Method for inducing primed pluripotent stem cells into naive pluripotent stem cells, method for producing naive pluripotent stem cells, kit for inducing naive pluripotent stem cells, and agent for inducing naive pluripotent stem cells

Abstract

Provided is a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, that includes a step in which primed pluripotent stem cells are cultured in a culture medium that includes an AMPK activator. Also provided is a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, that includes a step in which the primed pluripotent stem cell p38 MAPK pathway is activated. Also provided is a method for manufacturing naive pluripotent stem cells, which includes a step in which primed pluripotent stem cells are cultured in a culture medium that includes an AMPK activator. Also provided is a method for manufacturing naive pluripotent stem cells, that includes a step in which the primed pluripotent stem cell p38 MAPK pathway is activated. Also provided are a naive pluripotent stem cell induction kit for these methods and a naive pluripotent stem cell inducing agent.

Description

[Technical Field]

[0001] The present invention relates to a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, a method for producing naive pluripotent stem cells, a kit for inducing naive pluripotent stem cells, and an agent for inducing naive pluripotent stem cells. This application claims priority to U.S. Provisional Application No. 63 / 159,998, filed in the United States on March 12, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] Pluripotent stem cells, which can differentiate into any cell type in the body, can be broadly divided into two types: naive and primed. Naive pluripotent stem cells are thought to possess undifferentiated properties equivalent to those of the inner cell mass in blastocysts. After implantation into an embryo, naive pluripotent stem cells can contribute to chimera formation and differentiate into germ cell lineages. Primed pluripotent stem cells, on the other hand, are thought to correspond to the epiblast, a more advanced stage of development from the blastocyst, after implantation. Primed pluripotent stem cells retain pluripotency but have lost the ability to form chimeras and differentiate into germ cell lineages. Mouse pluripotent stem cells exist in both naive and primed forms. Primate pluripotent stem cells, on the other hand, are generally primed. Human naive pluripotent stem cells are expected to be used in new regenerative medicine applications, such as the generation of human organs in vivo, by utilizing their chimera-forming ability. Human naive pluripotent stem cells are also expected to be applied to various new research fields, such as the analysis of human developmental processes and the induction of germ cell lineages.

[0003] Several substances have been reported to be involved in the conversion of primed pluripotent stem cells to naive pluripotent stem cells (e.g., Non-Patent Document 1). It has also been reported that activation of adenosine monophosphate-activated protein kinase (AMPK) contributes to the maintenance of naive pluripotent stem cells (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Guo G et al., Epigenetic resetting of human pluripotency. Development. 2017 Aug 1;144(15):2748-2763. [Non-patent document 2] Liu Y, Yamashita JK., AMPK activators contribute to maintain naive pluripotency in mouse embryonic stem cells. Biochem Biophys Res Commun, 509:24-31, 2019. Summary of the Invention [Problem to be solved by the invention]

[0005] Although several factors that contribute to the conversion of primed pluripotent stem cells to naive pluripotent stem cells have been reported, the exact signals that are important remain unclear. Therefore, there is a need to develop technologies that enable the conversion of primed pluripotent stem cells to naive pluripotent stem cells.

[0006] Therefore, an objective of the present invention is to provide a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, a method for producing naive pluripotent stem cells from primed pluripotent stem cells, as well as a kit for inducing naive pluripotent stem cells and a naive pluripotent stem cell inducer that can be used in these methods. [Means for solving the problem]

[0007] The present disclosure includes the following aspects. [1] A method for inducing primed pluripotent stem cells into naive pluripotent stem cells, comprising the step of culturing primed pluripotent stem cells in a medium containing an AMPK activator. [2] The method according to [1], wherein the medium further contains LIF. [3] The method according to [1] or [2], wherein the AMPK activator is one or more substances selected from the group consisting of AICAR, A769662, and metformin. [4] The method according to any one of [1] to [3], wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells. [5] The method according to [4], wherein the primed pluripotent stem cells are human-derived primed iPS cells. [6] A method for inducing primed pluripotent stem cells into naive pluripotent stem cells, comprising the step of activating p38 MAPK in primed pluripotent stem cells. [7] The method according to [6], wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells. [8] The method according to [7], wherein the primed pluripotent stem cells are human-derived primed iPS cells. [9] A method for producing naive pluripotent stem cells, comprising the step of culturing primed pluripotent stem cells in a medium containing an AMPK activator.

[10] The method of producing according to [9], wherein the medium further contains LIF.

[11] The manufacturing method according to [9] or

[10] , wherein the AMPK activator is one or more substances selected from the group consisting of AICAR, A769662, and metformin.

[12] The production method according to any one of [9] to

[11] , wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

[13] The method of production described in

[12] , wherein the primed pluripotent stem cells are human-derived primed iPS cells.

[14] A method for producing naive pluripotent stem cells, comprising the step of activating p38 MAPK in primed pluripotent stem cells.

[15] The production method described in

[14] , wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

[16] The method of production described in

[15] , wherein the primed pluripotent stem cells are human-derived primed iPS cells.

[17] A kit for inducing naive pluripotent stem cells, which comprises an AMPK activator and is used to induce primed pluripotent stem cells into naive pluripotent stem cells.

[18] The kit for inducing naive pluripotent stem cells according to

[17] , further comprising LIF.

[19] The kit for inducing naive pluripotent stem cells according to

[17] or

[18] , wherein the AMPK activator is one or more substances selected from the group consisting of AICAR, A769662, and metformin.

[20] The kit for inducing naive pluripotent stem cells according to any one of

[17] to

[19] , wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

[21] The kit for inducing naive pluripotent stem cells according to

[20] , wherein the primed pluripotent stem cells are human-derived primed iPS cells.

[22] A naive pluripotent stem cell inducer, comprising a p38 MAPK activator, for inducing primed pluripotent stem cells into naive pluripotent stem cells. [Effects of the Invention]

[0008] The present invention provides a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, a method for producing naive pluripotent stem cells from primed pluripotent stem cells, as well as a kit for inducing naive pluripotent stem cells and a naive pluripotent stem cell inducer that can be used in these methods. [Brief explanation of the drawings]

