Method for producing primitive pluripotent stem cells

Abstract

The purpose of the present invention is to efficiently produce primitive pluripotent stem cells. The invention relates to a manufacturing method of primitive state pluripotent stem cells. The method comprises the following steps: (1) culturing primitive state pluripotent stem cells by using a primitive state pluripotent stem cell culture medium; and a step (2) for culturing the cells obtained in step (1) with a primitive state pluripotent stem cell culture medium, the method comprising at least one of the following steps: (i) a step for introducing miRNA selected from the group consisting of miRNA belonging to the let-7 miRNA family and a similar sequence thereof into the cells cultured in step (1) or (2), or expressing the miRNA in the cells, and a step (3) for culturing the cells obtained in step (1) with a primitive state pluripotent stem cell culture medium, and a step (4) for culturing the cells obtained in step (1) with a primitive state pluripotent stem cell culture medium. A step for introducing an activity-inhibiting substance of a protein of interest of the let-7 miRNA family into the cell cultured in step (1) or (2), or expressing the activity-inhibiting substance in the cell; and (ii) introducing an osteogenic protein into the cells cultured in step (1) or (2) to express the osteogenic protein in the cells, or adding the osteogenic protein to the culture medium in step (1) or (2). Or a step of adding an osteogenic protein activating substance to the culture medium in step (1) or (2).

Description

Technical Field

[0001] The present invention relates to a method for producing naive pluripotent stem cells, and more particularly to a method for producing naive pluripotent stem cells having transplantable safety from primed pluripotent stem cells at high efficiency. Background Art

[0002] Human iPS / ES cells are usually in a state similar to the post-implantation epiblast (epiblast) that is called the initial state. On the other hand, the original state iPS / ES cells are in a more undifferentiated state, for example, think that they can be differentiated into embryonic in vitro tissues such as placenta. Therefore, it is believed that the original state iPS / ES cells can be used for the research of people's early embryos or transplantation medical treatment. In addition, by using the original state iPS / ES cells, it is believed that the problem of differentiation resistance that can be expected to overcome the current initial state iPS cells has.

[0003] In the past, it is known that by expressing the genes KLF2 and NANOG in the naive state pluripotent stem cells, the naive state can be converted to the original state (for example, non-patent document 1). In addition, in recent years, a method for converting the naive state to the original state by adding a specified small molecule compound to the naive state pluripotent stem cells has been developed (for example, non-patent documents 2 and 3). The method of adding these small molecule compounds is also called the chemical reset method, which is advantageous in the following aspects: because there is no need to introduce genes and the risk of mutation to the genome is low, it is easy to apply to humans; because it only needs to be added to the culture medium, the method is simple, and because small molecule compounds are easy to produce in large quantities, the cost and availability are excellent. However, the methods disclosed in non-patent documents 2 and 3 require several passages when naive, so it takes a long time, and there is a problem of significantly poor efficiency of naive.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Takashima et al., Cell. 158, 1254-1269 (2014);

[0007] Non-patent document 2: Guo et al., Development. 144, 2748-2763 (2017);

[0008] Non-patent document 3: Szczerbinska et al., Stem Cell Rep. 13, 612-626 (2019). Summary of the invention

[0009] Problems to be solved by the invention

[0010] In the future, more efficient and safer methods of obtaining naive pluripotent stem cells will be needed not only for research but also for use in transplantation medicine.

[0011] Means for solving problems

[0012] As a result of in-depth research, the inventors have discovered a new method that can produce naive pluripotent stem cells without the need for gene introduction or the use of small molecule compounds such as valproic acid used in the chemical resetting method of the prior art. In addition, it was also discovered that the substances used in the new method promote the production of naive pluripotent stem cells based on the previous chemical resetting method. It was then thought that more naive pluripotent stem cell lines could be produced from naive pluripotent stem cells in a shorter period of time without using feeder cells, thereby completing the present invention.

[0013] That is, the present invention includes the following aspects.

[0014] [1] A method for producing naive pluripotent stem cells, the method comprising the following steps:

[0015] Step (1), culturing the primed pluripotent stem cells using a primed pluripotent stem cell culture medium; and

[0016] Step (2), culturing the cells obtained in the above step (1) using a native pluripotent stem cell culture medium,

[0017] And, at least one of the following steps is included:

[0018] (i) introducing a miRNA selected from the group consisting of miRNAs belonging to the let-7 miRNA family and sequences similar thereto into the cells cultured in the above step (1) or (2), or expressing the miRNA in the cells; or

[0019] A step of introducing an activity inhibitory substance of a target protein of the let-7 miRNA family into the cells cultured in the above step (1) or (2), or expressing the activity inhibitory substance in the cells; and

[0020] (ii) introducing an osteogenic protein into the cells cultured in step (1) or (2) above to express the osteogenic protein in the cells, or adding the osteogenic protein to the culture medium of step (1) or (2); or

[0021] A step of adding an osteogenic protein activating substance to the culture medium in the above step (1) or (2).

[0022] [2] The method described in [1] further comprises the step of adding a Wnt inhibitor to the culture medium of (1) or (2) before or simultaneously with the above step (ii).

[0023] [3] The method of [1], wherein (A) the naive pluripotent stem cell culture medium of step (2) above does not contain a histone deacetylase inhibitor, comprises step (i) above.

[0024] [4] The method described in [3] further comprises the above step (ii).

[0025] [5] The method of [1], wherein (B) the naive pluripotent stem cell culture medium of step (2) above contains a histone deacetylase inhibitor, including step (i) or step (ii) above.

[0026] [6] The method according to [1], wherein the histone deacetylase inhibitor comprises valproic acid.

[0027] [7][1] The method described in claim 1, wherein in the above step (i), the above miRNA belonging to the let-7 miRNA family and its similar sequences are selected from miR-98-5p, let-7a-5p, let-7b-5p, let-7c-5p, let-7e-5p, let-7f-5p, let-7g-5p, let-7i-5p or miRNA having a sequence identity of more than 80% therewith.

[0028] [8] The method described in [1], wherein in the above step (ii), the above osteogenic protein is selected from BMP2 or BMP4.

[0029] [9] The method of [5], wherein in the above step (ii), the above osteogenic protein activating substance is selected from SB-4 and isoliquiritigenin.

[0030]

[10] The method described in [1], wherein in the above step (i), the target protein of the above miRNA belonging to the let-7 miRNA family is NR6A1 and / or TRIM71.

[0031]

[11] The method described in [1] further comprises step (3): further culturing the cells obtained in the above step (2) using a naive pluripotent stem cell culture medium containing a tankyrase / Wnt / β-catenin inhibitor and a PKC inhibitor.

[0032]

[12] The method described in

[11] further comprises the step of subculturing the cells obtained in the above step (3) using the naive pluripotent stem cell culture medium described in

[11] .

[0033] Effects of the Invention

[0034] According to the manufacturing method of the naive state pluripotent stem cell involved in the present invention, it is not necessary to use feeder cells from mice, and the naive state pluripotent stem cell can be manufactured from the naive state pluripotent stem cell safely and in a short period of time. In addition, compared with the originalization efficiency (about 2%) based on the known chemical reset method by non-patent literature 2, 3, etc. in the past, the ratio of the naive state pluripotent stem cell can be sharply increased to up to 80% by the originalization treatment of about 2 weeks. In the manufacturing method involved in the present invention, it is also possible to avoid the step of necessarily introducing genes, and use RNA molecules or proteins, small molecule compounds to achieve the promotion of originalization. Thus, it can contribute to the research of promoting human early embryos. In addition, it is believed that the naive state pluripotent stem cell can be differentiated into various cells such as placenta, so compared with the current cell therapy using the naive state pluripotent stem cell, a wider range of cell therapy can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] [ Figure 1 ] Figure 1 This is a graph showing the results of flow cytometry analysis of translation activity (TE) obtained by introducing a miRNA-responsive RNA off switch into H9 EOS cells in the primed and naive states. The point indicated by the arrow is miR-98-5p, and the area surrounded by the dotted line is the area where the reduction in translation activity of the naive state is within 2^(1.1) times compared to the primed state.

[0036] [ Figure 2 ] Figure 2 This is a diagram for explaining the chemical reset culture scheme in the method for producing naive cells in the example. It shows a schematic diagram of H9EOS expressing GFP specifically for naive cells and the culture medium from day 0 to day 14.

[0037] [ Figure 3 ] Figure 3 Show in Figure 2 The reset efficiency of candidate miRNA mimics when the candidate miRNA mimics were transfected into primed H9EOS on day 1. The candidate miRNA mimics were based on Figure 1 miRNA mimics extracted in the form of miRNAs that show an activity difference of more than 2^(1.1) times between the initial state and the original state, ctrl_mimics are mimics that have not reacted, and chemical is the result of no miRNA added on the first day (only chemical reset).

[0038] [ Figure 4 ] Figure 4The resetting efficiency of the similar sequence of miR-98-5p when it was transfected into primed H9EOS is shown.

[0039] [ Figure 5 ] Figure 5 Show in Figure 2 On the first day, 13.6 ng / well of doxycycline-inducible piggyBac vector encoding 22 genes respectively and a total of 300 ng / well and 200 ng / well of transposase were transfected into H9EOS (all mix), the reset efficiency when tagBFP was transfected (tagBFP) and the reset efficiency when no transfection was performed (untransfected).

