formed by cells into which a reprogramming factor has been introduced is The method may not involve cloning the vector.

[0010] In the above method, colonies formed by cells into which reprogramming factors have been introduced are picked. It does not have to include backing up the data.

[0011] In the above method, the cells into which the reprogramming factors have been introduced are attached to the culture vessel. All cells that are present in the culture medium are collected, and at least a portion of the collected cells are seeded in a medium and subcultured. Good too.

[0012] In the above method, cells into which reprogramming factors have been introduced are distinguished by their gene expression status. They may be passaged without further treatment.

[0013] In the above method, cells into which reprogramming factors have been introduced are subjected to the reprogramming process. The cells may be passaged without distinction of passage frequency.

[0014] The above method allows cells transfected with reprogramming factors to be expanded in two-dimensional culture. It may further include.

[0015] The above method allows cells transfected with reprogramming factors to be expanded in three-dimensional culture. It may further include.

[0016] The above method further comprises establishing stem cells from cells into which reprogramming factors have been introduced. It may also be included in the above.

[0017] The above method further comprises freezing the cells into which the reprogramming factors have been introduced. It's fine.

[0018] The above method can be used to differentiate cells into which reprogramming factors have been introduced into endodermal, mesodermal, and The method may further comprise differentiating the cells into at least one selected from the ectodermal lineages.

[0019] The above method can be used to generate embryoid bodies, organoids, and other organoids from cells transfected with reprogramming factors. The method may further include forming at least one selected from a particle and a sphere.

[0020] The above method allows cells transfected with reprogramming factors to be transformed into somatic cells different from pluripotent stem cells. The method may further comprise directing the cell to express the gene.

[0021] The above method involves treating the cells to induce somatic cell differentiation and then cloning the treated cells. It may further include.

[0022] The above method involves gene editing of cells into which reprogramming factors have been introduced. It may further include:

[0023] In the above method, the cells into which the reprogramming factors have been introduced are blood cells or fibroblasts. It may be of cell origin.

[0024] In the above method, the cells to be transfected with the reprogramming factors are cells contained in urine. It's okay to have one.

[0025] In the above method, the cells to which the reprogramming factors are introduced are bladder epithelial cells. That's fine.

[0026] The above method further comprises collecting cells to be transfected with reprogramming factors from urine. It may also contain.

[0027] In the above method, the cells into which the reprogramming factors have been introduced are from a plurality of humans or a plurality of The antibody may be derived from a non-human animal.

[0028] In the above method, the cells into which the reprogramming factors have been introduced are cultured in a closed culture vessel. It may also be cultured.

[0029] In the above method, the cells may be seeded at a low density during subculture.

[0030] In the above method, the low concentration is 0.25×10 4 cells / cm 2 It can be the following stomach.

[0031] In the above method, the low concentration is a concentration at which 11 or more seeded cells do not come into contact with each other. It is also possible.

[0032] In the above method, the low concentration may be 5% or less confluent.

[0033] In the above method, the reprogramming factor may be RNA.

[0034] In the above method, reprogramming factors are introduced into cells by lipofection. That's fine.

[0035] In the above method, a reprogramming factor is introduced into a cell using a viral vector. That's fine.

[0036] In the above method, the viral vector may be an RNA viral vector.

[0037] In the above method, even if the RNA viral vector is a Sendai virus vector, good.

[0038] Furthermore, according to an aspect of the present invention, cells into which reprogramming factors have been introduced are cultured. The cells transfected with reprogramming factors can be transformed into somatic cells different from pluripotent stem cells without passage. The present invention provides a method for culturing cells into which reprogramming factors have been introduced, the method comprising: inducing the cells to form reprogramming cells. can be.

[0039] The above method allows cells transfected with reprogramming factors to be expanded in two-dimensional culture. It may further include.

[0040] The above method allows cells transfected with reprogramming factors to be expanded in three-dimensional culture. It may further include.

[0041] The above method further comprises freezing the cells into which the reprogramming factors have been introduced. It's fine.

[0042] The above method can be used to differentiate cells into which reprogramming factors have been introduced into endodermal, mesodermal, and The method may further comprise differentiating the cells into at least one selected from the ectodermal lineages.

[0043] The above method involves gene editing of cells into which reprogramming factors have been introduced. It may further include:

[0044] In the above method, the cells into which the reprogramming factors have been introduced are blood cells or fibroblasts. It may be of cell origin.

[0045] In the above method, the cells into which the reprogramming factors have been introduced are from a plurality of humans or a plurality of The antibody may be derived from a non-human animal.

[0046] In the above method, the cells into which the reprogramming factors have been introduced are cultured in a closed culture vessel. It may also be cultured.

[0047] In the above method, the reprogramming factor may be RNA.

[0048] In the above method, reprogramming factors are introduced into cells by lipofection. That's fine.

[0049] In the above method, a reprogramming factor is introduced into a cell using a viral vector. That's fine.

[0050] In the above method, the viral vector may be an RNA viral vector.

[0051] In the above method, even if the RNA viral vector is a Sendai virus vector, good. [Effects of the Invention]

[0052] According to the present invention, an efficient method for culturing cells into which reprogramming factors have been introduced can be provided. It is Noh. [Brief explanation of the drawings]

[0053] [Figure 1] 1 is a graph showing the measurement results obtained by a flow cytometer according to Example 1. [Figure 2] 1 is a graph showing the results of PCR according to Example 1. [Figure 3] 1 is a photograph showing TRA1-60 positive cells according to Example 1. [Figure 4] 1 is a graph showing clonal efficiency according to Example 1 and Comparative Example 1. [Figure 5] 1 is a graph showing the cell counts according to Example 2 and Comparative Example 2. [Figure 6] 1 is a graph showing the number of colonies according to Example 2 and Comparative Example 2. [Figure 7] 10 is a graph showing the number of clumps and the number of cells according to Example 3 and Comparative Example 3. [Figure 8] 10 is a table showing the presence or absence of myocardial pulsation in Example 4 and Comparative Example 4. [Figure 9] 1 is a graph showing the positive rates of cTnT in Example 4 and Comparative Example 4. [Figure 10] 1 is a graph showing the cell count and the positive rate of PSA-NCAM in Example 5 and Comparative Example 5. [Figure 11] 10 is a graph showing the positive rates of SOX1 and OTX2 according to Example 6 and Comparative Example 6. [Figure 12] 1 is a graph showing the positive rates of HAND1 and SOX17 according to Example 6 and Comparative Example 6. [Figure 13] 1 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 1. [Figure 14] 1 is a photograph showing TRA1-60 positive cells according to Reference Example 1. [Figure 15] 10 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 2. [Figure 16] 10 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 3. [Figure 17] 10 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 4. [Figure 18] 1 is a photograph showing cells 15 days after infection according to Reference Example 4. [Figure 19] 10 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 4. [Figure 20] 1 is a photograph showing cells at the first passage in Reference Example 4. [Figure 21] 10 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 5. [Figure 22] 10 is a photograph showing TRA1-60 positive cells according to Reference Example 5. [Figure 23] 10 is a graph showing the measurement results obtained by a flow cytometer according to Reference Example 6. [Figure 24] 1 is a graph showing the results of PCR in Reference Example 6. [Figure 25] 1 is a photograph showing TRA1-60 positive cells according to Reference Example 6. [Figure 26] 10 is a graph showing the results of PCR according to Example 7. [Figure 27] 10 is a photograph showing TRA1-60 positive cells according to Example 7. [Figure 28] 10 is a photograph of nervous system cells according to Example 8. [Figure 29] 10 is a photograph of a teratoma according to Example 9. [Figure 30] 10 is a photograph of an iPS cell-like colony according to Example 10. [Figure 31] 10 shows photographs of Oct3 / 4-positive cells and Nanog-positive cells according to Example 10. [Figure 32] 10 is a dot plot obtained by a flow cytometer according to Example 10. [Figure 33] 10 is a photograph of cardiomyocytes according to Example 10. [Figure 34] 1 shows photographs of Munch13-positive cells and vGlut-positive cells according to Example 10. [Figure 35] 10 is a photograph of urine-derived cells according to Example 11. [Figure 36] 10 is a photograph of urine-derived cells according to Example 11. [Figure 37] 12 is a photograph of urine-derived cells transfected with RNA encoding GFP according to Example 12. [Figure 38] 13 is a photograph of urine-derived cells transfected with reprogramming factors according to Example 13. [Figure 39] 13 is a photograph of urine-derived cells transfected with reprogramming factors according to Example 14. [Figure 40] 10 is a flow cytometer dot plot according to Example 15. [Figure 41] 16 is a photograph of urine-derived cells transfected with reprogramming factors according to Example 16. [Figure 42] 16 is a photograph of urine-derived cells transfected with reprogramming factors according to Example 16. DETAILED DESCRIPTION OF THE INVENTION

[0054] Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the above-mentioned technical concepts and should not be construed as limiting the scope of the present invention. The concept of this invention does not limit the combination of components to those described below. The concept can be modified in various ways within the scope of the claims.

[0055] The method for culturing cells into which reprogramming factors have been introduced according to the embodiment includes culturing cells containing reprogramming factors. Culturing the cells transfected with the reprogramming factors and culturing all the cells transfected with the reprogramming factors and recovering the cells, and seeding at least a portion of the recovered cells in a medium for passage. Cells into which reprogramming factors have been introduced are induced to become, for example, pluripotent stem cells. The stem cells are, for example, iPS cells.

[0056] The cells to which the reprogramming factors are introduced are not particularly limited. Examples include fibroblasts. follicles, blood cells, dental pulp stem cells, keratinocytes, hair papilla cells, oral epithelial cells, and somatic stem cells The cells into which the reprogramming factors are introduced are cells contained in urine. An example of cells found in urine is bladder epithelial cells. The cells into which the transfection factor is introduced may be derived from a human or a non-human animal. The cells into which the reprogramming factors are introduced may be derived from a single individual or from multiple individuals. The cells into which the reprogramming factors are introduced may be derived from several humans. The nucleic acid may be derived from a non-human animal or from multiple non-human animals.

[0057] Blood cells are separated from blood, such as peripheral blood and umbilical cord blood. The blood may be collected from an adult or a minor. When drawing blood, ethylenediaminetetraacetic acid (EDTA), heparin, and biologics standards are used. An anticoagulant such as blood preservation solution A (ACD-A) is used.

[0058] Blood cells include, for example, mononuclear cells (monocytes), neutrophils, macrophages, neutrophils, and Nucleated cells such as eosinophils, basophils, and lymphocytes, including red blood cells, granulocytes, and platelets Blood cells include, for example, endothelial progenitor cells, blood stem and progenitor cells, T cells, or B cells. The T cells may be, for example, αβ T cells.

[0059] Mononuclear cells are separated from blood using a blood cell separation medium and a centrifuge. When Ficoll (GE Healthcare) is used as a medium for separating blood cells, The method for isolating mononuclear cells is as follows.

[0060] Since the accuracy of mononuclear cell separation tends to decrease at low temperatures, the centrifuge should be kept at a temperature between 4°C and 42°C. The temperature is preferably set at 18°C. 10 μL to 50 mL of blood is collected from an adult or minor. The blood is then gently mixed with a chelating agent containing EDTA to prevent blood clotting. In addition, a medium for human lymphocyte separation (Ficoll-Paque PREMIUM, GE Healthcare) Dispense 5 mL of the solution (Ruscare Japan) into two 15 mL tubes. Add 5 mL of PBS to dilute the solution, and place 5 mL on top of the human lymphocyte isolation medium in the tube. At this time, let the diluted blood flow down the wall of the tube so as not to disturb the interface. Add slowly onto the medium.

[0061] The solution in the tube is stirred at 10×g to 1000×g, preferably 400×g, at 4°C to Centrifuge at 42°C, preferably 18°C, for 5 minutes to 2 hours, preferably 30 minutes. A cloudy white middle layer appears in the tube. This cloudy white middle layer contains mononuclear cells. Slowly remove the cloudy white layer from the tube using a pipette and transfer it to a new 15 mL tube. Transfer to a tube. Be careful not to remove the bottom layer. Approximately 1 mL can be recovered from the tube. Combine the middle layers from the two tubes and transfer them into one tube.