[0009] [Figure 1A] Scheme of naive reversion protocol. [Figure 1B] Mouse primed epithelial stem cells (mEpiSCs) maintained with FGF2 and Activin A. Upper panel: cell morphology and OCt4-GFP expression. Lower panel: FACS analysis of Oct4-GFP and PECAM1. SSC; side scatter. Scale bar, 200 μm. [Figure 1C]Cell morphology and Oct4-GFP expression levels after 16 days of treatment with Basal medium alone, 2 μL, AICAR, or AICAR + LIF. Four pairs of phase-contrast and Oct4-GFP images are shown for each condition. Scale bar, 200 μm. [Figure 1D] FACS analysis of Oct4-GFP and PECAM1 expression 16 days after reversion. The percentages in the figure represent the percentage of PECAM1-positive cells in the Oct4-GFP-positive population. [Figure 1E] Quantitative evaluation of the percentage of Oct4-GFP positive cells among all cells and the percentage of PECAM1 positive cells among Oct4-GFP positive cells (n=5; ND; not detected). [Figure 2A] Cell morphology and Oct4-GFP expression in AICAR-induced and AICAR+LIF-induced revertants. Scale bar, 200 μm. [Figure 2B] FACS analysis (d16+10p) of Oct4-GFP and PECAM1 expression in maintained primed mEpiSCs (Oct4GIP) and revertants induced with AICAR or AICAR+LIF. The percentages in the figure represent the percentage of Oct4-GFP- or PECAM1-positive cells among all cells. [Figure 2C] Proliferation of revertants induced by AICAR or AICAR + LIF. The cell numbers of revertants from passages 5 to 10 were compared with those of naive mESCs maintained at 2 μg / L (mean ± SD; n = 5). [Figure 2D] Alkaline phosphatase positive (AP+) colony formation assay. Mean ± SD; n=5, NS: not significant. [Figure 2E]Expression of naive and pluripotent marker genes in naive mESCs, primed mEpiSCs (Oct4GIP), and revertants induced with AICAR or AICAR + LIF. Naive mESCs: Rex1-GFP cells maintained in 2iL. Expression levels were normalized to GAPDH. Data are presented as mean ± SD (n = 5; with technical triplicates). The expression level in naive ESCs was set to 1. [Figure 2F] Immunofluorescence staining of naive and pluripotent markers in naive mESCs (Rex1-GFP), primed mEpiSCs (Oct4GIP), and revertants induced with AICAR or AICAR+LIF. Scale bar, 20 μm. [Figure 3A] Principal component analysis of PCF gene signatures (2036 genes) in different cell types. [Figure 3B] Heatmap of gene expression of pluripotency regulators and lineage markers. [Figure 4] Morphology of primed mEpiSC 129 / Ba1 and revertant 129 / Ba1 cells induced with AICAR or AICAR+LIF. Scale bar, 200 μm. [Figure 5A] FACS analysis of cells treated with AICAR+LIF or AICAR+LIF+p38 inhibitor (p38i; SB203580 (10 μM)). The percentages in the figure indicate the proportion of PECAM1-positive cells in the Oct4-GFP-positive population. [Figure 5B] FACS analysis of bulk cells cultured in 2iL for 7 days after reversion with AICAR+LIF or AICAR+LIF+p38i. The percentages in the figure indicate the proportion of PECAM1-positive cells in the Oct4-GFP-positive population. [Figure 5C] AP staining of bulk cells cultured in 2iL for 7 days after reversion with AICAR+LIF or AICAR+LIF+p38i. [Figure 5D]Representative cell morphology and Oct4-GFP expression levels after 16 days (d16) of Dox (1 μg / mL) and / or LIF treatment. Scale bar, 200 μm. [Figure 5E] FACS analysis of Oct4-GFP and PECAM1 expression 16 days (d16) after Dox and / or LIF treatment. The percentages in the figure indicate the proportion of PECAM1-positive cells in the Oct4-GFP-positive population. [Figure 5F] Representative cell morphology and Oct4-GFP expression of cells grown in 2iL conditions and then reverted to Dox+ or LIF+Dox+ conditions (d16+10p). Scale bar, 200 μm. [Figure 5G] FACS analysis of Oct4-GFP and PECAM1 in cells reverted with Dox+ or LIF+Dox+ (day 16+10p). The percentages in the figure indicate the proportion of Oct4-GFP- or PECAM1-positive cells among all cells. [Figure 5H] Immunofluorescence staining of naive and pluripotent markers in cells reverted with Dox+ or LIF+Dox+ (d16+10p). Scale bar, 20 μm. [Figure 6A] Quantitative evaluation of AMPK activation in primed mEpiSCs (Oct4GIP) by three AMPK activators: AICAR (1 mM), A769662 (50 μM), and metformin (1 mM). Western blot analysis results were quantified as the p-AMPK / AMPK density ratio. The value for the control sample was set to 1. p-AMPK: phosphorylated (activated) AMPK; AMPK: total AMPK. Mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, One-way ANOVA followed by Tukey's multiple comparison test. [Figure 6B] Cell morphology and Oct4GIP Oct-GFP expression after 16 days of treatment with A769662, A769662 + LIF, or metformin + LIF. Phase contrast and Oct4-GFP images. Scale bar, 200 μm. [Figure 6C]Quantitative evaluation of Oct4-GFP positive cells among all cells obtained by FACS analysis. n=4; ND: not detected. [Figure 6D] Quantitative evaluation of PECAM1-positive cells among Oct4-GFP-positive cells obtained by FACS analysis. n=4; ND: not detected. [Figure 7A] Cell morphology and Oct4-GFP expression after growth in 2iL of revertants with A769662, A769662 + LIF, or A769662 + LIF (d16 + 10p). Scale bar, 200 μm. [Figure 7B] FACS analysis of Oct4-GFP and PECAM1 in cells reverted with A769662, A769662 + LIF, or metformin + LIF (d16 + 10p). The percentages in the figure indicate the proportion of Oct4-GFP-positive or PECAM1-positive cells among all cells. [Figure 7C] Cell proliferation of revertants. The cell numbers of revertants during passages 5 to 10 were compared with those of naive mESCs maintained in 2 iL. Mean ± SD; n = 4. [Figure 7D] AP+ colony formation assay. Five hundred revertant cells (d16+10p) or naive mESCs were plated in 2 μL. After 5 days of culture, AP-stained colonies were counted. Mean ± SD; n = 4, NS: not significant. [Figure 7E] Expression of naive and pluripotent markers (qPCR) in naive mESCs, primed mEpiSCs (Oct4GIP), and reverted naive mESCs treated with A769662, A769662 + LIF, or metformin + LIF (d16 + 10p). Naive ESCs: Rex1-GFP cells maintained in 2iL. Expression levels were normalized to GAPDH. Data are shown as mean ± SD (n = 4; with technical triplicates). Results for naive mESCs were assigned a value of 1. [Figure 7F]Immunofluorescence staining of naive and pluripotent markers in revertants treated with A769662, A769662 + LIF, or metformin + LIF (d16 + 10p). Scale bar, 20 μm. [Figure 7G] Cell morphology of primed mEpiSC 129 / MSM and reverted 129 / MSM cells with AICAR or AICAR+LIF (d16+10p). Scale bar, 200 μm. [Figure 7H] AP staining of revertant 129 / MSM cells (d16+1p) cultured at 2i / L for 7 days. [Figure 8] Principal component analysis of the total gene signature (34,489 genes) in different cell types. [Figure 9A] Quantitative evaluation of p38 activation by AMPK activators. p-p38: phosphorylated p38 (Thr180 / Tyr182); p38: total p38. Western blot analysis results were quantified using the p-p38 / p38 density ratio. The value for the control sample was set to 1. p-AMPK: phosphorylated (activated) AMPK; AMPK: total AMPK. Mean ± SD, n=3, *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA followed by Tukey's multiple comparison test. [Figure 9B] FACS analysis of bulk cells cultured in 2iL for 7 days after reversion with A769662+LIF, A769662+LIF+p38i, metformin+LIF, or metformin+LIF+p38i. [Figure 9C] AP staining of bulk cells cultured in 2iL for 7 days after reversion with A769662+LIF, A769662+LIF+p38i, metformin+LIF, or metformin+LIF+p38i. [Figure 9D]Quantitative evaluation of p38 pathway activation by Dox-inducible (Dox-ON) constitutively active p38 (CA-p38). Western blot analysis of p-p38, p38, and β-actin was performed with or without doxycycline (1 μg / mL) treatment, and quantification was performed using the density ratio of p-p38 / p38. Control: Original Oct4GIP cells. The value of the control sample was set to 1. Mean ± SD, n=3, *p<0.05, **p<0.01, NS: not significant. [Figure 10] Naive reversion protocol using AICAR. [Figure 11] Images of naive hESCs (H1-EOS) induced by VPA (day 9 + 6p). VPA: valproic acid. Scale bar, 100 μm. [Figure 12] Flow cytometry analysis of EOS-GFP, SUSD2, CD75, and CD57 expression. [Figure 13] EOS-GFP-positive naive-like colony after growth in PXGL (day 14+14p). Scale bar, 100 μm. [Figure 14] RT-qPCR analysis of sorted SUSD2+CD75+ cells and parental primed hESCs (H1-EOS). [Figure 15] Immunofluorescence staining of OCT4, NANOG, KLF17, and TFE3 in primed and AICAR-induced cells. Scale bar, 50 μm. [Figure 16] TMRE staining of mitochondria depends on mitochondrial membrane activity. Scale bar, 50 μm. [Figure 17] Images of naive hiPSCs (Ff-I14-EOS) induced with AICAR (day 14 + 7p). Scale bar, 100 μm. [Figure 18] AICAR-induced differentiation of naive-like cells. (A) Immunofluorescent staining of mesodermal markers (THY1, PDGFRβ). (B) Immunofluorescent staining of endodermal markers (SOX17, CXCR4). (C) Immunofluorescent staining of ectodermal markers (MAP2, TUJ1). Scale bar, 100 μm. [Figure 19]Immunofluorescence staining for H3K9me3. Scale bar, 50 μm. [Figure 20] Immunofluorescence staining for 5mC, 5hmC, and NANOG. AICAR: day 14+14p. Scale bar, 100 μm. [Figure 21] X chromosome inactivation analysis. Xa: Active type. Xi: Inactive type. [Figure 22] Flow cytometry analysis of EOS-GFP, SUSD2, and CD75 after treatment with a p38 inhibitor (SB203580). [Figure 23] Tet-inducible CA-p38 expression system (upper panel). Western blot analysis of p38 protein after Dox treatment (lower panel). [Figure 24] p-p38 expression analysis by Western blotting. Error bars indicate SE. n=3. [Figure 25] Naive reversion protocol using p38 activation. [Figure 26] Flow cytometry analysis of EOS-GFP, SUSD2, CD75, and CD57 expression after induction of CA-p38. [Figure 27] EOS-GFP-positive naive-like colonies after growth in PXGL. Scale bar, 100 μm. [Figure 28] RT-qPCR analysis of sorted SUSD2+CD75+ naive hESCs and primed hESCs (CA-p38H1-EOS). [Figure 29] Immunofluorescence staining for OCT4, NANOG, KLF17, and TFE3 of naive-like revertant cells. [Figure 30] Principal component analysis of gene expression of all human genes analyzed using RNA sequencing in various naive and primed hPSCs. [Figure 31] Heatmap of RNA expression for naive and primed gene sets. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Method for inducing naive pluripotent stem cells> A first aspect of the present disclosure is a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, comprising culturing the primed pluripotent stem cells in a medium containing an AMPK activator.

[0011] (pluripotent stem cells) Pluripotent stem cells are stem cells that have the pluripotency to differentiate into many cells present in a living organism and also have the ability to proliferate. Specific examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, ntES cells (nuclear transfer embryonic stem cells), mGS cells (multipotent germline stem cells), and EG cells (embryonic germ cells). Preferably, the pluripotent stem cells are iPS cells or ES cells.

[0012] The type of organism from which pluripotent stem cells are derived is not particularly limited. Pluripotent stem cells may be derived from cells of mammals, birds, reptiles, amphibians, fish, insects, etc. Examples of mammals include, but are not limited to, humans, non-human primates (monkeys, chimpanzees, gorillas, common marmosets, cynomolgus monkeys, etc.), rodents (mice, rats, guinea pigs, hamsters, etc.), dogs, cats, rabbits, cows, pigs, horses, goats, sheep, etc.

[0013] Pluripotent stem cells can be obtained by known methods. For example, iPS cells can be produced by introducing reprogramming factors into any somatic cells. Reprogramming factors are factors that, when introduced into somatic cells, can induce the somatic cells to become iPS cells. Examples of reprogramming factors include genes such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, as well as their gene products. Reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include, for example, WO 2007 / 069666, WO 2008 / 118820, WO 2009 / 007852, WO 2009 / 032194, WO 2009 / 058413, WO 2009 / 057831, WO 2009 / 075119, WO 2009 / 079007, WO 2009 / 091659, WO 2009 / 101084, WO 2009 / 101407, WO 2009 / 102983, WO 2009 / 114949, WO 2009 / 117439, WO 2009 / 126250, WO 2009 / 126251, WO Publication No. 2009 / 126655, International Publication No. 2009 / 157593, International Publication No. 2010 / 009015, International Publication No. 2010 / 033906, International Publication No. 2010 / 033920, International Publication No. 2010 / 042800, International Publication No. 2010 / 050626, International Publication No. 2010 / 056831, International Publication No. 2010 / 068 955, WO 2010 / 098419, WO 2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO 2010 / 115050, WO 2010 / 124290, WO 2010 / 147395, WO 2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Huangfu D, et al. (2008), Nat.Biotechnol.26:1269-1275, Shi Y, et al.,(2008),Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11: 197-203, R. L. Judson et al. (2009), Nat. Biotechnol., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106: 8912-8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-?174?, Han J, et al. (2010), Nature.463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9 etc. The combinations described therein are exemplified.

[0014] 注:原文中“Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74”疑似有误,我按照合理推测翻译为“167-?174?”,你可根据实际情况调整。The type of somatic cells used to produce iPS cells is not particularly limited. Somatic cells include somatic cells from fetuses (offspring), newborns (offspring), and both healthy and diseased somatic cells from mature individuals. Somatic cells may be cultured cells, including primary culture cells, passaged cells, and established cell lines. Specific examples of somatic cells include: (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0015] (Naive pluripotent stem cells) Naive pluripotent stem cells are pluripotent stem cells that have properties identical to or similar to those of preimplantation embryos. Specifically, naive pluripotent stem cells have the following characteristics: (n1) Shows a dome-shaped colony morphology. (n2) Implantation into an embryo to form a chimera. (n3) Has alkaline phosphatase activity. (n4) Chromosomal DNA methylation levels are lower than those of primed pluripotent stem cells. (n5) Both X chromosomes are activated: XaXa. (n6) Lysine residues of histone H3 tend to be hypomethylated, and no foci are observed when immunostaining for H3K9me3 is performed. (n7) Naive markers (Rex1, Klf4, Klf2, Tfcp2l1, Stella, CD75, SUSD2, etc.; in mouse pluripotent stem cells, in addition to the above, PECAM1, Esrrb, etc.) are expressed. (n8) Higher mitochondrial activity than primed pluripotent stem cells. Stained with tetramethylrhodamine methyl ester (TMRM). (n9) Preferential use of the distal enhancer in transcription of the OCT3 / 4 gene. (n10) Expresses pluripotency markers (Oct3 / 4, Nanog, Sox2, etc.).

[0016] (primed pluripotent stem cells) Primed pluripotent stem cells are pluripotent stem cells that have properties identical to or similar to those of the epiblast of a post-implantation embryo. Conventional iPS cells and human ES cells, which are obtained by introducing reprogramming factors into somatic cells, are usually primed pluripotent stem cells. Specifically, primed pluripotent stem cells have the following characteristics: (p1) Shows flat colony morphology. (p2) Implantation into embryos does not result in chimeras. (p3) No alkaline phosphatase activity. (p4) Chromosomal DNA methylation levels are higher than in naive pluripotent stem cells. (p5) Only one X chromosome is activated: XaXi. (p6) Lysine residues of histone H3 tend to be methylated, and foci are identified when immunostaining for H3K9me3 is performed. (p7) Naive markers (PECAM1, Rex1, Klf4, Klf2, Esrrb, Tfcp2l1, Stella, CD75, SUSD2, etc.) are not expressed. (p8) expresses the prime marker (CD57 for human pluripotent stem cells). (p9) Mitochondrial activity is lower than that of naive pluripotent stem cells. They do not stain with tetramethylrhodamine methyl ester (TMRM). (p10)Preferential use of the proximal enhancer in transcription of the OCT3 / 4 gene. (p11) Expresses pluripotency markers (Oct3 / 4, Nanog, Sox2, etc.).

[0017] (n10) and (p11) are common features of naive pluripotent stem cells and primed pluripotent stem cells.