[0040] [ Figure 6 ] Figure 6 Show in Figure 2 The value obtained by normalizing the resetting efficiency in the case where one of the 22 vectors was removed and transfected into H9EOS on the first day of the experiment to the resetting efficiency in the case where no transfection was performed.

[0041] [ Figure 7 ] Figure 7 Show in Figure 2 The reset efficiency when the amount of the plasmid vector encoding hBMP2 introduced into H9EOS was changed on day 1.

[0042] [ Figure 8 ] Figure 8 The images are obtained under a phase contrast microscope on the 2nd, 5th, 7th and 14th days when the amount of the plasmid vector encoding hBMP2 introduced into H9EOS was 300 ng (upper section) and 15 ng (lower section).

[0043] [ Fig. 9A ] Fig. 9A The images are obtained under a phase contrast microscope on the first, second, third, and fifth days when the amount of the plasmid vector encoding hBMP2 introduced into H9EOS was 300 ng (upper panel) and 15 ng (lower panel).

[0044] [ Fig. 9B ] Fig. 9B The images are obtained under a phase contrast microscope on the 7th, 12th, and 14th days when the amount of the plasmid vector encoding hBMP2 introduced into H9EOS was 300 ng (upper section) and 15 ng (lower section).

[0045] [ Fig.10 ] Fig.10 Show in Figure 2The values ​​obtained by normalizing the H9EOS resetting efficiency when SB-4,4-hydroxychalcone (HyCh) and isoliquiritigenin (IsoLiq) were added to the culture medium at 1 μM, 5 μM or 10 μM on days 3-5 relative to the H9EOS resetting efficiency when these small molecules were not added (Chemical only).

[0046] [ Fig.11 ] Fig.11 Show in Figure 2 Phase contrast microscopy images (upper left) and fluorescence images (upper right) of H9EOS on day 14 when SB-4 was added to the culture medium once a day to reach 10 μM on days 3-5, and phase contrast microscopy images (lower left) and fluorescence images (lower right) of H9EOS on day 14 when SB-4 was not added (Chemical only).

[0047] [ Fig.12 ] Fig.12 This is a graph showing the difference in resetting efficiency caused by the substance transfected into H9EOS or the substance added to the culture medium and the timing of addition when valproic acid (VPA) as a histone deacetylase (HDAC) inhibitor was added to cRM-1 (VPA+) and when it was not added (VPA-). The timing of adding miR-98-5p or ctrl_mimetic was set to Figure 2 The timing of adding recombinant human osteogenic protein (BMP2 (rhBMP2) or BMP4 (rhBMP4)) to the culture medium is set as day 0 (0), day 1 (1), day 2 (2), day 3 (3) and / or day 4 (4). In the figure, the numbers 0 to 4 in brackets indicate that BMP is added multiple times at the timing (days) corresponding to the numbers.

[0048] [ Fig.13 ] Fig.13 Show in Figure 2 Phase contrast images (left) and fluorescence images (right) of H9EOS on day 14 when miR-98-5p or ctrl_mimic was transfected into H9EOS on day 1 and rhBMP4 was added to the culture medium on day 2.

[0049] [ Fig.14 ] Fig.14 The ratio of EGFP positive cells is expressed as Figure 2Figure 2 shows the resetting efficiency of H9EOS cells when siRNAs targeting endogenous proteins of the let-7 miRNA family were transfected into the cells on day 1. AllsiRNA represents the case of transfection of all 20 siRNAs, and miR-98-5p and ctrl_mimetic represent the resetting efficiency of H9EOS cells when siRNAs targeting endogenous proteins of the let-7 miRNA family were transfected into the cells on day 1. Figure 2 On day 1, miR-98-5p or ctrl_mimetic was transfected into H9EOS In the case of , chemical indicates the case of not transfecting siRNA or miR-98-5p, ctrl_mimetic. On the second day, the medium containing VPA was replaced and cultured in the presence of VPA.

[0050] [ Fig.15 ] Fig.15 The results of detailed siRNA studies on NR6A1 show that Figure 2 The reset efficiency when miR-98-5p, ctrl_mock, and siRNA_NR6A1 were transfected into H9EOS on day 1, and the reset efficiency when none of them were added (Chemical only). The experiment was performed using a 24-well plate with n=3.

[0051] [ Fig.16 ] Fig.16 The graph shows the resetting efficiency when iPS cells using AdiPS and each of H1, H9EOS, and H9 ES cells were cultured using cRM-1 containing VPA.

[0052] [ Fig.17 ] Fig.17 This is a graph showing the resetting efficiency when BMP and / or Wnt inhibitors are added. On the second day, cRM-1 containing VPA was replaced and cultured in the presence of VPA. The timing of adding BMP and / or Wnt inhibitors to the culture medium is set to day 0 (0), day 1 (1) and / or day 2 (2). In the figure, the numbers 0 to 2 in brackets are recorded together, which means that BMP and / or Wnt inhibitors are added to the culture medium multiple times at the timing (days) corresponding to the numbers.

[0053] [ Fig.18 ] Fig.18 This is a graph showing the resetting efficiency when miRNA, siRNA or BMP was added on day 1. On day 2, the cells were replaced with cRM-1 containing VPA and cultured in the presence of VPA.

[0054] [ Fig.19 ] Fig.19This is a graph showing the resetting efficiency when miRNA, siRNA and / or BMP were added on day 1. On day 2, the cells were replaced with cRM-1 without VPA, and culture was performed in the absence of VPA. DETAILED DESCRIPTION

[0055] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the embodiments described below.

[0056] According to one embodiment, the present invention relates to a method for producing naive pluripotent stem cells. The method for producing naive pluripotent stem cells comprises the following steps.

[0057] Step (1), culturing the primed pluripotent stem cells using a primed pluripotent stem cell culture medium; and

[0058] Step (2), culturing the cells obtained in the above step (1) using a naive pluripotent stem cell culture medium that does not contain a histone deacetylase (HDAC) inhibitor or contains an HDAC inhibitor.

[0059] The above step (1) or (2) further comprises at least one step selected from the following steps:

[0060] (i) introducing a miRNA selected from the group consisting of miRNAs belonging to the let-7 miRNA family and sequences similar thereto into the cells cultured in the above step (1) or (2), or expressing the miRNA in the cells; or

[0061] A step of introducing an activity inhibitory substance of a target protein of the let-7 miRNA family into the cells cultured in the above step (1) or (2), or expressing the activity inhibitory substance in the cells; and

[0062] (ii) introducing an osteogenic protein into the cells cultured in step (1) or (2) above to express the osteogenic protein in the cells, or adding the osteogenic protein to the culture medium of step (1) or (2); or

[0063] A step of adding an osteogenic protein activating substance to the culture medium in the above step (1) or (2).

[0064] The primitive state pluripotent stem cells manufactured by this embodiment are pluripotent stem cells in an undifferentiated and stable state, also known as a basal state, also known as reset cells. Primitive state pluripotent stem cells have the ability to differentiate into various cells including placenta. The manufactured cells are primitive state pluripotent stem cells, which can be confirmed by markers of primitive state pluripotent stem cells. As markers, for example, SUSD2, Klf17, CD75, Nanog can be listed, but are not limited to these. In addition, it can also be confirmed by qPCR and immunostaining. As a result of implementing the steps of the present invention, the obtained pluripotent stem cell group sometimes also includes cells other than primitive state pluripotent stem cells.

[0065] Step (1)

[0066] The starting material of the manufacturing method of this embodiment can be a primate pluripotent stem cell. The primate pluripotent stem cell can especially be a primate pluripotent stem cell, which can include: humans, monkeys, orangutans, chimpanzees, etc., but are not limited to these. In order to produce cells for transplantation in regenerative medicine, human pluripotent stem cells can preferably be used. In order to produce original drugs or experimental cells, any primate pluripotent stem cells can be used. The primate pluripotent stem cells can be somatic cells from primates such as humans or cells from biological secretions. As somatic cells, in addition to ordinary somatic cells, cells that can be contained in the blood and cells that can be contained in the urine (cells from the renal tubules or urethra) can also be listed.

[0067] Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells ("GS cells"), embryonic germ cells ("EG cells"), artificial pluripotent stem (iPS) cells, etc. Artificial pluripotent stem (iPS) cells are preferred.

[0068] In the past, the primate pluripotent stem cells obtained by common manufacturing methods showed the initial state. The pluripotent stem cells used as the starting material are the initial state pluripotent stem cells, which can be confirmed by alkaline phosphatase staining or a marker of a common pluripotent stem cell. As markers, for example, Nanog, Sox2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, etc. can be listed, but are not limited to these. Even if the original state pluripotent stem cells are included in the group of the initial state pluripotent stem cells used, there is no problem in the implementation of the present invention.