[0062] Add 1 mL to 48 mL, preferably 12 mL, of PBS to the collected mononuclear cells. The solution is then further stirred at 10×g to 1000×g, preferably 200×g, at 4°C to 42°C, Centrifuge at 18°C ​​for 1 to 60 minutes, preferably 10 minutes. Remove the supernatant by aspirating using a syringe and add 1 mL to 12 mL, preferably 3 mL, of known volume. The cells were suspended in a serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) and then mixed with a single A mononuclear cell suspension is obtained. 10 μL of the mononuclear cell suspension is stained with trypan blue. and count using a hemocytometer.

[0063] Method for isolating mononuclear cells when using Vacutainer (registered trademark, BD) as a blood collection tube The law is as follows:

[0064] Since the accuracy of mononuclear cell separation tends to decrease at low temperatures, the centrifuge is set at a temperature between 4°C and 42°C. The temperature is preferably set at 18°C. 8 mL of blood is collected using a syringe (BD) and mixed with the anticoagulant by inverting. Adjust the temperature and heat the solution at 4°C to 42°C, preferably 18°C, at 100 x g to 3000 x g. , preferably at 1500×g to 1800×g in a swinging bucket rotor for 1 minute to 60 minutes, After centrifugation, remove the upper layer, which is the plasma layer, and pipette the mononuclear The globular layer and the blood cells adhering to the gel are suspended to obtain a suspension. The resulting suspension is then separated into 15 Transfer to a mL tube.

[0065] Add 1 mL to 14 mL, preferably 12 mL, of PBS to the suspension in the 15 mL tube. The suspension is then stirred at 4°C to 42°C, preferably at 18°C, at 100 x g to 3000 x g, preferably at Centrifuge at 200 x g for 1 to 60 minutes, preferably 5 minutes. After centrifugation, aspirate the supernatant. The hemolytic agent (PharmLyse®, 10x concentration, BD) Dilute with sterile water to a concentration of 1x. Loosen the pellet in the 15mL tube by tapping. Add 1 mL to 14 mL, preferably 1 mL, of hemolysis agent. Then, keep the mixture at room temperature, protected from light, and incubate for 1 minute. The solution is allowed to stand for 60 minutes, preferably 1 minute.

[0066] Next, add 1 mL to 14 mL, preferably 12 mL, of PBS to a 15 mL tube. , 4°C to 42°C, preferably at room temperature, at 100 x g to 3000 x g, preferably at 200 Centrifuge at 1000 × g for 1 to 60 minutes, preferably 5 minutes. After centrifugation, remove the supernatant with an aspirator. Remove the cells and add 1 mL to 15 mL, preferably 3 mL, of chemically defined serum-free hematopoietic cell medium (X-VI Add VO (registered trademark) 10 (Lonza) and suspend to obtain a mononuclear cell suspension. 10 μL of the mononuclear cell suspension was stained with trypan blue and counted using a hemocytometer.

[0067] The method for separating mononuclear cells from blood is not limited to the above method, and may be, for example, a method using a dialysis membrane. Alternatively, mononuclear cells may be separated from blood using a PureCell Select system for enriching mononuclear cells from whole blood. Stem (registered trademark, PALL), blood cell removal purifier (Cellsorber E, registered trademark, Asahi Chemical (Sepacel PL, registered trademark, PLX-5B) and leukocyte removal filter for platelet preparations -SCD, Asahi Kasei) and other filters can also be used.

[0068] Mononuclear cells are isolated by gravity settling or centrifugation of red blood cells to separate nucleated cells. The red blood cells may be separated using a red blood cell separating agent capable of separating the red blood cells. Sep (registered trademark, STEMCELL Technologies) and HES40 (N IPRO) are examples.

[0069] In addition, mononuclear cells are available from Cellular Technology Limited. CTL-UP1 sold by Sanguine Biosciences PBMC-001 from the company may also be used.

[0070] Alternatively, for blood cells, CellBanker 1 and Stem CellBanker GMP Grade are available. and cell freezers such as Stem Cell Banker DMSO-free GMP grade (Zenoac) Blood cells that have been cryopreserved using a cryopreservation solution may be thawed and used.

[0071] When thawing mononuclear cells, first transfer 1 mL to 15 mL into a 15 mL tube, preferably Add 8 mL of chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) Keep the tube containing the frozen mononuclear cells at 4°C to 42°C, preferably 37°C. Then, with some ice remaining, place the mononuclear cells in a warm bath. The tube containing the mononuclear cells was removed from the bath and the mononuclear cells were incubated in a defined serum-free hematopoietic cell suspension. Transfer 10 μL of the mononuclear cell suspension to a tube containing the medium. Stain and count using a hemocytometer.

[0072] Blood cells may be separated based on cell surface markers. Blood stem and progenitor cells are CD3, CD4, or CD8 are positive for T cells. The cells are positive for either CD10, CD19, or CD20. Macrophages are positive for CD1 1b, CD68, or CD163 positive. Blood stem and progenitor cells, T cells, or B cells can be separated from blood cells using, for example, an automated magnetic cell separator and immunomagnetic beads. Alternatively, pre-isolated mononuclear cells may be prepared. Blood cells that have not been separated based on marker may also be used.

[0073] CD34+ cells are stem / progenitor cells and are prone to reprogramming. In addition, when iPS cells are produced using CD3-positive T cells, T cell-derived iPS cells S cells retain the TCR recombination pattern and can be efficiently induced to differentiate into T cells. It tends to be possible.

[0074] The method for isolating CD34-positive cells is as follows.

[0075] 10 mL of serum-free medium (StemSpan H3000, STEMCELLTechn ologies), 10 μL of IL-6 (100 μg / mL), 10 μL of SCF (3 10 μL of TPO (300 μg / mL), 10 μL of FLT3 ligand Add 10 μL of IL-3 (10 μg / mL) and 10 μL of IL-3 (300 μg / mL) to the hemocyte medium. Prepare (Blood Stem and Progenitor Cell Medium).

[0076] Place 1 mL to 6 mL, preferably 2 mL, of blood cell medium into each well of a 6-well plate. To prevent evaporation of the medium, add 1 mL to 6 mL of the desired amount of medium to each of the other 5 wells. Then, place the 6-well plate in a refrigerator at 4 to 42°C, preferably Place in a 37°C incubator to warm.

[0077] For 20 mL of PBS, add 10 μL to 1 mL, preferably 80 μL of EDTA (50 0 mmol / L) and 10 μL to 1 mL, preferably 200 μL, of FBS. Prepare a 1×10 4 From 1×10 9 pieces, preferably 2 x 10 7 Mononuclear cells Dispense the mononuclear cell suspension containing the cells into 15 mL tubes and store at 4°C. to 42°C, preferably 4°C, at 100 x g to 3000 x g, preferably 300 x g. After centrifugation, remove the supernatant and suspend the mononuclear cells in 100 μL to 1 mL, preferably Suspend in 300 μL of column buffer.

[0078] Add 10 μL to 1 mL, preferably 100 μL, to the mononuclear cell suspension in a 15 mL tube. L of FcR blocking reagent (Miltenyi Biotec) and 10 μL to 1 m L, preferably 100 μL of CD34 Microbead Kit (Miltenyi Bio The FcR blocking reagent is used to enhance the specificity of the microbead labeling. Then, the mononuclear cell suspension is mixed and the mixture is heated at 4 to 42°C, preferably at 4 The mixture is left standing at 5°C for 1 to 2 hours, preferably 30 minutes.

[0079] Next, add 1 mL to 15 mL, preferably 10 mL, of the mononuclear cell suspension in the 15 mL tube. Add 1 mL of column buffer to dilute and run at 4°C to 42°C, preferably at 4°C, 100 x g or Centrifuge at 1000 x g, preferably 300 x g, for 1 to 2 hours, preferably 10 minutes. After centrifugation, remove the supernatant from the 15 mL tube with an aspirator and dilute it in 10 μL to 10 mL, if desired. Preferably, 500 μL of column buffer is added to resuspend the particles.

[0080] A column for an automated magnetic cell separator (MS column, Miltenyi Biotec) was used. Automated magnetic cell separator (MiniMACS Separation Unit, Milt Attach the column to a tube (Enyi Biotec) and load 10 μL to 10 mL, preferably 50 mL. Add 100 μL of column buffer to wash the column. Next, add the mononuclear cells to the column. Add 10 μL to 10 mL, preferably 500 μL, of column buffer to the column. The column is washed 1 to 10 times, preferably 3 times. Then, the column is placed in an automated magnetic cell separator. Remove the column from its holder and place it in a 15 mL tube. Then, add 10 μL to 10 mL, preferably Add 1000 μL of column buffer and quickly press the syringe to extract the CD34-positive cells. Drain into a 15 mL tube.

[0081] 10 μL of the CD34-positive cell suspension was stained with trypan blue and the cell count was determined using a hemocytometer. Count the CD34-positive cells in the 15 mL tube. , preferably at 4°C, at 100 x g to 1000 x g, preferably at 300 x g, for 1 to 2 hours. Centrifuge for 10 minutes, preferably 10 minutes. After centrifugation, remove the supernatant with an aspirator. Resuspend the CD34-positive cells in the blood cell medium and seed the CD34-positive cells onto a culture plate. Then, incubate at 4°C to 42°C, preferably 37°C, in a 1% to 20% CO2 atmosphere, preferably 5% CO2. CD34-positive cells are cultured for 6 days without medium changes.

[0082] The method for isolating cells using markers other than CD34 is different from the method for isolating CD34-positive cells. The same is true.

[0083] The reprogramming factor introduced into the cell is, for example, RNA. , mRNA. The reprogramming factor introduced into the cells is, for example, OCT3 / 4, etc. OCT mRNA, SOX mRNA such as SOX2, KLF mRNA such as KLF4, and MYC mRNA, such as c-MYC. M3O, an improved version of T3 / 4, may also be used. IN28A, FOXH1, LIN28B, GLIS1, p53-dominant ne gative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4 , DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KL F5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A 1, consisting of NR5A2, TBX3, MBD3sh, TH2A, TH2B, and P53DD The mRNA may further comprise at least one factor selected from the group consisting of: The mRNA for this gene is available from TriLink. Although listed, capitalization is not intended to limit the species. For example, even if written in all capital letters, it does not exclude the inclusion of mouse or rat genes. However, in the examples, the gene descriptions are based on the actual organisms used. The number is indicated.

[0084] p53 is a tumor suppressor protein. Dominant-negative mutants of p53 cause myeloma. It acts competitively with wild-type p53 protein present in cells, inhibiting the function of wild-type p53 protein. There is no particular limitation as long as it can inhibit the function of p53. The p53 promoter at position 275 (position 278 in humans) is located in the DNA binding domain of mouse p53. p53P275S, a point mutation of phospho-serine, and the 14-301 position of mouse p53 (human p p53DD, which lacks the amino acids 11-304 of mouse p53; p53S58A, a human p53 with a point mutation of serine to alanine at position 8 (position 61 in humans). p5 with a point mutation of cysteine ​​at position 135 of 53 (position 132 in mouse) to tyrosine 3C135Y: Alanine at position 135 of mouse p53 (position 138 in humans) is changed to valine p53A135V, a point mutation at position 172 of mouse p53 (position 175 in humans), p53R172H, a point mutation of arginine to histidine, and p53R272H, a mutation at position 270 of mouse p53 (human p53R270H, in which arginine at position 273 in the case of p53A is mutated to histidine, and A point mutation of aspartic acid at position 278 (position 281 in humans) of mouse p53 to asparagine An example is p53D278N, which is a mutant form of the gene.

[0085] mRNA is modified with pseudouridine (Ψ) or 5-methyluridine (5meU). The mRNA may be polyadenylated.