[0018] When naive pluripotent stem cells are induced from primed pluripotent stem cells, the primed properties (p1) to (p10) above are lost and replaced by naive properties (n1) to (n9) above. The naive pluripotent stem cells obtained by the method of this embodiment have at least one of the properties (n1) to (n9) above, preferably three or more, more preferably five or more, even more preferably seven or more, and particularly preferably all of the properties (n1) to (n9). Furthermore, it is preferable that they have the characteristic (n10).

[0019] The primed pluripotent stem cells used in the method of this embodiment are not particularly limited, as long as they are pluripotent stem cells in a primed state. Preferred primed pluripotent stem cells include, for example, primed ES cells and primed iPS cells.

[0020] (AMPK activator-containing medium) The method of this embodiment includes a step of culturing primed pluripotent stem cells in a medium containing an AMPK activator (hereinafter also referred to as an "AMPK activator-containing medium"). The AMPK activator-containing medium may be a medium prepared by adding an AMPK activator to a basal medium used for animal culture.

[0021] <Basal medium> The basal medium is not particularly limited, and any medium commonly used in animal culture can be used without particular limitation. Examples of basal media include, but are not limited to, Glasgow's MEM (GMEM) medium, N2B27 medium (NDiff 227 medium), IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM) medium, Ham's F12 (F12) medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. The medium may also contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like.

[0022] The basal medium may be selected depending on the species from which the pluripotent stem cells are derived. For example, in the case of mouse pluripotent stem cells, GMEM medium may be used as the basal medium, or GMEM medium supplemented with serum, serum substitute, NEAA, pyruvic acid, etc. may be used. In the case of human pluripotent stem cells, N2B27 medium may be used as the basal medium.

[0023] AMPK activators An AMPK (5'-adenosine monophosphate-activated protein kinase) activator is a substance that activates AMPK. For example, an AMPK activator has the function of phosphorylating AMPK, thereby activating AMPK through phosphorylation.

[0024] Known AMPK activators can be used without particular limitation. Examples of AMPK activators include AICAR (5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-imidazole-4-carboxamide), A769662 (6,7-Dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile), and metformin (N,N-dimethylimidodicarbonimidic diamide), as well as derivatives thereof. Metformin may be in the form of an acid salt, such as metformin hydrochloride. These compounds may be commercially available or may be prepared by the user.

[0025] One AMPK activator may be used alone, or two or more may be used in combination. The concentration of the AMPK activator in the AMPK activator-containing medium can be appropriately selected depending on the type of AMPK activator. The concentration of the AMPK activator in the AMPK activator-containing medium is, for example, 0.001 to 100 mM, and preferably 0.01 to 10 mM. When the AMPK activator is AICAR or metformin, the concentration in the AMPK activator-containing medium is preferably 0.5 to 5 mM. When the AMPK activator is A769662, the concentration in the AMPK activator-containing medium is preferably 0.01 to 0.1 mM.

[0026] ≪Optional ingredients≫ The AMPK activator-containing medium may contain optional components in addition to the AMPK activator, such as LIF, MEK inhibitors, Wnt inhibitors, and PKC inhibitors.

[0027] LIF The AMPK activator-containing medium preferably contains leukemia inhibitory factor (LIF). The organism from which LIF is derived is not particularly limited. Examples of LIF that can be used include human (Japanese Patent Publication No. 1-502985), mouse (Japanese Patent Publication No. 1-502985), sheep (Japanese Patent Publication No. 4-502554), pig (Japanese Patent Publication No. 4-502554), and bovine (Japanese Patent Publication No. 8-154681). Among these, human or mouse LIF is preferred. Examples of human LIF (NCBI Gene ID: 3976) include proteins having the amino acid sequence of NCBI accession numbers NP_001244064.1 and NP_002300.1. Examples of mouse LIF (NCBI Gene ID: 16878) include proteins having the amino acid sequence of NCBI accession numbers NP_001034626.1 or NP_032527.1. LIF can be selected appropriately depending on the organism from which the pluripotent stem cells are derived. For example, mouse LIF may be used when the pluripotent stem cells are derived from mouse. Human LIF may be used when the pluripotent stem cells are derived from human. LIF may be a fragment or a functional variant as long as it retains its function. Commercially available LIF may be used, or a protein purified from cells or a protein produced by genetic recombination may be used.

[0028] When the AMPK activator-containing medium contains LIF, the concentration of LIF in the AMPK activator-containing medium is, for example, 0.01 to 1000 ng / mL, preferably 0.1 to 500 ng / mL, more preferably 1 to 100 ng / mL, and even more preferably 1 to 50 ng / mL.The concentration of LIF in the AMPK activator-containing medium is, for example, 10 to 5000 U / mL, preferably 100 to 3000 U / mL, and even more preferably 500 to 2000 U / mL.

[0029] MEK inhibitors MEK (MAPK / ERK kinase) inhibitors are substances that inhibit the function of MEK, a phosphorylating enzyme in the mitotic cell proliferation signaling pathway (MAP kinase pathway) that occurs when growth factors bind to cellular receptors and reach the nucleus.

[0030] Known MEK inhibitors can be used without particular limitation. Examples of MEK inhibitors include PD0325901, PD184352, PD98059, U0126, and SL327, as well as derivatives thereof. These compounds may be commercially available or may be prepared by the user. MEK inhibitors may be antisense nucleic acids against MEK, RNA interference-inducing nucleic acids (e.g., miRNA, siRNA, shRNA), dominant-negative mutants, and expression vectors thereof. PD0325901 is preferred as the MEK inhibitor.

[0031] One MEK inhibitor may be used alone, or two or more may be used in combination. The concentration of the MEK inhibitor in the AMPK activator-containing medium can be appropriately selected depending on the type of MEK inhibitor. When the AMPK activator-containing medium contains a MEK inhibitor, the concentration of the MEK inhibitor in the AMPK activator-containing medium is, for example, 0.001 to 100 μM, and preferably 0.01 to 50 μM. When the MEK inhibitor is PD0325901, the concentration in the AMPK activator-containing medium is preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM.

[0032] Wnt inhibitors Wnt inhibitors are substances that suppress Wnt production or inhibit the signal transduction that follows from Wnt binding to its receptor and the accumulation of β-catenin. Examples of Wnt inhibitors include substances that inhibit binding to Frizzled family receptors or substances that promote the degradation of β-catenin.

[0033] Known Wnt inhibitors can be used without particular limitation. Examples of Wnt inhibitors include DKK1 protein (e.g., in the case of humans, NCBI accession number: NM_012242), sclerostin (e.g., in the case of humans, NCBI accession number: NM_025237), IWR-1, IWP-2, IWP-3, IWP-4, IWP-L6, C59 (or Wnt-C59), ICG-001, LGK-974 (or NVP-LGK-974), FH535, WIKI4, KYO2111, PNU-74654, and XAV939, as well as derivatives thereof. Among these, tankyrase inhibitors such as XAV939 and IWR-1 are preferred, with XAV939 being more preferred. These compounds may be commercially available or may be prepared by the user. The Wnt inhibitor may be an antisense nucleic acid against tankyrase, an RNA interference-inducing nucleic acid (for example, miRNA, siRNA, shRNA), a dominant-negative mutant, or an expression vector thereof.

[0034] One type of Wnt inhibitor may be used alone, or two or more types may be used in combination. The concentration of the Wnt inhibitor in the AMPK activator-containing medium can be appropriately selected depending on the type of Wnt inhibitor. When the AMPK activator-containing medium contains a Wnt inhibitor, the concentration of the Wnt inhibitor in the AMPK activator-containing medium is, for example, 0.001 to 100 μM, and preferably 0.01 to 50 μM. When the Wnt inhibitor is XAV939, the concentration in the AMPK activator-containing medium is preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM.

[0035] PKC inhibitors PKC inhibitors are substances that inhibit the function of PKC (protein kinase C). PKC is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues in substrate proteins. There are at least 11 isozymes, forming a large family. PKC is involved in the regulation of many cellular functions, including cell proliferation, cell death, gene transcription and translation, cell morphology, and cell-cell contact.

[0036] Any known PKC inhibitor can be used without any particular limitation. The PKC inhibitor may be any substance that has the effect of inhibiting at least one of the PKC isozymes. Examples of PKC inhibitors include Go6983 (3-[1-[3-(Dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF109203X (2-[1-(3-Dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), LY-333531((9S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-di(m etheno)dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione), Staurosporine ([9S-(9α,10β,11β,13α)]-2,3,10,11,12,13-Hexahydro-10-methoxy-9-methyl-11-(methylamino)-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3',2',1'-lm]pyrrolo[3,4-j][1,7]benzodiazonin-1-one), and derivatives thereof. PKC inhibitors may be antisense nucleic acids against PKC, RNA interference-inducing nucleic acids (e.g., miRNA, siRNA, shRNA), dominant-negative mutants, and expression vectors thereof. These compounds may be commercially available or may be prepared by the user. The PKC inhibitor is preferably Go6983.

[0037] One type of PKC inhibitor may be used alone, or two or more types may be used in combination. The concentration of the PKC inhibitor in the AMPK activator-containing medium can be appropriately selected depending on the type of PKC inhibitor. When the AMPK activator-containing medium contains a PKC inhibitor, the concentration of the PKC inhibitor in the AMPK activator-containing medium is, for example, 0.001 to 100 μM, and preferably 0.01 to 50 μM. When the PKC inhibitor is XAV939, the concentration in the AMPK activator-containing medium is preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM.

[0038] When the primed pluripotent stem cells are mouse pluripotent stem cells, the AMPK activator-containing medium may contain an AMPK activator and LIF.When the primed pluripotent stem cells are human pluripotent stem cells, the AMPK activator-containing medium may contain an AMPK activator, LIF, a MEK inhibitor, a Wnt inhibitor (e.g., a tankyrase inhibitor), and a PKC inhibitor.

[0039] The AMPK activator-containing medium may contain, in addition to the above components, a GSK3β inhibitor, a ROCK inhibitor, a growth factor (FGF, BMP, etc.), and the like.

[0040] (Culture method) The culture method is not particularly limited. Primed pluripotent stem cells can be cultured under culture conditions typically used for culturing animal cells. The culture temperature is not particularly limited, but can typically be 25 to 40°C, preferably 30 to 40°C. A specific example of the culture temperature is about 37°C. Primed pluripotent stem cells can typically be cultured in an atmosphere of CO2-containing air. The CO2 concentration can typically be about 0.3 to 5%, preferably about 2 to 5%. A specific example of the CO2 concentration is about 5%.

[0041] The culture may be either adherent culture or suspension culture. In the case of adherent culture, the culture vessel may be coated. Examples of coating materials include gelatin, collagen, laminin, fibronectin, and Matrigel. In the case of adherent culture, the pluripotent stem cells may be co-cultured with feeder cells or the like. Examples of feeder cells include mitomycin C-treated mouse embryonic primary fibroblasts (MEF), STO cells, SNL cells, OP9 cells, and C3H10T1 / 2 cells.

[0042] The culture period is not particularly limited and can be any period. The culture period may be determined by monitoring the state of the cells during culture and continuing until naive pluripotent stem cells are induced. Examples of culture periods include 1 day or more, 3 days or more, 5 days or more, 10 days or more, and 14 days or more. The upper limit of the culture period is not particularly limited, and examples include 50 days or less, 40 days or less, 30 days or less, and 25 days or less.

[0043] The medium may be changed as appropriate during the culture period. Medium change can be performed by removing the old medium and replacing it with a new medium containing an AMPK activator. The cells may be passaged as appropriate during the culture period. For passage, the cells may be dissociated using a cell dissociation solution containing enzymes such as protease, collagenase, peptidase, and DNase, and then seeded in a new medium containing an AMPK activator. The interval between passages is not particularly limited, but can be, for example, about 1 to 10 days. In the case of human pluripotent stem cells, a ROCK inhibitor (e.g., Y27632) may be added to the medium for the first 24 hours or so after passage.