[0069] The culture medium for the pluripotent stem cells in the initial state of step (1) can use a culture medium that maintains pluripotency and allows the pluripotent stem cells to grow. As such a culture medium for pluripotent stem cells, it can be a culture medium that further contains bFGF (basic fibroblast growth factor) relative to the basal culture medium. The basal culture medium refers to a culture medium commonly used for culturing animal cells, for example, BME culture medium, BGJb culture medium, CMRL 1066 culture medium, Glasgow's Minimal Essential Medium (GMEM), modified MEM Zin Option culture medium, IMDM culture medium, Medium 199 culture medium, Eagle MEM culture medium, αMEM culture medium, DMEM culture medium, F-12 culture medium, DMEM / F12 culture medium, IMDM / F12 culture medium, Ham culture medium, RPMI 1640 culture medium, Fischer's culture medium, Neurobasal culture medium and their mixed culture medium. For example, it is preferred to use a culture medium (Scientific Reports Vol. 4, Article number: 3594 (2014)) containing bFGF and 20 essential amino acids, 10 vitamins (L-ascorbic acid, cobalamin, biotin, folic acid, I-inositol, nicotinamide, D-calcium pantothenate, pyridoxine hydrochloride, riboflavin and thiamine hydrochloride), and 5 minerals (copper sulfate, iron sulfate, iron nitrate, zinc sulfate and sodium selenite) relative to the basal culture medium. In particular, in the case of manufacturing primitive pluripotent stem cells for human transplantation purposes, it is preferably free of feeder cells. This is to avoid the mixing of feeder cells from mice. As a commercially available pluripotent stem cell culture medium suitable for culture without feeding, StemFit (registered trademark) AK02N or AK03N (manufactured by Ajinomoto Co., Ltd.) can be used, but it is not limited to a specific culture medium.

[0070] In the culture medium of the induced pluripotent stem cells, a ROCK inhibitor may be added to inhibit cell death. The ROCK inhibitor is not particularly limited as long as it is a substance that inhibits the function of Rho-kinase (ROCK), and examples thereof include: Y-27632 (for example, refer to Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (for example, refer to Uenata et al., Nature 389: 990-994 (1997)), H-1152 (for example, refer to Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (for example, refer to Nakajima et al., Cancer Chemother Pharmacol. 52(4):319-324(2003)) and their derivatives, as well as antisense nucleic acids, RNA interference-inducing nucleic acids (eg, siRNA), dominant negative mutants of ROCK and their expression vectors. In addition, as ROCK inhibitors, other known small molecule compounds can also be used (for example, refer to U.S. Patent Application Publication No. 2005 / 0209261, No. 2005 / 0192304, No. 2004 / 0014755, No. 2004 / 0002508, No. 2004 / 0002507, No. 2003 / 0125344, No. 2003 / 0087919, International Publication No. 2003 / 062227, No. 2003 / 059913, No. 2003 / 062225, No. 2002 / 076976, No. 2004 / 039796). In the present invention, one or more ROCK inhibitors can be used. As a preferred ROCK inhibitor, Y-27632 can be listed. The concentration of the ROCK inhibitor can be appropriately selected by a person skilled in the art according to the ROCK inhibitor used. For example, when Y-27632 is used as the ROCK inhibitor, the concentration is 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 20 μM. It is preferred that after culturing for a certain period in a medium containing a ROCK inhibitor, the cells are cultured for a certain period in a medium not containing a ROCK inhibitor.

[0071] Step (1) can be performed for about 1 to 4 days, preferably about 1 to 3 days, and more preferably about 2 days. In the most preferred embodiment, the steps of culturing for about 1 day with a primed pluripotent stem cell medium supplemented with a ROCK inhibitor and culturing for about 1 day with a primed pluripotent stem cell medium not supplemented with a ROCK inhibitor are sequentially included. However, the step of culturing for about 1 day with a primed pluripotent stem cell medium not supplemented with a ROCK inhibitor can be omitted.

[0072] Step (2)

[0073] Next, the cells obtained in the above step (1) are cultured in a naive pluripotent stem cell culture medium that does not contain an HDAC inhibitor or contains an HDAC inhibitor. Hereinafter, the production method in which the cells are cultured in a naive pluripotent stem cell culture medium that does not contain an HDAC inhibitor in step (2) is described as method (A), and the production method in which the cells are cultured in a naive pluripotent stem cell culture medium that contains an HDAC inhibitor in step (2) is described as method (B).

[0074] In methods (A) and (B), the naive pluripotent stem cell culture medium used in step (2) is a concept that includes both the induction medium used when inducing naive pluripotent stem cells from initiated pluripotent stem cells and the maintenance medium for maintaining the induced cells. In this specification, the naive pluripotent stem cell culture medium containing an HDAC inhibitor is sometimes referred to as a "naive pluripotent stem cell induction medium", and the naive pluripotent stem cell culture medium without an HDAC inhibitor is sometimes referred to as a "naive pluripotent stem cell maintenance medium".

[0075] The naive pluripotent stem cell culture medium can use a culture medium that contains one or more compounds selected from LIF (Leukemia inhibitory factor), MEK inhibitor, GSK3 inhibitor, cAMP production promoter, TGF-β inhibitor and PKC inhibitor, and anchor polymerase / Wnt / β-catenin inhibitor relative to the basal culture medium. The naive culture medium particularly preferably contains LIF, and preferably further contains LIF and GSK3 inhibitor. LIF is preferably hLIF. In addition, the naive culture medium preferably does not substantially contain bFGF. The concentration of LIF in the culture medium can be 1ng / ml to 100ng / ml or 5ng / ml to 50ng / ml, for example, about 10ng / ml. In step (2), it is particularly preferred to use a culture medium that contains LIF and a MEK inhibitor relative to the basal culture medium.

[0076] The basal medium for the naive pluripotent stem cell culture medium is not particularly limited, and examples thereof include: NDiff 227 medium as a neural differentiation medium, or a medium obtained by adding any combination of non-essential amino acids (NEAA), L-glutamine, 2-mercaptoethanol, antibiotics (e.g., streptomycin, penicillin, puromycin, mitomycin), bovine serum albumin (BSA) and the like to N2B27 medium (a medium prepared by mixing N2 medium obtained by adding N2 supplement to DMEM / F12 medium and B27 medium obtained by adding B27 supplement to Neurobasal medium in a ratio of 1:1).

[0077] The MEK inhibitor is not particularly limited, and examples thereof include PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide; CAS registration number: 391210-10-9), U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene; CAS registration number: 109511-58-2), PD98059 (2-(2-amino-3-methoxy)-1-nitropropoxy-2-nitropropoxy ... phenyl)-4H-1-benzopyran-4-one; CAS registration number: 167869-21-8), PD184352 (2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; CAS registration number: 212631-79-3. Among them, PD0325901 is preferred. When PD0325901 is used as a MEK inhibitor, its concentration in the culture medium can be 50nM to 100μM or 100nM to 10μM, for example, about 1μM.

[0078] The GSK3 inhibitor is not particularly limited, and examples thereof include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CAS registration number: 252917-06-9), BIO (6-bromoindirubin-3'-oxime; CAS registration number: 667463-62-9), Kenpaullone (9-bromo-7,12-dihydroindole[3,2-d][1]benzazepine) and -6(5H)-one; CAS registration number: 142273-20-9), IM-16 (3-(4-fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS registration number: 1129669-05-1). Among them, CHIR99021 is preferred. When CHIR99021 is used as a GSK3 inhibitor, its concentration in the culture medium can be 50nM to 100μM or 100nM to 10μM, for example, about 1μM.

[0079] The cAMP production promoter is not particularly limited, and examples thereof include forskolin. When forskolin is used as the cAMP production promoter, its concentration in the culture medium may be 50 nM to 100 μM or 100 nM to 10 μM, for example, about 1 μM.

[0080] As a TGF-β inhibitor. There is no particular limitation, and examples thereof include: A83-01 (3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolyl)-1H-pyrazole-1-thiocarboxamide), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide). Among them, A83-01 is preferred. When A83-01 is used as a TGF-β inhibitor, its concentration in the culture medium may be 10 nM to 100 μM or 100 nM to 10 μM, for example, about 1 μM.

[0081] The PKC inhibitor is not particularly limited, and examples thereof include Go6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS registration number: 133053-19-7) and GF109203X (3-(1-(3-dimethylamino)propyl)-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS registration number: 133052-90-1). Among them, Go6983 is preferred. When Go6983 is used as a PKC inhibitor, its concentration in the culture medium may be 50 nM to 100 μM or 100 nM to 10 μM, for example, about 1 μM to 3 μM.

[0082] There are no particular limitations on the tankyrase / Wnt / β-catenin inhibitor, and examples thereof include XAV939 (2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4(3H)-one; CAS registration number: 284028-89-3), IWT-2, IWP-3, and IWP-4. When XAV939 is used as a tankyrase / Wnt / β-catenin inhibitor, its concentration in the culture medium may be 0.5 μM to 100 μM, for example, about 2 μM.

[0083] The naive pluripotent stem cell culture medium can be commercially available, for example, t2iLGo (N2B27 + PD0325901 (1 μM) + CHIR99021 (1 μM) + LIF + Go6983 (2-3 μM)), 5iLAF (N2B27 + PD0325901 (1 μM) + CHIR99021 (1 μM) + SB590885 (0.5 μM) + WH-4-023 (1 μM) + Y-27632 (10 μM) + LIF + Activin A) or tt2iLGo (N2B27 + PD0325901 (1 μM) + LIF + Go6983 (2 μM) + XAV939 (2 μM)).