[0086] The RNA introduced into the cell is, for example, single-stranded RNA, and double-stranded RNA is substantially excluded. In addition, the RNA introduced into cells is free of impurities such as short-chain RNA and other contaminants. In order to substantially remove double-stranded RNA, it is preferable that the double-stranded RNA is qualitatively removed. The single-stranded RNA to be introduced may be purified and / or concentrated. As a method for purifying the compound, a purification method using high performance liquid chromatography (HPLC) is used. For example, double-stranded RNA may be detected by HPLC at a concentration of 70% or more, 75% or more, or 8% or more. 0%, 85%, or 90% or more of double-stranded RNA is removed. In order to efficiently remove the RNA introduced into cells, the RNA is treated with ribonuclease that degrades double-stranded RNA. It may also be treated with azeotropy.

[0087] The RNA introduced into cells is a MYOD gene directly linked to the full-length OCT3 / 4 RNA. The gene may further comprise RNA of a transcription activation domain (TAD).

[0088] Reprogramming factors are introduced into cells, for example, by lipofection. The infection method involves electrophoretically injecting a complex of a negatively charged substance, nucleic acid, and a positively charged lipid. The complex is then taken up into the cell by endocytosis or membrane fusion. The lipofection method causes little damage to cells and has excellent transfection efficiency. This method has the advantages of being simple to operate and not requiring much time.

[0089] Reprogramming factors are transfected using, for example, RNA transfection reagents. For example, if the cells are mononuclear cells, they are introduced into cells that have been isolated from blood. Immediately afterwards, the RNA may be introduced into the mononuclear cells.

[0090] As an RNA transfection reagent, Lipofectamine Messe IngerMAX (registered trademark, Thermo Fisher Scientific) is used Alternatively, the RNA transfection reagent may be, for example, Lipo fectamine® RNAiMAX (Thermo Fisher Scientific ENTIFIC), Lipofectamine StemTransfection Reagent (Thermo Fisher SCIENTIFIC), TransI T(Mirus), mRNA-In(MTI-GlobalStem), Stemfec t RNA Transfection Kit (ReproCELL), Jet M essenger (Polyplus), Lipofectamin®2 000, Lipofectamin® 3000, NeonTransfect ion System (Thermo Fisher SCIENTIFIC), Ste. mfect RNA transfection reagent(Stemfect) , NextFect® RNA Transfection Reagent ( BioScientific), Amaxa (registered trademark) Human T cell N Ucleofector (registered product) kit (Lonza, VAPA-1002), A maxa (registered product) Human CD34 cell Nucleofector (registered product) kit (Lonza, VAPA-1003), and ReproRNA (registered trademark (Trademark) Transfection reagent (STEMCELL Technologies) etc. A transfection reagent may also be used.

[0091] Alternatively, cells can be transduced with reprogramming factors using, for example, a viral vector. The viral vector may be an RNA viral vector. The vector may be a Sendai virus vector. Temperature-sensitive Sendai virus vectors, whose viral nucleic acid stability decreases above a certain temperature. The viral nucleic acid of the temperature-sensitive Sendai virus vector can be expressed at temperatures below a predetermined temperature. The viral nucleic acid may be viral DNA or viral RNA. The viral nucleic acid may be a viral genome. The term "decomposition of viral nucleic acid" refers to the decomposition of viral nucleic acid and the inhibition of replication or proliferation of viral nucleic acid. When the stability of the viral nucleic acid is reduced, the virus At least one of the following is reduced: nucleic acid proliferation, viral nucleic acid replication rate, and gene expression level The predetermined temperature is, for example, 36.5°C or higher and 37.5°C or lower, or 36.6°C or higher and 37.4°C or lower. 36.7℃ or higher, 37.3℃ or lower, 36.8℃ or higher and 37.2℃ or lower, 36.9℃ or lower The temperature is above 37.1°C or below 37°C. The stability of the nucleic acid, i.e., the growth, replication rate and / or gene expression level, , which is high below a certain temperature and low above a certain temperature. The vectors were compared with the growth rate or gene expression level in cells cultured at 32°C. In comparison, the growth rate or gene expression level in cells cultured at 37°C is 1 / It is 2 or less, 1 / 3 or less, 1 / 5 or less, 1 / 10 or less, or 1 / 20 or less.

[0092] Sendai virus has N gene, P gene, M gene, F / HN gene, and L gene. The HN protein encodes the cytoplasmic domain of the cytoplasmic domain of the Sendai virus. The F protein recognizes extracellular proteases and anchors the virus particles to the cells. The tethered Sendai virus envelope and the target cell are cleaved and activated by the enzyme. It catalyzes the fusion of the cell membrane of the vesicle, thereby establishing infection. After infection, the viral nucleic acid is replicated in the cytoplasm along with the P protein, resulting in multiple copies of the viral nucleic acid. It catalyzes the transcription of the nucleic acid from

[0093] By deleting the F gene in the Sendai virus vector, It is possible to suppress the production of infectious virus particles from the L gene. By introducing a mutation into at least one of the P gene and the P gene, the Sendai virus vector It is possible to make the detector temperature sensitive.

[0094] An example of a temperature-sensitive (TS) mutation in Sendai virus is TS7 (Y in the L protein). 942H / L1361C / L1558I mutation), TS12 (D433A / R434A / K437A mutation), TS13 (D433A / R434A / K of P protein 437A mutation and L1558I mutation in L protein), TS14 (D43 in P protein 3A / R434A / K437A mutation and L protein L1361C mutation), TS15( D433A / R434A / K437A mutations in the P protein and L1361 in the L protein C / L1558I mutation) and others.

[0095] For example, Sendai virus vectors have G69E, T116A, and A modifications in the M protein. It has the 183S mutation and the A262T, G264R, and K461G mutations in the HN protein. It has a mutation in the P protein, an L511F mutation in the L protein, and an N1197S and K1 An F gene-deleted (ΔF) Sendai virus vector having the 795E mutation, Sendaiwi strains with mutations in TS7, TS12, TS13, TS14, or TS15 However, the temperature-sensitive mutation of the Sendai virus vector Not limited.

[0096] Examples of Sendai virus vectors include SeV(PM) / TSΔF and SeV18+ / T SΔF, or SeV(HNL) / TSΔF, and the above TS7, TS12, TS13 , TS14, or TS15 mutations are introduced into the Sendai virus vector. Temperature-sensitive mutations of Sendai virus vectors are not limited to these.

[0097] The Sendai virus vector introduced into cells is a temperature-sensitive Sendai virus vector. Alternatively, a combination of a temperature-insensitive Sendai virus vector may be used. The only Sendai virus vectors introduced into cells are temperature-sensitive Sendai virus vectors. and may not include a temperature-insensitive Sendai virus vector. The introduced Sendai virus vector is TS7, TS12, TS13, TS14, or T Only temperature-sensitive Sendai virus vectors with S15 mutations introduced are available. It may not include a Sendai virus vector. The vector introduced mutations TS7, TS12, TS13, TS14, or TS15. A Sendai virus with temperature sensitivity equal to or higher than that of a temperature-sensitive Sendai virus vector The vector may be a vector only, and may not include a temperature-insensitive Sendai virus vector. For example, the Sendai virus vectors introduced into cells are TS7, TS12, TS13, and TS 14, or a temperature-sensitive Sendai virus vector containing a TS15 mutation. The only Sendai virus vectors that are sensitive to HIV infection are TS7, TS12, TS13, and Temperature-sensitive Sendai virus vectors carrying TS14 or TS15 mutations were used to It may not contain Sendai virus vectors, which are less sensitive.

[0098] The Sendai virus vector introduced into cells can carry any reprogramming factor. The Sendai virus vector introduced into cells is, for example, KLF mRNA, OCT mRNA, and SOX mRNA in this order, but no MYC mRNA. A sensitive Sendai virus vector containing MYC mRNA, KLF mRNA, and OC A temperature-sensitive Sendai virus vector that does not contain T mRNA or SOX mRNA. However, the reprogramming factors carried in the Sendai virus vector are The number, combination and order of are arbitrary and are not particularly limited.

[0099] The Sendai virus vector introduced into the cells contains KLF mRNA and OCT m It may also include a Sendai virus vector that does not contain RNA or SOX mRNA. Sendai contains KLF mRNA but does not contain OCT or SOX mRNA. The virus vector may be a temperature-sensitive Sendai virus vector or a temperature-insensitive However, according to the findings of the present inventors, the vector may be a temperature-sensitive Sendai virus vector. It is better not to introduce the insensitive Sendai virus vector than to introduce the Sendai virus vector. The Sendai virus vector disappears quickly from the transfected cells.

[0100] Temperature-sensitive senders including KLF mRNA, OCT mRNA, and SOX mRNA For example, the virus vector contains G69E, T116A, and A183S mutations in the M protein. and A262T, G264R, and K461G mutations in the HN protein. , which has the L511F mutation in the P protein and N1197S and K1795E in the L protein An F gene-deleted Sendai virus vector having a mutation, comprising the above-mentioned TS7 and TS1 2. Sendai virus vectors with TS13, TS14, or TS15 mutations The temperature-sensitive mutation is, for example, TS7 or TS12, or TS12.

[0101] Temperature-sensitive senders including KLF mRNA, OCT mRNA, and SOX mRNA The viral vector is, for example, SeV(PM)KOS / TS7ΔF or SeV(PM) KOS / TS12ΔF, or SeV(PM)KOS / TS12ΔF.

[0102] Temperature-sensitive Sendai virus vectors containing MYC mRNA are used to express, for example, M protein. It has G69E, T116A, and A183S mutations in the HN protein and A262T , G264R, and K461G mutations, and an L511F mutation in the P protein. F gene deletion type Sendai virus with N1197S and K1795E mutations in the protein Vectors that are variations of TS7, TS12, TS13, TS14, or TS15 above A Sendai virus vector containing a temperature-sensitive mutation, such as TS15. .

[0103] The temperature-sensitive Sendai virus vector containing MYC mRNA is, for example, SeV(H NL)MYC / TS12ΔF, SeV(HNL)MYC / TS13ΔF, or SeV(H NL)MYC / TS15ΔF or SeV(HNL)MYC / TS15ΔF be.

[0104] Sendai contains KLF mRNA but does not contain OCT or SOX mRNA. The viral vector contains, for example, G69E, T116A, and A183S mutations in the M protein. having mutations A262T, G264R, and K461G in the HN protein; The P protein has the L511F mutation, and the L protein has the N1197S and K1795E mutations. It is an F gene-deleted Sendai virus vector containing KLF mRNA. Sendai virus vectors that do not contain OCT mRNA and SOX mRNA are, for example, , the above-mentioned TS7, TS12, TS13, TS14, or TS15 mutations were introduced. Less temperature sensitive than viral vectors, allowing KLF gene expression above a certain temperature is.

[0105] Sendai contains KLF mRNA but does not contain OCT or SOX mRNA. The viral vector is, for example, SeV18+KLF4 / TSΔF.

[0106] When introducing multiple types of Sendai virus vectors into cells, for example, Alternatively, a certain type of Sendai virus can be introduced into the cells. Within 48 hours, within 36 hours, within 24 hours, and at 18:00 after transfection of the vector into the cells. Within 12 hours, 10 hours or less, 8 hours or less, 6 hours or less, 3 hours or more, 2 hours or more All types of Sendai virus vectors can be introduced into cells within one hour or less. It is preferable that:

[0107] The multiplicity of infection (MOI) of the Sendai virus vector when infecting cells is, for example, 0.1 or more, 0.3 or more, 0.5 or more, 1.0 or more, 2.0 or more, 3.0 or more, 4.0 or more or 5.0 or more. The MOI is, for example, 100 or less, 90 or less, or 80 or less. , 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less Below.

[0108] The temperature at which cells are infected with the Sendai virus vector is The temperature below which the stability of the viral nucleic acid of the vector decreases, i.e., the temperature-sensitive sensor The temperature may be a temperature at which the viral nucleic acid of the diviral vector is stable, or a temperature above a predetermined temperature. The Sendai virus vector may be a temperature-sensitive Sendai virus vector. If the cells contain only the temperature-insensitive Sendai virus vector, the cells are transformed with the Sendai virus vector. The temperature at which the vector is infected is the temperature-sensitive Sendai virus vector. Temperatures below which nucleic acid stability is reduced, i.e., temperature-sensitive Sendai virus Preferably, the temperature is one at which the viral nucleic acid of the vector is stable.