[0044] (Optional process) The method of this embodiment may include any optional steps in addition to the step of culturing in a medium containing an AMPK activator (hereinafter also referred to as the "AMPK activation step"). Examples of the optional steps include a pre-culture step and a maintenance culture step of naive pluripotent stem cells.

[0045] ≪Preculture step≫ The pre-culture step is a step of culturing primed pluripotent stem cells prior to the AMPK activation step. Examples of the medium (pre-culture medium) used in the pre-culture step include known pluripotent stem cell maintenance media. Examples include a medium obtained by adding Activin A and FGF2 to the above-mentioned basal medium. A commercially available pluripotent stem cell maintenance medium may be used as the pre-culture medium. Examples of commercially available pluripotent stem cell maintenance media include StemFit (registered trademark) AK02N (Ajinomoto). The pre-culture medium does not contain an AMPK activator.

[0046] The pre-culture step can be carried out in the same manner as the AMPK activation step, except that the medium used is a pre-culture medium.

[0047] <<Naive pluripotent stem cell maintenance step>> After the AMPK activation step, a naive pluripotent stem cell maintenance step may be carried out, which allows the naive state to be stably maintained.

[0048] The medium used in the naive pluripotent stem cell maintenance step includes known naive pluripotent stem cell maintenance media. Examples of naive pluripotent stem cell maintenance media include media obtained by adding LIF, a MEK inhibitor, and a GSK3β inhibitor to the above-mentioned basal medium. Alternatively, examples include media obtained by adding LIF, a MEL inhibitor, a Wnt inhibitor, and a PKC inhibitor to the above-mentioned basal medium.

[0049] GSK3β inhibitors GSK-3β inhibitors are substances that inhibit the function of GSK (Glycogen Synthase Kinase) 3β, for example, kinase activity (for example, the ability to phosphorylate β-catenin). Examples of GSK-3β inhibitors include the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), phenyl α-bromo Examples of such inhibitors include the methyl ketone compound GSK-3β inhibitor VII (4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), and CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile), as well as derivatives thereof. These compounds may be commercially available or prepared by the researcher. The GSK-3β inhibitor may be an antisense nucleic acid against GSK-3β, an RNA interference-inducing nucleic acid (e.g., miRNA, siRNA, shRNA), a dominant-negative mutant, or an expression vector thereof. The GSK-3β inhibitor is preferably CHIR99021. One GSK-3β inhibitor may be used alone, or two or more may be used in combination. When the GSK-3β inhibitor in the naive pluripotent stem cell maintenance medium is CHIR99021, the concentration is, for example, 0.01 to 100 μM, preferably 0.1 to 50 μM, more preferably 0.5 to 10 μM, and even more preferably 1 to 5 μM.

[0050] In the case of mouse pluripotent stem cells, the naive pluripotent stem cell maintenance medium may be, for example, a medium prepared by adding LIF, a MEK inhibitor (e.g., PD0325901), and a GSK3β inhibitor (e.g., CHIR99021) to a basal medium (e.g., the Basal medium used in the Examples). The concentrations of LIF and the MEK inhibitor in the naive pluripotent stem cell maintenance medium may be the same as those in the AMPK activator-containing medium.

[0051] In the case of human pluripotent stem cells, the naive pluripotent stem cell maintenance medium may be, for example, a basal medium (e.g., N2B27 medium) supplemented with LIF, a MEK inhibitor (e.g., PD0325901), a Wnt inhibitor (e.g., XAV939), and a PKC inhibitor (e.g., Go6983). The concentrations of LIF, MEK inhibitor, Wnt inhibitor, and PKC inhibitor in the naive pluripotent stem cell maintenance medium may be the same as those in the AMPK activator-containing medium.

[0052] The naive pluripotent stem cell maintenance medium may be a medium containing at least one inhibitor selected from the group consisting of a MEK inhibitor, a Wnt inhibitor, a PKC inhibitor, and a GSK3β inhibitor, and LIF. The naive pluripotent stem cell maintenance medium preferably contains two or more of the inhibitors. Examples of combinations of inhibitors include a combination of a MEK inhibitor and a GSK3β inhibitor, and a combination of a MEK inhibitor, a Wnt inhibitor, and a PKC inhibitor.

[0053] The naive pluripotent stem cell maintenance step can be carried out in the same manner as the AMPK activation step, except that the medium used is a naive pluripotent stem cell maintenance medium.

[0054] The method of this embodiment allows for the induction of naive pluripotent stem cells from primed pluripotent stem cells. Conventional methods for inducing naive pluripotent stem cells often use two or more drugs as inducers of the naive state. However, the method of this embodiment allows for the induction of naive pluripotent stem cells by using only one drug, an AMPK inhibitor, as the inducer of the naive state.

[0055] In one embodiment, the method of this aspect may be a method of inducing primed pluripotent stem cells to naive pluripotent stem cells, comprising the step of activating p38 MAPK in primed pluripotent stem cells.

[0056] p38 MAPK (Mitogen-activated Protein Kinase) is a downstream target of activated AMPK. Therefore, p38 MAPK can be activated by using an AMPK activator. p38 MAPK is a type of MAPK and is involved in cell differentiation, apoptosis, autophagy, etc. Activation of p38 MAPK occurs through phosphorylation of p38 MAPK. Therefore, activation of p38 MAPK can be confirmed by phosphorylation of p38 MAPK. Activation of p38 MAPK without the use of an AMPK activator can also induce naive pluripotent stem cells.

[0057] Examples of p38 MAPK activators include AMPK activators, inflammatory cytokines (e.g., IL-1β), endotoxin, etc. Alternatively, p38 MAPK activation may be induced by subjecting primed pluripotent stem cells to environmental stress (e.g., ultraviolet light, oxidative stress, heat shock stress), osmotic shock, or the like.

[0058] <Method for producing naive pluripotent stem cells> A second aspect of the present disclosure is a method for producing naive pluripotent stem cells, which includes a step of culturing primed pluripotent stem cells in a medium containing an AMPK activator (AMPK activator-containing medium) (AMPK activation step).

[0059] The AMPK activation step can be carried out in the same manner as the AMPK activation step in the induction method of the first embodiment.

[0060] The method of this embodiment may include any optional step in addition to the step of culturing in a medium containing an AMPK activator. Examples of optional steps include a pre-culture step and a naive pluripotent stem cell maintenance step. The pre-culture step can be carried out in the same manner as the pre-culture step in the induction method of the first embodiment. The naive pluripotent stem cell maintenance step can be carried out in the same manner as the naive pluripotent stem cell maintenance step in the induction method of the first embodiment.

[0061] The method of this embodiment may be a method for producing naive pluripotent stem cells, which includes the step of activating p38 MAPK in primed pluripotent stem cells. Methods for activating p38 MAPK include the same methods as those described above.

[0062] <Kit for inducing naive pluripotent stem cells> A third aspect of the present disclosure is a kit for inducing naive pluripotent stem cells. The kit of this aspect includes an AMPK activator. The kit of this aspect is used to induce primed pluripotent stem cells into naive pluripotent stem cells.

[0063] (AMPK activator) Examples of the AMPK activator include those similar to those mentioned above.

[0064] (Optional configuration) The kit of this embodiment may contain optional components in addition to the AMPK activator. Examples of optional components include a basal medium, LIF, a MEK inhibitor, a Wnt inhibitor (such as a tankyrase inhibitor), and a PKC inhibitor. Specific examples of these components include those listed above. These components can be used together with the AMPK activator to prepare an AMPK activator-containing medium.

[0065] The kit of this embodiment may include a GSK3β inhibitor. The GSK3β inhibitor can be used together with a basal medium, LIF, a MEK inhibitor, and the like to prepare a naive pluripotent stem cell maintenance medium.

[0066] The kit of this embodiment may include FGF2. The kit of this embodiment may include Activin A. These may be used together with the basal medium to prepare a pre-culture medium.

[0067] The kit of this embodiment may also include a ROCK inhibitor (Y27632), a cell dissociation agent, and the like, which can be used when passaging the cells. The kit of this embodiment may further include a culture plate, instructions for use, and the like.

[0068] The kit of this embodiment may contain, in addition to the above, other inhibitors such as an ERK (extracellular-signal related kinase) inhibitor or a RAF inhibitor.

[0069] The kit of this embodiment can be used to carry out the method of the first embodiment and the production method of the second embodiment.

[0070] <Naive pluripotent stem cell inducer> A fourth aspect of the present disclosure is a naive pluripotent stem cell inducer. The naive pluripotent stem cell inducer of this aspect contains a p38 MAPK activator as an active ingredient. The naive pluripotent stem cell inducer of this aspect is used to induce primed pluripotent stem cells into naive pluripotent stem cells. Examples of p38 MAPK activators include those similar to those described above. [Example]

[0071] The present invention will be described based on examples, but the embodiments of the present invention are not limited to the descriptions of these examples.

[0072] (1) Reversion of mouse epiblast stem cells to naive type Materials and Methods (reagents, etc.) The main reagents used are shown in Tables 1 to 4.

[0073] [Table 1]

[0074] [Table 2]

[0075] [Table 3]

[0076] [Table 4]

[0077] (cell culture) Mouse epiblast stem cells (mEpiSCs) Oct4GIP were cultured on fibronectin (Life technologies)-coated plates (10 μg / mL / cm ) as previously described (Ying and Smith, Methods Enzymol., 365 (2003), pp. 327-341; Guo et al., Development, 136 (2009), pp. 1063-1069). 2) for 1 hour at 37°C. Briefly, cells were cultured in Ndiff 227 (TaKaRa Bio) medium supplemented with FGF2 (12 ng / mL, WAKO) and activin A (20 ng / mL, R&D), dissociated with Accumax (Innovative Cell Technologies), and replated every 3–4 days. The medium was changed every 2 days. 129 / MSM was provided by Dr. Masaki Yagi (Yagi et al., Stem Cell Rep., 12 (2019), pp. 1113–1128). Briefly, male MSM / Ms mice were mated with female 129X1 / SvJ mice. Noon on the day when plugs were observed was considered embryonic day (E) 0.5. The day before embryo isolation, mitomycin C (Kyowa Hakko Kirin Co., Ltd.)-treated MEFs (feeders) were collected onto 0.2% gelatin-coated 24-well culture plates (True Line). At E6.5, the uterus was removed by cutting across the cervix and the junction of the two uterine tubes, and the embryos were placed in HEPES (GIBCO). The muscle layer was then removed, and the duodenum was dissected using a needle. Epiblasts were separated from the extraembryonic region and transferred to 24-well culture plates to induce EpiSCs. After epiblast outgrowth, they were passaged onto feeders in 24-well culture plates (p1), 6-well culture plates (p2), and 6 cm culture dishes (p3). 20% Knockout TM129 / MSM were cultured on MEF feeders in DMEM / F-12 medium (GIBCO) supplemented with serum replacement (KSR) (GIBCO), 0.1 mM MEM non-essential amino acids (NEAA) (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), penicillin / streptomycin, FGF2 (12 ng / mL), and Activin A (20 ng / mL). The medium was changed every 2 days. mEpiSCs 129 / Ba1 were obtained as previously described (Sugimoto et al., Stem Cell Rep., 4 (2015), pp. 744-757). Briefly, cells were grown on MEF feeders in DMEM / F-12 supplemented with 15% KSR, 0.1 mM NEAA, 0.1 mM 2-mercaptoethanol, penicillin / streptomycin, FGF2 (12 ng / mL), and activin A (20 ng / mL). The medium was changed every two days. Naive mouse embryonic stem cells (mESCs) and naive-like cells regenerated from mEpiSCs were cultured in Basal medium on 0.1% gelatin-coated dishes (Yamashita et al., Nature, 408 (2000), pp. 92-96; Ying et al., Nature, 453 (2008), pp. 519-523). Basal medium: GMEM (GIBCO) supplemented with 10% KSR (GIBCO), 1% fetal bovine serum (SAFC Biosciences), 0.1 mM NEAA, 1 mM sodium pyruvate (Sigma), 0.1 mM 2-mercaptoethanol, and penicillin / streptomycin. Alternatively, cells were cultured in Ndiff 227 medium supplemented with 1000 U / mL Lif (Millipore) and two small molecule inhibitors (1 μM PD0325901 (Sigma) and 3 μM CHIR99021 (Tocris)). Cultures were dissociated using Accumax (Innovative Cell Technologies) and passaged every 3–4 days. Medium was changed every 2 days.