[0084] It should be noted that the naive pluripotent stem cell medium and the primed pluripotent stem cell medium can be distinguished in that the primed pluripotent stem cell medium does not substantially contain LIF, and the naive pluripotent stem cell medium does not substantially contain bFGF.

[0085] In method (B), a naive pluripotent stem cell induction medium containing a histone deacetylase (HDAC) inhibitor is used. In this specification, an HDAC inhibitor refers to miRNA, siRNA, mRNA and / or small molecules that inhibit histone deacetylase or inhibit its gene. Specific examples of HDAC inhibitors include: valproic acid (VPA, alias: 2-propyl valeric acid), sodium butyrate, suberoylanilide hydroxamic acid, BRD4884 (N-(4-amino-4'-fluoro[1,1'-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide; CAS registration number: 1404559-91-6), BRD6688 (N-[2-amino-5-(4-pyridyl)phenyl]-1-pyrrolidinecarboxamide; CAS registration number: 1404562-17-9), but are not limited to these. When the HDAC inhibitor is a small molecule such as valproic acid, the concentration in the culture medium may be, for example, about 0.1 mM to 10 mM, preferably about 0.5 mM to 5 mM.

[0086] The culturing in step (2) using a naive pluripotent stem cell maintenance medium without or with an HDAC inhibitor can be performed for, for example, 1 to 5 days, and is particularly preferably performed for 3 days. Hereinafter, the method (A) of culturing using a naive pluripotent stem cell maintenance medium without an HDAC inhibitor and the method (B) of culturing using a naive pluripotent stem cell maintenance medium with an HDAC inhibitor are described in detail.

[0087] [(A) Production method without using an HDAC inhibitor]

[0088] The method (A) for producing naive pluripotent stem cells without including an HDAC inhibitor in the culture medium of (2) is described in more detail below. The method comprises the following steps.

[0089] Step (1), culturing primed pluripotent stem cells using a pluripotent stem cell culture medium; and

[0090] Step (2), culturing the cells obtained in the above step (1) using a naive pluripotent stem cell culture medium without an HDAC inhibitor, and comprising the following step (i).

[0091] (i) introducing a miRNA selected from the group consisting of miRNAs belonging to the let-7 miRNA family and sequences similar thereto into the cells cultured in the above step (1) or (2), or expressing the miRNA in the cells; or

[0092] A step of introducing a substance that inhibits the activity of a target protein of the let-7 miRNA family into the cells cultured in the above step (1) or (2), or expressing the substance in the cell.

[0093] In addition to step (i), the following step (ii) may be optionally included.

[0094] The method comprises (ii) introducing an osteogenic protein into the cells cultured in the above step (1) or (2) to express the osteogenic protein in the cells, or adding the osteogenic protein to the culture medium of (1) or (2); or

[0095] A step of adding an osteogenic protein activating substance to the culture medium in the above step (1) or (2).

[0096] Regarding steps (1) and (2), as described above, the naive pluripotent stem cell culture medium used in (2) does not need to contain an HDAC inhibitor such as VPA.

[0097] In step (i), the following steps are included: introducing a miRNA selected from the miRNA belonging to the let-7 miRNA family and its similar sequence into the cells cultured in step (1) or (2), or expressing the miRNA selected from the miRNA belonging to the let-7 miRNA family and its similar sequence in the cells cultured in step (1) or (2). In addition to or instead of the above steps, the following steps may be included: introducing an activity inhibitory substance of the target protein of the let-7 miRNA family into the cells cultured in step (1) or (2), or expressing the activity inhibitory substance of the target protein of the let-7 miRNA family in the cells cultured in step (1) or (2).

[0098] As miRNA belonging to the let-7miRNA family, there are listed: miR-98-5p, let-7a-5p, let-7b-5p, let-7c-5p, let-7e-5p, let-7f-5p, let-7g-5p, let-7i-5p. The sequences of these miRNAs are shown in Table 1 below. These similar sequences, especially sequences having a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more with these sequences, also maintain functional identity with the miRNA belonging to the let-7miRNA family, so they can be used. Among these, one type may be added, or two or more types may be added in combination. Hereinafter, in this specification, miRNA selected from miRNA belonging to the let-7miRNA family and its similar sequences are sometimes referred to as "reset miRNA". The specific sequence of the reset miRNA is shown in Table 1.

[0099] [Table 1]

[0100]

[0101] The activity inhibitory substance of the target protein of the let-7 miRNA family can be a substance that can inhibit the expression of the target protein of the let-7 miRNA family and / or reduce the activity of the target protein. As target proteins of the let-7 miRNA family, there are: NR6A1 (Nuclear Receptor Subfamily 6 Group A Member 1), TRIM71 (Tripartite Motif Containing 71, also known as LIN41), IGF2BP1 (Insulin-like growth factor 2mRNA-binding protein 1), VASH2 (Vasohibin-2), DPH3 (Diphthamide Biosynthesis 3). Among them, it is particularly preferred to inhibit the activity of NR6A1 and / or TRIM71.

[0102] There is no particular limitation on the active inhibitory substance, and examples include: substances that can inhibit the expression of proteins by knockdown methods, or substances that can change the genes encoding these proteins by knockout methods. As substances for knockdown methods, examples include: siRNA for the target protein of the cell or DNA constructs encoding shRNA. As DNA constructs encoding shRNA, plasmids encoding shRNA are preferably used, and plasmids encoding shRNA that can be induced by drug expression are preferably used. As substances for knockout methods, examples include: substances that knock out the target protein by genome editing technology of CRISPR / Cas9. In a preferred embodiment, the active inhibitory substance can be siRNA that can knock down NR6A1 and / or siRNA that can knock down TRIM71. The siRNA that can knock down NR6A1, for example, can be a combination of multiple siRNAs that can act on multiple sites of the NR6A1 gene, for example, it can be a combination of more than 4 siRNAs for the NR6A1 gene. As such siRNA, commercial products can be used, and siRNA can also be appropriately designed and synthesized by a person skilled in the art according to the sequence of the NR6A1 gene. The same is true for TRIM71.

[0103] Sometimes the previous reset miRNA, the siRNA that can knock them down, and the activity inhibitory substances of the target protein are collectively referred to as reset RNA.

[0104] Reset RNA can be artificially synthesized and introduced into cultured cells in the form of synthetic RNA molecules. As methods for introducing RNA molecules, examples include: lipid transfection, liposome method, electroporation, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method, etc., but are not limited to these. The advantages of introducing synthetic RNA molecules include: no need to insert into the genome, easy use of cells introduced with reset RNA for medical applications, etc.

[0105] Reset RNA can also be added to cultured cells in the form of DNA constructs such as vectors to biosynthesize reset RNA in cells. Expression vectors encoding sequences for resetting RNA can use commonly known expression vectors in the field, such as viral vectors, artificial chromosome vectors, plasmid vectors, expression systems using transposons (sometimes referred to as transposon vectors), etc. As viral vectors, examples include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai virus vectors, etc. As artificial chromosome vectors, for example, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc. can be listed. As plasmid vectors, all mammalian plasmids can be used, for example, episomal vectors (Episomal Vector). As transposon vectors, examples include expression vectors using piggyBac transposons, etc. The introduction method of DNA constructs can be roughly the same as the introduction method of RNA molecules.

[0106] The period of introducing the reset RNA into the cultured cells or expressing the reset RNA in the cultured cells can be substantially the same as the timing of the medium replacement of step (2) from 48 hours before the next step (2), preferably 48 hours before to 12 hours before (2). Therefore, in the case of adding RNA molecules composed of the reset RNA, the RNA molecules can be introduced into the culture medium from 48 hours before to 12 hours before the next step (2). In the case of adding a DNA construct encoding the reset RNA, the DNA construct can be added to the culture medium from 72 hours before to 24 hours before. In the case where the DNA construct is a drug-inducible shRNA, the DNA construct can also be introduced into the cells in advance, and in order to express miRNA, siRNA or shRNA in the cultured cells, the drug can also be added at the desired time point. It should be noted that in the case where step (1) does not include the step of culturing for about 1 day with a primed pluripotent stem cell culture medium without the addition of a ROCK inhibitor, it is preferred that the reset RNA be transfected into the cultured cells at the stage of inoculating the primed pluripotent stem cells in the primed pluripotent stem cell culture medium with the addition of a ROCK inhibitor.

[0107] In a preferred embodiment, in step (1), when the step of culturing for about 1 day with a pluripotent stem cell medium supplemented with a ROCK inhibitor (day 0) and the step of culturing for about 1 day with a pluripotent stem cell medium not supplemented with a ROCK inhibitor (day 1) are sequentially performed, the reset RNA is introduced into the cultured cells or expressed in the cultured cells from immediately after the replacement with the pluripotent stem cell medium not supplemented with a ROCK inhibitor to about 5 hours later. The amount of introduction in this case can be, for example, set to 8×10 4 The amount of each cell is 1pmol~10pmol.