[0109] The cells to which the reprogramming factors are introduced may be cultured in adhesion or suspension. It may also be used.

[0110] Somatic cells into which reprogramming factors are introduced are cultured on Matrigel (Corning), CELLstart (registered trademark, ThermoFisher), Laminin511( iMatrix-511, nippi), fibronectin, and vitrotin They may be cultured feeder-free using a membrane matrix.

[0111] The medium in which the cells into which the reprogramming factors are introduced are cultured may be, for example, Prim Human ES / iPS culture medium such as ReproCELL ES Cell Medium (ReproCELL) Stem cell culture media such as the above can be used.

[0112] However, the stem cell medium is not limited to this, and various stem cell media can be used. Primate ES Cell Medium, Reprostem, ReproF F, ReproFF2, ReproXF (Reprocell), mTeSR1, TeS R2, TeSRE8, ReproTeSR(STEMCELL Technologie s), PluriSTEM® Human ES / iPS Medium (Me rck), NutriStem® XF / FF Culture Medium m for Human iPS and ES Cells, Pluriton re programming medium(Stemgent), PluriSTEM(registration) Registered trademark), Stemfit AK02N, Stemfit AK03 (Ajinomot o), ESC-Sure® serum and feeder-free m edium for hESC / iPS(Applied StemCell), L7( Registered trademark) hPSC Culture System (LONZA), and Pluri Q (MTI-GlobalStem) or the like may be used. The cells are placed in an incubator such as a cup, well, or tube.

[0113] When cells are cultured in suspension or three-dimensionally, for example, a gel medium is used. For example, gellan gum is added to the stem cell medium at a final concentration of 0.001% by mass to 0.5% by mass. , 0.005% by mass to 0.1% by mass, or 0.01% by mass to 0.05% by mass It is prepared by adding

[0114] Gel media are made from gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, Tan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan Sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, deltaman sulfate, At least one polymeric compound selected from the group consisting of rhamnan sulfate and salts thereof The gel medium may also contain methylcellulose. By including cellulose, aggregation of cells is further suppressed.

[0115] Alternatively, the gel medium may be composed of poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl acrylate), opyl methacrylate) (PHPMA), Poly (N-isopropylacrylamide) (PNIPAM), amine termin ated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NH S) ester terminated, triethoxysilane terminated, Poly (N-isopropylacrylamide-co- acrylamide), Poly (N-isopropylacrylamide-co-acrylic acid), Poly (N-isopropylacry lamide-co-butylacrylate), Poly (N-isopropylacrylamide-co-methacrylic acid), Poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-Isopro The gel may contain at least one temperature-sensitive gel selected from pyracrylamide.

[0116] Gel medium is, for example, a basic fibroblast growth factor It may or may not contain growth factors such as r(bFGF). The medium contains growth factors such as bFGF at 400 μg / L or less, 40 μg / L or less, or 1 It may be contained at a low concentration of 0 μg / L or less.

[0117] The gel medium may contain TGF-β, or may not contain TGF-β. Contains low concentrations of 0 μg / L or less, 300 μg / L or less, or 100 μg / L or less That's fine.

[0118] The gel medium does not need to be stirred. The gel medium does not need to contain feeder cells. Good too.

[0119] The gel medium consists of cadherin, laminin, fibronectin, and vitronectin. The composition may contain at least one substance selected from the group:

[0120] After infecting the cells with the Sendai virus vector, the cells are incubated for at least 2 days, or for more than 2 days. Within 10 days, the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector decreases. The temperature-sensitive Sendai virus vector is The cells may be cultured at a temperature at which they are stable. Then, the cells may be cultured at a temperature above the predetermined temperature. While the cells are cultured at a predetermined temperature or higher, the medium may be changed, for example, once every two days.

[0121] After infecting the cells with the Sendai virus vector, the cells are incubated for at least 2 days, or for more than 2 days. 10 days or less, for example, 4.0℃ or higher, 10℃ or higher, 15℃ or higher, 20℃ or higher, 25℃ or higher , 30℃ or higher, 31.0℃ or higher, 32.0℃ or higher, 33.0℃ or higher, 33.1℃ or higher, 3 3.2℃ or higher, 33.3℃ or higher, 33.4℃ or higher, 33.5℃ or higher, 33.6℃ or higher, 3 3.7°C or higher, 33.8°C or higher, or 33.9°C or higher, but less than 37.0°C, 36 Less than 36.9℃, Less than 36.8℃, Less than 36.7℃, Less than 36.6℃, Less than 36.5℃, Less than 36 The cells may be cultured at temperatures below 3.0°C, below 35.0°C, or below 34.0°C. Then, the temperature is raised to 36.5°C or higher, 36.6°C or higher, 36.7°C or higher, 36.8°C or higher. ℃ or higher, 36.9℃ or higher, or 37.0℃ or higher, and 40.0℃ or lower, 39.0℃ Cells may be cultured at temperatures below 38.0°C or below 38.0°C. The temperature may be increased in one step. Alternatively, the temperature may be increased stepwise. After increasing the temperature, the temperature may be increased, for example, once every two days while the cells are being cultured. The medium may be changed once a day.

[0122] After infecting cells with Sendai virus vectors, stem cell-like colonies began to appear. At a predetermined temperature at which the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector decreases less than 100°C, i.e., at a temperature at which the viral nucleic acid of the temperature-sensitive Sendai virus vector is stable. The cells may be cultured. After stem cell-like colonies begin to appear, the cells are cultured at a predetermined temperature or higher. While culturing the cells at a predetermined temperature or higher, the medium may be changed, for example, once every two days. Good too.

[0123] After infecting cells with Sendai virus vectors, stem cell-like colonies began to appear. For example, 4.0℃ or more, 10℃ or more, 15℃ or more, 20℃ or more, 25℃ or more, 30℃ above 31.0℃, above 32.0℃, above 33.0℃, above 33.1℃, above 33.2℃ above 33.3℃, ​​above 33.4℃, above 33.5℃, above 33.6℃, ​​above 33.7℃ or above, 33.8℃ or above, or 33.9℃ or above, but below 37.0℃, or below 36.9℃ Less than 36.8℃, Less than 36.7℃, Less than 36.6℃, Less than 36.5℃, More than 36.0℃ The cells may be cultured at temperatures below 35.0°C, or below 34.0°C. After colonies begin to appear, the temperature is raised to 36.5°C or higher, 36.6°C or higher, and 36. 7°C or higher, 36.8°C or higher, 36.9°C or higher, or 37.0°C or higher, and The cells may be cultured at temperatures below 39.0°C, below 39.0°C, or below 38.0°C. The temperature can be increased in one step or in stages. After increasing the temperature, the cells are cultured. The medium may be changed periodically, for example, once every two days.

[0124] Reprogramming factors are introduced into cells, the cells are cultured, and then the reprogramming factors are introduced. All the cells that were introduced were collected, and at least a portion of the collected mixed cells was seeded into the culture medium. After that, all the cells that had been transfected with the reprogramming factors were The cells are collected, and at least a portion of the collected mixed cells is seeded in a medium and passaged. The above steps may be repeated several times. All the cells that were collected were then collected, and at least a portion of the collected mixed cells was seeded in a medium for subculture. Alternatively, all of the collected mixed cells may be seeded onto a medium.

[0125] Here, all the cells that have been transfected with the reprogramming factors are collected and mixed. The phrase "seeding at least a portion of the cells in a medium and passage" refers to, for example, the introduction of reprogramming factors into the medium. This refers to the passage of cells into which the gene has been introduced without distinguishing between the gene expression states. During passage, cells transfected with reprogramming factors are differentiated by their gene expression status. Alternatively, all the cells transfected with the reprogramming factors may be seeded in the same culture vessel. The cells are collected, and at least a portion of the collected mixed cells is seeded in a medium and passaged. For example, cells transfected with reprogramming factors are differentiated based on the degree of reprogramming. For example, when a reprogramming factor is introduced during the passaging, The reprogrammed cells may be seeded in the same culture vessel without distinction based on the degree of reprogramming.

[0126] Alternatively, all cells transfected with reprogramming factors can be collected and mixed together. The phrase "seeding at least a portion of the cells in a medium and passage" refers to, for example, the introduction of reprogramming factors into the medium. This refers to the passage of cells into which a gene has been introduced without distinguishing them by morphology. The cells transfected with the reprogramming factors were plated in the same culture vessel without distinguishing them by morphology. Alternatively, all cells into which the reprogramming factors have been introduced can be collected and then reseeded. The phrase "passaging at least a portion of the mixed cells by seeding them in a medium" refers to, for example, reproducibility. This refers to the passage of cells into which gramming factors have been introduced without distinguishing between different sizes. For example, during passage, cells into which reprogramming factors have been introduced are divided into the same group without size discrimination. The cells may be inoculated into the same incubator.

[0127] Alternatively, all cells transfected with reprogramming factors can be collected and mixed. The term "seeding and passage of at least a portion of the cells that have interacted with each other" means that the reprogramming factors are introduced into the cells. This refers to the passage of introduced cells without cloning. For example, cloning When cells are passaged without transfection, the colonies formed by the transfected cells are For example, if you are subculturing without cloning, The multiple colonies formed by cells transfected with programming factors do not need to be separated from each other. For example, when subculturing, cells that have formed multiple different colonies can be mixed and cultured in the same culture medium. Alternatively, for example, when subculturing without cloning, It is not necessary to clone the single colonies formed by cells transfected with laming factors. For example, when subculturing, colonies may be mixed and inoculated into the same culture vessel.

[0128] For example, when cells transfected with reprogramming factors are cultured in adherent culture, All cells that have been collected are harvested and at least a portion of the harvested mixed cells are seeded into culture medium. For example, at the time of subculture, all the cells may be detached from the culture vessel, detached and mixed. At least some of the cells may be seeded in the same culture vessel. All cells may be detached from the vessel and the detached mixed cells may be passaged in their entirety. For example, Cells that do not form colonies may be passaged. When the cells are cultured in suspension, the cells may be passaged as a whole in suspension culture.

[0129] When cells transfected with reprogramming factors are passaged, the cells are grown in a low concentration in the medium or in the culture medium. Here, low density means, for example, 1 cell / cm 2 It is more than or equal to 0. 25×10 4 cells / cm 2 Below, 1.25 x 10 3 cells / cm 2 Below, 0.25 x10 3 cells / cm 2 Below, 0.25 x 10 2 cells / cm 2 Less than or equal to 0. 25×10 1 cells / cm 2 Alternatively, low concentration means 10 or less, 9 or more Below, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less This is the concentration at which 11 or more cells can come into contact with each other, but 11 or more cells cannot come into contact with each other. There may be multiple cell clusters with 0 or less cells in contact with each other. A state in which the plate is covered with cells is considered 100% confluent, and a low concentration is 5% or less confluent. Confluent, 4% or less confluent, 3% or less confluent, 2% or less confluent, Less than 1% confluence, less than 0.5% confluence, less than 0.1% confluence, The cells are less than 0.05% confluent, or less than 0.01% confluent. Alternatively, a low concentration is, for example, a concentration at which single cells do not come into contact with each other when seeded. For example, single cells may be seeded in the wells of a well plate. The plate may be a 12-well plate or a 96-well plate. According to the study, when cells transfected with reprogramming factors are passaged, the cells are cultured at low concentrations. By seeding the cells onto the surface of the cells, the residual Sendai virus in the pluripotent stem cells derived from the cells was detected. In induced pluripotent stem cells, Sendai virus can suppress the proliferation of The percentage of remaining cells is, for example, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less. Or 0%.