[0078] (Reversion of mRpiSCs to naive form by AMPK activators) mEpiSCs dissociated with Accumax were plated on MEF feeders in Ndiff 227 medium supplemented with FGF2 (12 ng / mL) and Activin A (20 ng / mL) for 1 day (d-1-d0). On d0, the medium was changed to Basal medium with the combination of reagents shown in Figure 1A and Table 6, and cultured for 16 days (d0-d16). The AMPK activators used were AICAR (1 mM, diluted in D2W, WAKO), A769662 (50 μM, diluted in DMSO, ADooQ), and metformin (1 mM, diluted in D2W, TCI). The medium was changed every 2 days. Because AICAR has antiproliferative properties and prolonged culture with AICAR reduces cell numbers, the AICAR concentration was reduced to 0.5 mM for the first 6 days (d0-d6). After 16 days, all cells were dissociated using Accumax for further analysis. Alternatively, cells were cultured in gelatin-coated 6-well plates using Basal medium or Ndiff227 medium supplemented with 2 μL (1000 U / mL LiF (Millipore)) and two small molecule inhibitors: 1 μM PD0325901 (Sigma) and 3 μM CHIR99021 (Tocris). The medium was changed every other day, and cells were passaged at least three times, every 4–7 days, until stable colonies emerged. The p38 inhibitor SB203580 (10 μM, Wako) was added 1–2 h before AMPK activator treatment.

[0079] (FACS analysis) The cells were washed twice with PBS, collected with Accumax, and stained with allophycocyanin (APC)-conjugated anti-CD31 (PECAM1) MoAb (BD) and DAPI (Invitrogen). Flow cytometry analysis was performed using a FACSAria. TM All FACS experiments were repeated at least three times.

[0080] (Immunostaining and alkaline phosphatase staining) Immunostaining was performed as previously described (Yamashita et al., Nature, 408 (2000), pp. 92–96). Briefly, cells were fixed with 4% paraformaldehyde for 15–20 min, washed three times with PBS, and blocked with 2% skim milk (BD) for 30 min. Cells were incubated with primary antibodies overnight at 4°C. The next day, cells were washed three times with PBST (PBS + 0.02% Tween 20, Nacalai Tesque) and treated with 1:500 diluted Alexa488- or Alexa546-conjugated secondary antibodies (anti-mouse rabbit or goat IgG antibodies) at room temperature for 1 h. Nuclei were stained with DAPI. The following antibodies were used at the indicated dilutions: anti-Oct3 / 4 (Santa Cruz, sc-5279, 1:200), anti-Nanog (ReproCell, RCAB002P-F, 1:300), anti-KLF4 (R&D, AF3158, 1:500), anti-ESRRB (Perseus Proteomics, PP-H6705-00, 1:500), anti-TFCP2l1 (Invitrogen, PA5-34361, 1:400), anti-TFE3 (Sigma, HPA023881, 1:300), anti-FOXA2 (Merck Millipore, 07-633, 1:500), anti-Brachyury (R&D, AF2085, 1:500), and anti-Nestin (SemCell Technologies, 01418, 1:500). Alkaline phosphatase staining was performed using the AP Staining Kit II (Stemgent). Following the manufacturer's instructions, cells were fixed with 4% paraformaldehyde for 5 minutes, washed once with PBST, stained with Solution A+B+C for 10 minutes, and washed three times with PBS. For single-cell colony formation, 500 cells were plated in a 12-well plate and cultured for 5 days.

[0081] (RNA isolation and quantitative PCR) Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). cDNA was reverse transcribed from 1 μg of RNA using SuperScript III (Invitrogen). Quantitative PCR analysis was performed in duplicate using 1 / 50 of the StepOnePlus (Applied Biosystems) reverse transcription reaction with SYBR Green Master Mix (Applied Biosystems). All qPCR reactions were performed in triplicate in at least three independent experiments. GAPDH, an endogenous control, was used to normalize gene expression. All results are presented as mean ± SD.

[0082] (Western blot analysis) Cells were lysed in a sample buffer solution containing 2-mercaptoethanol (Nacalai Tesque). The cell lysate was treated with e-PAGEL Gel (ATTO) and electrophoretically transferred to a nitrocellulose membrane. Next, cells were blocked with Blocking One (Nacalai Tesque) for 30 minutes and incubated overnight at 4°C with primary antibodies targeting the following: phospho-AMPK (Thr172, Cell Signaling, 2531S, 1:1000); AMPK (Cell Signaling, 2532S, 1:1000); phospho-p38 (Thr180 / Tyr182, Cell Signaling, 9215S, 1:1000); p38 (Cell Signaling, 9212S, 1:1000); and β-actin (Sigma, A5441, 1:5000). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-mouse (Invitrogen, 62-6520, 1:50,000) and rabbit IgG (Cell Signaling, 7074S, 1:3,000-1:1,000). After 2 hours of incubation at room temperature, the antibodies were visualized using Immobilon western chemiluminescent substrate (Millipore) and detected using an ImageQuant LAS4000. All primary and secondary antibodies were diluted with the Can Get Signal Immunoreaction Enhancer Solution Kit (Toyobo). Blot results were quantified using Image J.

[0083] (Generation of cell lines with constitutively active p38) p38 cDNA containing the D176A and F327S mutations (Xu et al., Cell Res., 23 (2013), pp. 131–141; Diskin et al., J. Biol. Chem., 279 (2004), pp. 47040–47049) was inserted into a PiggyBac (PB) vector carrying rtTA expression coupled with mCherry (Tanaka et al., PLoS One, 8 (2013), p. e61540). The Tet-On CA-p38 with mCherry gene was introduced into the Oct4GIP cell line using the Nucleofector transfection system (Amaxa). Cells were plated in 6-cm dishes and maintained with FGF2 (12 ng / ml) and Activin A (20 ng / ml) for 3 days, after which G418 (400 μg / ml) was applied for selection. The induction of CA-p38 was confirmed by measuring mCherry expression. After 24 hours of Dox treatment from day 8 after transfection, mCherry + The cells were selected and maintained in Ndiff 227 medium supplemented with FGF2, Activin A, and G418 in the absence of Dox. Western blot analysis confirmed that the p38 pathway was activated under the dox+ condition (Figure 9D).

[0084] (Chimeric blastocyst injection and germline transmission) All animal experimental protocols were approved by the Kyoto University Animal Care and Use Committee. All animal experiments were conducted in accordance with the Kyoto University Animal Experiment Guidelines, which are in accordance with Japanese law and the "Guide for the Care and Use of Laboratory Animals." The male cell line 129 / Ba1, derived from 129 / Sv × C57BL / 6N mice, was used (Sugimoto et al., Stem Cell Rep., 4 (2015), pp. 744-757). Cells were precultured in MEF supplemented with FGF2 and Activin A for 1 day (d(-1)-d0), then switched to Basal medium supplemented with AICAR or AICAR + LIF (d0-d16). After 16 days, the cells were subcultured several times in 2iL conditions and then used for blastocyst injection. Host blastocysts were isolated from Slc:ICR female mice, and up to 12 revertant cells were injected into the blastocyst cavity using a piezo-electric micromanipulator (Primetech, Japan). Injected blastocysts were implanted into pseudopregnant females at 2.5 days after conception. Chimeric mice were identified by coat color. When the chimeric mice reached sexual maturity at 8 weeks after conception, they were mated with wild-type ICR mice to confirm germline transmission. The offspring, gray-coated mice, were considered to have undergone germline transmission.

[0085] (Image analysis) An inverted microscope equipped with a U-RFL-T (Olympus IX71) was used for bright-field and live-cell fluorescence imaging. An inverted confocal microscope (LSM700) (Zeiss) equipped with 405 nm, 488 nm, 556 nm, and 635 nm lasers was used for immunostaining imaging with a ×10 or ×20 objective.

[0086] (RNA sequencing) Sequencing libraries were constructed using 500 ng of total RNA using TruSeq Stranded mRNA Library Prep (Illumina, Inc.) and sequenced on a HiSeq2500 in 79-cycle single-read mode. All reads that passed the quality filter were extracted to FASTQ format and demultiplexed into individual cells by barcode using BCL2 FASTQ Conversion Software v2.20.0.422. FASTQ-converted reads were mapped to Ensembl GRCm38 release 100 reference cDNA and ncRNA sequences using Bowtie2 v2.2.5 with the very-sensitive-local option. Of the 68,196,501 reads, 62,621,611 reads (91.8%) were mapped and quantified as genes with a MAPQ score ≥ 1 threshold. A total of 34,489 genes (average 21,500) were detected in the 22 samples. The following analyses were performed in R ver. 3.6.3 after unclassified Limma Voom normalization (Law et al., 2014). A total of 22 samples were analyzed: naive ESCs, primed EpiSCs, revertants (d16+2p, 3p, 10p), and revertant cells (d8, 16). Principal component analysis (PCA) of the 22 samples for 2,036 (excluding BC052688) and 4,489 total genes of pluripotent cell fate (PCF) genes (Fidalgo et al., Cell Stem Cell, 19 (2016), pp. 1-15) is shown in Figure 3A and Figure 8, respectively. A heatmap (Figure 3B) was generated for the gene set selected for pluripotency regulators and lineage markers (Takashima et al., Cell, 158 (2014), pp. 1254-1269).

[0087] (quantification analysis, statistical analysis) For experiments comparing differences between different groups, one-way analysis of variance with Tukey's multiple comparison test was used. Differences were considered significant when p-values ​​were <0.05. All experiments were performed independently at least three times. Image J was used to quantify Western blot analysis results. Error bars indicate mean ± SD (standard deviation).