[0108] In method (A), at least step (i) is performed to introduce at least any one of reset miRNA, siRNA or shRNA into cells, thereby producing naive pluripotent stem cells from primed pluripotent stem cells even without using an HDAC inhibitor.

[0109] The arbitrarily selected step, i.e., step (ii), includes the following steps: introducing osteogenic protein into the cells cultured in the above step (1) or (2), or expressing osteogenic protein in the cells; or, in the above step (1) or (2), adding osteogenic protein to the culture medium. In addition to or in place of the above steps, the step of adding an osteogenic protein activating substance to the culture medium in the above step (1) or (2) may be included.

[0110] Examples of bone morphogenetic proteins (BMPs) include BMP2, BMP4, or analogs or derivatives thereof, but are not limited thereto. Preferably, the BMP may be human BMP. The BMP added to the culture medium may be a recombinant protein.

[0111] Regarding the introduction method of BMP, in addition to the method of directly transfecting the synthetic protein molecule into the cell, it can also be listed as follows: the method of introducing the mRNA encoding BMP into the cell in the form of a synthetic RNA molecule, the method of introducing the DNA construct encoding BMP into the cell so that it is expressed in the cell, and the method of adding BMP to the culture medium. The method of directly transfecting the synthetic protein molecule into the cell can use any transfection reagent. In addition, regarding the introduction of the synthetic RNA molecule, the introduction of the DNA construct, it can be the same as the previous step (i).

[0112] The period for introducing BMP and expressing it, or adding it to the culture medium can be any period of step (1) or (2). In a certain embodiment, it can be set within 1 to 3 days from the start of culture, preferably within 24 hours from the start of culture in step (2), and more preferably, BMP is introduced and expressed, or added to the culture medium at the same time as step (2). The introduction period may vary depending on the form of the introduced substance, and preferably, within 24 hours from the start of culture in step (2), BMP exists in an active state in the culture medium or cultured cells. The introduction amount in this case can be set, for example, to 5ng / mL to 100ng / mL, preferably to 5ng / mL to 10ng / mL.

[0113] In another embodiment, the introduction of BMP can be set at any time in step (1) and within 24 hours from the start of culturing in step (2). In particular, when step (1) is performed within 2 days, it can be set at any time from just after the start of culturing in step (1) to 3 days. During this period, BMP can be introduced only once, or it can be introduced twice or more. In the case of multiple introductions, it can also be introduced in both steps (1) and step (2).

[0114] In step (1), when BMP is introduced and expressed or added, it is preferred to add a Wnt inhibitor to the culture medium in advance or simultaneously. The reason is to inhibit the differentiation induction of BMP. It should be noted that when BMP is introduced at least once during the culture of step (1) and BMP is introduced multiple times, it is sufficient to add the Wnt inhibitor before or at the same time as the initial introduction, and the Wnt inhibitor may or may not be added during the subsequent introduction. Specific examples of Wnt inhibitors can be selected from the same options as the Wnt inhibitors recorded as any component of the original pluripotent stem cell culture medium. In particular, IWT-2, IWP-3, IWP-4 or any combination thereof are preferred, but are not limited to these. The amount of Wnt inhibitor added can be set to 0.1μM to 100μM, preferably 0.25μM to 5μM. When the Wnt inhibitor is added multiple times, the amount added is also preferably a concentration within the above range.

[0115] Even when the culture medium did not contain an HDAC inhibitor, the addition of BMP increased the resetting efficiency by about 7 times compared to the case where no BMP was added.

[0116] In the step of adding a bone-forming protein (BMP) activating substance to the culture medium in step (1) or (2), an endogenous BMP of the cells or a substance that increases the activity of BMP added externally as described above is added to the culture medium. The BMP activating substance can typically be a small molecule compound. Preferred BMP activating substances include, but are not limited to, SB-4 (CAS registration number: 100874-08-6) and isoliquiritigenin (CAS registration number: 961-29-5).

[0117] The BMP activating substance can be directly added to the culture medium of step (1) or (2). The addition period can be arbitrary and can be the same as the introduction or addition period and frequency of BMP. In a certain embodiment, the addition period is preferably set to within 1 day to 3 days from the start of the culture in step (2), and sometimes it is further preferably set to 3 consecutive days from the start of step (2). In addition, when adding a BMP activating substance to the culture medium of step (1), it is preferred to add a Wnt inhibitor to the culture medium in advance or at the same time. The addition period and frequency of the Wnt inhibitor can be the same as the introduction and addition of BMP. In this case, the amount of BMP activating substance introduced can be set to 7μM to 50μM, for example. By implementing the step of adding a BMP activating substance, the efficiency of the production of naive pluripotent stem cells can be significantly improved, and the production of naive pluripotent stem cells can be increased within a short culture period compared to the past.

[0118] The step of introducing BMP or the step of adding BMP activating substances may be performed in either one or both steps. Step (ii) can also be regarded as a step of activating the BMP signal transduction pathway of cells. By implementing these steps, the BMP signal transduction pathway of cells can be activated and the resetting efficiency can be improved.

[0119] In method (A), it is not necessary to substantially include HDAC inhibitors, especially VPA, in the culture medium. By introducing reset RNA into the cells at a specified time in the culture step and arbitrarily and selectively introducing BMP or BMP activating substances, primitive state pluripotent stem cells can be produced. In particular, in an embodiment in which both reset RNA and BMP or BMP activating substances are introduced, the production of primitive state pluripotent stem cells can be increased by about 2 to 20 times in a short culture period compared to the previous culture using HDAC inhibitors represented by VPA without using reset RNA and BMP or BMP activating substances. In the past, when making primitive state pluripotent stem cells, it was believed that genes KLF2 and NANOG must be introduced or cultured in the presence of small molecule compounds such as VPA, i.e., HDAC inhibitors. According to method (A), without using HDAC inhibitors, there is an advantage that primitive state pluripotent stem cells can be produced more cheaply and efficiently.

[0120] [(B) Production method using HDAC inhibitor]

[0121] The method for producing naive pluripotent stem cells by containing an HDAC inhibitor in a culture medium (method (B)) is described in more detail below. The method comprises the following steps.

[0122] Step (1), culturing primed pluripotent stem cells using a pluripotent stem cell culture medium; and

[0123] Step (2), culturing the cells obtained in the above step (1) with a naive pluripotent stem cell culture medium containing an HDAC inhibitor, and comprising the following step (i) or (ii).

[0124] Step (i): introducing a miRNA selected from the miRNA belonging to the let-7 miRNA family and its similar sequence into the cells cultured in the above step (1) or (2), or expressing the miRNA in the cells; or

[0125] Introducing an activity inhibitory substance of a target protein of the let-7 miRNA family into the cells cultured in the above step (1) or (2), or expressing the activity inhibitory substance in the cells;

[0126] Step (ii): introducing osteogenic protein into the cells cultured in step (1) or (2) above to express the osteogenic protein in the cells, or adding the osteogenic protein to the culture medium of (1) or (2); or

[0127] An osteogenic protein activating substance is added to the culture medium in the above step (1) or (2).

[0128] Both steps (i) and (ii) may be optionally included.

[0129] Step (i) of method (B) can be carried out in the same manner as step (i) of method (A), and either or both of the miRNA resetting substance and the target protein activity inhibitory substance are introduced into the cell.

[0130] In method (B), even if the step of introducing the reset miRNA in step (i) is performed alone without performing step (ii), the efficiency of producing naive pluripotent stem cells can be significantly improved, and the amount of naive pluripotent stem cells produced can be increased by at least 2 to nearly 20 times in a short culture period compared with the past. In addition, even if the step of introducing an activity inhibitory substance of the target protein, such as siRNA, in step (i) is performed alone without performing step (ii), the efficiency of producing naive pluripotent stem cells can be significantly improved, and the amount of naive pluripotent stem cells produced can be increased in a short culture period compared with the past.

[0131] Step (ii) of method (B) can be implemented in the same manner as step (ii) of method (A), and any one or both of the steps of introducing BMP into cells or adding it to a culture medium or adding a BMP activating substance to the culture medium can be implemented. Even if the BMP introduction step of step (ii) is implemented alone without performing step (i), the efficiency of producing naive pluripotent stem cells can be significantly improved, and the amount of naive pluripotent stem cells produced can be increased by about 24 times within a short culture period compared to the conventional chemical resetting method using a culture medium containing VPA without using osteogenic protein. In addition, even if the BMP activating substance addition step of step (ii) is implemented alone without performing step (i), the efficiency of producing naive pluripotent stem cells can be significantly improved, and the amount of naive pluripotent stem cells produced can be increased by about 3 times within a short culture period compared to the conventional method.

[0132] The method (B) can be performed by combining steps (i) and (ii). In particular, by combining steps (i) and (ii), the production amount can be expected to increase by about 2 to 20 times compared to when these steps are not performed.

[0133] In both (A) the manufacturing method without using an HDAC inhibitor and (B) the manufacturing method using an HDAC inhibitor, after implementing step (1) or (2), the step (3) can be further optionally included: further culturing the cells obtained in step (2) with a naive pluripotent stem cell culture medium (naive pluripotent stem cell maintenance medium) that does not contain an HDAC inhibitor. The naive pluripotent stem cell maintenance medium used in step (3) preferably contains LIF, a MEK inhibitor, an end anchor polymerase / Wnt / β-catenin inhibitor, and a PKC inhibitor relative to the basal culture medium. More specifically, it preferably contains hLIF, PD0325901, XAV939, and Go6983. The naive pluripotent stem cell maintenance medium used in step (3) does not substantially contain an HDAC inhibitor such as VPA.