[0130] When a temperature-sensitive Sendai virus vector is used, the temperature-sensitive Sendai virus The cells may be cultured at a temperature above a predetermined temperature at which the stability of the viral nucleic acid of the vector decreases. After the subculture, the cells are cultured at a temperature of, for example, 36.5°C or higher and lower than 38.0°C. For example, cells are cultured at a temperature of 36.5°C or higher but lower than 38.0°C until cell-cell adhesion begins. After the onset of cell-cell adhesion, a higher temperature, e.g., 37.5°C or higher, is required until cell-cell adhesion begins. and above 42.0°C, 41.5°C, 41.0°C, 40.5°C, or After subculture, cells may be cultured at temperatures below 40.0°C before cell-cell adhesion begins. , 37.5℃ or higher, 42.0℃ or lower, 41.5℃ or lower, 41.0℃ or lower, 40.5 The cells may be cultured at temperatures below 40.0°C, or below 40.0°C.

[0131] The cells transfected with the reprogramming factors are cultured and passaged in a closed culture vessel. The closed incubator may be used to isolate the culture medium from the outside, for example, gases, viruses, microorganisms, impurities, etc. In addition, cells transfected with reprogramming factors are expanded in two-dimensional culture. Alternatively, the cells may be expanded in a three-dimensional culture.

[0132] Cells into which reprogramming factors have been introduced are induced to become pluripotent stem cells, which then become pluripotent stem cells. After establishment, the whole cells in the adherent culture may be cryopreserved as pluripotent stem cells. For example, the entire cells detached from the culture vessel using a detachment solution may be cryopreserved as pluripotent stem cells. In addition, after cells transfected with reprogramming factors were induced to become pluripotent stem cells, they were cultured in suspension. The cultivated whole cells may be cryopreserved as pluripotent stem cells.

[0133] The induced pluripotent stem cells form flat colonies similar to those of ES cells and grow in alkaline water. The induced pluripotent stem cells express Na+, an undifferentiated cell marker. The induced pluripotent stem cells can express TERT, nog, OCT4, and SOX2. The induced pluripotent stem cells may exhibit telomerase activity.

[0134] In addition, whether or not the cells were induced to become pluripotent stem cells was determined by measuring the number of undifferentiated cells with a flow cytometer. TRA-1-60, TRA-1-81, and SSEA- 1, and SSEA5. TRA-1-60 is an antibody specific to iPS / ES cells. Since iPS cells can only be produced from the TRA-1-60 positive fraction, -60 positive cells are considered to be the seeds of iPS cells.

[0135] Even if induced pluripotent stem cells are induced to become somatic cells in a state different from that of pluripotent stem cells, Examples of somatic cells include nerve cells, retinal epithelial cells, liver cells, beta cells, kidney cells, and dental pulp stem cells. Cells, mesenchymal stem cells, somatic stem cell progenitor cells, keratinocytes, hair papilla cells, oral epithelial cells, soft tissue These include bone cells, muscle cells, vascular cells, epithelial cells, cardiac muscle cells, blood cells, and immune cells. Examples of blood cells include erythroblasts, red blood cells, megakaryocytes, and platelets. Examples of leukocytes include monocytes, neutrophils, eosinophils, basophils, B cells, T cells, NK cells, and NK The induced stem cells can be differentiated into endodermal, mesodermal, or ectodermal lines. The stem cells may be allowed to form embryoid bodies, organoids, and spheres.

[0136] Examples of factors that induce cells into neural cells include the ASCL family and DLX family. -, MYT family, NeuroD family, SOX family, and NGN family Examples of the ASCL family include ASCL1 and DLX. Examples of the family include DLX2 and MYT. Examples of NGN family members include NGN2. Examples of cells include neurons, neural stem cells, and neural progenitor cells. These include inhibitory neurons, excitatory neurons, and dopamine-producing neurons, cerebral nerves, and interneurons. Alternatively, the nervous system cells may include motor neurons, oligodendrocytes, and optic nerves. The cells may be prodrug cells, astrocytes, oligodendrocytes, and the like.

[0137] Examples of factors that induce cells into cardiomyocytes include the GATA family and the MEF family. , TBX family, MYOCD family, MESP family, and miR-133 An example of the GATA family is GATA4A. An example of the MEF family is MEF2C. An example of the TBX family is An example of the MESP family is MESP1. do.

[0138] In the present disclosure, induction includes reprogramming, reprogramming, transformation, transdifferentiation (T transdifferentiation or lineage reprogramming), differentiation induction and cell fate change (Cell f This refers to programs such as program reprogramming.

[0139] The established pluripotent stem cells are then treated to induce them into somatic cells different from pluripotent stem cells. The derived cells may be cloned.

[0140] After gene editing of the established pluripotent stem cells, the gene-edited cells are cloned. You may also use the following.

[0141] In addition, the cells into which the reprogramming factors were introduced were not passaged at all, and the reprogramming factors were then added to the cells. The cells into which the gene has been introduced may be induced to become somatic cells other than pluripotent stem cells. The method for introducing a reprogramming factor is as described above. The cells transfected with the reprogramming factors were then cultured without any passage, and the differentiation-inducing factors were then added to the cells transfected with the reprogramming factors. The cells into which the reprogramming factors have been introduced are transformed into somatic cells that are different from pluripotent stem cells. Alternatively, the cells transfected with the reprogramming factors may be induced to undergo reprogramming without any passage. The cells into which the reprogramming factors have been introduced are then given hormones or chemicals to induce reprogramming. The cells into which the migrating factor has been introduced may be induced to become somatic cells other than pluripotent stem cells. In this case, cells transfected with reprogramming factors are not cloned but are induced to become somatic cells. The somatic cells to be induced are as described above. [Example]

[0142] (Example 1, Comparative Example 1) The dish coated with laminin 511 was used for inducing pluripotent stem cells. In addition, human peripheral blood mononuclear cells were suspended in blood medium, and the number of mononuclear cells was counted using a hemocytometer. The number of mononuclear cells in the blood medium was then adjusted. The cells were cultured in two dimensions on a dish for pluripotent stem cell induction for 1 to 7 days.

[0143] Two-dimensionally cultured mononuclear cells were incubated with SeV(PM)hKOS / TS12ΔF and Se V(HNL)hC-Myc / TS15ΔF was added at an MOI of 5 to induce pluripotent stem cells. The cell induction dish was placed in an incubator at 34°C and the cells were cultured. Two days after infection, the blood medium was replaced with iPS cell medium. The medium was changed every two days using the same medium. The temperature was gradually increased to 37°C and then 38°C. Geta.

[0144] Eight days after infection, stem cell-like cell clusters were formed. On the day 1, almost all cells were TRA1-60 positive and showed an iPS cell-like morphology. 14 days after infection, Triple Select, a cell detachment agent, was added to the dish. After that, the cell-containing solution was aspirated and incubated at 37°C for 5 minutes. After that, iPS cell medium was added and the cells were incubated for 10 minutes. The cell culture medium was collected in a 15 mL tube. The cell number was measured using a hemocytometer. Adjust the concentration of the solution so that the concentration is 0.25 x 10 4 cells / cm 2 The cells are In Example 1, the cells were seeded on a well plate and subcultured for the first time. Detach all cells from the plate and transfer the detached and mixed cells to the next well without discrimination. In contrast, in Comparative Example 1, colonies were picked at the first subculture. In both Example 1 and Comparative Example 1, 11 or more cells were obtained during the subculture. The cells were seeded so that the cells were not in contact with each other.

[0145] Next, the well dish was placed in a 37°C incubator and the cells were cultured in two dimensions. After the cells began to divide, the culture temperature was raised to 38°C. In both cases, all cells were harvested and collected when they reached 60% to 80% confluence. At least a portion of the collected and mixed cells was seeded in a medium and subcultured. Even when the concentration is 0.25 × 10 4 cells / cm 2 Plate the cells into wells as follows: Again, no more than 11 cells were in contact with each other.

[0146] As shown in Figure 1, cells that had been passaged only once were stained with anti-Sendai virus antibodies. The Sendai virus remaining in the cells of Example 1 was evaluated using a flow cytometer. As shown in Figure 2, the Sendai virus in the cells was eliminated by PCR. However, no Sendai virus was detected remaining in the cells of Example 1. When cells were seeded at a high density where 11 or more cells adhered to each other, the cells The virus remained. Immunostained photographs of the TRA1-60 positive cells obtained are shown in Figure 3.

[0147] In addition, the number of colonies formed 5 days after the disappearance of the intracellular Sendai virus was counted. Furthermore, the number of colonies was divided by the number of cells seeded to calculate the clonal efficiency. The results of three tests are shown in Figure 4. The first subculture was performed using mTeSR Plus medium. At some point, all the cells were collected, and some of the collected mixed cells were seeded in a medium and passaged. In all cases, the clonal efficiency was approximately 5% to 8%, with little variation. Colonies were picked and cloned at the first passage using mTeSR Plus. When using a clonal efficiencies, the clonal efficiency is sometimes less than 1% and sometimes about 6%. The clonal efficiency varied. At the 3rd passage, all cells were collected and a portion of the collected mixed cells was seeded in the medium. When subcultured, the clonal efficiency was approximately 10% to 15%, with little variation. The colonies were picked and cloned at the first subculture using StemFit medium. When the clonal efficiency was increased, it was sometimes less than 1% and sometimes about 16%. There were times when the clonal efficiency was inconsistent.

[0148] Therefore, all cells transfected with reprogramming factors were collected at the first passage. At least a portion of the collected and mixed cells is seeded in a medium and passaged to obtain a clone. It was shown that local efficiency increased and became stable.

[0149] (Example 2, Comparative Example 2) The iPS cell-like cells obtained by subculture in the same manner as in Example 1 and Comparative Example 1 were detached and dissociated. Next, about 1 × 10 5 The cells were transferred to STEM-CELLBANKER (registered trademark, The frozen cells were then thawed and stored at approximately 1 × 10 4 Individual cells The cells were seeded in wells and cultured to grow. The number of cells and colonies was counted 7 days after seeding. The results of three tests are shown in Figures 5 and 6.

[0150] As shown in Figure 5, all cells were collected at the first passage, and the collected mixed cells were When a portion of the cells was seeded in a medium and subcultured, the number of cells cultured for 7 days after freezing and thawing increased by approximately 10x 10 4 From about 15 x 10 4 On the other hand, during the first passage, The colony-picked and cloned cells were cultured for 7 days after freezing and thawing. is about 0.4 × 10 4 From about 15 x 10 4 There was a large variation. All cells transfected with reprogramming factors at the first passage were collected and mixed. At least a portion of the cells that have been mixed together are seeded in a medium and subcultured, thereby preventing the freezing and thawing of the cells. It was shown that the cell proliferation rate was high and stable.

[0151] As shown in Figure 6, all cells were collected at the first passage, and the collected mixed cells were When at least a portion of the cells was seeded and subcultured, the cells cultured for 7 days after freezing and thawing The number of colonies ranged from about 500 to about 800, with little variation. The cells cloned by colony picking during subculture were cultured for 7 days after freezing and thawing. The number of cell colonies obtained varied widely, ranging from about 30 to about 600. At the first passage, all cells that had been transfected with reprogramming factors were collected and mixed. At least a portion of the cells that have been mixed together are seeded in a medium and subcultured, thereby forming a cell that can be frozen and thawed. It was shown that the colony formation rate of the cells was high and stable.

[0152] (Example 3, Comparative Example 3) iPS cell-like cells obtained by subculture in the same manner as in Example 1 and Comparative Example 1 were detached and .5×10 5 The cells were suspended in a gel medium, cultured in three dimensions, and clumps were formed on the cells. The number of clumps and cells was counted 13 days after the cells were seeded on the gel medium. The cells obtained in Example 1 were tested twice, and the cells obtained in Comparative Example 1 were tested four times. The results are shown in FIG.

[0153] At the first passage, all cells were collected and a portion of the collected mixed cells was seeded into the medium. When the cells were passaged, they were cultured in three dimensions and formed clumps of around 3,000 cells. In addition, all cells were collected at the first passage and mixed. When some of the cells that had been mixed together were seeded in a medium and subcultured, the cells were cultured in three dimensions for approximately 200 0×10 2The number of cells was small, and the variability between tests was small. Colony-picked and cloned cells formed approximately 7,000 clumps. However, there were also tests in which the bacteria died and hardly formed any clumps, and there was variability between tests. In addition, the cells that were cloned by colony picking during the first passage were , and three-dimensionally cultured to approximately 5000 × 10 2 There were tests where the number of bacteria was reduced to 0, but they died out and the number was reduced to almost 0. There were some trials where the results were different, and there was a lot of variation between trials.