[0088] <Result> AMPK activators induce primed mEpiSCs to revert to naive-like cells. We used mEpiSCs, Oct4GIP, harboring an Oct4 promoter / enhancer-driven eGFPiresPuro transgene (Oct4-GFP), which is expressed in primed and naive pluripotent stem cells (Guo et al., Development, 136 (2009), pp. 1063-1069; Wray et al., 2010). Oct4GIP cells were cultured in Basal medium with various combinations of reagents (Figure 1A). Before switching to the medium for naive reversion, Oct4GIP cells were maintained in mEpiSC culture conditions, including serum-free Ndiff 227 medium supplemented with FGF2 and Activin A (Guo et al., Development, 136 (2009), pp. 1063-1069). Nearly all cells (99.2 ± 0.7%) were positive for Oct4-GFP and negative for PECAM1 / CD31, a naive ESC marker. These results indicated that these cells were primed (day 0; d0) (Figure 1B). When cells were cultured in Basal medium alone or in Basal medium supplemented with 2 μL of Basal medium (naive maintenance conditions), GFP expression disappeared within 5 days. After 16 days of culture (d16), all cells exhibited differentiated morphology (Figure 1C), and GFP expression disappeared (Figure 1D, Figure 1E). On the other hand, when cells were cultured with the AMPK activator AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), GFP expression disappeared around day 10, but some GFP expression reappeared by d16. +Colonies appeared (Fig. 1C). Under AICAR and LIF conditions, GFP + The emergence of colonies was more significantly induced (Fig. 1C). FACS analysis showed that in AICAR-only cultures, approximately 5% (5.34±3.27%) of the total cells expressed Oct4-GFP on day 16 (Fig. 1D, Fig. 1E). In the AICAR and LIF conditions, approximately 17% (17.1±11.6%) of the total cells expressed OCT4-GFP. Expression of naive ESC markers, PECAM1 / CD31 (Illich et al., Cell Rep., 15 (2016), pp. 1-14), was confirmed in Oct4-GFP-positive cells. OCT4-GFP + Approximately 9% (9.04 ± 3.88%) of the cells were PECAM1 positive in the AICAR-only condition, whereas approximately 72% (72.6 ± 9.23%) were PECAM1 positive in the AICAR and LIF-treated condition (Figure 1D, Figure 1E). The appearance of these double-positive cells indicates that AICAR treatment reverts primed mEpiSCs to a naive state.

[0089] Other AMPK activators, A769662 and metformin, were also tested and resulted in activation of the AMPK pathway (Figure 6A). On day 16 after treatment with A769662 alone, A769662 and LIF, or metformin and LIF, Oct4-GFP + The number of Oct4-GFP and PECAM1 double-positive cells was observed (Figure 6B). FACS analysis revealed that, although fewer than with AICAR treatment, Oct4-GFP and PECAM1 double-positive cells were detectable under A769662 alone, A769662 + LIF, or metformin + LIF treatment. These AMPK activators also demonstrated reversion activity to naive primed mEpiSCs (Figures 6C and 6D). These results suggest that AMPK activators, when used in combination with LIF, can more effectively induce reversion of primed mEpiSCs to the naive state.

[0090] (Revertant cells show characteristics of naive pluripotent stem cells) Next, we confirmed whether naive-like cells emerged from primed mEpiSCs treated with an AMPK activator fulfilled the criteria for naive mESCs. + On day 16, when cells emerged, the AMPK activator-supplemented medium was replaced with 2iL of Basal medium, which allows for the growth and maintenance of naive cells. After 10 passages (day 16 + 10p), compact, dome-shaped naive-like colonies uniformly expressing Oct4-GFP selectively grew under the 2iL condition (Figure 2A). FACS analysis revealed that primed mEpiSCs:Oct4GIP were positive for OCT4-GFP but negative for the naive marker PECAM1. Naive-like cells (revertants, day 16 + 10p) induced with AICAR alone or AICAR and LIF were mostly homogeneously positive for PECAM1 (Figure 2B). Examination of mRNA expression of pluripotency genes confirmed that many naive-specific genes, such as Rex1, Klf4, Klf2, Esrrb, and Tfcp2l1, were expressed in revertants at levels comparable to those in naive MESCs (Figure 2E). These naive-specific proteins were clearly and homogeneously expressed in the nuclei of revertants, similar to those in naive MESCs, but not in primed MEpiSCs (Figure 2F). Furthermore, we evaluated naive-like cells induced with other AMPK activators. Similar to those induced with AICAR, Oct4-GFP was expressed in revertants induced with A769662 alone, A769662 + LIF, or metformin + LIF (d16 + 10p). + Compact, dome-shaped, naive-like colonies that homogeneously expressed Oct4-GFP selectively emerged from the cells (Figure 7A). FACS analysis revealed that revertants induced by A769662 alone, A769662 + LIF, or metformin + LIF treatment (d16 + 10p) were mostly homogeneously positive for PECAM1 (Figure 7B). These cells were well maintained and proliferated under 2iL conditions (Figure 7C), and showed AP expression comparable to that of control naive mESCs. + Colony formation was observed (Figure 7D). Many naive-specific mRNAs and proteins were expressed at levels comparable to those in naive MESCs (Figures 7E and 7F).

[0091] Next, we evaluated the global gene expression of AMPK-induced revertants by RNA sequencing (Figure 8). The revertants were clearly distinct from primed mEpiSCs and similar to naive mESCs. Furthermore, the identified PCF (pluripotent cell fate) gene signature has been reported to clarify the separation between naive and primed pluripotent stem cells more clearly than global transcriptomes (Fidalgo et al., Cell Stem Cell, 19 (2016), pp. 1-15). Principal component analysis (PCF) of 2,036 PCF genes successfully distinguished naive from primed cells. After several passages of AMPK-induced cells under 2iL conditions (d16+2p, 3p, 10p), all of these revertants belonged to a cluster similar to naive mESCs. This cluster was mutually exclusive with primed mEpiSCs and the bulk cell population treated with an AMPK activator before culture in 2iL conditions (d8 and d16) (Figure 3A). The first principal component (PC1) captured much of the variability, demonstrating that revertants clearly corresponded to naive cells but not to primed cells (Figure 3A). Revertants showed a short distance from naive mEpiSCs after two or three passages in 2iL conditions (d16 + 2p, 3p), but after 10 passages (d16 + 10p), they were nearly identical to naive mEpiSCs. This result suggests that sufficient passage in 2iL conditions allows revertants to fully maintain their naive nature. Naive-like cells double-positive for Oct4-GFP and PECAM1 were also observed on d16 (Figure 1D, Figure 1E, Figure 6C, Figure 6D). The gene expression pattern revealed that PC1 was a small subset prior to passaging and expansion in 2iL and did not reflect the appearance of naive-like cells in the bulk. Indeed, by day 16, PC1 largely represented a major cell population differentiating and evolving from naive or primed pluripotent stem cells.A heatmap of mRNA expression from a separate gene panel for pluripotency regulators and lineage markers (Takashima et al., Cell, 158 (2014), pp. 1254–1269) revealed that naive-like cells induced with different AMPK activators shared expression patterns similar to those of naive mESCs but distinct from those of primed mEpiSCs (Figure 3B). For example, naive markers such as Esrrb, Zfp42 (also known as Rex1), Prdm14, Nr5a2, Tfcp2l1, and Klf2 were expressed at levels comparable to those of naive mESCs. On the other hand, lineage markers such as Emoes, T, Foxa2, GATA4, GATA6, and Sox17 were expressed at lower levels in revertants than in primed mEpiSCs. In addition to Oct4GIP mEpiSCs, we also examined another mEpiSC line, the 129 / MSM cell line, derived from male MSM / MSM mice mated with female 129X1 / SvJ mice (Yagi et al., Stem Cell Rep., 12 (2019), pp. 1113-1128). Both PCA analysis and heatmap analysis clearly demonstrated that 129 / MSM mEpiSCs were also successfully reverted to a naive state (Figures 3A, 3B, and 8). Therefore, it was confirmed that naive-like revertants induced with AMPK activators at least fulfill the in vitro criteria for naive mESCs.

[0092] (Revertants contribute to chimera formation and germline transmission) Next, we confirmed that AMPK-induced cells more clearly exhibit a naive state by contributing to chimera formation and germline transmission. We used two primed mEpiSC lines, the 129 / Ba1 and 129 / MSM cell lines, derived from 129 / Sv × C57BL / 6N (Sugimoto et al., Stem Cell Rep., 4 (2015), pp. 744-757). After treatment with AICAR alone or AICAR plus LIF, 129 / Ba1 cells maintained at 2iL for several passages exhibited naive-like colony morphology similar to Oct4GIP cells (Figure 4). The reverted 129 / Ba1 cells were injected into blastocysts of ICR female mice to examine their contribution to chimera formation. As a result, 129 / Ba1 cells exhibited naive-like colony morphology similar to Oct4GIP cells (Figure 4). Most offspring were successfully coat color chimeras. When male chimeric mice were mated with female ICR mice, Agouti-colored mice were born, demonstrating germline transmission of the 129 / Ba1 cell genome (Table 5). Cell morphology and AP staining assays of 129 / MSM cells showed normal naive-like cell appearance (Figures 7G and 7H), and chimera formation was observed (data not shown). These results suggest that AMPK activation can induce the reversion of primed mEpiSCs to naive mESCs.

[0093] [Table 5]

[0094] The reversion efficiencies of AMPK activators in several cell lines are summarized in Table 6. Successful reversion was defined as naive-like colonies obtained from primed mEpiSCs that could be maintained under 2iL conditions. AMPK activators (AICAR or A769662) alone were able to induce the emergence of naive-like cells from primed mEpiSCs. Addition of LIF, especially in combination with AMPK activators, increased the reversion efficiency to 100% in all primed mEpiSCs tested (Oct4GIP, 129 / Ba1, and 129 / MSM cell lines). These results demonstrate that AMPK activation contributes to the reversion of primed mEpiSCs to a naive pluripotent state.

[0095] [Table 6]

[0096] p38 is a key downstream target in the reversion of AMPK by AMPK activators. Next, we investigated the molecular mechanism underlying AMPK activator-induced reversion to naive cells. We previously demonstrated that p38 is a functional downstream pathway of AMPK signaling for maintaining naive pluripotency (Liu and Yamashita, Biochem. Biophys. Res. Commun., 509 (2019), pp. 24-31). Activation of p38 has been reported to promote the reprogramming of somatic cells into pluripotent stem cells (Xu et al., 2013). Therefore, we hypothesized that p38 may be involved downstream in AMPK activator-induced reversion. AMPK activators increased p38 phosphorylation compared with control cells (Figure 9A). First, we examined the effect of p38 inhibition on AICAR+LIF-induced reversion to naive cells. OCT4-GFP + The appearance of cells did not change significantly, but OCT4-GFP + PECAM1 in cells +After 16 days of reversion, the cells were significantly reduced by the p38 inhibitor (p38i), SB203580 (Figure 5A). Seven days after the medium was changed to 2iL conditions, the AICAR+LIF-induced cells expressed Oct4-GFP. + / PECAM1 + While the cells began to be condensed, the cells treated with AICAR, LIF, and p38i did not express Oct4-GFP. + The cells were not maintained (Figure 5B). AP staining, which was negative when primed mEpiSCs were cultured under 2iL conditions, was negative in AICAR+LIF+p38i-treated cells (Figure 5C). The appearance of naive-like cells induced by A769662+LIF or metformin+LIF was also blocked by SB203580 (Figures 9B and 9C). These results suggest that p38 signaling is involved in the reversion to naive cells, which involves AMPK activation.

[0097] Furthermore, we performed a gain-of-function experiment for p38. We generated the Oct4GIP cell line, which contains a tetracycline-inducible (Tet-ON) constitutively active form of p38 containing the D176A and F327S mutations (Xu et al., Cell Res., 23 (2013), pp. 131-141; Diskin et al., J. Biol. Chem., 279 (2004), pp. 47040-47049), and activated the p38 pathway with Dox treatment (Figure 9D). p38 activation alone or in combination with LIF significantly reduced the expression of Oct4-GFP cells after 16 days in basal medium. + The appearance of colonies was induced (Figure 5D). + In cells, PECAM1 + The cells were then cultured in 2 μL of medium (Figure 5E). Similar to the AMPK activator treatment experiment, homogeneous Oct4-GFP expression was observed even with p38 activation alone (dox+). +Naive-like colonies selectively appeared (Figure 5F). These cells were uniformly double-positive for Oct4-GFP and PECAM1 (Figure 5G) and showed clear expression of naive marker proteins (Figure 5H). As summarized in Table 6, the reversion efficiency of p38 activation was not as good as that of AMPK activators. These results indicate that p38 is an important downstream gene of AMPK for reversion to the naive state, but can partially reproduce AMPK-induced reversion.