[0134] Step (3) can be performed for 8 to 130 days, and is preferably performed for 9 days. By performing step (3), primordialization is performed on the 5th day, and the formation of primordial-like cell masses is observed on the 14th day. If the culture is continued thereafter, the maturation of the primordial cells is considered to have progressed.

[0135] The total culture period of steps (1) and (2) and the optional step (3) is at least 14 days, and can be up to about 135 days, and most preferably about 25 to 30 days, for example 28 days.

[0136] The step of subculturing the cells obtained in step (2) or step (3) may be further included. In the case of subculturing, a naive pluripotent stem cell culture medium (naive pluripotent stem cell maintenance medium) that does not contain an HDAC inhibitor may be used to culture the cells for 1 to 10 to 20 subcultures or more. Thus, a cell population in which the content of naive pluripotent stem cells is increased to about 90% can be obtained.

[0137] Example

[0138] The present invention will be described in more detail below using examples. The following examples do not limit the present invention.

[0139] [Experimental methods]

[0140] [Activity assay based on miRNA-responsive disconnect switch library]

[0141] Preparation of miRNA-responsive switch libraries

[0142] The template of the library was prepared by PCR amplification using TOYOBO KOD Plus Neo polymerase using synthetic oligo DNA (KEC879) containing a T7 promoter, synthetic oligo DNA (723 species) containing a complementary sequence of miRNA, ORF containing TagBFP, and a reverse primer (KEC883) containing poly-T. The template was purified using SpeedBeads magnetic carboxylate-modified particles or MonarchDNA purification columns and MinElute reaction purification kits. The mRNA used as an internal standard (reference) was prepared by PCR amplification of synthetic oligo DNA (KEC001, KEC062, KEC063, KEC065) containing a sequence of UTR, ORF of hmAG1, and a reverse primer (KEC883) containing poly-T using TOYOBOKOD Plus Neo polymerase. Alternatively, a plasmid obtained by cloning UTR and hmAG1 into a pUC19 vector was prepared using a synthetic oligo DNA (YF0771) containing a T7 promoter and UTR and a reverse primer (KEC883) containing poly-T. The primers used in the experiment are shown in Table 2 below.

[0143] [Table 2]

[0144]

[0145] The synthesis of mRNA was performed using the MEGAscript T7 transcription kit (Catalog No. AMB13345) from Thermo Fisher Scientific. The transcription reaction used pseudouridine-5'-triphosphate (TriLink, N-1019-10, Ψ) and 5-methylcytidine-5'-triphosphate (TriLink, N-1014-10) to replace UTP and CTP, respectively. In addition, GTP was mixed with a CAP analog (TriLink, N-7003-10, ARCA) in a ratio of 1:4 to attach a Cap structure. After transcription, the template was degraded with TURBO DNase (Thermo Fisher Scientific, AM2238), and after dephosphorylation, the mRNA was purified using any of SpeedBeads magnetic carboxylate-modified particles, RNAClean XP (Bechman Coulter, A63987, and FavorPrep total RNA extraction columns (FAVORGEN, FARBC-C50).

[0146] Introduction and fluorescence assay of miRNA-responsive off-switch

[0147] A stable strain (H9EOS(NK)) was used in which GFP and doxycycline-inducible Klf2 and Nanog with EOS (early transposon promoter, Oct-4 (Pou5f1) and Sox2 enhancer) upstream were inserted into the genome of H9 cells. The H9EOS(NK) cells in the initial state and the original state were seeded in 96-well plates, and the next day, the miRNA-responsive disconnect switch and mRNA encoding hmAG1 fluorescent protein as an internal standard were co-introduced using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher Scientific, LMRNA015). On the second day of transfection, Accumax (Nacalai tesque, 17087-54) was added, cultured at 37°C, and cells were separated from the plate and analyzed by flow cytometry (LSR fortessa, BD). The value obtained by dividing the tagBFP fluorescence value of the switch by the internal standard hmAG1 was normalized by dividing the tagBFP fluorescence value of the normal tagBFP mRNA by the fluorescence value of the internal standard hmAG1 to quantify the activity of the miRNA in the cell. 1.1 The above miRNAs.

[0148] [Cell culture]

[0149] Each ES cell of H9EOS, H9EOS, H9, H1 and each iPS cell of AdiPS were cultured in AK02N (Ajinomoto, AK02N) under feeder-free conditions, and the medium was changed every few days for maintenance. For passaging, after washing once with PBS, Accutase (Fujifilm, 12679-54) was added, and the cells were cultured at 37°C for about 10 minutes, and then peeled off by pipetting. After centrifuging the cell suspension, the supernatant was removed, and the cells were resuspended in AK02N, and the cell number was measured using a Countess automatic cell counter, etc., and 16,000-30,000 cells / well were inoculated in AK02N medium containing Y-27632 (Nacalai tesque, 08945-84) and iMatrix-511silk (nippi, 387-10131) for passaging.

[0150] [Chemical Reset]

[0151] H9EOS and H9EOS (NK) cells were recovered using the same method as for passaging, and after measuring the cell number, they were diluted with AK02N medium (initial pluripotent stem cell medium) containing Y-27632 (Nacalai tesque, 08945-84) and iMatrix-511silk (nippi, 387-10131) and inoculated into multi-well plates (modified the method of G. Guo et al., 144, 2748-2763 (2017)). On the first day, the medium was replaced with AK02N. From the second to the fifth day, the cells were cultured with cRM-1 (NDiff 227 (Takara, Y40002) containing 1μM PD0325901, 10ng / mL hLIF, and 1mMVPA) or cRM-1 without VPA (naive pluripotent stem cell medium). Thereafter, the cells were cultured with cRM-2 (NDiff227 containing 1μM PD0325901, 10ng / mL hLIF, 2μM Go6983, and 2μM XAV939) until the 14th day. The reset efficiency was quantified by counting the number of cells using a flow cytometer, using cells expressing EGFP as the original cells. For an overview, see Figure 2 .

[0152] [Improving the efficiency of miRNA remodeling]

[0153] The mimics corresponding to each miRNA use mirVana miRNA mimics (Thermo Fisher Scientific). In addition, as controls, mirVana miRNA mimics and negative controls (Thermo Fisher Scientific, 4464058) are used. On any one day or combination of the 1st, 2nd, 3rd, and 4th days of the usual reset process, miRNA mimics are introduced using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, 13778075). Afterwards, culture is performed in the same manner as the conventional reset procedure, and EGFP-positive cells are quantified using a flow cytometer on the 14th day.

[0154] [scRNA-seq]

[0155] Single-cell RNA-seq was performed using the Chromium Next GEM single-cell 3' library and the Single Index Silica Bead Kit v3.1 (10×Genomics, 1000128) according to the attached protocol. H9EOS cells into which miRNA mimics (miR-98-5p or negative control) were introduced on day 1 were recovered on days 3, 8, and 14. After the cells were washed twice with PBS, Accumax was added and cultured for 15 minutes. The cells were then detached by pipetting and suspended in PBS (0.04% BSA). After centrifugation at 300×g for 5 minutes, the supernatant was removed and slowly suspended in PBS (0.04% BSA) and washed repeatedly 2-3 times. The cell number of the cell suspension was measured using Countess, and the target cells were diluted to recover 5,000 cells for scRNA-seq. For the library, the average size was determined using the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626) or QSep, and the concentration was determined using the Qubit dsDNAHS Assay Kit (Thermo Fisher Scientific, Q32851). For the library, NextSeq500 (Illumina) or NovaSeq (Illumina) was used for sequencing. Cell Ranger was used for analysis to screen for genes whose expression levels were specifically increased or decreased in cells introduced with miR-98-5p.

[0156] [Plasmid construction]

[0157] Total RNA of cells on day 3 after reset was recovered using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, AM1561). cDNA was prepared from total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-201). Genes with increased expression in miR-98-5p were amplified from cDNA by PCR using KOD Plus Net DNA polymerase and then cloned into a doxycycline-inducible piggyBac vector (pPB-TRE2-PshAI-HygR) using the InFusion Cloning Kit (takara, Z9650N). The sequence was confirmed using a sequencer, and the plasmid was purified using the PureYieldPlasmid Midiprep System (Promega, A2495). The following 22 forced expression vectors were prepared: pPB-TRE2-ANGPT1-HygR, pPB-TRE2-ANXA2-HygR, pPB-TRE2-BAMBI-HygR, pPB-TRE2-BMP2-HygR, pPB-TRE2-FTL-HygR, pPB-TRE2-GATA2-HygR, pPB-TRE2-HPGD-HygR, pPB-TRE2-ID1-HygR, pPB-TRE2-ID3-HygR, pPB-TRE2-LAYN-variant1-HygR, pPB-TRE2-PCAT1-HygR 4-HygR, pPB-TRE2-PCDH17-HygR, pPB-TRE2-SHISA9-HygR, pPB-TRE2-SLC6A8-HygR, pPB-TRE2-SLC7A3-HygR, pPB-TRE2-SOX2-OT-HygR, pP B-TRE2-SOX4-HygR, pPB-TRE2-SPP1-HygR, pPB-TRE2-TAGLN-HygR, pPB-TRE2-TFAP2A-HygR, pPB-TRE2-TWIST1-HygR, pPB-TRE2-VIM-HygR.