[0154] Therefore, all cells transfected with reprogramming factors were collected at the first passage. At least a portion of the collected mixed cells is seeded in a medium and passaged, thereby The proliferation rate and clump formation ability of the cells in the three-dimensional culture after the treatment are high and stable. It was shown that:

[0155] (Example 4, Comparative Example 4) The iPS cell-like cells obtained by subculture in the same manner as in Example 1 and Comparative Example 1 were detached and then transplanted into the heart. Cardiomyocyte Differentiation Kit (PSC) iPS cell-like cells were transformed into cardiomyocytes using the iPS Cell Transformation Kit (Gibco, registered trademark). It was differentiated.

[0156] Specifically, the bottom surface was treated with a basement membrane matrix (Corning Matrigel, registered trademark). Approximately 2 × 10 iPS cell-like cells were added to a 12-well plate. 4 From about 6 x 10 4 individual sowing The cells were cultured using mTeSR1 medium. Days later, Cardiomyocyte Differentiation Medium The medium was changed in A. Two days later, Cardiomyocyte Differentiation The medium was replaced with Cardiomyosis Medium B. Two days later, The medium was replaced with cyte Maintenance Medium. When necessary, replace the medium with Cardiomyocyte Maintenance Medium. The cells were cultured for 22 days after seeding the iPS cell-like cells while carrying out the above steps.

[0157] As a result, as shown in Figure 8, before differentiation into cardiomyocytes, reprogramming factors were After the introduction of β-glucan, all of the cells were collected at the first passage. Cells that were seeded and passaged in culture showed beating on day 22 in all tests. Before differentiation into muscle cells, reprogramming factors were introduced and the cells were cultured for the first time. Colony-picked and cloned cells were only viable by day 22 in less than half of the studies. showed pulsation.

[0158] In addition, the positive rate of cardiac troponin T (cTnT), a marker of cardiomyocytes, was measured by FACS. As shown in Figure 9, before differentiation into cardiomyocytes, reprogramming After the introduction of the factor, all of the cells were collected at the first passage. The cells that were seeded in the medium and passaged showed a stable cTnT positive rate of approximately 20%. On the other hand, after the introduction of reprogramming factors before differentiation into cardiomyocytes, The cells cloned by colony picking at the first passage showed a high cTnT positivity rate. The variation was large, ranging from approximately 1% to approximately 37%.

[0159] Therefore, all cells transfected with reprogramming factors were collected at the first passage. At least a portion of the collected mixed cells is seeded in a medium and passaged to establish stem cells. By doing so, the differentiation ability into somatic cells such as cardiomyocytes is enhanced and stabilized. It was shown that

[0160] (Example 5, Comparative Example 5) iPS cell-like cells obtained by subculture in the same manner as in Example 1 and Comparative Example 1 were detached and .5×10 5 The cells were placed in a cell culture plate with a round bottom suitable for cell cluster formation. The cells were inoculated onto a tray (Kuraray, RB 500 400 NA 6) and cultured.

[0161] The culture medium contains TGF-β1 activin receptor-like kinase (ALK)-4, -5, - 7 selective inhibitor (500 nmol / L, A-83-01, Stemgent) and BM Inhibitor of membrane permeability of P Type I receptor (ALK2, ALK3) (100 nm 8GMK medium (8% K, 0.5% CO₂O, 0.5% mol / L, LDN193189, Stemgent) was added. nockOut Serum Replacement (Life Technolo gies), 1% non-essential amino acids (NEAA, Life Technologies), 1% sodium pyruvate (Sigma), 100 nmol / L 2-mercaptoethanol In the presence of the above inhibitors, It is known that pluripotent stem cells cultured in vitro can be induced to differentiate into neural progenitor cells.

[0162] The medium was changed 5, 8, and 11 days after seeding the cells. After 14 days, cell masses were formed. As shown in Figure 10, the number of reprogenitor cells was counted before they were induced to differentiate into neural progenitor cells. After transfection with the graminizing factor, all cells were collected at the first passage and mixed. At least a portion of the cells that have been cultured are seeded in a medium and the cells are passaged at a density of about 25 to about 55 On the other hand, differentiation into neural progenitor cells was induced. Before the reprogramming factors were introduced, colonies were picked at the first passage. The cloned cells formed clusters of approximately 10 to 80 cells, with a large variation. It was.

[0163] In addition, the cells after 14 days were collected in a tube and centrifuged, and then the cells were treated with a cell dissociation agent (TrypLE Disaggregate cell clumps into single cells using ThermoFisher Select (registered trademark). After counting the number of cells, an antibody that detects polysialylated molecules of neural cell adhesion molecule (N-CAM) was used. Using the PSA-NCAM antibody, cells were immunostained and analyzed by flow cytometry. The positive rate of SA-NCAM was analyzed. As a result, as shown in Figure 10, differentiation into neural progenitor cells was confirmed. Before induction of reprogramming, all cells were transfected at the first passage after the introduction of reprogramming factors. and recovering the mixed cells, and then seeding at least a portion of the recovered mixed cells in a medium to obtain passaged cells. The PSA-NCAM positivity rate was approximately 25% to 30%, with little variation. On the other hand, before differentiation into neural progenitor cells, reprogramming factors were introduced and then the cells were reprogrammed once. The colony picked and cloned cells at the first passage were positive for PSA-NCAM. The rates varied widely, ranging from approximately 5% to approximately 15%.

[0164] Therefore, at the first passage, all cells transfected with reprogramming factors were collected. At least a portion of the collected mixed cells is seeded in a medium and passaged to establish stem cells. This increases the ability to induce differentiation into somatic cells such as neurons, and also increases the stability of the differentiation. It was shown that this is the case.

[0165] (Example 6, Comparative Example 6) The iPS cell-like cells obtained by subculture in the same manner as in Example 1 and Comparative Example 1 were detached and 1 x 10 cells in a non-adherent dish 5 The medium was supplemented with bFGF. The medium used was a human ES cell medium containing no ES cells. The medium was changed every two days. After 3 days, the formed embryoid bodies (EBs) were replated onto gelatin-coated 6-well plates. After that, the medium was changed every two days, and trypsin treatment was performed on the 24th day after reseeding. The collected cells were fixed with 4% paraformaldehyde and stained with SOX1 antibody, The cells were stained with OTC2 antibody, HAND1 antibody, and SOX17 antibody, respectively. The stained cells were analyzed using a flow cytometer. is a marker for ectoderm, HAND1 is a marker for mesoderm, and SOX17 is a marker for endoderm. is a marker.

[0166] As a result, as shown in Figures 11 and 12, after the introduction of the reprogramming factors, At the 3rd passage, all cells were collected and a portion of the collected mixed cells was seeded in the medium. The passaged cells showed little variation in the positive rate of each marker. After the introduction of the factor, colonies were picked and cloned at the first passage. The positive rate of each marker varied widely. All cells that have been transfected with programming factors are collected, and the remaining cells are removed. By seeding some of them in culture and subculturing them, they can be differentiated into endoderm, mesoderm, and ectoderm. It was shown that the differentiation potential of the cells was increased and stabilized.

[0167] Reference Example 1 Two-dimensionally cultured fibroblasts were incubated with SeV(PM)hKOS / TS12ΔF and Se V18+hKLF4 / TSΔF and SeV(HNL)hC-Myc / TS15ΔF. Add the cells so that the MOI is 5, and incubate the dish for pluripotent stem cell induction at 34°C. Two days after infection, the blood medium was transferred to the iPS cell culture medium. The medium was then replaced with iPS cell medium every two days. By day 14 after the transfection, the culture temperature was gradually increased to 37°C and then 38°C.

[0168] Ten days after infection, stem cell-like cell clusters were formed. On day 4, almost all cells were TRA1-60 positive and showed iPS cell-like morphology. 14 days after infection, Triple Select, a cell detachment agent, was added to the dish. The solution containing the added cells was incubated at 37°C for 5 to 10 minutes. Culture medium was added, and the iPS cell culture medium containing the cells was collected in a 15 mL tube. Measure the cell number using PBS and adjust the concentration of the cell-containing solution so that the concentration is 0.25 x 10. 4 cel ls / cm 2 The cells were seeded on a well plate as follows and the first passage was carried out. When the cells were mixed, no more than 11 cells came into contact with each other. After the cells began to divide, the culture temperature was increased to 38°C. Thereafter, the cells were passaged every time they reached 60% to 80% confluence. At the second and subsequent passages, the concentration was 0.25 × 10 4 cells / cm 2 The cells were then placed in a well. The cells were seeded onto a plate. Again, no more than 11 cells were allowed to come into contact with each other.

[0169] As shown in Figure 13, cells that had been passaged only once were stained with anti-Sendai virus antibody. The remaining Sendai virus in the cells was evaluated using a flow cytometer. The Sendai virus in the cells had almost completely disappeared. Shown in 14.

[0170] Reference Example 2 Two-dimensionally cultured mononuclear cells were incubated with SeV(PM)hKOS / TS12ΔF and Se V18+hKLF4 / TSΔF and SeV(HNL)hC-Myc / TS15ΔF. Add the cells so that the MOI is 5, and incubate the dish for pluripotent stem cell induction at 37°C. Two days after infection, the blood medium was transferred to the iPS cell culture medium. The medium was then replaced with iPS cell medium every two days.

[0171] Eight days after infection, stem cell-like cell clusters were formed. After 3 days, Triple Select, a cell detachment agent, was added to the dish, and the dish was left to stand at room temperature for 1 minute. Aspirate the cell-containing solution and incubate the cell-containing solution at 37°C for 5 to 10 minutes. Then, iPS cell culture medium was added, and the iPS cell culture medium containing the cells was placed in a 15 mL tube. The concentration of the solution containing the cells was adjusted to a concentration such that 11 or more cells adhered to each other. Degrees are 0.25 x 10 4 cells / cm 2 Cells were seeded in well plates so that they were taller The well dish was then placed in a 37°C incubator. After the cells began to divide, the culture temperature was raised to 38°C. After that, the cells were passaged every time they reached 60% to 80% confluence. From the 1st to the 5th passage, the concentration was 0.25 × 10 4 ce lls / cm 2 Cells were seeded in well plates so that the cells were taller. has a concentration of 0.25×10 4 cells / cm 2 Cells were seeded onto well plates as follows: In this case, no more than 11 cells were in contact with each other.

[0172] As shown in Figure 15, cells were stained with anti-Sendai virus antibody, and the remaining When the Sendai virus was evaluated using a flow cytometer, up until the sixth passage, Sendai virus remained in the cells, but at a concentration of 0.25 × 10 4 cells / cm 2 After the sixth passage, the cells were seeded on the well plate so that the following conditions were met. The Ndai virus disappeared quickly.

[0173] Reference Example 3 Sendai virus vector kit, CytoTune-iPS2.0 (registered trademark, CytoTune-iPS2.0 was prepared using the reprogramming factor Temperature-sensitive chromosomes carrying the KLF4 gene, OCT3 / 4 gene, and SOX2 gene as The Sendai virus vector SeV(PM)hKOS / TS12ΔF and the reprogrammed SeV, a temperature-sensitive Sendai virus vector carrying KLF4 as a binding factor 18+hKLF4 / TSΔF and temperature carrying c-MYC as a reprogramming factor and the sensitive Sendai virus vector SeV(HNL)hC-Myc / TS15ΔF. , including.

[0174] Mononuclear cells were infected with SeV(PM)hKOS / TS12ΔF and SeV18+hKLF4 / TSΔF and SeV(HNL)hC-Myc / TS15ΔF at an MOI of 5. The dish for pluripotent stem cell induction was placed in a 37°C incubator to allow the cells to grow. Two days after infection, the blood medium was replaced with iPS cell medium (mTeSR P lus (STEMCELL Technologies) or StemFit (Ajinomoto) The medium was then replaced with iPS cell medium every two days.