[0098] (2) Reversion of human pluripotent stem cells to the naive state Materials and Methods (cell culture) The hiPSC line (FfI14) was provided by the Center for iPS Cell Research and Application, Kyoto University, and the hESC line (H1) was provided by WiCELL. Primed PSCs were maintained on Matrigel (Invitrogen, 1:60 dilution)-coated dishes in StemFit AK02N (Ajinomoto) (Kubara et al., 2018. Stem Cell Reports. 14;11(2):380-394). Cells were cultured in TrypLE. TM Select CTS TM The cells were passaged every 3 to 7 days using a 1000kJ / mL PBS (Gibco) as single cells. For the first 24 hours after passage, the ROCK inhibitor Y27632 (Fujifilm, 10 μM) was added to the medium.

[0099] Naive human PSCs were cultured in PXGL medium (Ndiff227 (Takara Bio) medium supplemented with PD0325901 (Sigma, 1 μM), XAV939 (Millipore, 2 μM), Go6983 (Fujifilm, 2 μM) (Bredenkamp et al., 2019. Stem Cell Reports. 1212-1222), human Lif (Fujifilm, 10 ng / ml), and penicillin / streptomycin (Meiji). Culture was performed on Matrigel-coated 6-well plates using mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeders.

[0100] AICAR (Fujifilm, 1 mM) was added to PXGL medium (Liu et al., 2019. Biophys. Res. Commun. 509, pp. 24-31; Liu et al., 2021. iScience. 25;24(7):102783), and VPA (Guo et al., 2017. Development. 144, 2748-2763) was added to Ndiff227 basal medium (supplemented with PD0325901 (Sigma, 1 μM), human Lif (Fujifilm, 10 ng / ml), and penicillin / streptomycin) to induce a naive state. Cells were then cultured in TrypLE. TM Select CTS TM The cells were passaged as single cells every 5–10 days on MEF feeder layers using a 1000 kJ / ml PBS (Gibco). ROCK inhibitor Y27632 (Fujifilm, 10 μM) was added to the medium for the first 24 hours after passage. The medium was changed daily.

[0101] (EOS-GFP and CA-p38 transfection) It has been reported that naive mouse ES cells and iPS cells, as well as naive human ES cells and iPS cells, preferentially utilize the distal enhancer (DE) for OCT4 transcription (Yeom et al., 2014, Development. 122, 881-894; Theunissen et al., 2014, Cell Stem Cell. 15, 471-487; Choi et al., 2016, Stem Cell Reports. 7, pp. 911-926). Primed mouse Epi-SCs, human ES cells, and iPS cells preferentially utilize the proximal enhancer (PE) (Tesar et al., 2007, Nature. 448, 196-199; Gafni et al., 2013, Nature. 504, 282-286). PB-EOS-C(3+)-EiP (EGFP-IRES-Puro) has been described as a reporter of DE-OCT4 transcription and has been used as a naive marker (Takashima et al., 2014, Cell. 158, 1254-1269; Hotta et al., 2009, Nat. Protoc. 4, 1828-1844). To monitor the conversion from naive to primed states in live human PSCs, we transfected the H1 human ES cell line (H1-EOS) and the Ff-I14 human iPS cell line (Ff-I14-EOS) with PB-EOS-C(3+)-EiP (EGFP-IRES-Pro). 24 hours after transfection, cells were selected with puromycin (Nacalai, 10 μg / ml) for 5 days. Transfected cells were maintained on Matrigel-coated dishes with StemFit AK02N (Ajinomoto).

[0102] To generate the CA-p38 H1-EOS cell line, H1-EOS cells were transfected with a tetracycline-inducible (Tet-ON) constitutively active form of p38 (CA-p38), containing the cDNA for mutant p38 (D176A and F327S) in a piggyBac (PB) vector carrying rtTA expression linked to mCherry (Xu et al., 2013. Cell Res. 23: 131-141; Liu et al., 2019. Biophys. Res. Commun. 509, pp. 24-31). After doxycycline (DOX) treatment, mCherry-positive cells were purified by FACS. Transfected cells were maintained on StemFit AK02N (Ajinomoto) Matrigel-coated dishes.

[0103] For piggyBac transposase, pHL-EF1a-hcPBase-iC-A was used. For electroporation, NEPA21 (NEPA GENE) was used.

[0104] (Flow cytometry) TrypLE TM Select CTS TM Cells were dissociated into single cells and stained with conjugated antibodies and 4',6-diamidino-2-phenylindole (DAPI). SUSD2 clone W5C5 (SUSD2-PE, BioLegend 327406), CD75 / CD75s clone ZB55 (CD75-APC, BD 566350), and CD57 clone NK-1 (CD57-PV421, BD 563896) were used for flow cytometry.

[0105] (immunostaining) Immunostaining was performed as previously described (Yamashita et al., 2000. Nature. 408, 92-96). Cells were fixed with 4% paraformaldehyde for 15 minutes and blocked with Blocking One Histo (Nacalai, 1:20 dilution) in PBS + 0.5% Triton for 1 hour. Cells were stained with primary antibodies diluted in PBS + 0.5% Triton and incubated overnight at 4°C. Secondary antibodies (anti-rabbit, mouse, or goat IgG antibodies) conjugated with Alexa488 or Alexa546 (Thermo) were diluted in PBS + 0.5% Triton and incubated for 1 hour at room temperature. Phosphate-buffered serine with Tween-20 was used for washing, and DAPI was used for nuclear staining.

[0106] (Western blotting) CA-p38 H1-EOS cells were lysed in a sample buffer containing 2-mercaptoethanol (ME) (Nacalai). Proteins in the whole cell lysate were separated using Blot™ Gel (Invitrogen) and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with Blocking One (Nacalai) for 30 minutes. The membrane was then incubated overnight at 4°C with the following primary antibodies: p38 (Cell Signaling (9212S), 1:1000); phosphorylated p38 (Thr180 / Tyr182, Cell Signaling (9215S), 1:1000); and β-actin (Sigma (A5441), 1:10000). Horseradish peroxidase (HRP)-conjugated anti-mouse-rabbit IgG antibody (Cell Signaling, 1:3000–1:1000) was used as the secondary antibody. The antibody was diluted using the Can Get Signal Immunoreaction Enhancer Solution kit (Toyobo). After incubation with the secondary antibody for 2 hours at room temperature, detection was performed using Immobilon western chemiluminescent substrate (Millipore).

[0107] (RNA isolation and RT-qPCR) Total RNA was isolated using the RNeasy Mini kit (Qiagen) and reverse transcribed using Super-Script III (Invitrogen). All qPCR reactions were performed using SYBR Green Master Mix (Applied Biosystems). Cells were normalized to the endogenous control RPS18.

[0108] (Cellular metabolic assay) Mitochondria were stained with tetramethylrhodamine, ethyl ester (TMRE, final concentration 20 nM, Life Technologies) for 10 minutes and analyzed by confocal microscopy.

[0109] (RNA sequencing) RNA sequencing was performed on primed H1-EOS, primed CA-p38 H1-EOS, AICAR-induced naive H1-EOS, CA-p38-induced naive CA-p38 H1-EOS, and VPA-induced naive H1-EOS. TM Select CTS TMNaive CD75+ / SUSD2+ cells were dissociated and sorted by FACS. Total RNA was isolated using the RNeasy Mini kit (Qiagen) and purified using the RNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer's instructions. Polyadenylated RNA (polyA) was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). RNA-seq libraries were generated using the SMART-Seq Stranded Kit (TaKaRa Bio) and sequenced on a Novaseq 6000. Reads were aligned to GRCh38.p13, and RSEM (RNA-Seq by Expectation-Maximization) was used to calculate reads per kilobase of transcript and per million mapped reads (RPKM).

[0110] For comparison, data from accessions ERP006823, SRP059279, SRP045911), SRP055810, and SRP074076 were downloaded from the European Nucleotide Archive (ENA).

[0111] All 29 fastq files were adapter-trimmed using cutadapt-1.15 using trim_galore-0.4.4_dev with the -stringency 3 option and mapped to Ensembl GRCh38 release 100 reference cDNA and ncRNA sequences using Bow-tie2 v2.2.5 with the very-sensitive-local option. Of a total of 1,800,071,774 reads (single or paired), 1,254,839,844 (69.7%) were successfully mapped and quantified as genes with a MAPQ score ≥ 1 threshold. A total of 61,222 genes (average 43,813) were detected across the 29 samples.

[0112] The following analysis was performed using R version 4.0.3 after unclassified limma voom normalization. A total of 26 samples from naive hESCs, primed hESCs, AICAR- and CA-p38-induced naive hPSCs were analyzed. Heat maps (Figure 28) were analyzed to depict selected gene sets of pluripotency regulators, naive-state, and primed-state related genes.

[0113] (In vitro differentiation) Naive cells were "reprimed" prior to in vitro differentiation. Naive cells were passaged on Matrigel-coated dishes in StemFit AK02N (Ajinomoto) for approximately 1 month. For endoderm differentiation, the medium was switched from StemFit AK02N (Ajinomoto) to mTeSR1 (STEMCELL Technologies), and the reprimed cells were cultured for 1 week. The mTeSR (STEMCELL Technologies) medium was then replaced with RPMI1640 (Gibco) + B27 medium supplemented with Activin A (R&D, 100 ng / ml) and Wnt3A (Proteintech, 25 ng / ml).

[0114] The next day, the medium was replaced with RPMI medium containing Activin A (R&D, 100 ng / ml) and 0.2% serum.

[0115] For mesoderm differentiation, reprimed cells were cultured to confluence in StemFit AK02N (Ajinomoto) media and then overlaid with Matrigel (Invitrogen, 1:60 dilution) diluted in mouse embryonic fibroblast conditioned medium (MEF-CM) supplemented with hbFGF (Fujifilm, 4 ng / ml) for 1 day. The next day, MEF-CM was replaced with RPMI 1640 (Gibco) + B27 medium (RPMI 1640, 2 mM L-glutamine, 1x1 B27 supplement without insulin) supplemented with Activin A (R&D, 100 ng / ml) for 24 hours. Then, human bone morphogenetic protein 4 (R&D, 10 ng / ml) and hbFGF (Fujifilm, 10 ng / ml) were added. The medium was then left unexchanged for 4 days. Finally, the medium was switched to RPMI1640 (Gibco) + B27 medium (RPMI1640, 2 mM L-glutamine, x1 B27), and the cells were cultured for one week.

[0116] For ectodermal differentiation, the reprimed cells were cultured in Ndiff227 (Takara Bio) supplemented with hbFGF (Fujifilm, 10 ng / ml), SB431542 (Tocris, 20 mM), and Noggin (R&D, 260 ng / ml) for 4 days, after which the medium was switched to Ndiff227 (Takara Bio) supplemented with hbFGF (Fujifilm, 10 ng / ml) and SB431542 (Tocris, 20 mM).

[0117] <Result> (AICAR treatment transforms human pluripotent stem cells into a naive state) We used PXGL medium (Ndiff227 medium supplemented with PD0325901 (1 μM), XAV939 (2 μM), Go6983 (2 μM), and human LIF (10 ng / ml)) as a maintenance medium for naive human PSCs. We investigated the effect of AMPK activation under these culture conditions (Figure 10).