[0158] [Improving the efficiency of plasmid remodeling]

[0159] exist Figure 2On the first day of the reset protocol, 22 plasmids were removed from each of the 22 plasmids and mixed, and transfected with a plasmid expressing transposase (pCAG-PBase) using Lipofectamine Stem transfection reagent (ThermoFisher Scientific, STEM00003). Doxycycline was added to cRM-1 at a final concentration of 250 ng / mL on days 2-5 to induce gene expression. After that, the cells were cultured in the same manner as the conventional reset protocol, and EGFP-positive cells were quantified using flow cytometry on day 14.

[0160] [Resetting efficiency of small molecule BMP activators]

[0161] Towards Figure 2 SB-4 (Tocris, 6881 / 50), 4-hydroxychalcone (Fuji Film Wako Chemical, 320-75521) or isoliquiritigenin (Fluorochem, 462708) were added to cRM-1 containing VPA on days 2-5 of the resetting protocol. After that, the cells were cultured in the same manner as in the conventional resetting protocol, and EGFP-positive cells were quantified on day 14 using flow cytometry.

[0162] [Replacement efficiency of rhBMP4]

[0163] Towards Figure 2 On days 1 to 5 of the resetting protocol, 10-100 ng / mL of rhBMP4 (Reprocell, 314-BP-010) was added to any one or more cRM-1 containing or not containing VPA. After that, the cells were cultured in the same manner as the conventional resetting protocol, and EGFP-positive cells were quantified on day 14 using flow cytometry.

[0164] [Improving the efficiency of siRNA remodeling]

[0165] exist Figure 2On the first day of the reset protocol, siRNA of any one of NR6A1, TRIM71, LRIG1, DNMT3B, IGF2BP1, CACHD1, PCDH8, L1TD1, SFRP1, VASH2, LIN28B, USP44, MAPK10, DICER1, DPH3, PRTG, MAPK6, HMGA2, LINC01356 (n491112), and LINC01356 (n549768) (all purchased from Horizon Discovery / Dharmacon) was introduced using Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific, 13778075). Afterwards, the cells were cultured in the same manner as the conventional reset protocol, and EGFP-positive cells were quantified using flow cytometry on the 14th day.

[0166] [Procedure for SUSD2 staining]

[0167] Towards Figure 2 AccuMax was added to the cells on the 14th day of the reset protocol, and the cells were cultured at 37°C for about 10 to 15 minutes and then stripped. In order to inhibit nonspecific binding of the antibody, the cells were suspended in 1% BSA-PBS and blocked on ice for 30 minutes. The cells were mixed with the cell suspension to a final concentration of 1:500 for SUSD2 antibody, and the reaction was carried out on ice for 30 minutes. After washing with PBS, the cells were measured by flow cytometry.

[0168] [result]

[0169] Figure 1 The graph shows the results of introducing a miRNA-responsive RNA off switch into H9 EOS (NK2) cells in the primed and naive states and analyzing the translation activity (TE) by flow cytometry. The translation activity TE was determined by the following formula.

[0170] TE=(Switch tagBFP / hmAG1) / (Ctrl tagBFP / hmAG1)

[0171] Here, Switch tagBFP is the expression level of tagBFP from the miRNA-responsive RNA switching off switch encoding tagBFP, hmAG1 is the expression level of hmAG1 from mRNA encoding hmAG1 without the miRNA target site, and Ctrl tagBFP is the expression level of tagBFP from mRNA encoding tagBFP without the miRNA target site. The values ​​of Switch tagBFP / hmAG1 and Ctrl tagBFP / hmAG1 were measured by flow cytometry. Figure 1 As shown in Figure 2, in addition to the miRNAs known in previous reports (origin-related miRNAs), several novel miRNAs with high activity in protocells were discovered ( Figure 1 mRNA in the lower right region).

[0172] Figure 3 is Figure 2 On day 1, candidate miRNA mimics were transfected into primed H9EOS cells, and the resetting efficiency of candidate miRNA mimics in the presence of VPA in cRM-1 was shown by EGFP-positive cells. Figure 3 It was confirmed that among the candidate miRNA mimics, miR-98-5p had a significantly higher resetting efficiency. Figure 3 In the figure, chemical shows the resetting efficiency when the cells were cultured with cRM-1 containing VPA without transfection of miRNA mimics or ctrl_mimics.

[0173] Figure 4 Show in Figure 2 On the first day of transfection, the miR-98-5p analogous sequence was transfected into the primed H9EOS, and the reset efficiency of the analogous sequence in the presence of VPA in cRM-1 was compared. As analogous sequences, let-7a-5p, let-7b-5p, let-7c-5p, let-7e-5p, let-7f-5p, let-7g-5p, let-7i-5p, and miR-4500 were used. Figure 4 It was found that the sequence with 100% identity between the seed sequence and miR-98-5p showed the same high resetting efficiency as miR-98-5p. Figure 2 The resetting efficiency of the cells transfected on the second, third or fourth day of the experiment was not shown (not shown). The resetting efficiency of the cells transfected with the let-7 miRNA family at these times was lower than that of the cells transfected on the first day, but higher than that of the cells transfected without transfection with miRNA mimics or ctrl_mimics and cultured with cRM-1 containing VPA, i.e., chemical resetting efficiency.

[0174] Figure 5 The reset efficiency when all 22 genes are expressed in H9EOS, the reset efficiency when tagBFP is transfected, and the reset efficiency when no transfection is performed are shown. In either case, VPA acid is contained in cRM-1 and culture is performed. Even when the tagBFP expression plasmid is introduced to confirm the effect of transfection, a tendency of increased reset efficiency is seen compared to the case where no introduction is made, but no significant difference is obtained. On the other hand, when 22 genes are introduced, the reset efficiency increases. The reason why the ratio increases by introducing tagBFP alone is speculated as follows: due to the toxicity of transfection, the total number of cells decreases, while the original cells grow, and the denominator of [original cells / total cells] becomes smaller preferentially.

[0175] Figure 6 Show in Figure 2 The first day will start from Figure 5 The value obtained by normalizing the resetting efficiency in the case where one of the 22 vectors used in the experiment was removed and transfected into H9EOS and cRM-1 contained VPA to the resetting efficiency in the case where no transfection was performed. Figure 6 It was confirmed that when hBMP2 was removed, the resetting efficiency (standard value) was significantly reduced.

[0176] Figure 7 Show in Figure 2 The resetting efficiency when the amount of the plasmid vector encoding hBMP2 introduced into H9EOS was changed on the first day and cRM-1 contained VPA. Figure 8 Phase contrast images of the 2nd, 5th, 7th, and 14th days when the amount of plasmid vector encoding hBMP2 introduced into H9EOS was set to 300 ng (upper section) and 15 ng (lower section). The reset efficiency peaked when the amount of hBMP2 expression plasmid introduced was about 15 ng. In addition, under the condition of excess hBMP2, almost all cells died at the 14th day.

[0177] Fig. 9A , Fig. 9B The phase contrast images are taken on the 1st, 2nd, 3rd, 5th, 7th, 12th and 14th days when the amount of plasmid vector encoding hBMP2 introduced into H9EOS was 300 ng (upper section) and 15 ng (lower section). When the amount of hBMP2 introduced was 300 ng, almost all cells died on the 14th day, and almost no EGFP-positive cells were observed.

[0178] Fig.10 Show in Figure 2The values ​​obtained by normalizing the resetting efficiency of H9EOS in the case where SB-4, 4-hydroxychalcone (HyCh) and isoliquiritigenin (IsoLiq) were added to the culture medium at 1, 5 or 10 μM on days 3-5 and VPA was contained in cRM-1 to the resetting efficiency of H9EOS in the case where these small molecules were not added but the cRM-1 containing VPA was used for culture (Chemical only). In addition, Fig.11 Show in Figure 2 Phase contrast image (upper left) and fluorescence image (upper right) of H9EOS on day 14 when SB-4 was added to the culture medium at 10 μM on days 3-5, and phase contrast image (lower left) and fluorescence image (lower right) of H9EOS on day 14 when no small molecules such as SB-4 were added (Chemical only). Fig.10 , 11 It was confirmed that SB-4 and isoliquiritigenin are small molecule compounds that can activate BMP.

[0179] Fig.12 Show in Figure 2 In the protocol shown, the differences in resetting efficiency caused by the substances transfected into H9EOS or the substances added to the culture medium and the timing of addition when culturing with cRM-1 containing VPA (VPA+) or cRM-1 without VPA (VPA-) are shown. Chemical means resetting without adding miR-98-5p or ctrl_mimetic or BMP. Fig.12 In the culture using cRM-1 containing VPA, the addition of BMP2 or BMP4 on the second day significantly improved the resetting efficiency compared to chemical resetting. Under this condition, the resetting efficiency tended to be further improved by transfecting miR-98-5p on the first day. In addition, even in the culture using cRM-1 without VPA, the resetting efficiency was improved by transfecting miR-98-5p on the first day and adding BMP2 or BMP4 on the second day compared to chemical resetting, confirming the induction of protocells by chemical resetting without using HDAC inhibitors.