[0175] Eight days after infection, stem cell-like cell clusters were formed. On the day 1, almost all cells were TRA1-60 positive and showed an iPS cell-like morphology. 14 days after infection, Triple Select, a cell detachment agent, was added to the dish. After that, the cell-containing solution was aspirated and incubated at 37°C for 5 minutes. After that, iPS cell medium was added and the cells were incubated for 10 minutes. The cell culture medium was collected in a 15 mL tube. The cell number was measured using a hemocytometer. Adjust the concentration of the solution so that the concentration is 0.25 x 10 4cells / cm 2 The cells are The cells were seeded on a well plate and the first passage was performed. At this time, the cells were allowed to stand for 11 or more times without contact with each other. It was.

[0176] Next, the well dish was placed in a 37°C incubator and the cells were cultured in two dimensions. Thereafter, the cells were passaged every time they reached 60% to 80% confluence. At the second and subsequent passages, the concentration was 0.25 × 10 4 cells / cm 2 The cells were then placed in a well. The cells were seeded onto a plate. Again, no more than 11 cells were allowed to come into contact with each other.

[0177] As shown in Figure 16, cells were stained with anti-Sendai virus antibody, and the remaining antibodies in the cells were When Sendai virus was evaluated using a flow cytometer, intracellular serine serotonin levels were significantly increased at the eighth passage. The Ndai virus has almost disappeared.

[0178] Reference Example 4 Two-dimensionally cultured mononuclear cells were incubated with SeV(PM)hKOS / TS12ΔF and Se V(HNL)hC-Myc / TS15ΔF was added at an MOI of 5 to induce pluripotent stem cells. The cell induction dish was placed in an incubator at 34°C and the cells were cultured. Two days after infection, the blood medium was replaced with iPS cell medium. The medium was replaced with new medium every two days, and the temperature was raised to 38°C.

[0179] Eight days after infection, stem cell-like cell clusters were formed. On the day 1, almost all cells were TRA1-60 positive and showed an iPS cell-like morphology. 15 days after infection, Triple Select, a cell detachment agent, was added to the dish. After that, the cell-containing solution was aspirated and incubated at 37°C for 5 minutes. After that, iPS cell medium was added and the cells were incubated for 10 minutes. The cell culture medium was collected in a 15 mL tube. The cell number was measured using a hemocytometer. Adjust the concentration of the solution so that the concentration is 0.25 x 10 4 cells / cm 2 The cells are The cells were seeded on a well plate and the first passage was performed. At this time, the cells were allowed to stand for 11 or more times without contact with each other. Next, the well dish was placed in an incubator at 38°C to grow the cells in two dimensions. The cells were then passaged every time they reached 60% to 80% confluence. From the second passage onwards, the concentration was 0.25 × 10 4 cells / cm 2 The cells are as follows: The cells were seeded onto a well plate. In this case, no more than 11 cells were allowed to come into contact with each other.

[0180] Using anti-Sendai virus antibody, cells were cultured 15 days after infection before subculture. The cells were stained and the residual Sendai virus in the cells was evaluated using a flow cytometer. The results are shown in Figure 17. Also, a photograph of the cells 15 days after infection is shown in Figure 18. The cells were stained with Sendai virus antibody after one passage to detect the residual Sendai virus in the cells. The results of evaluating the virus using a flow cytometer are shown in Figure 19. After the first passage, the Sendai virus in the cells had almost disappeared. Shown in Figure 20.

[0181] Reference Example 5 SeV(PM)hKO cells were cultured in three-dimensional culture in polymer-containing blood medium. S / TS12ΔF, SeV18+hKLF4 / TSΔF, and SeV(HNL)hC-M yc / TS15ΔF and α-glucan were added at an MOI of 5, and the cells were cultured in a dish for pluripotent stem cell induction. The tube was placed in an incubator at 37°C and the cells were cultured. After that, the polymer-containing blood medium was replaced with a polymer-containing iPS cell medium. The medium was replaced with iPS cell medium containing the ATP every two days.

[0182] 14 days after infection, stem cell-like cell clusters were formed. On day 4, almost all cells became TRA1-60 positive. When some of the 60-positive cells were seeded in a culture vessel and cultured in two dimensions, iPS cell-like colonies were formed. In addition, cell aggregates were collected using a mesh, and the collected cells were treated with a cell detachment agent. Add Triple Select, and let stand at room temperature for 5 minutes. Then aspirate the cell-containing solution and The solution containing the cells was incubated at 37°C for 5 to 10 minutes. The iPS cell medium containing the cells was collected in a 15 mL tube. Measure the cell count and adjust the concentration of the cell-containing solution so that the concentration is 0.25 x 10 4 cells / cm 2 The cells were seeded on a well plate as follows and the first passage was carried out. The well dish was then incubated at 37°C. The cells were then placed in a 2D culture tube and cultured in 2D culture until the cells reached 60% to 80% confluence. The cells were passaged every time the number of cells reached 10. From the second passage onwards, the concentration was kept at 0.25 × 10 4 cel ls / cm 2 The cells were seeded on a well plate as follows: The cells did not come into contact with each other. The temperature was raised to 38°C during the process.

[0183] Cells that had been passaged twice were stained with anti-Sendai virus antibody to detect the residual Sendai virus in the cells. When the divirus was evaluated using a flow cytometer, as shown in Figure 21, intracellular The Sendai virus was almost completely eradicated. Figure 22 shows a photograph of the cells after two passages.

[0184] Reference Example 6 Two-dimensionally cultured fibroblasts were incubated with SeV(PM)hKOS / TS12ΔF and Se V(HNL)hC-Myc / TS15ΔF was added at an MOI of 5 to induce pluripotent stem cells. The cell induction dish was placed in an incubator at 34°C and the cells were cultured. Two days after infection, the blood medium was replaced with iPS cell medium. The medium was replaced with new medium every two days.

[0185] Eight days after infection, stem cell-like cell clusters were formed. On the day 1, almost all cells were TRA1-60 positive and showed an iPS cell-like morphology. 14 days after infection, Triple Select, a cell detachment agent, was added to the dish. After that, the cell-containing solution was aspirated and incubated at 37°C for 5 minutes. After that, iPS cell medium was added and the cells were incubated for 10 minutes. The cell culture medium was collected in a 15 mL tube. The cell number was measured using a hemocytometer. Adjust the concentration of the solution so that the concentration is 0.25 x 10 4 cells / cm 2 The cells are The cells were seeded on a well plate and the first passage was performed. At this time, the cells were allowed to stand for 11 or more times without contact with each other. The well dish was then placed in a 37°C incubator to allow the cells to grow in a secondary culture. After that, the cells were passaged every time they reached 60% to 80% confluence. The concentration was 0.25 × 10 4 cells / cm 2 The following details are required: The cells were seeded in well plates, and again, no more than 11 cells were allowed to come into contact with each other.

[0186] As shown in Figure 23, cells were stained with anti-Sendai virus antibody, and the remaining antibodies in the cells were Sendai virus was evaluated by flow cytometry and after the first passage, intracellular As shown in Figure 24, the Sendai virus remaining in the cells was also eliminated by PCR. No Sendai virus was detected. Shown in Figure 25.

[0187] (Example 7, Comparative Example 7) DMEM containing 10% FBS was used as the fibroblast medium. Fibroblasts were suspended in a fibroblast medium to obtain a fibroblast suspension.

[0188] For each well of a 6-well dish, add 1.5 mL of PBS and 4.8 μL of silkworm-derived Next, a mixture of laminin (iMatrix-511 silk, Nippi) was added, and the cells were incubated at 37°C. The dish was placed in an incubator for 1 hour. Then, PBS and laminar The minin mixture was removed from the wells, and 1.5 mL of fibroblast suspension was added to each well. The number of fibroblasts in one well is 0.5 x 10 5 to 2.0 x 10 5 There were 100. Thereafter, the fibroblasts were cultured in an incubator at 37°C for one day.

[0189] The medium was then replaced with stem cell induction medium, and the volume of the replaced medium was also 1.5 mL.

[0190] Prepare tubes A and B. Add 125 μL of OCT4 to 125 μL of PBS in tube A. A mixture of RNA, SOX2 mRNA, KLF4 mRNA, and c-MYC mRNA (100ng / μL) was added in an amount of 0.1μL to 100μL. In tube B, add 0.1 μL to 100 μL of PBS. Next, mix the solution in tube A with the solution in tube B. The mixture was then left at room temperature for 10 minutes, and the entire mixture was added to the medium in one well. Afterwards, place the dish in a 37°C incubator for one day to allow the RNA to be transfected into the cells. After that, RNA transfection was repeated 11 times using the same procedure. .

[0191] The day after the 11th RNA transfection, adjust the concentration of the solution containing the cells. is 0.25 x 10 4 cells / cm 2 The cells were plated on laminin-coated wafers as follows: In Example 7, the cells were seeded on a well plate for the first passage. Collect all the cells that have detached from the plate and at least some of the collected mixed cells. In contrast, in Comparative Example 7, the cells of the first subculture were seeded in the next well plate without distinction. In both Example 7 and Comparative Example 7, the colonies were picked and cloned. When seeding, the cells were seeded so that no more than 11 cells came into contact with each other. The tube was placed in an incubator at 37°C and the cells were cultured in two dimensions. Cells were passaged only once when they reached 80% to 80% confluence. Even when the concentration is 0.25 × 10 4 cells / cm 2 Plate the cells into wells as follows: At this time, no more than 11 cells were in contact with each other.

[0192] As shown in Figure 26, the reprogramming factors were almost completely removed from the cells within one day of the first passage. The reprogramming factors were almost completely lost from the cells by the second day after the first passage. On the 10th day after infection, almost all cells became TRA1-60 positive. The cells exhibited a PS cell-like morphology. Immunostained photographs of TRA1-60 positive cells are shown in Figure 27.

[0193] The stem cells established by the method according to Example 7 were used to generate clonal efficiencies similar to those in Example 1. The stem cells established by the method according to Example 7 were similar to those in Example 2, and the variation in the number of stem cells was small. Similarly, the proliferation rate and colony formation ability were stable. As in Example 3, there was little variation in the number of clumps and cells after three-dimensional culture. The stem cells established by the method according to Example 7 were induced to differentiate into cardiomyocytes in the same manner as in Example 4. The stem cells established by the method according to Example 7 were able to induce neural The stem cells established by the method according to Example 7 were stable in their ability to induce differentiation into progenitor cells. As in Example 6, the differentiation potential into endoderm, mesoderm, and ectoderm was stable.

[0194] The stem cells established by the method of Comparative Example 7 showed clonal efficiency similar to that of Comparative Example 1. The stem cells established by the method of Comparative Example 7 were similar to those of Comparative Example 2, and the variation in the number of stem cells was large. Similarly, there was a large variation in the proliferation rate and colony formation ability. As in Comparative Example 3, the number of clumps and cells after three-dimensional culture varied greatly. The stem cells established by the method according to Comparative Example 7 were able to differentiate into cardiomyocytes, as in Comparative Example 4. The stem cells established by the method according to Comparative Example 7 had the same differentiation induction ability as those of Comparative Example 5. Similarly, the ability to induce differentiation into neural progenitor cells was not stable. The established stem cells, as in Comparative Example 6, had stable differentiation potential into endoderm, mesoderm, and ectoderm. It wasn't there.

[0195] Example 8 Reprogramming factors were introduced into mononuclear cells in the same manner as in Example 1. On the 14th day after the initial incubation, almost all cells became TRA1-60 positive and showed an iPS cell-like morphology. After that, the cells were cultured in Ngn at an MOI of 20 before passage. Sendai virus capable of expressing 2-Puro mRNA was used to induce iPS cell-like cells. Two days after infection with Sendai virus, the medium in the wells was changed to 2 μg / mL of The medium was replaced with neural induction medium (N3 medium) containing puromycin at a concentration of 1000 mg / mL to kill uninfected cells. N3 medium was prepared by adding 10 mL of B27 and 5 mL of N to 500 mL of DMEMF12. 2 and 1.6 mL of insulin with a concentration of 6.25 mg / mL.