[0118] The emergence of human naive PSCs was initially monitored by expression of the EOS-GFP reporter gene as a naive state marker. Human PSCs expressing the EOS-GFP reporter gene were generated using H1 human ESCs (H1-EOS) and Ff-I14 human iPSCs (Ff-I14-EOS). Treatment of H1-EOS cells with valproic acid (VPA), which has been reported to induce the transition from primed to naive human PSCs, successfully induced GFP-positive cells (Figure 11), demonstrating the validity of the EOS-expressing cell line. When these cells were treated with 1 mM AICAR in PXGL medium for 14 days, the emergence of a small population of GFP-positive cells was observed. Flow cytometry analysis confirmed the emergence of a small but distinct population of GFP-positive cells, distinct from cells treated with PXGL medium alone. Most cells were negative for the primed state marker CD57, while a subset of cells (less than 2%) were positive for the naive state markers CD75 and SUSD2 (Figure 12). After the emergence of GFP-positive cells (day 14 of induction), the cells were passaged and cultured for an additional week in a reduced concentration of AICAR (0.5 mM) (Figure 10). AICAR was then removed, and the cells were continued to grow in naive maintenance PXGL medium alone on MEF feeder cells. GFP-positive cells proliferated for over 4 months in PXGL medium without AICAR and exhibited naive-type, domed colonies (Figure 13). Under these culture conditions (PXGL with MEF feeder cells), their doubling time was 4–5 days. The CD75+ / SUSD2+ / GFP+ cell population was enriched after passage and clearly identified by flow cytometry. RT-qPCR analysis of FACS-purified CD75- and SUSD2-positive naive-like cells showed that the pluripotency markers Oct4 and Nanog were expressed in both primed H1-EOS, VPA-induced, and AICAR-induced naive-like cells. Meanwhile, the naive-state markers Klf4, Tfcp2l1, Stella, and Klf2 were expressed exclusively in naive-like cells (Figure 14). Immunofluorescence staining also revealed that OCT4 and NANOG were expressed in both primed H1-EOS and naive-like cells, whereas the naive marker KLF17 was expressed only in naive-like cells. Nuclear translocation of TFE3 has been reported to occur in naive-like cells (Betschinger et al., 2013. Cell. 153(2):335-47).Consistent with this, VPA- and AICAR-induced naive-like cells showed nuclear localization of TFE3, whereas primed H1-EOS cells showed cytoplasmic localization (Figure 15). TMRE (tetramethylrhodamine methyl ester) staining revealed increased mitochondrial activity, a characteristic of naive PSCs, in AICAR-induced naive-like cells compared with primed H1-EOS cells (Figure 16). These results suggest that AICAR-induced cells possess various characteristics of the naive state. Similarly, AICAR treatment successfully induced CD75+ / SUSD2+ / GFP+ dome-shaped naive-like colonies from hiPSCs (Ff-I14-EOS) (Figure 17).

[0119] (AICAR-induced naive-like human pluripotent stem cells have differentiation potential) The differentiation potential of AICAR-induced naive-like cells was evaluated by in vitro differentiation. Because the induction protocol used was developed for primed PSCs, AICAR-induced naive-like cells were "reprimed" by culturing them in primed cell culture medium AK02N for more than three passages. These reprimed cells showed differentiation into three germ cell lineages. When differentiated using a mesoderm induction method (a modified DD protocol (Uosaki et al., 2011. Nat. Rev. Mol. Cell Biol. 17, 155-169)), THY1- or PDGFRβ-positive mesodermal cells emerged (Figure 18(A)). Endoderm (SOX17- or CXCR4-positive) was induced by treatment with Activin A and Wnt3A (Kroon et al., 2008. Nat. Biotechnol. 26, 443-452) (Figure 18(B)). Neural differentiation was achieved using Noggin and SB431542 (Chambers et al., 2009. Nat. Biotechnol. 27, 275-280) and confirmed by immunostaining for TUJ1 and MAP2 (Figure 18C).

[0120] (state of the epigenome) Histone 3 lysine 9 trimethylation (H3K9me3) is a heterochromatin marker. H3K9me3 staining reveals foci formation in primed PSCs, whereas it disappears in naive PSCs (Takashima et al., 2014. Cell. 158, 1254-1269). Consistent with this, H3K9me3 foci were observed in primed H1-EOS but not in AICAR-induced naive-like cells (Figure 19). Human ICM cells, naive mouse ES cells, and naive human PSCs exhibit global DNA hypomethylation, whereas primed mouse Epi-SCs and primed human PSCs exhibit hypermethylation. Immunofluorescence staining of the DNA methylation markers 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) confirmed that their expression was significantly reduced in AICAR-induced naive-like cells (Figure 20). The X chromosome was XaXi (Xa: active X chromosome; Xi: inactive X chromosome) in primed cells, whereas it was XaXa in naive cells. Analysis of the X chromosome in AICAR-induced naive-like cells confirmed that it was XaXa (Figure 21). These results confirm that the addition of AICAR alone to PXGL maintenance conditions can induce primed human PSCs into cells that fulfill multiple criteria for the naive state.

[0121] p38 is a downstream gene of AICAR and can convert human pluripotent stem cells to a naive state. Next, we investigated the involvement of p38 in the AICAR-induced conversion to naive-like cells. Addition of the p38 inhibitor SB203580 together with AICAR suppressed AICAR-induced naive-like cell transformation. These results indicated that p38 is involved in the reversion to naive-like cells (Figure 22). Next, we examined whether p38 could induce reversion to the naive state. We generated human PSCs capable of activating the p38 pathway by drug-induced expression of constitutively active p38 (CA-p38). Tetracycline-inducible (Tet-ON) CA-p38 was transfected into H1-EOS (CA-p38-H1-EOS) cells. In these cells, p38 could be phosphorylated and activated by doxycycline (DOX) treatment (Figures 23 and 24). After 5 days of DOX treatment in PXGL medium to activate p38 (Figure 25), EOS-GFP-positive cell clusters began to be observed. Flow cytometry analysis confirmed the emergence of CD75+ / SUSD2+ / CD57- / GFP-dull cells (less than 2%), similar to AICAR treatment (Figure 26). CA-p38-induced GFP-positive cells were then cultured and expanded in PXGL medium on MEF feeder cells without DOX. CA-p38-induced cells were maintained for more than 4 months with a doubling time of approximately 4 days. After expansion, CA-p38-induced GFP-positive cells formed dome-shaped, naive-like colonies (Figure 27), and flow cytometry analysis confirmed EOS-GFP positivity and CD75 and SUSD2 positivity. CA-p38-induced CD75 and SUSD2-double positive cells were sorted by FACS and analyzed by RT-qPCR. These cells showed comparable expression of pluripotency markers (Oct4, Nanog) to primed cells (H1-EOS-GFP) and higher expression of naive markers (Klf4, Tfcp2l1, Stella, Klf2) than primed cells (Figure 28). These CA-p38-induced cells showed expression of pluripotency markers, naive markers, and nuclear localization of TFE3 (Figure 29). CA-p38-induced GFP-positive cells showed mitochondrial activation as determined by TMRE staining and naive-state characteristics in the epigenome similar to those of AICAR-induced naive-like cells (data not shown).The reprimed CA-p38-induced cells were confirmed to have the potential to differentiate into all three germ layers (data not shown), confirming that CA-p38 activation successfully induced naïve hESCs.

[0122] (Global gene expression analysis of primed and naive hPSCs) Previously published analytical data were obtained from: authentic naive HNES1 cells derived from human ICM (Guo et al., 2016); naive-like hPSCs converted from primed cells by various methods (Takashima et al., 2014. Cell. 158, 1254-1269; Theunissen et al., 2014. Cell Stem Cell. 15, 471-487; Sperber et al., 2015. Nat. Cell Biol. 17, 1523-1535; Guo et al., 2017. Development. 144, 2748-2763; Yang et al., 2017. Cell. 169, 243-257.e25); and their respective primed cells. We collected and compared RNA-seq data from naive-like cells induced with AICAR, CA-p38, or VPA with those from their parental primed cells. Principal component analysis (PCA) confirmed that HNES1 and chemically reset (VPA) cells were closely located. These cells were established in the same laboratory (Guo et al., 2016, Stem Cell Rep. 6, 437-446; Guo et al., 2017, Development. 144, 2748-2763). AICAR, CA-p38, or VPA-induced naive-like cells, as well as 5iL / A-induced naive-like cells (Theunissen et al., 2014, Cell Stem Cell. 15, 471-487), formed tightly packed clusters near the clusters of HNES1 and cR cells (Figure 30). EPS cells (Yang et al., 2017, Cell. 169, 243-257, e25) and NHSM / 4i-derived cells (Gafni et al., 2013, Nature. 504, 282-286), previously reported as types of naive-like cells, were located close to the primed cells. Heatmap analysis of 66 genes related to naive and primed pluripotency clearly confirmed that AICAR-induced naive-like cells and CA-p38-induced naive-like cells exhibited similar gene expression profiles to other naive-like cells, including HNES1 cells (Figure 31).These data indicated that the various naive-like cells formed distinct clusters from each of the parental primed cells (Fig. 31). These results demonstrate that activation of AICAR or p38 can induce reversion to the naive state in human primed PSCs. [Industrial Applicability]

[0123] The present invention provides a method for inducing primed pluripotent stem cells into naive pluripotent stem cells, a method for producing naive pluripotent stem cells from primed pluripotent stem cells, as well as a kit for inducing naive pluripotent stem cells and a naive pluripotent stem cell inducer that can be used in these methods.

[0124] While preferred embodiments of the present invention have been described and illustrated, it should be understood that these are exemplary of the present invention and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the scope of the appended claims.

Claims

1. A method for inducing primed pluripotent stem cells into naive pluripotent stem cells, comprising the step of culturing primed pluripotent stem cells in a medium containing one or more AMPK activators selected from the group consisting of AICAR, A769662, and metformin.

2. The method of claim 1 , wherein the medium further comprises LIF.

3. The method according to claim 1 or 2, wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

4. The method according to claim 3 , wherein the primed pluripotent stem cells are human-derived primed iPS cells.

5. The method according to claim 1 or 2, wherein p38 MAPK is activated in primed pluripotent stem cells.

6. The method according to claim 5 , wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

7. The method according to claim 6, wherein the primed pluripotent stem cells are human-derived primed iPS cells.

8. A method for producing naive pluripotent stem cells, comprising a step of culturing primed pluripotent stem cells in a medium containing one or more AMPK activators selected from the group consisting of AICAR, A769662, and metformin.

9. The method of claim 8 , wherein the culture medium further comprises LIF.

10. The production method according to claim 8 or 9, wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

11. The production method according to claim 10 , wherein the primed pluripotent stem cells are human-derived primed iPS cells.

12. The production method according to claim 8 or 9, wherein p38 MAPK in primed pluripotent stem cells is activated.

13. The production method according to claim 12 , wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

14. The production method according to claim 13 , wherein the primed pluripotent stem cells are human-derived primed iPS cells.

15. A kit for inducing naive pluripotent stem cells, for inducing primed pluripotent stem cells into naive pluripotent stem cells, comprising one or more AMPK activators selected from the group consisting of AICAR, A769662, and metformin.

16. The kit for inducing naive pluripotent stem cells according to claim 15, further comprising LIF.

17. The kit for inducing naive pluripotent stem cells according to claim 16, wherein the primed pluripotent stem cells are primed ES cells or primed iPS cells.

18. The kit for inducing naive pluripotent stem cells according to claim 17, wherein the primed pluripotent stem cells are human-derived primed iPS cells.

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