[0180] Fig.13 Shown in Figure 2 When miR-98-5p or ctrl_mimetic was transfected into H9EOS on day 1 and rhBMP4 or rhBMP2 was added to the culture medium on day 2, the resetting efficiency and phase contrast images (left) and fluorescence images (right) of H9EOS on day 14. Fig.12 , 13It was confirmed that the highest resetting efficiency was shown by setting the period of adding recombinant osteogenic protein to the culture medium to the time point when the culture was started with the original pluripotent stem cell culture medium. On the other hand, if rhBMP4 is added on day 0, the resetting efficiency is reduced. It is speculated that this is because if rhBMP4 is allowed to act on ordinary iPS cells, mesoderm induction begins, so before resetting by adding cRM-1 containing VPA, differentiation into mesoderm begins and pluripotency is lost. Once it begins to develop in the direction of differentiation, pluripotency has been lost, so it is believed that even if cRM-1 containing VPA is added, it will not be reset. rhBMP4 has two effects, promoting mesoderm differentiation and resetting. It is speculated that the presence or absence of VPA determines whether it will proceed in the direction of differentiation or dedifferentiation (resetting).

[0181] The percentage of EGFP-positive cells generated by knocking down a specific protein by siRNA and inhibiting the activity (expression) of the protein is shown in Fig.14 Mimic NC is a negative control mimic that is not related to any miRNA. When the activity was inhibited by knocking down NR6A1 and TRIM71, the reset efficiency was significantly improved compared to the case where the knockdown was not performed and the cells were cultured with cRM-1 containing VPA (chemical). In addition, knockdown of IGF2BP1, VASH2, and DPH3 showed a tendency to improve the reset efficiency compared to the control (ctrl_mimic). Fig.15 This is the result of a detailed study of NR6A1 using siRNA. Knockdown of NR6A1 using siRNA resulted in a higher resetting efficiency than chemical knockdown.

[0182] Fig.16This is a graph showing the resetting efficiency when iPS cells using AdiPS and ES cells of H1, H9EOS, and H9 were cultured using cRM-1 containing VPA. For mimics_ctrl and mimics-98-5p, control mimics or miR-98-5p mimics were transfected into each cell on day 1, cultured with cRM-1 containing VPA from day 2 to day 4, and cultured with cRM-2 from day 5 to day 14. For BMP-4, BMP4 was added to cRM-1 containing VPA on day 2, cultured until day 4, and cultured with cRM-2 from day 5 to day 14 without transfecting each cell with control mimics or miR-98-5p mimics. These cells were peeled off using Accumax, suspended in 1% BSA-PBS, blocked on ice for 30 minutes, mixed with the cell suspension to a final concentration of 1:500 for SUSD2 antibody solution, reacted on ice for 30 minutes, washed with PBS, and measured by flow cytometry. In any cell type, the proportion of SUSD2-positive cells tended to increase after treatment with miR-98-5p or BMP4.

[0183] Fig.17 This is a diagram showing the effect of BMP and Wnt inhibitors on the resetting efficiency when H9EOS cells are used and cultured with cRM-1 containing VPA. H9EOS cells were inoculated with AK02N (lock inhibitor, containing iMatrix-511) on day 0, and after the culture medium was replaced with AK02N on day 1, miR-98-5p was transfected with RNAiMax. On day 2, the medium was replaced with cRM-1 containing VPA, and on day 5, the medium was replaced with cRM-2. Culture with cRM-2 continued until day 14, and then GFP-positive cells were counted. BMP4 and / or IWP mix were added to the culture medium at any one or more time points on day 0, day 1, and day 2. IWP mix is ​​a cocktail of IWP-2, IWP-3, and IWP-4, and the amount added is set to 1 μM. In the figure, IWP+BMP4 refers to the simultaneous addition of IWP mix and BMP4 to the culture medium. By adding a Wnt inhibitor to the culture medium simultaneously with or before BMP4, the resetting efficiency can be maintained unchanged and the BMP4 addition period can be accelerated, so that even when BMP is added during the culture step using a primed pluripotent stem cell culture medium, a high resetting efficiency can be obtained.

[0184] Fig.18This is a graph showing the reset efficiency when H9EOS cells are used and cultured with cRM-1 containing VPA. H9EOS cells were seeded with AK02N (locked inhibitor, containing iMatrix-511) on day 0, and after the medium was replaced with AK02N on day 1, various siRNAs or miR-98-5p were transfected with RNAiMax. On day 2, it was replaced with cRM-1 containing VPA. The BMP4-treated sample was prepared by adding BMP4 to the cRM-1 medium on day 2. On day 5, the medium was replaced with cRM-2, and culture was continued with cRM-2 until day 14, after which GFP-positive cells were counted. Fig.18 In the above, all mix refers to a mixture of miR-98-5p, siNR6A1, and siTRIM71. Resetting miRNA, resetting siRNA, and BMP can significantly improve the resetting efficiency individually.

[0185] Fig.19 The graph shows the resetting efficiency when H9EOS cells were cultured with cRM-1 without VPA. Fig.18 The same procedure was followed. Fig.19 In the table, only Chemical indicates the result of culture without VPA and without any of reset miRNA, reset siRNA, or BMP, and all mix refers to a mixture of miR-98-5p, siNR6A1, and siTRIM71. Even when cultured in a medium without VPA, reset miRNA, reset siRNA, and / or BMP can improve the reset efficiency.

[0186] Experiments were performed to knock down NR6A1 or TRIM71 as targets of miR-98-5p, and the results showed that in the manufacture of naive pluripotent stem cells, knocking down NR6A1 would promote the induction of naive pluripotent stem cells from naive pluripotent stem cells, and knocking down TRIM71 would remove differentiated cells other than naive pluripotent stem cells (data not shown). In addition, experiments were performed to overexpress NR6A1, and the results confirmed that NR6A1 hindered the induction of naive pluripotent stem cells from naive pluripotent stem cells (data not shown). Moreover, experiments were performed to add BMP inhibitors, and the result was that the induction efficiency of naive pluripotent stem cells was reduced, thereby showing that BMP signals promote the induction of naive pluripotent stem cells (data not shown).

Claims

1. A method for producing naive pluripotent stem cells, the method comprising the following steps: Step (1), culturing the primed pluripotent stem cells using a primed pluripotent stem cell culture medium; as well as Step (2), culturing the cells obtained in the above step (1) using a native pluripotent stem cell culture medium, And, at least one of the following steps is included: (i) introducing into the cells cultured in the above step (1) or (2) a miRNA selected from miRNAs belonging to the let-7 miRNA family and sequences similar thereto, or expressing the miRNA in the cells; or A step of introducing an activity inhibitory substance of a target protein of the let-7 miRNA family into the cells cultured in the above step (1) or (2), or expressing the activity inhibitory substance in the cells; and (ii) a step of introducing an osteogenic protein into the cells cultured in step (1) or (2) above to express the osteogenic protein in the cells, or adding the osteogenic protein to the culture medium of (1) or (2); or A step of adding an osteogenic protein activating substance to the culture medium in the above step (1) or (2).

2. The method of claim 1, further comprising: A step of adding a Wnt inhibitor to the culture medium of (1) or (2) before or simultaneously with the above step (ii).

3. The method of claim 1, wherein: (A) The naive pluripotent stem cell culture medium of the above step (2) does not contain a histone deacetylase inhibitor, and includes the above step (i).

4. The method according to claim 3, further comprising the above step (ii).

5. The method of claim 1, wherein: (B) The naive pluripotent stem cell culture medium of the above step (2) contains a histone deacetylase inhibitor, including the above step (i) or step (ii).

6. The method of claim 1, wherein: The above-mentioned histone deacetylase inhibitors include valproic acid.

7. The method of claim 1, wherein: In the above step (i), the above miRNA belonging to the let-7 miRNA family and its similar sequences are selected from miR-98-5p, let-7a-5p, let-7b-5p, let-7c-5p, let-7e-5p, let-7f-5p, let-7g-5p, let-7i-5p or miRNA having a sequence identity of more than 80% therewith.

8. The method of claim 1, wherein: In the above step (ii), the above osteogenic protein is selected from BMP2 or BMP4.

9. The method of claim 5, wherein: In the above step (ii), the above osteogenic protein activating substance is selected from SB-4 and isoliquiritigenin.

10. The method of claim 1, wherein: In the above step (i), the target protein of the above miRNA belonging to the let-7 miRNA family is NR6A1 and / or TRIM71.

11. The method according to claim 1, further comprising step (3): further culturing the cells obtained in the above step (2) with a naive pluripotent stem cell culture medium containing a tankyrase / Wnt / β-catenin inhibitor and a PKC inhibitor.

12. The method of claim 11, further comprising: The step of subculturing the cells obtained in the above step (3) using the naive pluripotent stem cell culture medium according to claim 11.

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