[0196] A micrograph of the cells on day 14 after infection with Sendai virus is shown in Figure 28. Thus, it was morphologically confirmed that the cells were induced to become neural cells after infection with Sendai virus. It was recognized.

[0197] Example 9 Reprogramming factors were introduced into mononuclear cells in the same manner as in Example 1. On the 14th day after the initial incubation, almost all cells became TRA1-60 positive and showed an iPS cell-like morphology. Subsequently, iPS cell-like cells were isolated from immunodeficient mice without any subculture. After a few weeks, the teratomas were extracted from the mice, and the cells were then transplanted into the testes of the mice. Histological sections were prepared from the tissue, stained with hematoxylin and eosin (HE), and observed under a microscope. The results are shown in Figure 29. In the tissue sections, secretory tissue-like structures, neural tube-like structures, intestinal structures, and Cartilage and bone-like structures were observed.

[0198] Example 10 The same method as in Example 1 was used except that chimpanzee-derived fibroblasts were used. As a result, iPS cell-like cells were obtained as shown in Figure 30. Colony formation was observed. The cells were maintained in culture and immunostained with an antibody against Oct3 / 4. As shown in Figure 31(a), the cells were positive for Oct3 / 4. The cells were immunostained with an antibody against Nanog, and the results shown in Figure 31(b) were obtained. As shown in Figure 32, the cells were positive for Nanog. -1-60 was confirmed positive.

[0199] The established chimpanzee-derived stem cells were induced to differentiate into cardiomyocytes. A photograph of this is shown in Figure 33.

[0200] Furthermore, the established chimpanzee-derived stem cells were induced to differentiate into neural cells. A photograph of muscle cells is shown in Figure 34. Neurons were Munch13-positive and vGlut-positive. It was confirmed that this is the case.

[0201] Example 11 300 mL of urine was collected from a healthy subject and dispensed into six 50 mL Falcon tubes. The tube was centrifuged at 400 G for 5 minutes. After centrifugation, the supernatant was removed from the tube and 30 mL PBS was added to the tube and centrifuged at 400G for 5 minutes. Remove the supernatant from the tube, add 30 mL of primary culture medium, and mix at 400 G for 5 minutes. The tube was centrifuged. The primary medium was DMEM / Ham's F12 (Gibco, 11320-033) with fetal bovine serum (Gibco, 10437028, final concentration 10%) ), SingleQuots Kit CC-4127 REGM(Lonza, 100 0 dose), and Antibiotic-Antimycotic (Gibco, 15 After centrifugation, remove the supernatant from the tube. Suspend the cells in 1 mL of primary medium and plate them on a gelatin-coated 24-well plate. The cells were seeded in one well of each plate and incubated in an incubator at 37°C. For two days after cell seeding, 300 μL of primary medium was added to the wells, and from the third day onwards, The medium was replaced with a cell culture medium. The epithelial cell culture medium was a kidney epithelial cell basal medium (Lonza ) and add SingleQuots Kit CC-4127 REGM (Lonza) The microscopic images of the cells after 6 days of expansion culture are shown in Figure 35. The cells were passaged for the first time on the day 1, and then expanded. The cells were cultured for the first time on the 7th day after the first passage. The cells were passaged a second time. A microscopic image of the cells 6 days after the second passage is shown in Figure 36.

[0202] Example 12 The dish coated with laminin 511 was used for inducing pluripotent stem cells. 1 × 10 cells derived from urine prepared in Example 11 were placed in a dish for pluripotent stem cell induction. 4 pieces From 1×10 5 The cells were seeded and incubated at 37°C. The medium used was a medium for epithelial cells. The next day, transfection reagent and RNA encoding green fluorescent protein (GFP) were added. The mixture was added to the medium, and the medium was replaced with the new medium. The mixture was then incubated at 37°C. The microscopic image of the cells taken the next day is shown in Figure 37. It was shown that urine-derived cells can be transfected.

[0203] Example 13 The dish coated with laminin 511 was used for inducing pluripotent stem cells. 1 × 10 cells derived from urine prepared in Example 11 were placed in a dish for pluripotent stem cell induction. 4 pieces From 1×10 5 The cells were seeded and incubated at 37°C. The medium used was a medium for epithelial cells. The next day, prepare tubes A and B. Add 125 μL of PBS to tube A and add MO. mRNA, SOX2 mRNA, KLF4 mRNA, c-MYC mRNA, and L Mixture of IN28 mRNA (100 ng / µL) from 0.1 µL to 10 2 μL was added. These RNAs were modified with pseudouridine (Ψ). The RNA was purified by HPLC and concentrated to single-stranded RNA. The ratio of absorbance at nm (A 260 / A 280 ) is 1.71 to 2.1, and the protein is substantially Furthermore, dot blot analysis using anti-double-stranded RNA antibody J2 confirmed that the RNA was not contaminated. The 125% dsRNA in tube B was removed by more than 90%. 0.1 μL to 100 μL of lipofection reagent was added to 1 μL of PBS. Mix the solution in tube A with the solution in tube B, leave the mixture at room temperature for 10 minutes, and then add the total amount The mixture was added to the transfection medium without using B18R etc., and the transfection The medium was replaced with the medium for transfection, and the cells were incubated at 37°C. The experiment was carried out once a day for 10 days. After cell seeding, observations were made on days 1, 5, 7, and 14. As shown in Figure 38, as the days progressed, the cell morphology changed to that of ES cells. It was recognized.

[0204] Example 14 As in Example 13, transfection was carried out on urine-derived cells for 10 days. From the 11th day after seeding, the cells were cultured in stem cell medium (StemFit, Ajinomoto) and then cultured for 14 days. All cells were removed from the dish, and a portion of the mixed cells was seeded in the culture medium. During subculture, the entire cells on the dish were collected without picking up the colonies. Collected, 1 x 10 2 Pieces to 1×10 5 Cells were seeded in a dish. Six days after passage A microscopic image of the eye cells is shown in Figure 39. ES cell-like cells were confirmed.

[0205] Example 15 As in Example 13, transfection was carried out on urine-derived cells for 10 days. From the 11th day after seeding, the cells were cultured in stem cell medium (StemFit, Ajinomoto) and then cultured for 14 days. The cells were removed from the dish and some were analyzed by flow cytometry. As shown in Figure 40(a), it was confirmed that the cells were TRA-1-60 positive. Cells were detached from the dish on day 4, passaged, and analyzed by flow cytometry 7 days later. As a result, it was confirmed that the cells were TRA-1-60 positive, as shown in Figure 40(b). .

[0206] Example 16 As in Example 13, transfection was carried out on urine-derived cells for 10 days. From the 11th day after seeding, the cells were cultured in stem cell medium (StemFit, Ajinomoto) and then cultured for 14 days. All cells were detached from the dish, and some of the detached mixed cells were seeded and subcultured. After subculturing, StemFit (registered trademark) was used as the medium. Seven days after subculturing, The cells were fixed and stained with anti-OCT3 / 4 and anti-NANOG antibodies. In addition, chemical staining of the nuclei was also performed using Hoechst (registered trademark). As a result, the nuclei were stained as shown in FIG. Thus, the expression of OCT3 / 4 and NANOG, which are specific markers of pluripotent stem cells, is important for the development of pluripotent stem cells. Therefore, pluripotent stem cells can be induced from urine-derived cells using RNA. It was shown that this is possible. Figure 41(d) shows the results of cells stained with anti-OCT3 / 4 antibody. Photographs of cells stained with anti-NANOG antibody and Hoechst (registered trademark) This is a composite photograph of cells stained with the marker.

[0207] Example 17 As in Example 13, transfection was carried out on urine-derived cells for 10 days. From the 11th day after seeding, the cells were cultured in stem cell medium (StemFit, Ajinomoto) and then cultured for 14 days. Use Triple Select to remove all cells from the dish, dislodge them, and mix them. A portion of the cells was seeded in a medium and passaged. After passage, the medium contained StemFit (registered trademark). Seven days after subculture, the cells were fixed and stained with anti-LIN28 antibody. The nuclei were also chemically stained with Hoechst (registered trademark). As shown in 2, the expression of LIN28, a specific marker of pluripotent stem cells, was confirmed in the cell nucleus. Therefore, it was demonstrated that pluripotent stem cells can be induced from urine-derived cells using RNA. It was shown that the cells stained with anti-LIN28 antibody were stained with anti-LIN28 antibody. 1 is a composite photograph of a cell stained with Hoechst® and a cell stained with Hoechst®.

Claims

1. Culturing the cells into which the reprogramming factors have been introduced; All cells into which the reprogramming factors have been introduced are collected, and at least Also, some of the cells are seeded on a medium and subcultured. A method for culturing cells into which a reprogramming factor has been introduced, comprising:

2. The method of claim 1 , wherein the cells are not cloned in the passaging.

3. The colonies formed by the cells into which the reprogramming factors have been introduced are separated from each other.

3. The method of claim 1 or 2, wherein the method does not include:

4. Cloning a single colony formed by the cells into which the reprogramming factors have been introduced. The method of any one of claims 1 to 3, wherein the method does not include:

5. All cells that have been introduced with the reprogramming factors and are attached to the culture vessel The method according to any one of claims 1 to 4, wherein the cells are harvested and at least a portion of the harvested cells is seeded in a medium.

1. The method according to claim 1.

6. The cells into which the reprogramming factors have been introduced are passaged without distinguishing between the gene expression states. The method according to any one of claims 1 to 5,

7. Distinguishing the cells into which the reprogramming factors have been introduced based on the degree of reprogramming. The method according to any one of claims 1 to 6, wherein the cells are passaged without any further passage.

8. The method according to claim 1, further comprising freezing the cells into which the reprogramming factors have been introduced.

8. The method according to any one of claims 7 to 7.

9. The cells into which the reprogramming factors have been introduced are selected from the endodermal, mesodermal, and ectodermal lineages.

9. The method according to claim 1 , further comprising differentiating the cells into at least one selected from the group consisting of:

2. The method according to claim 1.

10. Embryoid bodies, organoids, and spheres are produced from the cells into which the reprogramming factors have been introduced. Any of claims 1 to 8, further comprising forming at least one selected from 2. The method according to claim 1.

11. The cells into which the reprogramming factors have been introduced are induced to become somatic cells different from pluripotent stem cells.

11. The method of claim 1, further comprising:

12. After the treatment to induce the somatic cells, the treated cells are further cloned. The method of claim 11 , further comprising:

13. The method further comprises subjecting the cells into which the reprogramming factors have been introduced to a gene editing treatment.

13. The method according to any one of claims 1 to 12.

14. The cells introduced with the reprogramming factor are derived from blood cells or fibroblasts.

14. The method of any one of claims 1 to 13.

15. The cells to be transfected with the reprogramming factors are cells contained in urine.

14. The method according to any one of claims 13 to 13.

16. 1. The method according to claim 1, wherein the cells to be transfected with the reprogramming factors are bladder epithelial cells.

3. The method according to any one of claims 1 to 3.

17. and collecting the cells transfected with the reprogramming factors from urine. Item 17. The method according to item 15 or 16.

18. The cells into which the reprogramming factors have been introduced are derived from multiple humans or multiple non-human animals.

18. The method of any one of claims 1 to 17, wherein

19. The cells into which the reprogramming factors have been introduced are cultured in a closed culture vessel.

19. The method of any one of 1 to 18.

20. Culturing the cells into which the reprogramming factors have been introduced; The cells into which the reprogramming factors have been introduced are not subcultured, but are transformed into somatic cells different from pluripotent stem cells. Inducing cells A method for culturing cells into which a reprogramming factor has been introduced, comprising:

21. 21. The method of claim 20, further comprising freezing the cells into which the reprogramming factors have been introduced. The method described below.

22. The cells into which the reprogramming factors have been introduced are selected from the endodermal, mesodermal, and ectodermal lineages.

22. The method of claim 20 or 21, further comprising: directing the method.

23. The cells into which the reprogramming factors have been introduced are cultured in a closed culture vessel.

23. The method of any one of claims 20 to 22.

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